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Bioprocess Development for Asymmetric Reductions by Saccharomyces Cerevisiae Enzymes

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BIOPROCESS DEVELOPMENT FOR ASYMMETRIC REDUCTIONS BY Sachharomyces cerevisiae ENZYMES By PARAG ANIL PAREKH A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLOR IDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2006

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Copyright 2006 by PARAG ANIL PAREKH

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This document is dedicated to my Family.

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iv ACKNOWLEDGMENTS I would like to thank my parents for th eir continuous encouragement for my graduate education. I would also like to tha nk my brother, Nitin. Thanks go to all the Stewart group members.

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v TABLE OF CONTENTS page ACKNOWLEDGMENTS.................................................................................................iv LIST OF FIGURES..........................................................................................................vii ABSTRACT....................................................................................................................... ..x CHAPTER 1 BACKGROUND..........................................................................................................1 Oxidoreductases............................................................................................................1 Cofactors...................................................................................................................... .3 Mechanism of Hydride Delivery..................................................................................3 Asymmetric Reductions Usi ng Bakers’ Yeast Enzymes..............................................4 Applications of Baker’s Yeast Enzymes......................................................................5 Isolated Enzyme Catalysis.....................................................................................5 Substrate-coupled regeneration......................................................................5 Enzyme-coupled regeneration........................................................................6 Whole Cell Biocatalysis........................................................................................7 Long Term Objectives..................................................................................................8 2 RESULTS.....................................................................................................................9 Biotransformations.......................................................................................................9 Section I: Bioprocess Development fo r Reduction of Ethyl butyrylacetate.................9 Biotransformations with Non-growing Cells......................................................10 Strategy I: Centrifugation.............................................................................12 Strategy II: In situ product removal (ISPR).................................................13 Strategy III: Biphasic reactor (ATPS)..........................................................17 Bioprocess Development for the Redu ction of Ethyl butyrylacetate..................19 Section II: Bioprocess Development for Re duction of Ethyl 4-chloroacetoacetate...20 Biotransformations with Non-growing Cells......................................................21 Strategies to overcome substrate inhibition.................................................21 Slow substrate feed.......................................................................................21 Slow-release biocatalysis.............................................................................22 Addition of organic solvent to increase the solubility of the ECAA...........22 Addition of surfactants to the whole cells....................................................23 Biotransformations with Cell-free Extracts:........................................................24

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vi Biotransformation with cell free ex tracts under controlled pH conditions..26 Biotransformation using cell free extract in a two-phase system.................26 Section III: Bioprocess Development for Reduction of Ethyl acetoacetate...............28 Biotransformation with Non-growing Cells........................................................28 Metabolic Engineering........................................................................................30 Section IV: Bioprocess Development us ing Immobilised Cells and Enzymes..........32 Optimal Total Si: Buffer Ratio for Better Product Formation............................35 Different Composition of the Silica Gel Matrix for Better Product Formation..38 Effect of Amount of Cell Loading on the Product Formation.............................40 Conclusion..................................................................................................................42 3 EXPERIMENTAL......................................................................................................46 Materials.....................................................................................................................4 6 Gas Chromatography..................................................................................................46 Sample Preparation for GC.........................................................................................47 Glucose Assay............................................................................................................47 Media Preparation.......................................................................................................47 Cell Culturing.............................................................................................................48 Biotransformations with Non-growing Cells..............................................................48 Biotransformation with In situ Product Removal External Column..........................49 Biotransformation in Biphasic Reactor......................................................................49 Biotransformation with Cell-free Extracts..................................................................49 Biotransformation in Biphasic Medium with Cell-free Extracts................................49 Biotransformation with Immobilized Cells................................................................49 Product Recovery........................................................................................................50 LIST OF REFERENCES...................................................................................................52 BIOGRAPHICAL SKETCH.............................................................................................57

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vii LIST OF FIGURES Figure page 1-1 Enzyme catalyzed reductions.....................................................................................2 1-2 Enzyme catalyzed oxidations.....................................................................................2 1-3 Nicotinamide cofactors NAD+ and NADP+...............................................................3 1-4 Stereochemistry of hydride transfer from NAD(P)H to the carbonyl carbon on the substrate (S = small and L = large group) ...........................................................4 1-5 Cofactor recycling by th e coupled-substrate method ................................................6 1-6 Co-factor recycling by c oupled – substrate method ..................................................6 1-7 Cofactor recycling by coupled – enzyme method......................................................7 1-8 Principle of whole-cell reduction of carbonyl compounds .......................................8 2-1 Reduction of ethyl butyrylacetate..............................................................................9 2-2 Cell growth in a 4L New Brunswick fe rmentor and bioconversion carried out in 1L Braun Bioststat B fermentor...............................................................................10 2-3 Increasing substrate (EBA) feed rate.......................................................................11 2-4 No additional product formed af ter bolus addition of ethyl 3-hydroxy butyrylacetate at 1.25 hrs.........................................................................................12 2-5 Product removal by centrifugation. Whole cells resuspended in fresh aqueous media........................................................................................................................13 2-6 Experimental setup for in situ product removal.......................................................16 2-7 In situ product removal using an external co lumn packed with resin Amberlite XAD-4......................................................................................................................16 2-8 Biphasic reactor (aque ous two phase system)..........................................................18 2-9 Bioconversion in a biphas ic reactor containing BEHP as the organic phase...........19

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viii 2-10 Bioprocess development for the re duction of ethyl butyrylacetate..........................19 2-11 Reduction of ethyl 4-chloro acetoacetate.................................................................20 2-12: Slow decomposition of ECAA at pH 7 in aqueous medium.................................21 2-13 Reduction of ECAA by E.coli whole cells expressing different Bakers’Yeast reductases.................................................................................................................22 2-14 Comparison of product formation with and without organic solvent......................23 2-15 Coupled enzyme regeneration of NADP H using glucose dehydrogenase from B.subtilis...................................................................................................................24 2-16 Reduction of ECAA with cell free extracts with different ratios of reductase and glucose dehydrogenase (GDH)................................................................................25 2-17 Reduction ECAA with cell free extracts under pH controlled conditions...............26 2-18 Reduction of ECAA with cell free extracts in biphasic conditions..........................27 2-19 Bioprocess development for reduction of ECAA....................................................27 2-20 Reduction of ethyl acetoacetate................................................................................28 2-21 Major pathways of glucose metabolism in E.coli ....................................................29 2-22 Comparison of wild type and pgi knockout for product formation and glucose consumed per mole of product formed. A) OD 600 = 11. B) OD 600 = 14. C) OD 600 = 17......................................................................................................................31 2-24 Reactions occurring during formati on of sol-gel synthesis of silica........................34 2-25 Product mM / gm catalyst for the re duction of ethyl acetoacetate using immobilized whole cells in silica gel glass with different Si:Buffer ratio...............35 2-26 Productivity for the reduction of ethy l acetoacetate using immobilized whole cells in silica gel glass w ith different Si:Buffer ratio ..............................................36 2-27 Reuse of immobilized cells for the bi otransformation with different Si: Buffer ratio.......................................................................................................................... .36 2-28 Productivity of the reused immobilized cells with different Si:Buffer ratio............37 2-29 2nd Reuse of immobilized cells with different Si: Buffer ratio................................37 2-30 Productivity of the im mobilized cells after 2nd reuse with different Si: Buffer ratio.......................................................................................................................... .38

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ix 2-31 Product mM / gm catalyst for the re duction of ethyl acetoacetate using Immobilized whole cells in silica gel glass with di fferent NaSi:Ludox ratio..........38 2-32 Productivity for the reduction of ethy l acetoacetate using immobilized whole cells in silica gel glass with different NaSi:Ludox ratio..........................................39 2-33 Reuse of immobilized cells for the bi otransformation with different NaSi:Ludox ratio.......................................................................................................................... .39 2-34 Productivity of the reused immobilized cells different NaSi:Ludox ratio...............40 2-35 Product mM / gm catalyst for the re duction of ethyl acetoacetate using immobilized whole cells in silica gel glass with different amount of cell loading..41 2-36 Productivity for the reduction of ethy l acetoacetate using immobilized whole cells in silica gel glass with different amount of cell loading..................................41 2-37 Plug –flow column reactor......................................................................................42 3-1 Experimental set-up for conti nuous extraction for product recovery.......................51

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x Abstract of Thesis Presen ted to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science BIOPROCESS DEVELOPMENT FOR ASYMMETRIC REDUCTIONS BY Saccharomyces cerevisiae ENZYMES By Parag Anil Parekh May 2006 Chair: Jon D. Stewart Major Department: Chemistry Biotechnological methods are becoming increasingly important in industrial production of fine chemicals. But, the total number of compounds made from biocatalysis on an industrial scale is still limited due to disadvantages like long reaction times and low productivity associated with bioprocesses. We used Saccharomyces cerevisiae enzymes to asymmetrically reduce keto esters to chiral hydr oxyl esters, which are important building blocks as versatile chiral synthons for asymmetric synthesis of pharmaceuticals and agrochemicals. Different levels of consideration for de velopment of bioprocess were applied to achieve the maximum bioprocess efficiency. Biocatalyst Enzyme vs. Whole cells Form Free vs. Immobilized Medium Water vs. Organic

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xi We focused on reduction of three different substrates, ethyl butyryl acetate, ethyl 4chloro acetoacetate and ethyl acetoacetate. Different problems varying from product inhibition, substrate toxicity and poor substrat e solubility were tack led using techniques like in situ product removal, aqueous two-phase system reactions with cell-free extracts, etc. Reduction in the operating costs of the process was achieved by using immobilized cells and application of metabo lic engineering tools. Studies of these bioprocesses in nonconventional media like ionic liquids or other immobilization techniques like cross-linked enzyme aggregates will further improve the bioprocess efficiency of the system.

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1 CHAPTER 1 BACKGROUND Biocatalysis is an attractive alte rnative to chemical conversions.1-4 The reaction conditions are mild, the solvent is genera lly water and the bio catalysts are easily decomposed after use. Biocatalysis is ther efore environmentally fr iendly green chemistry and a sustainable technology. Additionally, bioc atalysis often circumvents protection and deprotection steps in the synthesis of products with chiral centers. These properties give biocatalysis an edge over chemical conversions. One of the main disadvantages encountered in biocatalysis is the application of biocatalysis at large scale, i.e. the scale-up of reacti ons and low space-time yield. 5 The biochemical engineering required to develop a mature process from early observations in the lab is demanding and ther efore the number of compounds made from biocatalysis is relatively limited. 6 The aim of this study is to apply various techniques to overcome these limitations and make a model bioprocess more efficient. Oxidoreductases Oxidoreducatases comprise approximate ly 25% of presently known enzymes. 7 Almost 30% of all industrial biocataly tic processes involve oxidoreductases. 6 They perform interesting reactions such as reduction, epoxi dation, hydroxylation, dihydroxylation etc. There are two major cate gories of oxidoreductases: dehydrogenases, also known as reductases, and oxygenases.

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2 Reductases are enzymes that reduce carbonyl groups yielding ch iral products like alcohols, acids or their esters or amino acids respectively. 8 Examples of these reactions are shown in Figure1-1. R R H OH + NAD R R O + NADH + H R H OH + NAD R O + NADH + H COOR1 COOR1 R H OH + NAD R O +NADH + H COOH COOH Figure 1-1. Enzyme catalyzed reductions Oxygenases use molecular oxygen as co-subs trate and are used for oxidation of non-functionalised C-H or C C bonds yielding hydroxyl or epoxy products. 9 Examples of these reactions are shown in Figure1-2. R R1 R R1 O monooxygenase R R1 R R1 diooxygenase OH OH or R R OH OH R O or or or R O O O Baeyer-Villiger Monooxygenases n n Epoxide Hydrolase H2O Figure 1-2. Enzyme catalyzed oxidations

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3 Cofactors Oxidoreductases require redox cofactors, or coenzymes, which accept or donate chemical equivalents for reduction or oxidation. Most oxidoreductases require Nicotinamide Adenine Dinucleotide (NAD+) or Nicotinamide Adenine Dinucleotide Phosphate (NADP+) as the cofactor; some require Flavin Adenine Dinucleotide (FAD+) or Flavin Mononucleotide (FMN). These cofact ors are very expensive and are relatively unstable. In the enzymatic reduction of a substrate, these cofactors participate in the redox reaction via the direct transfer of hydride ( H-) ions either to or from the cofactor and a substrate. Oxidoreductases acting as oxygena ses abstract a hydrid e ion from the donor and transfer it to the nicotinamide moiety. Ox idoreductases acting as reductases abstract a hydride ion from the reduced nicotinamide and transfer it to the carbonyl to reduce the substrate. P O P O O O O OH HO O O O O HO OR N N N N NH2 H N H2NOC NAD+ R = H NADP+ R = PO 3 -2 Figure 1-3. Nicotinamide cofactors NAD+ and NADP+ Mechanism of Hydride Delivery Hydride transfer is highly stereoselective. The delivery of the hydride from the si or re face of the carbonyl results in its reduction to yield either (R) or (S) alcohol. There are four possible stereochemical patterns that enab le transfer of hydride from the cofactor to

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4 the substrate. This hydride transfer is well st udied and results from the specific binding of the substrates as well as coenzymes on the chiral enzyme surface in close proximity and defined geometry. 10, 11 The four different hydride transfers, two from the re face and two from the si -face, result in either ( R) or (S) alcohols corresponding to transfer of pro-R and pro-S hydrides from the cofactors. O L N HS SHRCONH2 R N HRHSCONH2 R si face re face NAD(P)H NAD(P)H Figure 1-4. Stereochemistry of hydride transf er from NAD(P)H to the carbonyl carbon on the substrate (S = small and L = large group) 12 Asymmetric Reductions Using Bakers’ Yeast Enzymes Baker’s Yeast (BY), Saccharomyces cerevisiae, is cheap, readily available and easy to use. Thus it is a very popular cataly st for asymmetric reductions of carbonyl compounds. 13 Baker’s Yeast has more than 40 different carbonyl reductases in its genome which may have overlapping substr ate specificities, but with different stereoselectivites, yielding a mixture of stereosiomeric alcohols. 14 To overcome this, Iwona Kaluzna in the Stewart lab made a library of twenty -keto ester reductases from Bakers’ Yeast, S.cerevisae, over expressed in E.coli cells with a Glutathione Stransferase tag. 15 These reductases are used for the synthesis of hydroxyl esters which hold a very important place in organic chemistr y as they can be transformed into various functionalities, without racemi zation, to synthesize industrially important chemicals. 16

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5 Applications of Baker’s Yeast Enzymes Isolated Enzyme Catalysis Isolated dehydrogenases, when used as biocatalysts, give cl eaner reactions, less side-products and less purification pr oblems than the whole cell reactions. 17 The main disadvantage of using isolated dehydrogenases is the cofactor requirement, which in most of the Baker’s yeast carb onyl reductases is NADPH. 18 NADPH is relatively unstable and expensive and thus is impractical to us e in stochiometric amounts. Therefore an in-situ regeneration of NADPH is a pr erequisite for large-scale appl ications to be economically feasible. Recycling of cofactors can be done chemically, electrochemically, enzymatically, etc. 19-21 The efficiency of recycling is measured by the number of cycles that can be achieved before a cofactor molecu le is destroyed. It is expressed as total turnover number (TTN), which is the total number of moles of product formed per mole of cofactor during the complete course of the reaction. The mo st successful application of cofactor regeneration is by enzymatic methods either by coupled-substrate or coupledenzyme processes. 22 Substrate-coupled regeneration In substrate-coupled regeneration, a si ngle enzyme, a dehydrogenase, accepts an additional substrate that regene rates the cofactor for further use. This additional substrate is generally an aliphatic alcohol which is oxidized to a ketone to regenerate the cofactor. 23 Though this approach is elegant it has several disadvantages such as enzyme deactivation and co-substrate inhibition due to presence of large amounts of auxiliary substrate. 17

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6 SubstrateSubstrate H2Auxillary Substrate Auxillary Substrate H2Single Enzyme NAD(P)H NAD(P) Figure 1-5. Cofactor recycling by the coupled-substrate method 17 Enzyme-coupled regeneration In enzyme-coupled regeneration, two enzymes are employed. In this case, two parallel redox reactions are ca talyzed by two different enzymes, i.e. conversion of main substrate and cofactor recycling. 24 For best results, both en zymes should have different specificities for their respective substrates so that the substrates do not have to compete for the active site of a single enzyme, but ar e efficiently converted independently by the two biocatalysts. SubstrateSubstrate H2Auxillary Substrate Auxillary Substrate H2 NAD(P)H NAD(P) Enzyme A Enzyme B Figure 1-6. Co-factor recycling by coupled – substrate method 17 Formate dehydrogenase, which oxidizes form ate to carbon dioxide, is the enzyme of choice for NADH regeneration. Carbon dioxi de is easily remove d and not toxic to protein with concomitant reduction of NAD+ to NADH. 25 Recycling of NADPH with glucose dehydrogenase or glucose 6-phospha te dehydrogenase is widely employed. 26, 27

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7 Glucose is oxidized to gluconolactone that spontaneously hydrolyzes to gluconic acid. These regeneration techniques require pure pr oteins. Special equipment and training is required to purify proteins furthe r increasing costs of the process. CO2 NAD(P)NAD(P)H FormateDeh y drogenase O H HO H HO H OH OH H H OR O H HO H HO H OH H O OR NAD(P)NAD(P)H Glucose 6-Phosphate Dehydrogenase Glucose Dehydrogenase OH COOH H HO H HO H H OR OH spontaneous R = H R = Phosphate Gluconic Acid H O O Figure 1-7. Cofactor recycli ng by coupled – enzyme method Whole Cell Biocatalysis The cofactor regeneration problems can be circumvented by in vivo application of dehydrogenases since the host ce ll regenerates cof actors as a part of its normal metabolism. 28 The whole cell biocatalysts functi on as miniaturized reaction vessels, which produce functional enzyme, regenerate co factor, take up the substrate and convert substrate to product that diffuses out of the cell. Cofactor recycling in vivo requires metabolically active cells; when reducing equi valents are no longer generated, the desired reaction also stops. Therefore the cells must be maintained in a state that permits high cofactor regeneration. A minimum requirement for this is that the cell membrane remains intact during the biocatalysis process.

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8 Major drawbacks of whole cell biocatal ysis are formation of by-products and purification of the product. Essentially no special training is required to run whole cell mediated reactions. Metabolism Reductase Carbonyl Compound Reduced Chiral Product Co-substrate By-product NAD(P)HNAD(P) E. c oli Figure 1-8. Principle of wholecell reduction of carbonyl compounds 29 Long Term Objectives We want to study reductions of -keto esters and increase the bioprocess efficiency of the system. The bioprocess efficiency is defined as the main parameters influencing the cost of the process. It is addressed in terms of yield, final pr oduct concentration (g/L of product), volumetric producti vity (i.e. the space time yield g/L/hr) and catalyst consumption (product/catalyst ratio gm/g m or biocatalyst/su bstrate ratio g/g). The goal of this work is to standardi ze protocols and develop a more efficient bioprocess using genetic tec hniques and optimization of reaction conditions and apply them to make a pool of chiral products with in an infrastructure /technology platform.

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9 CHAPTER 2 RESULTS We have classified our work in four di fferent sections for reductions of three different -keto esters viz. ethyl butyrylacetate (EBA), 4-chloro ethyl acetoacetate (ECAA) and ethyl acetoacetate (EAA) and solv ed different problems encountered in the bioprocess development. Biotransformations Section I: Bioprocess Development for Reduction of Ethyl butyrylacetate O O O O O OH E.Coli BL21(DE3) pAA3 Gre2p Ethyl butyrylacetate Ethyl 3-hydroxy butyryl acetate Figure 2-1. Reduction of Ethyl butyrylacetate Enzymatic reduction of ethyl butyryl acetate to ethyl 3-hydroxy butyryl acetate (EHBA) was carried out in our lab yieldi ng an enantiomeric excess of >98% using several of the yeast re ductases found in Bakers Yeast. Of the enzymes present in our library, Gre2p was selected and over expressed in E.coli strain BL21 (DE3) containing the plasmid pAA3 to give the S alco hol with >98% enantiomeric excess. 16 The plasmid contained genes for kanamycin resistance and Gre2p, whose expression is under the control of phage T-7 RNA polymerase. When induced by isopropy-thiogalactopyranoside (IPTG), the efficient T-7 promoter over expresses Gre2p that equals about 20% of the cell dry weight.

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10 Biotransformations with Non-growing Cells The whole cell reductions were carried out with non-growing cells using the general procedure of Walton and Stewart. 30 The cells were grown in Luria-Bertani medium (LB) with 4g/L glucose and were harvested just before the cells reached stationary phase. The cells were resuspe nded in 1 L of minimal composed of M-9 medium without NH4Cl to prevent cell growth. Cell GrowthBiocatalysis 4:1 Figure 2-2. Cell growth in a 4L New Brunswi ck fermentor and bioconversion carried out in 1L Braun Bioststat B Fermentor Parameters such as temperature, pH, a nd dissolved oxygen tension were optimized to give the best results and then were kept constant. The reductions were carried out at 30 C, pH = 7.0, and the dissolved oxygen tens ion at 75% saturation. The bioconversions were conducted at one-liter scale with biomass loading at OD 600 18. Different substrate feed rates to the react or with a pump was used to find the maximum rate of bioconversion as shown in Figure 2-3.

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11 0 10 20 30 40 50 60 010203040506070 Time hrsEHBA mM 11.5mM/h r 8.5mM/h r 1.6mM/h r Figure 2-3. Increasing subs trate (EBA) feed rate The product formation stopped after the pr oduct reached a concentration of about 50 mM. Even after increasing the cell loadi ng in the system, there was no observable increase in the product formation. This led us to believe that th e product was inhibiting the biocatalyst, i.e. the product is toxic to the catalyst. Product i nhibition was confirmed by performing an experiment wherein along with a substrate addition of 15 mM/hr we did bolus addition of 50 mM at 1.25hrs. No further product formation was observed confirming the product inhibition of the biocatalyst (Figure2-4).

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12 50mM EHBA @ 1.25hrs 0 10 20 30 40 50 60 70 80 90 02468 Time hrsEHBA mM Figure 2-4. No additional product formed after bolus addition of ethyl 3-hydroxy butyrylacetate at 1.25 hrs. When substrates or products are inhibito rs of biocatalysts, the eventual product concentrations are very low, making the product recovery laborious and expensive. Therefore, strategies were developed to ma intain sub-toxic product concentration in the reactor to attain hi gh process yields. Strategy I: Centrifugation A very basic strategy was used where the biocatalyst was resuspended in fresh aqueous buffer after the product reached the critical concentration. The reaction media was centrifuged at 6000 rpm and the biocatal yst then resuspended in a new aqueous medium for a new round of bioconversion. Subs trate was added at rate of 15 mM/hr.

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13 0 10 20 30 40 50 60 70 80 90 0510152025 Time hrsEHBA mM Total Before centrifugation After centrifugation Figure 2-5. Product removal by centrifugation. W hole cells resuspended in fresh aqueous media. While the final product con centration achieved was much higher, this strategy proved to be only of limited use and could not be applied due to the inconsistent behavior of the whole cells after centrifugation. During the whole cell catalysis, the cell membrane is affected due to long exposure to the substrate and the produc t. These cells cannot withstan d the high forces exerted on it during centrifugation. This causes many of the cells to become meta bolically inactive, which in turn causes the biotrans formation to be less productive. Strategy II: In situ product removal (ISPR) As a result of the product inhibition, the adverse effect on the biocatalyst can be minimized by the removal of the product as s oon as it is formed. This also helps to increase the productiv ity of the system. In situ product removal techniques address this

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14 problem by removing product (or maintaining lo w concentrations of substrate) formed from the vicinity of the biocatalyst. 31 The separation of product from the reactor can be carried out in different modes. Different techniques are available for ba tch, fed-batch or continuous process. 32,33 The separation step can occur within the biorea ctor or outside it. Different physical and chemical properties like volat ility, molecular size, solubil ity, charge, hydrophobicity, etc are exploited as the driving force for separa tion of the product (or substrate). Different techniques like distillation, gas stripping, memb ranes (micro filtration and ultra filtration techniques are available), pervaporation or perstraction, extraction, supercritical carbondioxide, precipitation, crystallization, ion-exchange, electr odialysis, hydrophobicinteraction chromatography, adsorption and di fferent affinity methods can be employed for the separation of product. 34 The most commonly used separation technique for in situ product removal relies on adsorption of produc t (or substrate) to a hydrophobic resin. The hydrophobic Amberlite XAD resins have been re ported to be used most widely in industrial context. 35, 36 We selected XAD 4 from XAD 4, 16 and 18, because it had the highest capacity to adsorb ethyl butyrylacetate. The easiest way to perform an ISPR is to preadsorb substrate onto a resin and add the resin to the reactor where the resin w ould act as a second phase. The resin would adsorb the product formed, and during the re action, the concentra tion of the starting material or the product would not increase beyond a certain point due to adsorption and desorption of the product and substrate. 37 This approach is easy but has its own limitations. More resin added to the reactor results in more mass transfer and oxygen

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15 transfer limitation of the substrate with the bi ocatalyst, affecting its performance. Also, increased stirring rate may l ead to cell lysis by shearing. To avoid this we used a reac tor called a “Recycle Reactor”. 38 Here, the bioconversions and product extraction were carried out in two separate units. The charged resin was placed as a fixed bed into a column in an external loop. In the bioconversion unit, substrate was added and when the product concen tration neared the product inhibition levels the ce ll broth was passed through the resin by a peristaltic pump. The resin could be reused because the so lid phase extraction was performed in the external loop to avoid the continuous and vigorous stirring conditions in the reactor. Unfortunately, ethyl butytrylacetate is le ss polar than the hydroxyl product and was thus preferentially adsorbed on to the column, lowering the resin’s capacity for the hydroxyl product. Therefore all the substrate added was exhausted prior to the product removal. The medium was then recirculated through the resin column until all the product and substrate were removed from the medium. A new batch of biotransformation with the same biocatalyst was carried out enabling a transition to semi-continuous process from a fed-batch process. The substrate wa s added at the rate of 15 mM/hr. Detailed studies of this system can lead to a much more efficient continuous process for the reduction of ethyl butytrylacet ate by adding the substrate at limiting rate; under these conditions all substrate is convert ed to product, which can be continuously concentrated in the extracti on unit by adjusting the rate of the cell broth through the external loop.

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16 Figure 2-6. Experimental setup for in situ product removal 0 10 20 30 40 50 60 70 80 90 100 0510152025303540 Time hrsEHBA mM Total Product Formed After Each XAD Adsorption Figure 2-7. In situ product removal using an external co lumn packed with resin Amberlite XAD-4 In Situ Product Removal ( ISPR )

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17 Strategy III: Biphasic reactor (ATPS) Problems arise when conducting biocatalys is in aqueous medium due to poorly soluble substrate and or products Low concentrations of the pr oduct or substrate, that are hydrophobic can be maintained in the reactor by use of a “Biphasic Reactor” or an “Aqueous Two-Phase System” (ATPS). 39, 40 This can be achieved by addition of an immiscible organic solvent or other non-c onventional media like i onic liquids to the reaction mixture which act as a substrat e reservoir or a pr oduct extractant. 41, 42 The solvent acts as a bulk extractan t or sink, which effectively w ithdraws product or substrate from the aqueous phase, and keeps the effec tive concentration of these compounds near the biocatalyst at low levels. As products are concentrated in organic phase, they can be easily separated from the biocatalyst suspen sion and the extractants can be recycled, therefore making the process more cost-effective. The most important task when using the biphasic reactor system is to find a biocompatible organic solvent. In selecti on of the auxiliary organic phase in the bioprocess, the most important parameter is the log P value of the organic phase. 43,44 Log P is the value indicating the pa rtition coefficient of the solvent in the octanol-water twophase system. Microbial whole cells are generally compatible with solvents with log P value greater than 4. Another factor desirable in the organic phase is a favorab le distribution coefficient for the biotransformation product. The orga nic phase should show high product recovery capacity and high selectivity. These are quan tified by the partition co efficient (Kp) and the separation factor ( ). Kp is defined as the ratio between product concentration in solvent and its concentration in aque ous medium; the separation factor ( ) is the ratio

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18 between partition coefficient of the product and the other contaminant from which we want to isolate the product, e.g. substrate. We tried two organic phases: hexadecane and bis ethyl hexyl phthalate (BEHP) with BEHP giving better results. 45, 46 BEHP is cheaper and higher boiling than hexadecane, has low flammability and shows no toxicity towards E.coli cells. Therefore we carried out our further studies with BE HP, efficiently overcoming product inhibition. The reaction was conducted in a fed-batch two-liquid phase reactor. The reduction was carried out with BEHP as the organic phase with a ratio of 0.5, i.e. the apolar solvent consisted of 50 % of the reactor volume. The reactions were carried out with OD600 of 18, 22 and 25 with no significant increase in to tal final product formation for higher biomass loading. The substrate was added at the rate of 50 mM/hr. Figure 2-8. Biphasic reactor (aqueous two phase system) substrate air acid base Anti foam level Stirrer pH pO2Organic Aqueous

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19 0 20 40 60 80 100 120 140 051015202530 Time hrsEHBA mM AqueousOrganicTotal Figure 2-9. Bioconversion in a bi phasic reactor containing BE HP as the organic phase Bioprocess Development for the Re duction of Ethyl butyrylacetate The bioprocess efficiency for the reducti on of ethyl butyryl acetate was increased using techniques like in situ product removal and biphasic reactor. 2.78 4.87 5.450 1 2 3 4 5 6 AqueousISPRBiphasic TechniquesProductivity mM / OD 600 Figure 2-10. Bioprocess development for th e reduction of Ethyl Butyryl Acetate

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20 Section II: Bioprocess Development for Re duction of Ethyl 4-chloroacetoacetate Cl O O O Cl O O OH E.Coli BL21(DE3) pIK x Ketoreductase Ethyl 4-chloro Acetoacetate Ethyl 3-hydroxy 4-chloro Acetoacetate Figure 2-11. Reduction of et hyl 4-chloro acetoacetate Asymmetric reduction of ethyl 4-chloro ace toacetate leads to the formation of (R) or (S) ethyl 4-chloro 3-hydroxy butanoate (E CHB). Both the (R) and (S) enantiomers are important chiral synthons in the synthesis of various pharmaceuticals and agrochemicals. 47 For example, the Renantiomer is an impor tant chiral building block for the synthesis of ( )-macrolactin A, L-carnitine and (R)-amino-hydroxybutyric acid. 48 On the other hand the S enantiomer is a key chiral intermed iate in the enantioselective synthesis of slagenins B and C and in the total synthesi s of a class of HMG-Co A reductase inhibitors. 49 Baker’s Yeast reduction of ethyl 4-chloro ac etoacetate results in products with low optical purity owing to the presence of forty different reductases of opposing stereoselectivites. 50 An in-house fusion protein library of 20 -keto ester ketoreductases from Sacchromyces cerevisae over expressed in E.coli was screened for the reduction of ethyl 4-chloro acetoacetate. A group of y east enzymes denoted by their genetic codes YJR096w, YDL124w, YHR104w, YGL185c, YAL061w and YOL151w bearing the plasmids pIK-9, pIK-8, pIK-29, pAKS-1, pI K-28 and pIK-3 yielded an enantiomeric excess of >99% to give the optically pure (S) enantiomer. 16

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21 Biotransformations with Non-growing Cells The whole cell reductions were carried out with non-growing cells using our standard procedure wherein cells were harves ted just before reaching stationary phase were resuspended in non-growing minima l media (M-9 media composition without NH4Cl) with a 4:1 dilution with an OD600 18. Temperature and dissolved oxygen were kept constant at 30C and 75 % saturation. Ethyl 4-chloro-acetoacetate slowly d ecomposes in aqueous medium to 4chloroacetoacetic acid which is known to be a toxic compound for E.coli cells. 51 This decomposition is accelerated at pH 7 and high concentration (the mechanism of decomposition is not known) but no decomposition was observed if the pH was lowered to 6.00.Therefore, a pH of 6.00 was ma intained during the bioreduction. Cl O O O Cl OH O O + OH Aqueous pH > 7 Slow Figure 2-12: Slow decomposition of ECAA at pH 7 in aqueous medium Strategies to overcome substrate inhibition Slow substrate feed Different rates for substrate feed from 1mM/ hr to as low as 60 M/hr were tested to avoid substrate inhibition. As soon as the substr ate is added it is conve rted to the product, but this prolonged the reaction time and made it an extremely slow process. The reactions were carried out for 24 hrs. Also, product formation never exceeded 18 mM.

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22 0 2 4 6 8 10 12 14 16 18pik-8pik-8pik-9pik-9pik-28pik-28pik-29pik29pik-3pik-3paks1 paks1ECHB mM OD 18 OD 20 OD 25 Figure 2-13. Reduction of ECAA by E.coli whole cells expressing different Bakers’Yeast reductases Slow-release biocatalysis In reactions that show substrate inhib ition, the substrate conc entration cannot be too high. Absorbing resins have been used for in situ product removal to prevent product inhibition and cell toxicity. Sim ilarly, ion exchange resins can absorb a large quantity of substrate and then release it slowly to the reaction solution. 37 This technique is effective in eliminating substrate inhibition. Te n grams of resin Amberlite XAD-16 were preadsorbed with 40mM of ECAA so that the substrate is slowly released into the solution. The slow-release biocatalysis did no t work as intended yielding 25mM product formation at much slower rate than without the resin. Addition of organic solvent to in crease the solubility of the ECAA The solubility of ethyl 4-chloro acetoacetate is very in low in water, and this limits the substrate available to the biocatalyst making the bioprocess inefficient. Organic solvents can be added in small amou nts to dissolve the hydrophobic compound and increase its availabil ity to the whole cells. 52 Ethyl 3-hydroxy butyrate (50mM), which

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23 was readily available in our lab from reductions of et hyl acetoacetate by the same biocatalyst, was added as an or ganic solvent to dissolve ECAA. 53 The addition of organic solvent worked to a smaller extent increasing the product formation to 34 mM. 0 5 10 15 20 25 30 35 40 0102030405060 Time hrsECHB mM With organic solvent Without organic solvent Figure 2-14. Comparison of product forma tion with and without organic solvent Addition of surfactant s to the whole cells When whole cells are used, the cell membrane acts as a barrier to the transport of substrate to the enzyme and hampers optimal biotransformation. Different surfactants like Tween 20 and Triton-X have been used to increase the permeability of the cells, facilitating the transport of substrat e and product across the cell membrane. 54 We used Tween 20 and Triton-X in 0.5 % w/v in the reac tor but product was not formed in greater amount than in the absence of the surfactant. Further studies are required to understand the effect of ethyl-4 chloroacetoacetate on whole cells.

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24 Biotransformations with Cell-free Extracts: Since we were not able to increase the bioprocess efficiency with whole cells, we turned to reductions of ECAA with cell free extracts. 55-57 Here the complex network of cofactor regeneration within an active cell breaks down. All our reductases are NADPH dependant and cannot accept NADH as a cofact or. Due to the high cost and relative instability of NADPH, it cannot be added in stochiometric amounts and needs to be regenerated. We tried the c oupled-substrate regeneration using isopropanol as an auxiliary substrate but none of our enzymes accepted it as a co-substrate. We then tried the coupled-enzyme regeneration system for NADPH using glucose dehydrogenase (GDH) from B.subtilis over expressed in E.coli as the second enzyme. It oxidizes glucose to gluconolactone that spont aneously hydrolyses to gluc onic acid. This GDH did not accept ECAA as a substrate. Cl O O O Cl O O OH E .Coli BL21(DE3)pIKx Ketoreductase O H HO H HO H OH OH H H OR O H HO H HO H OH H O OR NAD(P)NAD(P)H Glucose Dehydrogenase OH COOH H HO H HO H H OH OH spontaneous Figure 2-15. Coupled enzyme regeneration of NADPH using glucose dehydrogenase from B.subtilis over expressed in E.coli BL21(DE3) cells for the reduction of ECAA using Bakers’ Yeast reductases over expressed in E.coli BL21(DE3) Cells over expressing YDL124w gene pr oduct and glucose dehydrogenase were grown separately in a 4-liter fermentor and we re harvested just before they reached the

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25 stationary phase. These cells were lysed usi ng a French Press. These cell-free extracts were then mixed together in M-9 minimal medium (without NH4Cl) and the biotransformation performed by addition of the substrates ECAA and glucose. The volume of the reactions was kept constant at 50ml in all cases Different biomass ratios of ketoreductase to GDH, viz. 8:1, 4:1, and 2:1 we re applied to find the best ratio to give better productivity. 200 mM substrate was added to the shake flask reactor. Final product concentrations of 100mM were reached. 0 20 40 60 80 100 120 051015202530Time hrsECHB mM 8:1 4:1 2:1 Figure 2-16. Reduction of ECAA with cell free extracts with di fferent ratios of reductase and glucose dehydrogenase (GDH) A small increase in the final product tite r was observed by using higher ratio of ketoreductase but the amount of catalyst consumed was more per mole of product formed.

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26 Biotransformation with cell free extr acts under controlled pH conditions The formation of gluconic acid via gl uconolactone from glucose oxidation by GDH used for regeneration of the nicotinamide co -factors caused the pH to drop, making the conditions acidic. The acidic conditions, though good for ECAA (no d ecomposition at pH 6.00 and lower), are harmful to NADPH causing it to degrade. The bioconversions were carried out under controlled pH conditions at pH 7.00 yielding higher final product concentrations. 200 mM substrate was added to the shake flask reac tor. A final product concentration of 160mM was obtained. 0 20 40 60 80 100 120 140 160 180 051015202530 Time hrsECHB m M Figure 2-17. Reduction ECAA with cell free extracts under pH controlled conditions Biotransformation using cell free extract in a two-phase system ECAA is poorly soluble a nd unstable at neutral pH in water thus limiting the product formation. To overcome this, an organi c solvent is added to the medium so both the product and substrate can be ex tracted in the organic phase. 58 The bioconversions were carried out with benzene and ethyl acetate as the orga nic phase and compared with

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27 reductions in the aqueous medium. 200 mM substrate was added to the shake flask reactor. 0 10 20 30 40 50 60 70 80 0510152025 Time hrsECHB mM Benzene Normal Aq Et Ac Figure 2-18. Reduction of ECAA with cell fr ee extracts in biphasic conditions To compare the values of final product concentration reached in each case i.e. whole cells, cell free extracts, pH controlled cell-free extract, twophase cell free extract the product formation was calculated as pr oduct mM per gm of biocatalyst used. 0.50 1.00 2.50 3.75 7.18 0 1 2 3 4 5 6 7 8 Whole cellsWhole cells + Organic Solvent Cell-free extracts Cell-free extracts pH Control Cell-free extracts organic Type of system for reductionProduct mM / g cells Figure 2-19. Bioprocess devel opment for reduction of ECAA

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28 Section III: Bioprocess Development fo r Reduction of Ethyl acetoacetate O O O O O OH E.Coli BL21(DE3) pAA3 GRE2p Ethyl acetoacetate Ethyl (S) 3-hydroxy butyrate Figure 2-20. Reduction of ethyl acetoacetate Biotransformation with Non-growing Cells Reduction of ethyl acetoacetate was done with non-growing E.coli BL21(DE3) harboring the plasmid pAA3 over expressi ng the ketoreductase Gre2p from Baker’s Yeast. The reaction was carried out in a r eactor with 1L minimal media (M9 without NH4Cl) yielding an OD600 18 at 30C, pH 7.00 dissolved oxygen tension 75%. The maximum product formation observed was 285 mM, and it consumed 228 mM glucose producing 1.25mole of NADPH per mole of glucose consumed. Stewart and Walton published a basic study of reducti on of ethyl acetoacetate where in they established that NADPH dependent reactions could be carri ed out under non-growing conditions using glucose-fed bath conditions. 30 It was proposed in this study that the efficiency of the biocatalytic process can be further enhan ced and the costs reduced. The central carbon metabolism in E.coli proceeds via the Embden-Meyerhoff-Parnas (EMP) pathway (glycolysis), the Phosphate pentose pathwa y (PPP) and the Entner–Doudoroff pathway (EDP). 59 The EDP pathway links together the "sta rting" points of th e EMP, ED and PPP pathways. The pyruvate formed is decarboxylat ed and is further metabolized by Krebs cycle.

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29 Figure 2-21. Major pathways of glucose metabolism in E.coli Glycolysis (EmbdenMeyerhoff-Parnas Pathway), Pentos e Phosphate pathway (PPP) and the Entener-Doudoroff pathway (EDP).Gene names are italicized. 60 pts ,PEP:glucose phospho transferase system; glk glucose kinase; pgi phosphoglucose isomerase; pfk phosphofructokinase; fbp, fructose-1,6-bisphosphatase; fba fructose-1,6-bisphosphate aldolase; tpi triosephosphate isomerase; gap glyceraldehyde 3-phosphate dehydrogenase; pgk phosphoglycerate kinase; pgm phosphoglyceratemutase; eno enolase; pyk pyruvate kinase; pps PEP synthase; ppdk pyruvate phosphate dikinase; gcd glucose dehydrogenase; gntK gluconate kinase; zwf glucose-6-phosphate dehydrogenase; pgl 6-phosphogluconolactonase; edd 6-phosphogluconate dehydratase; eda 2-keto-3-deoxy-6-phosphogluconate aldolase; gnd 6-phosphogluconate dehydrogenase; rpe ribulose-5-phosphate epimerase; tkt transketolase; tal transaldolase; rpi ribose-5-phosphateisome rase transketolase; tal transaldolase; rpi ,ribose-5-phosphateisomerase

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30 Metabolic Engineering The tools of metabolic engineering we re applied for the optimization of biocatalysts to lower the bi oprocess costs. The targeted change of flux rates in the metabolism for the purpose of optimizati on of product yield is called metabolic engineering. In this case glucose was used as the source of reducing equivalents. In E. coli, 80% of the glucose is consumed via the EM pathway. The other 20% is sent via the HMP shunt and consumed through the pentose pathway. 61 The NADPH produced per mole of glucose consumed can be impr oved by increasing the carbon flux through the pentose phosphate pathway. This could be achieved by over expr essing the two NADP+ producing dehydrogenases of this pathway or by knocking out the phosphoglucoisomerase ( pgi ) gene and forcing the car bon flux through the phosphate pentose pathway. 62,63 We selected the later and th e strain with the knocked out pgi gene was made by Despina Bouigioukou in our lab. The pgi knockouts were compared with the cells with the pgi gene (referred to as the wild type) for the NADPH produced per mole of glucose consumed. The biotransformation was carried out with our usual conditions of temperature 30C, pH 7.00 and dissolved oxygen tension 75%. The reactions were carried out at different biomass loading giving an OD 600 of 11, 14 and 17. The substrate was fed with a pump at the rate of 20 mM/hr.

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31 0 2 4 6 8 10 12 14 16 051015202530Time hrsProduct mM /OD 600 110 0.5 1 1.5 2 2.5Product / Glucose (P/G) WT pgi pgiP/G WT P/G A) 0 2 4 6 8 10 12 14 0510152025303540 Time hrsProduct/OD 600 140 0.5 1 1.5 2 2.5Product / Glucose (P/G) pgi WT pgi P/G WT P/G B)

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32 0 2 4 6 8 10 12 14 16 18 05101520253035 Time hrsProduct mM/ OD 600 170 0.5 1 1.5 2 2.5Product / Glucose (P/G) pgi WT pgi P/G WT P/G C) Figure 2-22. Comparison of wild type and pgi knockout for product formation and glucose consumed per mole of product formed. A) OD 600 = 11 B) OD 600 = 14 C) OD600 = 17. The delta pgi knockout strain was better than th e wild type. It produced more NADPH per mole of glucose consumed and the final product concentration per gm of cells was higher than the wild type. So,the efficiency of the bioprocess was increased. Section IV: Bioprocess Development us ing Immobilised Cells and Enzymes. Biocatalyst costs are often important. Imm obilization of biocatalysts is a method where catalyst is recycled or its prolonged us e leads to significan t reduction in catalyst cost. 64 Also, improved performance such as activity, stability, selectivity, and productivity can be achieved by immobilization. Immobilized biocatalysts can be considered as a composite consisting of tw o essential components: the non catalytic structural component (carrie r) which is designed to aid separation and reuse of the catalyst, and the catalytic functional com ponent (enzyme or whole cell), which is designed to convert the desired s ubstrates into desired products. 65 Immobilization can be

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33 either carrier bound or carrier free. The crit eria for effective immobilized systems are defined by requirements such as mechanical and chemical stability, high volume activity, operation stability, reusabilit y, easy disposal and safety depending on the non-catalytic and catalytic function of the system. 66 The catalytic functions are responsible for the desired activity, selec tivity, substrate specificity, producti vity and space-time yields. The non-catalytic functions are dependent on reac tor configurations (batch, stirred-tank, column, plug-flow), the type of reaction medium (aqueous, organic, biphasic), the process conditions (tempe rature, pressure, pH). 67 The selected parameters should facilitate downstream processi ng and control of the process. A carrier can be considered as a modifier of the biocatalyst as it is not only the support on which the biocatalyst can be attach ed but its surface chemistry, pore size and hydrophilicity or hydrophobicity dictates the ac tivity, selectivity a nd stability of the biocatalyst. 68 A large number of synthetic or natural carriers with different characteristics are available according to th e requirements. Enzyme carriers can be of different types like beads, hollow fibers, capsules, films, membranes, sol-gels, mesoporous molecular sieves and inorganic nanocomposites. 66 Beads Fibers CapsulesFilms Membranes Figure 2-23. Different types of carrier s for immobilizati on of biocatalyst 66 Bioencapsulations with silica sol-gel gl asses, which is biologically inert and imparts better mechanical strength and chemi cal stability and does not swell in aqueous or organic solvents, is a new and interesting field. 69, 70 This process of immobilization

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34 involves no high temperature and harsh chem ical reactions; it has the ability to immobilize biomolecules without modifyi ng their structure or function greatly. Moreover, the porosity can be controlled with selection of proper precursors, modifiers and polymerization conditions. Sol-gel entrapment techniques often us e orthosilicate such as tetramethyl orthosilicate (TMOS), tetraethyl orthosili cate (TEOS) or methyl trimethoxy silane (MTMS) as precursors. 71 In aqueous media, the hydrolysis of precursor results in the formation of an oligomer that is furthe r hydrolyzed to form an aqueous sol. The occurrence of gelation process leads to de velopment of a three dimensional network where the biomolecules are trapped. A major disa dvantage of this process is that the high concentration of ethanol or methanol pr oduced during the immobilization process is harmful to the activity of enzymes or cells. Recently a sodium silicate silica based process for sol-gel formation was reported which avoids the formation of alcohol completely. 72 Si OR RO OR OR + 4 H2O Si(OH)4+4 ROH SiOH + SiOH SiO2 +H20 Figure 2-24. Reactions occurring during fo rmation of sol-gel synthesis of silica We decided to study the immobilization of whole cells within the silica gel glass due to the dependence of our enzymes on NADP H cofactors. Sodium silicate silica and colloidal silica (LUDOX HS40) was used for s ynthesis of silica gel. Phosphoric acid was added to decrease the pH to 7.0 before addi ng the whole cells. The gel was air-dried for 75 hrs and then crushed. This crushed sili ca gel glass containing the biocatalyst was resuspended in non-growing mi nimal M-9 medium without NH4Cl for the

PAGE 46

35 biotransformation. The reduction of ethyl a cetoacetate was used as the model reaction and E.coli BL21(DE3) expressing the enzyme GRE 2p harboring the plasmid pAA3 was used for the bioconversions. Different expe riments were performed to improve the productivity of the biotransformation usi ng immobilized cells. 50 mM substrate was added in all the reactions. Also, in all the experiments the reusability of the immobilized cells was checked. The immobilized cells were centrifuged and then resuspended in fresh buffer for repeated use. Optimal Total Si: Buffer Ratio for Better Product Formation. The ratio of the total silica content to th e buffer added with a fixed amount of cell loading yielding gels with different properties was studied .The volume ratios used were 3:1, 2:1, 1:1 respectively. The gelati on occurred within one minute. 0 1 2 3 4 5 6 7 8 9 10 02468 Time daysProduct mM / Catalyst gm0 5 10 15 20 25 30 35Product mM / Catalyst gm 3:01 2:01 1:01 Control Figure 2-25. Product mM / gm catalyst for the reduction of ethyl acetoacetate using immobilized whole cells in silica gel glass with different Si:Buffer ratio. Secondary axis corresponds to control

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36 0 5 10 15 20 25 30 35 02468Time daysProductivity mM / day0 100 200 300 400 500 600Productivity mM / day 3:01 2:01 1:01 control Figure 2-26. Productivity for the reduction of ethyl acetoacetate using immobilized whole cells in silica gel glass w ith different Si:Buffer ratio Secondary axis corresponds to control. The immobilized cells were checked for th eir reusability. The free and immobilized cells were centrifuged and resuspended in a fresh medium and glucose and substrate was added to the system for a new round of bioconversion. 0 2 4 6 8 10 12 14 16 18 02468101214 Time daysProduct mM / Catalyst gm control 3:01 2:01 1:01 Figure 2-27. Reuse of immobilized cells for the biotransformation with different Si: Buffer ratio

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37 0 20 40 60 80 100 120 02468101214Time daysProductivity mM / day control 3:01 2:01 1:01 Figure 2-28. Productivity of the reused immob ilized cells with different Si:Buffer ratio These cells were again centrifuge d and reused for another round of biotransformation. 0 2 4 6 8 10 12 14 051015 Time daysProduct mM / Catalyst gm control 3:01 2:01 1:01 Figure 2-29. 2nd Reuse of immobilized cells w ith different Si: Buffer ratio

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38 0 20 40 60 80 100 120 0246810121416Time daysProductivity mM / day control 3:01 2:01 1:01 Figure 2-30. Productivity of th e immobilized cells after 2nd reuse with different Si: Buffer ratio For all the above experiments Si: Buffer ratio of 1:1 gave the best results and was kept constant for the next experiments. Different Composition of the Silica Gel Matrix for Better Product Formation Once we determined the ratio of total Si:Buffer, we decided to optimize the silica matrix by varying the Si:Ludox ratio. This ratio would the change the composition of the gel and thus affect the charac teristics of the matrix affecting the biotransformation. Figure 2-31. Product mM / gm catalyst for the reduction of ethyl acetoacetate using Immobilized whole cells in silica gel glass with different NaSi:Ludox ratio. Secondary axis corresponds to control. 0 2 4 6 8 10 12 024681012Time daysProduct mM/ gm catalyst 0 5 10 15 20 25Product mM/ gm catalyst 1:01 1:02 1:03 1:04 1:05 control

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39 0 5 10 15 20 25 30 35 40 45 024681012 Time daysProductivity mM / day 0 50 100 150 200 250 300Productivity mM / day 1:01 1:02 1:03 1:04 1:05 Control Figure 2-32. Productivity for the reduction of ethyl acetoacetate using immobilized whole cells in silica gel glass with diffe rent NaSi:Ludox ratio. Secondary axis corresponds to control. 0 1 2 3 4 5 6 7 8 9 051015 Time daysProduct mM / gm catalyst control 1:01 1:03 1:04 1:05 Figure 2-33. Reuse of immobilized cells fo r the biotransformation with different NaSi:Ludox ratio

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40 0 10 20 30 40 50 60 70 80 90 100 0246810121416 Time daysProductivity mM / day control 1:01 1:03 1:04 1:05 Figure 2-34. Productivity of the reused immobilized cells different NaSi:Ludox ratio NaSi:Ludox with a ratio 1:3 gave the best re sults. The gels used for further tests had the Si:Buffer ratio 1:1 and NaSi:Ludox ratio 1:3. Effect of Amount of Cell Load ing on the Product Formation We studied the effect of different amounts of cell loading in th e silica-gel matrix. This was an important parameter affecting the bioconversions. There definitely is an optimum loading of cells beyond which resu lts in lower productivity. The loading amount varied from 0.24 gm to 12 gm of E.coli BL21 (DE3) pAA3 (wet weight) in 200ml total volume of the system.

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41 0 5 10 15 20 25 30 0510152025 Time daysProduct mM / gm Catalyst 0 10 20 30 40 50 60 70Product mM / gm catalyst control 2.4 1.2 2.4 4.8 12 0.24 Figure 2-35. Product mM / gm catalyst for the reduction of ethyl acetoacetate using immobilized whole cells in silica gel glass with different amount of cell loading. Secondary axis corresponds to sample with 0.24 g biocatalyst. 0 5 10 15 20 25 30 35 40 0510152025Time daysProductivity mM / lit / day 0.24 1.2 2.4 4.8 12 control 2.4 Figure 2-36. Productivity for the reduction of ethyl acetoacetate using immobilized whole cells in silica gel glass with different amount of cell loading. We also studied the stability of immobilized cells. The immobili zed cells stored at room temperature for three weeks showed no deterioration and were perfectly normal. The product formation was exactly the same as in the case of the immobilized cells used

PAGE 53

42 immediately without storing. The free cells cannot be stored at room temperature for more than a day. All previously mentioned r eactions were carried out in shake flask reactors. We tried to use a plug-flow column reactor and we re able to get the product, but the product formation was low. We suspect that due to diffusion barrier created by the silica gel glass around the biocatalyst. Further studies are required to make the si lica gel glasses with the right porosity to enhance diffusion and increase the product forma tion rate. Also, this would enable us to use the immobilized cells in plug-flow colu mn type reactors further increasing the productivity of the system Substrate Product Immobilised Biocatalyst Figure 2-37. Plug –flow column reactor Conclusion In conclusion, we have tried to overcome th e disadvantages of biocatalysis such as the scale up of reactions, very low space-time yields and product or substrate inhibition, etc. using different methods for each problem.

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43 Production of optically active alcohols usi ng either whole cells or enzymes by the enantioselective reduction of ketones is a ve ry important activity in biocatalysis. We studied the reduction of ethyl butyryl acetate, ethyl 4-chloro acetoacetate and ethyl acetoacetate. Each reduction was accompanied by its own unique set of problems that were overcome using simple bioprocess solutions. Ethyl butyrylacetate reduc tion yielding ethyl 3-hydr oxy butyrylacetate was limited due to the product inhibition of the biocat alyst. We applied simple techniques like in situ product removal using solid resins and e nhanced the productivity of the system. Additional increases in the productiv ity can be achieved by using the in situ SFPR technique i.e. the in-situ substrate feedi ng and product removal techniques wherein the substrate feed is adjusted so that all the s ubstrate added is converted to product which is removed promptly from the reactor. Thus a steady concentration of the product and substrate is maintained at all times, and l onger reaction times can be achieved resulting in higher productivities. Another method that was explored to a void product inhibition was the use of a Biphasic reactor also known as an Aqueous Two Phase System (ATPS). In this method, we added an organic solvent to the reactor and the product and substrate would partition in the two phases. The total co ncentration of the inhibitory substance, the product in our case, around the biocatalyst is reduced en abling more product formation. We used BisEthyl-Hexyl Phthalate (BEHP) as the orga nic phase because it is inexpensive and increased the final product concentration of the product. BEHP also made product recovery easier because most of the product can be removed from the organic phase by

PAGE 55

44 simple distillation as the bo iling point of BEHP is very high, allowing for the separation of organic phase and the a queous phase by centrifugation Using solvents with better partition rate s can optimize the biphasic approach. Ionic liquid solvents are a good example because they are environmentally friendly, have essentially no vapor pressure and can be reused, making them very attractive for biocatalysis. Reduction of ethyl-4 chloro acetoacetate presented different problems, such as toxicity of the substrate to the whole cel ls. The amount of substrate tolerated by the whole cells was very low, thus making the use of whole cells unattractive as it slowed the reaction time substantially. The reactions w ith cell free extracts with a cofactor regenerating system were tr ied with moderate success. Th e final product concentration increased from 18 mM to 100 mM. pH control of this reaction system further increased the product formation to 165 mM. Some basic studies regarding bipha sic reactions using benzene and ethyl acetate as organic solvents with the cell-free extracts helped increase the rate of reaction. More studies with these biphasic organic reactions with pH and temperature control are require d to increase the final produc t titer and the productivity. Application of metabolic engineering to further reduce the costs of the bioprocess was successfully achieved. The glucose metabolism of the E.coli cells was routed through the pentose phosphate pathway rather than the Krebs cycle, thus increasing the amount of NADPH/glucose consumed. This was achieved by knocking out the phosphoglucoisomerase ( pgi ) gene from the E.coli The engineered E.coli consumed less glucose per mol NADPH produced and thus reducing the operating costs of the biotransformation.

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45 Reduction of ethyl acetoacetate was used to confirm the validity of the knockout. The wild type produced 1.25mol NADPH pe r mole of glucose consumed and the engineered pgi knockout produced 2.25mol NADPH pe r mole of glucose consumed. Immobilized cell systems wherein the whol e cells were encapsulated in silica matrix were studied. This system needs furt her studies to be developed by altering the porosity of the silica gel glasses formed to enhance the diffusion of the substrate and the product. This system has its advantages regard ing the reuse and storag e of the biocatalyst. We have thus improved the bioprocess effi ciency for the asymmetric reduction of -keto esters viz. ethyl butyryl acetate, ethyl 4chloroacetoacetate, and ethyl acetoacetate using various techniques.

PAGE 57

46 CHAPTER 3 EXPERIMENTAL Materials Ethyl acetoacetate, ethyl butyrylacetate, et hyl 4-chloro acetoacetate were purchased from Sigma. (R, S) Ethyl 3-hydroxy butyr ate, (R, S) ethyl 3-hydroxy 4-chloro acetoacetate, ethyl 3-hydroxy butyryl acetate purchased from Aldrich. Resins Amberlite XAD 4, 8, 16 purchased from Aldrich and bis-ethyl hexyl phthalate (BEHP) from TCI America. Gas Chromatography GC analysis was done on Hewlett-Packard model 5890 GC/FID with a Chirasil-Val and DB17 columns. Ethyl butyrylacetate was analyzed on a 25m Chirasil-Val column. The temperature program consisted of an initial time and temperature of 2 minutes and 80 C, a gradient of 1 C/min until the temperature reached 110 C, and final time of 2 minutes at 180 C attained with a rate of 10 C / min. Ethyl 4-chloro acetoacetate was analy zed on a 25m Chirasil-Val column. The temperature program consisted of an initial time and temperature of 2 minutes and 60 C, a gradient of 1 C/min until a temperature of 90 C was reached and then with a gradient of 10 C/min final temperature of 180 C with a final time 2 minutes. Ethyl acetoacetate was analyzed on a 30m DB-17 column. The temperature program consisted of an initial time and temperature of 2 minutes and 60 C, a gradient of 10 oC /min until the temperature reached 180 C, and a final time of 2 minutes.

PAGE 58

47 Sample Preparation for GC 200 l of aqueous reaction media was extracted with 600 l of ethyl acetate containing 1 mM methyl benzoa te as the internal standar d. After one more extraction with 600 l ethyl acetate and the two organic ph ases were combined. Samples were mixed using a vortex mixer. Standard cu rves for ethyl 3-hydr oxy butyrate, ethyl 3hydroxy 4-chloro acetoacetate, and ethyl 3hydroxy butyrylacetate were prepared by making aqueous solutions of different con centrations and extracted identically. The starting materials, i.e. the -keto esters ethyl acetoacetate, ethyl-4chloroacetoacetate and ethyl butyryl acetate decomposed during th e GC analysis and were therefore not quantified. Glucose Assay The glucose concentration in the reactor was determined offline using a Trinder assay kit commercially available from Di agnostic Chemicals Limited, Canada. The absorbance of the reaction, which consists of 5 l of the reaction media and 1ml of the Trinder reagent mixed by inve rsion and incubated at 37 oC for 15 minutes, was measured at 505 nm. The concentration of the glucose in the reactor was measure by comparing it to the standard reference containing 0.4g/L glucose. Media Preparation Cell growth was always in Luria-Bert ani (LB) medium. Composition of LB medium: 10g/L tryptone, 5g/L Yeast Extract, 10g/L NaCl. For LB plates 15g/L of agar was used for solidification. The medium was sterilized by autoclav e. This medium was supplemented with 4g/L glucose for cell grow th. The glucose additi on was done with 20 % glucose solution that is autoclaved separately.

PAGE 59

48 Non-growing medium for biotransformati on was always M-9 minimal medium that was autoclaved before use. It consis ted of di-sodium hydrogen phosphate 12.8 g/L, Potassium di-hydrogen phosphate 3 g/L, NaCl 0.5g/L and NH4Cl that is omitted to maintain non-growing conditions. 2 mM of CaCl2 and 0.1 mM of MgSO4 and 4g/L glucose are separately sterilized and added to the total volume. Cell Culturing The different E.coli BL21 (DE3) strain expressing a ketoreductase used for the biotransformation were streaked fresh weekly from a 15% glycerol stock stored at -80 C, onto LB plates with 40 M kanamycin, incubated overnight until colonies formed. Single colonies were picked to prep are inocula for cell growth. Fr esh stock of the appropriate strain was prepared whenever necessary us ing the correct plasmi d via electroporation. Biotransformations with Non-growing Cells 50ml of LB was inoculated with a sing le colony of E.coli expressing the required ketoreductase with kanamycin (40 mg/L) a nd shaken overnight. The preculture was added to 4L of LB media with 4g/L glucos e, 40 mg/L kanamycin and 0.25ml of antifoam AF-204 (Sigma) in New Brunswick ferm entor. The cells were grown at 37 C until the OD600 reached ~1 .The temperature was then lowered to 30 C and the cells were induced with 0.1mM isopropyl thio ga lactoside (IPTG). The cells were harvested after 6.00hrs.During cell growth the agitation speed was 800 rpm and the aeration rate 1 lit/min was kept constant. The cells were centrifuged at 6000 rpm fo r 30minutes and then resuspended in 100ml of M-9 medium without NH4Cl.For biotransformation, the cell slurry was added to 1L of medium to a final OD600 of about 18.

PAGE 60

49 Biotransformation with In situ Product Removal External Column The external in situ product removal column contained 200gms of XAD-4 resin packed in a glass column attached to th e fermentor. The aqueous reaction media was passed through it when the pr oduct neared inhibitory levels. The product free medium was subjected to another cycle of biotransformation. Biotransformation in Biphasic Reactor The biotransformation is carri ed out as usual but with 50% of the aqueous reaction phase is replaced by an organic phase. Hexa decane and Bis-ethyl hexyl phthalate were used as the organic phase. BEHP yielded bett er results and was used for further studies. Biotransformation with Cell-free Extracts The biotransformation carried out with cell free extracts involved cell growth as usual. The cell were lysed using an ultrasonica tor or a French press. The results obtained using with either method were similar. The lysed cell extract susp ension was centrifuged at 600 rpm for 15 minutes and the supernatan t used for bioconversion. The total volume of the shake flask reactor wa s kept constant at 50 ml. Biotransformation in Biphasic Medium with Cell-free Extracts The supernatant obtained from the above-described method was then resuspended with organic solvents forming 50% of the tota l reactor volume. The organic solvents used were benzene, ethyl acetate. The total volum e of the system was constant at 50 ml. Biotransformation with Immobilized Cells Cells grown were harvested and washed twice with minimal media M-9 without NH4Cl. To maintain non-growing conditions they were resuspended in the M-9 minimal media without NH4Cl and 10 % (by volume) glycerol. An aqueous sol using molar solutions of sodium silicate silica (27 wt % SiO2 with 14 wt % NaOH from Sigma) and

PAGE 61

50 colloidal silica (Ludox HS 40 from Aldrich) was synthesized. Phosphoric acid was used to maintain a pH of 7.00 .The slurry containing E.coli (Buffer) was immediately added to the sol. Gelation time varied from instantane ous to 1 minute. The total volume for sol-gel was kept constant at 200ml. This silica gel wa s air-dried for 75 hrs. The resultant silica gel glass encapsulating the whole E.coli cells was crushed with a pestle and mortar and resuspended in minimal media M-9 without NH4Cl. Different ratios of total Si content and the buffer (Si:Buffer) from 3:1, 2:1, 1: 1 were used for the reaction. Once this parameter was fixed then the composition of th e gel was varied by varying the silica ratio of sodium silicate silica with the colloidal silica Ludox (HS 40) viz. 1:1, 1:2, 1:3, 1:4 and 1:5 yielding gels with different characteristic s. The immobilised cells with Si:Buffer ratio 1:1 and the composition of gel with Si:Ludox ra tio 1:3 gave best resu lts. The effect of loading of cells onto the silica matrices wa s studied by loading diffe rent amount of cells in the system. The wet weight of the cel ls loaded on the silica matrix was 0.24 gm, 1.2gm, 2.4gm, 4.8gm, and 12 gm. The biotransfo rmation was carried out in a shake flask reactor by resuspending the crushed silica gel glass in 50 ml M-9 media without NH4Cl and adding 50mM of ethyl acetoacetate as a substrate and glucose for recycling the cofactors. Centrifuging the reaction mixture and resuspending the catalyst in a fresh medium tested reusability of the immobilized cells for undergoing an additional round of biotranformations. Product Recovery In case of whole cell reacti ons the substrate was convert ed to product completely and then the aqueous reaction media was contin uously extracted with ethyl acetate in a continuous extractor. The arrange ment was as in the figure.

PAGE 62

51 Figure 3-1. Experimental set-up for con tinuous extraction for product recovery The ethyl acetate was concentrated and re moved over a rotary evaporator leaving the pure product behind. Distil lation was used to separate product and substrate if present. In the case of the ISPR colum n, passing acetone through the column and the acetone evaporating the acetone over a rota ry evaporator washed out the product adsorbed on the column. In case of a bipha sic system involving BEHP, the aqueous and the organic phase were separated using centrifugation and the product from BEHP was extracted by distillation. Produc t from the aqueous phase wa s recovered using the usual method. In the case of product recovery fr om cell-free extract systems, (NH4)2SO4 was added to the medium to preci pitate the proteins and the product then extracted using chloroform from the reaction mixture. In the case of biphasic reactions with cell-free extracts, the organic phase was removed us ing a separatory funnel and concentrated giving the product, which was pooled with the product obtained from the aqueous medium using the above-described method. Pum p Or g an Aq ueo

PAGE 63

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54 (33) Yang, L.; Wei, D.; Zhang, Y. J. Chem. Tech. & Biotechnol. 2004 79 480-485. (34) Stark, D.; von Stockar, U. In Situ Product Removal (ISPR) in Whole Cell Biotechnology During the Last Twenty Years ; Adv. in Biochem. Eng./Biotechnol., Springer: Berlin/Heidelberg, 80 ed., 2003; pp 149-175. (35) Nakamura, K.; Fuji, M.; Ida, Y. J. Chem. Soc., Perkin Trans. 1 2000 3205 3211. (36) Lye, G. J.; Woodley, J. M. Trends in Biotechnology 1999 17 395-402. (37) Houng, J. Y.; Liau, J. S. Biotechnology Letters 2003 25 17-21. (38) Hilker, I.; Alphand, V.; Wohlgemuth, R.; Furstoss, R. Adv. Synth. Catal. 2004 346 203-214. (39) Wubbolts, M. G.; Favre-Bulle, O.; Witholt, B. Biotechnol.Bioeng. 1996 52 301308. (40) Kwon, Y. J.; Kaul, R.; Mattiasson, B. Biotechnol.Bioeng. 1996 50 280-290. (41) Cull, J. D. H.; Vargas-Mora, V.; Seddon, K. R.; Lye, G.J. Biotechnol.Bioeng. 2000 69 227-233. (42) Panke, S.; Wubbolts, M. G.; Schmid, A.; Witholt, B. Biotechnol.Bioeng. 2000 69 91-100. (43) Straathof, A. J. J. Biotechnol. Prog. 2003 19 755-762. (44) Laane, C.; Boeren, S.; Vos, K.; Veeger, C. Biotechnol.Bioeng. 1987 30 81-87. (45) Bhler, B.; Bollhalder, I.; Ha uer,B; Witholt, B; Schmid, A. Biotechnol.Bioeng. 2003 82 833-842. (46) Bhler, B.; Bollhalder, I.; Ha uer,B; Witholt, B; Schmid, A. Biotechnol.Bioeng. 2003 81 683-694. (47) Shieh, W.; Gopalan, A. S.; Sih, C. J. J. Am. Chem. Soc. 1985 107 2993-2994. (48) Zhou, B.N.; Gopalan, A. S.; VanMiddlesworth, F.; Shieh, W.; Sih, C. J. J. Am. Chem. Soc. 1983 105 5925-2926. (49) Patel, R. N.; McNamee, C. G.; Banerjee, A.; Howell, J. M.; Robison, R. S.; Szarka, L. J. Enz. Microbiol. Tech. 1992 14 731-738. (50) Kita, K.; Kataoka, M.; Shimizu, S. J. Biosci. Bioeng. 1999 88 591-598.

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55 (51) Shimizu, S.; Kataoka, M.; Katoh, M.; Morikawa, T.; Miyoshi, T.; Yamada, H. Appl Environ Microbiol. 1990 56 2374–2377. (52) R. Len, Fernandes, P.; Pinheiro, H. M.; Cabral, J. M. S. Enz. Microbiol. Technol. 1998 23 483-500. (53) Chin-Joe, I.; Straathof, A.J.J.; Pr onk, J.T.; Jongejan, J.A.; Heijnen, J.J. Biocatal. Biotrans. 2002 20 337-345. (54) Gong, P. F.; Xu, J. H.; Shen, D.; Liu, Y.Y. Appl. Microbiol. Biotechnol. 2002 58 728 734. (55) Yamamoto, H.; Kobayashi, Y. Appl. Microbiol. Biotechnol. 2003 61 133-139. (56) Liu, Y.; Xu, Z.; Jing, K.; Jia ng, X.; Lin, J.; Wang, F.; Cen, P. Biotechnol. Lett. 2005 27 119-125. (57) Shimuzu, S.; Kataoka, M.; Kita, K. Ann NY Acad Sci 1998 864 87-95. (58) Rotthaus, O.; Krger, D.; Demuth, M.; Schaffner, K. Tetrahedron 1997 53 935938. (59) Munoz-Elias, E. J.; McKinney, J. D. Cellular Microbiol. 2006 8 10-22. (60) Fraenkel, D. G. Glycolysis. Escherichia co li and Salmonella: Cellular and Molecular Biology. ASM Press: Washington, DC, 1996, pp 189-198 (61) Fischer, E.; Sauer, U. Eur J Biochem 2003 270 880-891. (62) Lim, S. J.; Jung, Y. M.; Shi, H. D.; Lee, Y. H. J. Biosci.Bioeng. 2002 93 543549. (63) Mascarenhas, D.; Ashworth, D. J.; Chen, C. S. Appl Environ Microbiol. 1991 57 2995–2999. (64) Ferreira, B. S.; Fernandez, P.; Cabral, J. M. S. Multiphase Bioreactor Design ; Taylor & Francis: London, 2001, pp 85-114. (65) Klibanov, A. M. Science 1983 219 722-727. (66) Hartmeier, W. Immobilized Biocatalysts: An Introduction ; Springer-Verlag: Berlin, 1988; pp 1-35. (67) Mosbach, K. Immobilized Enzymes and Cells ; Academic Press: Orlando, 1987; pp 1-584.

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56 (68) Cao, L. In Carrier-bound Immobilized Enzymes: Principles, Application and Design. WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim, 2005, pp 1-52. (69) Livage, J.; Coradin, T.; Roux, C. J. Phys.: Condens. Matter 2001 R673-R691. (70) Nassif, N.; Bouvet, O.; Rager, M. N.; Roux, C.; Coradin, T.; Livage, J. Nat. Mat. 2002 1 42–44. (71) Coiffier, A.; Coradin, T.; Roux, C.; Bouvet; O.; Livage, J. J. Mater. Chem. 2001 11 2039 2044. (72) Bhatia, R. B.; Brinker, C. J.; Gupta, A. K.; Singh, A. K. Chem. Mater. 2000 12 2434-2441.

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57 BIOGRAPHICAL SKETCH Parag Anil Parekh is from Pune, India. He is a graduate of the Fergusson College, Pune, with a Master of Science in organic chemistry. He joined the National Chemical Laboratory as a Research A ssistant. He was assigned to a project sponsored by Department of Biotechnology, Government of India, investiga ting the oxidation of polyunsaturated fatty acids. He joined the Depa rtment of Chemistry at the University of Florida to advance his career gaining expe rtise in the field of bioprocess chemistry.


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Copyright Date: 2008

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Title: Bioprocess Development for Asymmetric Reductions by Saccharomyces Cerevisiae Enzymes
Physical Description: Mixed Material
Copyright Date: 2008

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Source Institution: University of Florida
Holding Location: University of Florida
Rights Management: All rights reserved by the source institution and holding location.
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BIOPROCESS DEVELOPMENT FOR ASYMMETRIC REDUCTIONS BY
Sachharomyces cerevisiae ENZYMES













By

PARAG ANIL PAREKH


A THESIS PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
MASTER OF SCIENCE

UNIVERSITY OF FLORIDA


2006




























Copyright 2006

by

PARAG ANIL PAREKH




























This document is dedicated to my Family.
















ACKNOWLEDGMENTS

I would like to thank my parents for their continuous encouragement for my

graduate education. I would also like to thank my brother, Nitin. Thanks go to all the

Stewart group members.




















TABLE OF CONTENTS


page

ACKNOWLEDGMENT S .............. .................... iv

LI ST OF FIGURE S .............. .................... vii


AB S TRAC T ......_ ................. ............_........x


CHAPTER


1 BACKGROUND ................. ...............1....._.._ ......

Oxidoreductases............... .............
Cofactors .................. ... ......_.__ ...............3.....
Mechanism of Hydride Delivery ........._.... ...._._ .....__ ......__ ...........3
Asymmetric Reductions Using Bakers' Yeast Enzymes..........._..._. ........._.__........4
Applications of Baker' s Yeast Enzymes ................. ...............5...............
Isolated Enzyme Catalysis............... ...............5
Substrate-coupled regeneration ................ ...............5.................
Enzyme-coupled regeneration............... ..............
W hole Cell Biocatalysis .............. ...............7.....
Long Term Obj ectives ........._..... ...............8......... .....

2 RE SULT S .............. ...............9.....


Biotransform nations .............. .............. ...... ... ..................9
Section I: Bioprocess Development for Reduction of Ethyl butyrylacetate .................9
Biotransformations with Non-growing Cells .............. ...............10....
Strategy I: Centrifugation............... ...................1
Strategy II: In situ product removal (ISPR) .............. ....................1
Strategy III: Biphasic reactor (ATPS) ................. ..... ........ ................ 17
Bioprocess Development for the Reduction of Ethyl butyrylacetate .................. 19
Section II: Bioprocess Development for Reduction of Ethyl 4-chloroacetoacetate ...20
Biotransformations with Non-growing Cells .............. ...............21....
Strategies to overcome substrate inhibition .............. ....................2
Slow substrate feed............... ...............21..
Slow-release biocatalysis ............ .. ............. .......................2
Addition of organic solvent to increase the solubility of the ECAA ...........22
Addition of surfactants to the whole cells ....._____ .........__ ..............23
Biotransformations with Cell-free Extracts: ....._ ....___ .....__ .24











Biotransformation with cell free extracts under controlled pH conditions ..26
Biotransformation using cell free extract in a two-phase system ...............26
Section III: Bioprocess Development for Reduction of Ethyl acetoacetate ...............28
Biotransformation with Non-growing Cells............... ...............28.
M etabolic Engineering ............... .. ..... ...... .. .. .......3
Section IV: Bioprocess Development using Immobilised Cells and Enzymes. .........32
Optimal Total Si: Buffer Ratio for Better Product Formation. ...........................35
Different Composition of the Silica Gel Matrix for Better Product Formation ..3 8
Effect of Amount of Cell Loading on the Product Formation. ................... .........40
Conclusion ................. ...............42......._ ......

3 EX PE RIMENT AL ................. ...............46..._.__.......


M materials .............. .. ...............46...
Gas Chromatography ............ ....._._. ...............46.....
Sample Preparation for GC............... ...............47...
Glucose Assay .............. ...............47....
Media Preparation ........._._.._......_.. ...............47.....
Cell Culturing ............... ... ..... ...... ........4
Biotransformations with Non-growing Cells............... .......... ..........4
Biotransformation with In situ Product Removal External Column ..........................49
Biotransformation in Biphasic Reactor .............. ...............49....
Biotransformation with Cell-free Extracts............... ....... .... ........4
Biotransformation in Biphasic Medium with Cell-free Extracts .............. ..............49
Biotransformation with Immobilized Cells .............. ...............49....
Product Recovery ............ ...... ..._. ...............50....

LI ST OF REFERENCE S ........._._.._......_.. ...............52....

BIOGRAPHICAL SKETCH ............. .............57......


















LIST OF FIGURES


Figure pg

1-1 Enzyme catalyzed reductions ..........._......_ .....__ ..........2

1-2 Enzyme catalyzed oxidations ........ ................ ...............2......

1-3 Nicotinamide cofactors NAD' and NADP' ............. ...............3.....

1-4 Stereochemistry of hydride transfer from NAD(P)H to the carbonyl carbon on
the substrate (S = small and L = large group) ............. ...............4.....

1-5 Cofactor recycling by the coupled-substrate method .............. .....................

1-6 Co-factor recycling by coupled substrate method .............. .....................

1-7 Cofactor recycling by coupled enzyme method ...._._._._ ........____ ...............7

1-8 Principle of whole-cell reduction of carbonyl compounds ................... ...............8

2-1 Reduction of ethyl butyrylacetate .............. ...............9.....

2-2 Cell growth in a 4L New Brunswick fermentor and bioconversion carried out in
1L Braun Bioststat B fermentor .............. ...............10....

2-3 Increasing substrate (EBA) feed rate ................. ...............11...............

2-4 No additional product formed after bolus addition of ethyl 3-hydroxy
butyrylacetate at 1.25 hrs. ............. ...............12.....

2-5 Product removal by centrifugation. Whole cells resuspended in fresh aqueous
m edia. ............. ............... 13....

2-6 Experimental setup for in situ product removal .............. ...............16....

2-7 In situ product removal using an external column packed with resin Amberlite
XAD-4 ............ .. ..__. ...............16...

2-8 Biphasic reactor (aqueous two phase system) ...._._._.. .......___.. ........_._.......18

2-9 Bioconversion in a biphasic reactor containing BEHP as the organic phase...........19











2-10 Bioprocess development for the reduction of ethyl butyrylacetate ................... .......19

2-11 Reduction of ethyl 4-chloro acetoacetate ................ ...............20...............

2-12: Slow decomposition of ECAA at pH > 7 in aqueous medium ............... ... ............_21

2-13 Reduction of ECAA by E. coli whole cells expressing different Bakers'Yeast
reductases .............. ...............22....

2-14 Comparison of product formation with and without organic solvent ................... ...23

2-15 Coupled enzyme regeneration of NADPH using glucose dehydrogenase from
B. subtili s ................ ...............24................

2-16 Reduction of ECAA with cell free extracts with different ratios of reductase and
glucose dehydrogenase (GDH) .............. ...............25....

2-17 Reduction ECAA with cell free extracts under pH controlled conditions ...............26

2-18 Reduction of ECAA with cell free extracts in biphasic conditions..........................27

2-19 Bioprocess development for reduction of ECAA .............. ....................2

2-20 Reduction of ethyl acetoacetate............... ..............2

2-21 Maj or pathways of glucose metabolism in E. coli. ................ ........................29

2-22 Comparison of wild type and Apgi knockout for product formation and glucose
consumed per mole of product formed. A) OD 600 = 1 1. B) OD 600 = 14. C) OD
600 = 17 ............... ...............3 1

2-24 Reactions occurring during formation of sol-gel synthesi s of silica.............._._......3 4

2-25 Product mM / gm catalyst for the reduction of ethyl acetoacetate using
immobilized whole cells in silica gel glass with different Si:Buffer ratio. ..............35

2-26 Productivity for the reduction of ethyl acetoacetate using immobilized whole
cells in silica gel glass with different Si:Buffer ratio. ............... ...................3

2-27 Reuse of immobilized cells for the biotransformation with different Si: Buffer
ratio............... ...............36.

2-28 Productivity of the reused immobilized cells with different Si:Buffer ratio............37

2-29 2nd Reuse of immobilized cells with different Si: Buffer ratio .............. .... ........._..37

2-30 Productivity of the immobilized cells after 2nd TOUSe with different Si: Buffer
ratio............... ...............38.










2-31 Product mM / gm catalyst for the reduction of ethyl acetoacetate using
Immobilized whole cells in silica gel glass with different NaSi:Ludox ratio.. ........38

2-32 Productivity for the reduction of ethyl acetoacetate using immobilized whole
cells in silica gel glass with different NaSi:Ludox ratio. ................ ................. ..39

2-33 Reuse of immobilized cells for the biotransformation with different NaSi:Ludox
ratio............... ...............39.

2-34 Productivity of the reused immobilized cells different NaSi:Ludox ratio ..............40

2-35 Product mM / gm catalyst for the reduction of ethyl acetoacetate using
immobilized whole cells in silica gel glass with different amount of cell loading. .41

2-36 Productivity for the reduction of ethyl acetoacetate using immobilized whole
cells in silica gel glass with different amount of cell loading. ............. .................41

2-37 Plug -flow column reactor ........._..... ...............42._.........

3-1 Experimental set-up for continuous extraction for product recovery.......................51
















Abstract of Thesis Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Master of Science

BIOPROCESS DEVELOPMENT FOR ASYMMETRIC REDUCTIONS BY
Saccharomyces cerevisiae ENZY ME S

By

Parag Anil Parekh

May 2006

Chair: Jon D. Stewart
Maj or Department: Chemistry

Biotechnological methods are becoming increasingly important in industrial

production of fine chemicals. But, the total number of compounds made from biocatalysis

on an industrial scale is still limited due to disadvantages like long reaction times and low

productivity associated with bioprocesses. We used Saccharomyces cerevisiae enzymes

to asymmetrically reduce P keto esters to chiral hydroxyl esters, which are important

building blocks as versatile chiral synthons for asymmetric synthesis of pharmaceuticals

and agrochemicals.

Different levels of consideration for development of bioprocess were applied to

achieve the maximum bioprocess efficiency.

Biocatalyst Enzyme vs. Whole cells

Form Free vs. Immobilized

Medium Water vs. Organic









We focused on reduction of three different substrates, ethyl butyryl acetate, ethyl 4-

chloro acetoacetate and ethyl acetoacetate. Different problems varying from product

inhibition, substrate toxicity and poor substrate solubility were tackled using techniques

like in situ product removal, aqueous two-phase system, reactions with cell-free extracts,

etc. Reduction in the operating costs of the process was achieved by using immobilized

cells and application of metabolic engineering tools. Studies of these bioprocesses in non-

conventional media like ionic liquids or other immobilization techniques like cross-linked

enzyme aggregates will further improve the bioprocess efficiency of the system.















CHAPTER 1
BACKGROUND

Biocatalysis is an attractive alternative to chemical conversions.1-4 The reaction

conditions are mild, the solvent is generally water and the biocatalysts are easily

decomposed after use. Biocatalysis is therefore environmentally friendly green chemistry

and a sustainable technology. Additionally, biocatalysis often circumvents protection and

deprotection steps in the synthesis of products with chiral centers. These properties give

biocatalysis an edge over chemical conversions.

One of the main disadvantages encountered in biocatalysis is the application of

biocatalysis at large scale, i.e. the scale-up of reactions and low space-time yield.

The biochemical engineering required to develop a mature process from early

observations in the lab is demanding and therefore the number of compounds made from

biocatalysis is relatively limited. 6 The aim of this study is to apply various techniques to

overcome these limitations and make a model bioprocess more efficient.

Oxidoreductases

Oxidoreducatases comprise approximately 25% of presently known enzymes.

Almost 30% of all industrial biocatalytic processes involve oxidoreductases. 6 They

perform interesting reactions such as reduction, epoxidation, hydroxylation,

dihydroxylation etc. There are two maj or categories of oxidoreductases: dehydrogenases,

also known as reductases, and oxygenases.









Reductases are enzymes that reduce carbonyl groups yielding chiral products like

alcohols, acids or their esters or amino acids respectively. Examples of these reactions

are shown in Figurel-1.

O H OH+
R R + NADH +H R R + NAD




R C~OOR1 + NADH + H R CO1 A




R COOOH + NDH + H R COOH + NAD

Figure 1-1. Enzyme catalyzed reductions

Oxygenases use molecular oxygen as co-substrate and are used for oxidation of

non-functionalised C-H or C=C bonds yielding hydroxyl or epoxy products. 9 Examples

of these reactions are shown in Figurel1-2.

R R




H20Epoxide
Hydrolase

R R

RR1 ordiooxygenase RR1OH orOH





)nBaeyer-Villiger


Figure 1-2. Enzyme catalyzed oxidations









Cofactors

Oxidoreductases require redox cofactors, or coenzymes, which accept or donate

chemical equivalents for reduction or oxidation. Most oxidoreductases require

Nicotinamide Adenine Dinucleotide (NAD ) or Nicotinamide Adenine Dinucleotide

Phosphate (NADP ) as the cofactor; some require Flavin Adenine Dinucleotide (FAD )

or Flavin Mononucleotide (FMN). These cofactors are very expensive and are relatively

unstable.

In the enzymatic reduction of a substrate, these cofactors participate in the redox

reaction via the direct transfer of hydride (H-) ions either to or from the cofactor and a

substrate. Oxidoreductases acting as oxygenases abstract a hydride ion from the donor

and transfer it to the nicotinamide moiety. Oxidoreductases acting as reductases abstract a

hydride ion from the reduced nicotinamide and transfer it to the carbonyl to reduce the

sub state.

N)-H2




H2NOC O----

HO OH HO OR

NAD+ R= H
NADP+ R= PO3-2

Figure 1-3. Nicotinamide cofactors NAD+ and NADP+

Mechanism of Hydride Delivery

Hydride transfer is highly stereoselective. The delivery of the hydride from the si or

re face of the carbonyl results in its reduction to yield either (R) or (S) alcohol. There are

four possible stereochemical patterns that enable transfer of hydride from the cofactor to









the substrate. This hydride transfer is well studied and results from the specific binding of

the substrates as well as coenzymes on the chiral enzyme surface in close proximity and

defined geometry. 10, 11 The four different hydride transfers, two from the re face and two

from the si-face, result in either (R) or (S) alcohols corresponding to transfer of pro-R and

pro-S hydrides from the cofactors.






Hs HR
Hs NHfae re face CONH2




NARD(P)HNADR(P)H


Figure 1-4. Stereochemistry of hydride transfer from NAD(P)H to the carbonyl carbon
on the substrate (S = small and L = large group) 12

Asymmetric Reductions Using Bakers' Yeast Enzymes

Baker' s Yeast (BY), Saccharomyces cerevisiae, is cheap, readily available and easy

to use. Thus it is a very popular catalyst for asymmetric reductions of carbonyl

compounds. 13 Baker' s Yeast has more than 40 different carbonyl reductases in its

genome which may have overlapping substrate specifieities, but with different

stereoselectivites, yielding a mixture of stereosiomeric alcohols. 14 To overcome this,

Iwona Kaluzna in the Stewart lab made a library of twenty P-keto ester reductases from

Bakers' Yeast, S. cerevisae, over expressed in E. coli cells with a Glutathione S-

transferase tag. 15 These reductases are used for the synthesis of hydroxyl esters which

hold a very important place in organic chemistry as they can be transformed into various

functionalities, without racemization, to synthesize industrially important chemicals. 16









Applications of Baker's Yeast Enzymes

Isolated Enzyme Catalysis

Isolated dehydrogenases, when used as biocatalysts, give cleaner reactions, less

side-products and less purification problems than the whole cell reactions. 17 The main

disadvantage of using isolated dehydrogenases is the cofactor requirement, which in most

of the Baker' s yeast carbonyl reductases is NADPH. Is NADPH is relatively unstable and

expensive and thus is impractical to use in stochiometric amounts. Therefore an in-situ

regeneration of NADPH is a prerequisite for large-scale applications to be economically

feasible. Recycling of cofactors can be done chemically, electrochemically,

enzymatically, etc. 19-21 The efficiency of recycling is measured by the number of cycles

that can be achieved before a cofactor molecule is destroyed. It is expressed as total

turnover number (TTN), which is the total number of moles of product formed per mole

of cofactor during the complete course of the reaction. The most successful application of

cofactor regeneration is by enzymatic methods, either by coupled-substrate or coupled-

enzyme processes. 2

Substrate-coupled regeneration

In substrate-coupled regeneration, a single enzyme, a dehydrogenase, accepts an

additional substrate that regenerates the cofactor for further use. This additional substrate

is generally an aliphatic alcohol which is oxidized to a ketone to regenerate the cofactor.

23 Though this approach is elegant it has several disadvantages such as enzyme

deactivation and co-substrate inhibition due to presence of large amounts of auxiliary

substrate. 17




















































Auxiliary Auxillary
Substrate EnyeBSubstrate H2


Figure 1-6. Co-factor recycling by coupled substrate method 1

Formate dehydrogenase, which oxidizes format to carbon dioxide, is the enzyme

of choice for NADH regeneration. Carbon dioxide is easily removed and not toxic to

protein with concomitant reduction of NAD+ to NADH. 25 Recycling of NADPH with

glucose dehydrogenase or glucose 6-phosphate dehydrogenase is widely employed. 26' 27


NAD(P)H Single NAD(P)
Enzyme


SEnzyme A




NAD(P)H NAD(P)


Substrate


Substrate


Substrate H2


Substrate H2


Auxiliary J Auxillary
Substrate Substrate H2


Figure 1-5. Cofactor recycling by the coupled-substrate method 1

Enzyme-coupled regeneration

In enzyme-coupled regeneration, two enzymes are employed. In this case, two

parallel redox reactions are catalyzed by two different enzymes, i.e. conversion of main

substrate and cofactor recycling. 24 For best results, both enzymes should have different

specificities for their respective substrates so that the substrates do not have to compete

for the active site of a single enzyme, but are efficiently converted independently by the

two biocatalysts.










Glucose is oxidized to gluconolactone that spontaneously hydrolyzes to gluconic acid.

These regeneration techniques require pure proteins. Special equipment and training is

required to purify proteins further increasing costs of the process.

H O ~Formate Dehydrogenase CO



NAD(P) NAD(P)H

HOR HOR
Glucose 6-Phosphate Dehydrogenase -
HO YO Glucose Dehydrogenase H~O ~O
HOH 1 HO tneuO
R=H

R =Phosphate NAD(P) NAD(P)H
HR
HOH
HO COOH
Gluconic Acid OH


Figure 1-7. Cofactor recycling by coupled enzyme method

Whole Cell Biocatalysis

The cofactor regeneration problems can be circumvented by in vivo application of

dehydrogenases since the host cell regenerates cofactors as a part of its normal

metabolism. 28 The whole cell biocatalysts function as miniaturized reaction vessels,

which produce functional enzyme, regenerate cofactor, take up the substrate and convert

substrate to product that diffuses out of the cell. Cofactor recycling in vivo requires

metabolically active cells; when reducing equivalents are no longer generated, the desired

reaction also stops. Therefore the cells must be maintained in a state that permits high co-

factor regeneration. A minimum requirement for this is that the cell membrane remains

intact during the biocatalysis process.










Maj or drawbacks of whole cell biocatalysis are formation of by-products and

purification of the product. Essentially no special training is required to run whole cell

mediated reactions.

E. coli


Carbonyl Reduced
Compound Chiral Product
NAD(P)H NAD(P)+

Co-substrate I By-product



Figure 1-8. Principle of whole-cell reduction of carbonyl compounds 29

Long Term Objectives

We want to study reductions of P-keto esters and increase the bioprocess efficiency

of the system. The bioprocess efficiency is defined as the main parameters influencing

the cost of the process. It is addressed in terms of yield, final product concentration (g/L

of product), volumetric productivity (i.e. the space time yield g/L/hr) and catalyst

consumption (product/catalyst ratio gm/gm or biocatalyst/substrate ratio g/g).

The goal of this work is to standardize protocols and develop a more efficient

bioprocess using genetic techniques and optimization of reaction conditions and apply

them to make a pool of chiral products within an infrastructure /technology platform.















CHAPTER 2
RESULTS

We have classified our work in four different sections for reductions of three

different P-keto esters viz. ethyl butyrylacetate (EBA), 4-chloro ethyl acetoacetate

(ECAA) and ethyl acetoacetate (EAA) and solved different problems encountered in the

bioprocess development.

Biotransformations

Section I: Bioprocess Development for Reduction of Ethyl butyrylacetate




O ~E.Coli BL21(DE3) pAA3 O
Ethyl butyrylacetate Gre2p Ethyl 3-hydroxy butyryl acetate

Figure 2-1. Reduction of Ethyl butyrylacetate

Enzymatic reduction of ethyl butyryl acetate to ethyl 3-hydroxy butyryl acetate

(EHBA) was carried out in our lab yielding an enantiomeric excess of >98% using

several of the yeast reductases found in Bakers Yeast. Of the enzymes present in our

library, Gre2p was selected and over expressed in E. coli strain BL21 (DE3) containing

the plasmid pAA3 to give the S alcohol with >98% enantiomeric excess. 16 The plasmid

contained genes for kanamycin resistance and Gre2p, whose expression is under the

control of phage T-7 RNA polymerase.

When induced by isopropy-P-thiogalactopyranoside (IPTG), the efficient T-7 promoter

over expresses Gre2p that equals about 20% of the cell dry weight.









Biotransformations with Non-growing Cells

The whole cell reductions were carried out with non-growing cells using the

general procedure of Walton and Stewart. 30 The cells were grown in Luria-Bertani

medium (LB) with 4g/L glucose and were harvested just before the cells reached

stationary phase. The cells were resuspended in 1 L of minimal composed of M-9

medium without NH4Cl to prevent cell growth.


Cell Growth 41 Biocatalysis






Air
S Lstrai -2 p tre












IAirSulpp~l yL


Figure 2-2. Cell growth in a 4L New Brunswick fermentor and bioconversion carried out
in 1L Braun Bioststat B Fermentor

Parameters such as temperature, pH, and dissolved oxygen tension were optimized

to give the best results and then were kept constant. The reductions were carried out at

30 o C, pH = 7.0, and the dissolved oxygen tension at 75% saturation. The bioconversions

were conducted at one-liter scale with biomass loading at OD 600 18. Different substrate

feed rates to the reactor with a pump was used to find the maximum rate of bioconversion

as shown in Figure 2-3.





10 20 30 40 50

Time hrs

-4 11.5mM/hr +, 8.5mM/hr 1.6mM/hr


60 70


Figure 2-3. Increasing substrate (EBA) feed rate

The product formation stopped after the product reached a concentration of about

50 mM. Even after increasing the cell loading in the system, there was no observable

increase in the product formation. This led us to believe that the product was inhibiting

the biocatalyst, i.e. the product is toxic to the catalyst. Product inhibition was confirmed

by performing an experiment wherein along with a substrate addition of 15 mM/hr we did

bolus addition of 50 mM at 1.25hrs. No further product formation was observed

confirming the product inhibition of the biocatalyst (Figure2-4).











90

80

70

60

E 50

a 40 _1 50mM EHBA @1.25hrs

30

20


10

0 2 4 6 8
Time hrs



Figure 2-4. No additional product formed after bolus addition of ethyl 3-hydroxy
butyrylacetate at 1.25 hrs.

When substrates or products are inhibitors of biocatalysts, the eventual product

concentrations are very low, making the product recovery laborious and expensive.

Therefore, strategies were developed to maintain sub-toxic product concentration in the

reactor to attain high process yields.

Strategy I: Centrifugation

A very basic strategy was used where the biocatalyst was resuspended in fresh

aqueous buffer after the product reached the critical concentration. The reaction media

was centrifuged at 6000 rpm and the biocatalyst then resuspended in a new aqueous

medium for a new round of bioconversion. Substrate was added at rate of 15 mM/hr.











90
80
70
60
~50
40
30
20
10


0 5 10 15 20 25
Time hrs

-M Total -* Before centrifugation -A After centrifugation


Figure 2-5. Product removal by centrifugation. Whole cells resuspended in fresh aqueous
media.

While the final product concentration achieved was much higher, this strategy

proved to be only of limited use and could not be applied due to the inconsistent behavior

of the whole cells after centrifugation.

During the whole cell catalysis, the cell membrane is affected due to long exposure

to the substrate and the product. These cells cannot withstand the high forces exerted on it

during centrifugation. This causes many of the cells to become metabolically inactive,

which in turn causes the biotransformation to be less productive.

Strategy II: In situ product removal (ISPR)

As a result of the product inhibition, the adverse effect on the biocatalyst can be

minimized by the removal of the product as soon as it is formed. This also helps to

increase the productivity of the system. In situ product removal techniques address this









problem by removing product (or maintaining low concentrations of substrate) formed

from the vicinity of the biocatalyst. 31

The separation of product from the reactor can be carried out in different modes.

Different techniques are available for batch, fed-batch or continuous process. 32,33 The

separation step can occur within the bioreactor or outside it. Different physical and

chemical properties like volatility, molecular size, solubility, charge, hydrophobicity, etc

are exploited as the driving force for separation of the product (or substrate). Different

techniques like distillation, gas stripping, membranes (micro filtration and ultra filtration

techniques are available), pervaporation or perstraction, extraction, supercritical carbon-

dioxide, precipitation, crystallization, ion-exchange, electrodialysis, hydrophobic-

interaction chromatography, adsorption and different affinity methods can be employed

for the separation of product. 34 The most commonly used separation technique for in situ

product removal relies on adsorption of product (or substrate) to a hydrophobic resin. The

hydrophobic Amberlite XAD resins have been reported to be used most widely in

industrial context. 35, 36 We selected XAD 4 from XAD 4, 16 and 18, because it had the

highest capacity to adsorb ethyl butyrylacetate.

The easiest way to perform an ISPR is to preadsorb substrate onto a resin and add

the resin to the reactor where the resin would act as a second phase. The resin would

adsorb the product formed, and during the reaction, the concentration of the starting

material or the product would not increase beyond a certain point due to adsorption and

desorption of the product and substrate. 37 This approach is easy but has its own

limitations. More resin added to the reactor results in more mass transfer and oxygen









transfer limitation of the substrate with the biocatalyst, affecting its performance. Also,

increased stirring rate may lead to cell lysis by shearing.

To avoid this we used a reactor called a "Recycle Reactor". 38 Here, the

bioconversions and product extraction were carried out in two separate units. The

charged resin was placed as a fixed bed into a column in an external loop. In the

bioconversion unit, substrate was added and when the product concentration neared the

product inhibition levels the cell broth was passed through the resin by a peristaltic pump.

The resin could be reused because the solid phase extraction was performed in the

external loop to avoid the continuous and vigorous stirring conditions in the reactor.

Unfortunately, ethyl butytrylacetate is less polar than the hydroxyl product and was

thus preferentially adsorbed on to the column, lowering the resin's capacity for the

hydroxyl product. Therefore all the substrate added was exhausted prior to the product

removal. The medium was then recirculated through the resin column until all the product

and substrate were removed from the medium. A new batch of biotransformation with the

same biocatalyst was carried out enabling a transition to semi-continuous process from a

fed-batch process. The substrate was added at the rate of 15 mM/hr.

Detailed studies of this system can lead to a much more efficient continuous

process for the reduction of ethyl butytrylacetate by adding the substrate at limiting rate;

under these conditions all substrate is converted to product, which can be continuously

concentrated in the extraction unit by adjusting the rate of the cell broth through the

external loop.


























Figure 2-6. Experimental setup for in situ product removal


~x*",


r f


0 5 10 15 20
Time hrs


25 30 35 40


-Total Product Formed -*After Each XAD Adsorption
Figure 2-7. In situ product removal using an external column packed with resin Amberlite
XAD-4


In Situ Product Remnoval (ISPR1









Strategy III: Biphasic reactor (ATPS)

Problems arise when conducting biocatalysis in aqueous medium due to poorly

soluble substrate and or products. Low concentrations of the product or substrate, that are

hydrophobic can be maintained in the reactor by use of a "Biphasic Reactor" or an

"Aqueous Two-Phase System" (ATPS). 39, 40 This can be achieved by addition of an

immiscible organic solvent or other non-conventional media like ionic liquids to the

reaction mixture which act as a substrate reservoir or a product extractant. 41, 42 The

solvent acts as a bulk extractant or sink, which effectively withdraws product or substrate

from the aqueous phase, and keeps the effective concentration of these compounds near

the biocatalyst at low levels. As products are concentrated in organic phase, they can be

easily separated from the biocatalyst suspension and the extractants can be recycled,

therefore making the process more cost-effective.

The most important task when using the biphasic reactor system is to find a

biocompatible organic solvent. In selection of the auxiliary organic phase in the

bioprocess, the most important parameter is the log P value of the organic phase. 43,44 Log

P is the value indicating the partition coefficient of the solvent in the octanol-water two-

phase system. Microbial whole cells are generally compatible with solvents with log P

value greater than 4.

Another factor desirable in the organic phase is a favorable distribution coefficient

for the biotransformation product. The organic phase should show high product recovery

capacity and high selectivity. These are quantified by the partition coefficient (Kp) and

the separation factor (a). Kp is defined as the ratio between product concentration in

solvent and its concentration in aqueous medium; the separation factor (a) is the ratio










between partition coefficient of the product and the other contaminant from which we

want to isolate the product, e.g. substrate.

We tried two organic phases: hexadecane and bis ethyl hexyl phthalate (BEHP)

with BEHP giving better results. 45, 46 BEHP is cheaper and higher boiling than

hexadecane, has low flammability and shows no toxicity towards E. coli cells. Therefore

we carried out our further studies with BEHP, efficiently overcoming product inhibition.

The reaction was conducted in a fed-batch two-liquid phase reactor. The reduction was

carried out with BEHP as the organic phase with a ratio of 0.5, i.e. the apolar solvent

consisted of 50 % of the reactor volume. The reactions were carried out with OD600 Of 18,

22 and 25 with no significant increase in total final product formation for higher biomass

loading. The substrate was added at the rate of 50 mM/hr.




substrate

pO2











SAqueous


Figure 2-8. Biphasic reactor (aqueous two phase system)





























10 15 20 25 30
Time hrs
-C Organic +e Total


SAqueous


Figure 2-9. Bioconversion in a biphasic reactor containing BEHP as the organic phase

Bioprocess Development for the Reduction of Ethyl butyrylacetate

The bioprocess efficiency for the reduction of ethyl butyryl acetate was increased

using techniques like in situ product removal and biphasic reactor.


61 5.45
8 4.87


2.78


Aqueous


ISPR
Techniques


Biphasic


Figure 2-10. Bioprocess development for the reduction of Ethyl Butyryl Acetate









Section II: Bioprocess Development for Reduction of Ethyl 4-chloroacetoacetate




Cl O ~E.Coli BL21(DE3) plK x C

Ethyl 4-chloro Acetoacetate Ketoreductase Ethyl 3-hydroxy 4-chloro Acetoacetate

Figure 2-11i. Reduction of ethyl 4-chloro acetoacetate

Asymmetric reduction of ethyl 4-chloro acetoacetate leads to the formation of (R)

or (S) ethyl 4-chloro 3-hydroxy butanoate (ECHB). Both the (R) and (S) enantiomers are

important chiral synthons in the synthesis of various pharmaceuticals and agrochemicals.

47 For example, the R- enantiomer is an important chiral building block for the synthesis

of (-)-macrolactin A, L-carnitine and (R)-y-amino-P-hydroxybutyric acid. 48 On the other

hand the S enantiomer is a key chiral intermediate in the enantioselective synthesis of

slagenins B and C and in the total synthesis of a class of HMG-CoA reductase inhibitors.



Baker' s Yeast reduction of ethyl 4-chloro acetoacetate results in products with low

optical purity owing to the presence of forty different reductases of opposing

stereoselectivites. 5o An in-house fusion protein library of 20 P-keto ester ketoreductases

from Sacchromyces cerevisae over expressed in E. coli was screened for the reduction of

ethyl 4-chloro acetoacetate. A group of yeast enzymes denoted by their genetic codes

YJRO96w, YDL124w, YHR104w, YGL185c, YALO61w and YOL151w bearing the

plasmids plK-9, plK-8, plK-29, pAKS-1, plK-28 and plK-3 yielded an enantiomeric

excess of >99% to give the optically pure (S) enantiomer. 16









Biotransformations with Non-growing Cells

The whole cell reductions were carried out with non-growing cells using our

standard procedure wherein cells were harvested just before reaching stationary phase

were resuspended in non-growing minimal media (M-9 media composition without

NH4C1) with a 4: 1 dilution with an OD600 18. Temperature and dissolved oxygen were

kept constant at 30oC and 75 % saturation.

Ethyl 4-chloro-acetoacetate slowly decomposes in aqueous medium to 4-

chloroacetoacetic acid which is known to be a toxic compound for E.coli cells. 51 This

decomposition is accelerated at pH >7 and high concentration (the mechanism of

decomposition is not known) but no decomposition was observed if the pH was lowered

to 6.00.Therefore, a pH of 6.00 was maintained during the bioreduction.

O Ouou O O7 l
Cl~~ ~ ~~~~~ O qeu H>7 lw4C H OH

Figure 2-12: Slow decomposition of ECAA at pH > 7 in aqueous medium

Strategies to overcome substrate inhibition

Slow substrate feed

Different rates for substrate feed from 1mM/hr to as low as 60 CIM/hr were tested to

avoid substrate inhibition. As soon as the substrate is added it is converted to the product,

but this prolonged the reaction time and made it an extremely slow process. The reactions

were carried out for 24 hrs. Also, product formation never exceeded 18 mM.









18
16
14
g 12
S10
I 8-


(1 6


pik-8 pik-8 pik-9 pik-9 pik-28 pik-28 pik-29 pik29 pik-3 pik-3 paks- paks-
1 1
MOD 18 OOD 20 HOD 25

Figure 2-13. Reduction of ECAA by E. coli whole cells expressing different Bakers'Yeast
reductases

Slow-release biocatalysis

In reactions that show substrate inhibition, the substrate concentration cannot be

too high. Absorbing resins have been used for in situ product removal to prevent product

inhibition and cell toxicity. Similarly, ion exchange resins can absorb a large quantity of

substrate and then release it slowly to the reaction solution. 37 This technique is effective

in eliminating substrate inhibition. Ten grams of resin Amberlite XAD-16 were

preadsorbed with 40mM of ECAA so that the substrate is slowly released into the

solution. The slow-release biocatalysis did not work as intended yielding 25mM product

formation at much slower rate than without the resin.

Addition of organic solvent to increase the solubility of the ECAA

The solubility of ethyl 4-chloro acetoacetate is very in low in water, and this limits

the substrate available to the biocatalyst making the bioprocess inefficient. Organic

solvents can be added in small amounts to dissolve the hydrophobic compound and

increase its availability to the whole cells. 52 Ethyl 3-hydroxy butyrate (50mM), which










was readily available in our lab from reductions of ethyl acetoacetate by the same

biocatalyst, was added as an organic solvent to dissolve ECAA. 53 The addition of

organic solvent worked to a smaller extent increasing the product formation to 34 mM.


40

35

30

a 25

m 20




10


0 10 20 30 40 50 60

Time hrs
-oWith organic solvent -.Without organic solvent


Figure 2-14. Comparison of product formation with and without organic solvent

Addition of surfactants to the whole cells

When whole cells are used, the cell membrane acts as a barrier to the transport of

substrate to the enzyme and hampers optimal biotransformation. Different surfactants like

Tween 20 and Triton-X have been used to increase the permeability of the cells,

facilitating the transport of substrate and product across the cell membrane. 54 We used

Tween 20 and Triton-X in 0.5 % w/v in the reactor but product was not formed in greater

amount than in the absence of the surfactant.

Further studies are required to understand the effect of ethyl-4 chloroacetoacetate

on whole cells.










Biotransformations with Cell-free Extracts:

Since we were not able to increase the bioprocess efficiency with whole cells, we

turned to reductions of ECAA with cell free extracts. 55-57 Here the complex network of

cofactor regeneration within an active cell breaks down. All our reductases are NADPH

dependant and cannot accept NADH as a cofactor. Due to the high cost and relative

instability of NADPH, it cannot be added in stochiometric amounts and needs to be

regenerated. We tried the coupled-substrate regeneration using isopropanol as an

auxiliary substrate but none of our enzymes accepted it as a co-substrate. We then tried

the coupled-enzyme regeneration system for NADPH using glucose dehydrogenase

(GDH) from B.subtilis over expressed in E.coli as the second enzyme. It oxidizes glucose

to gluconolactone that spontaneously hydrolyses to gluconic acid. This GDH did not

accept ECAA as a substrate.

E. Coli BL21(D E3) plK x
Cl O ~Ketoreductase > C


NAD(P)+ NAD(P)H

H OR O OHO OR O

HO H
Glucose Dehydrogenase


Spontaneous O
OH
OH

OH


Figure 2-15. Coupled enzyme regeneration of NADPH using glucose dehydrogenase
from B.subtilis over expressed in E.coli BL21(DE3) cells for the reduction of
ECAA using Bakers' Yeast reductases over expressed in E.coli BL21(DE3)

Cells over expressing YDL124w gene product and glucose dehydrogenase were

grown separately in a 4-liter fermentor and were harvested just before they reached the










stationary phase. These cells were lysed using a French Press. These cell-free extracts

were then mixed together in M-9 minimal medium (without NH4C1) and the

biotransformation performed by addition of the substrates ECAA and glucose. The

volume of the reactions was kept constant at 50ml in all cases. Different biomass ratios of

ketoreductase to GDH, viz. 8:1, 4:1, and 2:1 were applied to find the best ratio to give

better productivity. 200 mM substrate was added to the shake flask reactor. Final product

concentrations of 100mM were reached.


120 1


10 15 20

Time hrs


25 30


Figure 2-16. Reduction of ECAA with cell free extracts with different ratios of reductase
and glucose dehydrogenase (GDH)

A small increase in the final product titer was observed by using higher ratio of

ketoreductase but the amount of catalyst consumed was more per mole of product

formed.










Biotransformation with cell free extracts under controlled pH conditions

The formation of gluconic acid via gluconolactone from glucose oxidation by GDH

used for regeneration of the nicotinamide co-factors caused the pH to drop, making the

conditions acidic. The acidic conditions, though good for ECAA (no decomposition at pH

6.00 and lower), are harmful to NADPH causing it to degrade. The bioconversions were

carried out under controlled pH conditions at pH 7.00 yielding higher final product

concentrations. 200 mM substrate was added to the shake flask reactor. A final product

concentration of 160mM was obtained.


180-

160-

140-

120-

E 100-

a 80-

60-

40-


20

0 5 10 15 20 25 30
Time hrs



Figure 2-17. Reduction ECAA with cell free extracts under pH controlled conditions

Biotransformation using cell free extract in a two-phase system

ECAA is poorly soluble and unstable at neutral pH in water thus limiting the

product formation. To overcome this, an organic solvent is added to the medium so both

the product and substrate can be extracted in the organic phase. 58 The bioconversions

were carried out with benzene and ethyl acetate as the organic phase and compared with





































































Whole cells + Cell-free Ce
Organic extracts extr
Solvent co

Type of system for reduction


C


reductions in the aqueous medium. 200 mM substrate was added to the shake flask


reactor.


80

70

60

S50

40 -

S30

20

10


Time hrs

-* Benzene Normal Aq -- Et Ac


Figure 2-18. Reduction of ECAA with cell free extracts in biphasic conditions

To compare the values of final product concentration reached in each case i.e.


whole cells, cell free extracts, pH controlled cell-free extract, two-phase cell free extract


the product formation was calculated as product mM per gm of biocatalyst used.


V5


E4
S3


3.75


Whole cells


lIl-free Cell-free
acts pH extracts
,ntrol organic


Figure 2-19. Bioprocess development for reduction of ECAA









Section III: Bioprocess Development for Reduction of Ethyl acetoacetate


O ~E. Coli BL21(DE3) pAA3 O
Ethyl acetoacetate GRE2p Ethyl (S) 3-hydroxy butyrate

Figure 2-20. Reduction of ethyl acetoacetate

Biotransformation with Non-growing Cells

Reduction of ethyl acetoacetate was done with non-growing E. coli BL21(DE3)

harboring the plasmid pAA3 over expressing the ketoreductase Gre2p from Baker' s

Yeast. The reaction was carried out in a reactor with 1L minimal media (M9 without

NH4C1) yielding an OD600 18 at 30oC, pH 7.00 dissolved oxygen tension 75%. The

maximum product formation observed was 285 mM, and it consumed 228 mM glucose

producing 1.25mole of NADPH per mole of glucose consumed. Stewart and Walton

published a basic study of reduction of ethyl acetoacetate where in they established that

NADPH dependent reactions could be carried out under non-growing conditions using

glucose-fed bath conditions. 30 It was proposed in this study that the efficiency of the

biocatalytic process can be further enhanced and the costs reduced. The central carbon

metabolism in E. coli proceeds via the Embden-Meyerhoff-Parnas (EMP) pathway

(glycolysis), the Phosphate pentose pathway (PPP) and the Entner-Doudoroff pathway

(EDP). 59 The EDP pathway links together the "starting" points of the EMP, ED and PPP

pathways. The pyruvate formed is decarboxylated and is further metabolized by Krebs

cycle.











Glucorse~ Glucose is Gluconate
PEP ATP glk1 ATP gt

Pyrurat GI case-6-P nL G Blcniate-8-P
pg? NADP NADrPHIc~l&
F~llrtn rNADPM


2-Ke o-3-deox y-6-
P-GIucsfata


EDP


Figure 2-21. Major pathways of glucose metabolism in E.coli. Glycolysis (Embden-
Meyerhoff-Parnas Pathway), Pentose Phosphate pathway (PPP) and the
Entener-Doudoroff pathway (EDP).Gene names are italicized. 60


pts,PEP:glucose phosphotransferase system:
glk, glucose kinase:
pgi, phosphoglucose isomerase:
ppk, phosphofructokinase:
fbp, fructose-1 ,6-bisphosphatase:
fba, fructose-1,6-bisphosphate aldolase:
tpi, triosephosphate isomerase:
gap, glyceraldehyde 3-phosphate
dehydrogenase:
pgk, phosphoglycerate kinase:
pgin, phosphoglyceratemutase:
eno, enolase;
pvk, pyruvate kinase:
pps, PEP synthase:
ppdk, pyruvate phosphate dikinase:


gcd, glucose dehydrogenase:
gntK, gluconate kinase:
zw~f glucose-6-phosphate dehydrogenase:
pgl, 6 -pho sphogluconolactonase ;
edd, 6-phosphogluconate dehydratase;
eda, 2 -keto-3 -deoxy -6 -pho sphogluconate
aldolase:
gnd, 6-phosphogluconate dehydrogenase:
rpe, ribulose-5-phosphate epimerase:
tkt, transketolase:
tal, transaldolase:
rpi, ribose-5-phosphateisomerase transketolase;
tal, transaldolase:
rpi,ribo se -5-pho sphateisomerase


PI, NA~D IJ

Glycerat-1,3-P2
nocAD
ATP~
GClyce rate-3-P

Spgmr
Gl~ycerate-2-P









Metabolic Engineering

The tools of metabolic engineering were applied for the optimization of

biocatalysts to lower the bioprocess costs. The targeted change of flux rates in the

metabolism for the purpose of optimization of product yield is called metabolic

engineering. In this case glucose was used as the source of reducing equivalents. In E.

coli, 80% of the glucose is consumed via the EM pathway. The other 20% is sent via the

HMP shunt and consumed through the pentose pathway. 61 The NADPH produced per

mole of glucose consumed can be improved by increasing the carbon flux through the

pentose phosphate pathway. This could be achieved by over expressing the two NADP+

producing dehydrogenases of this pathway or by knocking out the

phosphoglucoi somerase (pgi) gene and forcing the carbon flux through the phosphate

pentose pathway. 62,63 We selected the later and the strain with the knocked out pgi gene

was made by Despina Bouigioukou in our lab.

The pgi knockouts were compared with the cells with the pgi gene (referred to as

the wild type) for the NADPH produced per mole of glucose consumed. The

biotransformation was carried out with our usual conditions of temperature 30oC, pH 7.00

and dissolved oxygen tension 75%. The reactions were carried out at different biomass

loading giving an OD 600 Of 11, 14 and 17. The substrate was fed with a pump at the rate

of 20 mM/hr.











16

14



o 10

E 8
E
S6

4

2

0


2-



* 1.5 o


1
b

0.5 0.


0


5 10 15 20 25
Time hrs

-* WT ~ pgi--*- pgi- P/G -m WT P/G


2.5


2-






1 0


0.5


0


12 -

1" 10



06

4

2


as-


I4




Ia


0 5 10 15 20 25 30 35 40
Time hrs

Spgi WT --pgi P/G -- -WT P/G










18 12.:5
16
S14 -1 ---;C~ 2




E8 -
I 1


a. 0.5 a.


0' 0
0 5 10 15 20 25 30 35
Time hrs

~pgi -m-WT -- -pgi P/G -- -WT P/G

C)
Figure 2-22. Comparison of wild type and Apgi knockout for product formation and
glucose consumed per mole of product formed. A) OD 600 = 11 B) OD 600 = 14
C) OD600 = 17.

The delta pgi knockout strain was better than the wild type. It produced more

NADPH per mole of glucose consumed and the final product concentration per gm of

cells was higher than the wild type. So,the efficiency of the bioprocess was increased.

Section IV: Bioprocess Development using Immobilised Cells and Enzymes.

Biocatalyst costs are often important. Immobilization of biocatalysts is a method

where catalyst is recycled or its prolonged use leads to significant reduction in catalyst

cost. 64 Also, improved performance such as activity, stability, selectivity, and

productivity can be achieved by immobilization. Immobilized biocatalysts can be

considered as a composite consisting of two essential components: the non catalytic

structural component (carrier) which is designed to aid separation and reuse of the

catalyst, and the catalytic functional component (enzyme or whole cell), which is

designed to convert the desired substrates into desired products. 65 Immobilization can be









either carrier bound or carrier free. The criteria for effective immobilized systems are

defined by requirements such as mechanical and chemical stability, high volume activity,

operation stability, reusability, easy disposal and safety depending on the non-catalytic

and catalytic function of the system. 66 The catalytic functions are responsible for the

desired activity, selectivity, substrate specifieity, productivity and space-time yields. The

non-catalytic functions are dependent on reactor configurations (batch, stirred-tank,

column, plug-flow), the type of reaction medium (aqueous, organic, biphasic), the

process conditions (temperature, pressure, pH). 67 The selected parameters should

facilitate downstream processing and control of the process.

A carrier can be considered as a modifier of the biocatalyst as it is not only the

support on which the biocatalyst can be attached but its surface chemistry, pore size and

hydrophilicity or hydrophobicity dictates the activity, selectivity and stability of the

biocatalyst. 68 A large number of synthetic or natural carriers with different

characteristics are available according to the requirements. Enzyme carriers can be of

different types like beads, hollow fibers, capsules, films, membranes, sol-gels,

mesoporous molecular sieves and inorganic nanocomposites. 66







Beads Fibers Capsules Filrns Membranes

Figure 2-23. Different types of carriers for immobilization of biocatalyst 66

Bioencapsulations with silica sol-gel glasses, which is biologically inert and

imparts better mechanical strength and chemical stability and does not swell in aqueous

or organic solvents, is a new and interesting field. 69, 70 This process of immobilization










involves no high temperature and harsh chemical reactions; it has the ability to

immobilize biomolecules without modifying their structure or function greatly.

Moreover, the porosity can be controlled with selection of proper precursors, modifiers

and polymerization conditions.

Sol-gel entrapment techniques often use orthosilicate such as tetramethyl

orthosilicate (TMOS), tetraethyl orthosilicate (TEOS) or methyl trimethoxy silane

(MTMS) as precursors. n1 In aqueous media, the hydrolysis of precursor results in the

formation of an oligomer that is further hydrolyzed to form an aqueous sol. The

occurrence of gelation process leads to development of a three dimensional network

where the biomolecules are trapped. A maj or disadvantage of this process is that the high

concentration of ethanol or methanol produced during the immobilization process is

harmful to the activity of enzymes or cells. Recently a sodium silicate silica based

process for sol-gel formation was reported which avoids the formation of alcohol

completely. 72

OR
RO-Si--OR + 4 H20 Si(OH)4 + 4 ROH
OR


Si-OH + Si-OH SiO2 + H20

Figure 2-24. Reactions occurring during formation of sol-gel synthesis of silica

We decided to study the immobilization of whole cells within the silica gel glass

due to the dependence of our enzymes on NADPH cofactors. Sodium silicate silica and

colloidal silica (LUDOX HS40) was used for synthesis of silica gel. Phosphoric acid was

added to decrease the pH to 7.0 before adding the whole cells. The gel was air-dried for

75 hrs and then crushed. This crushed silica gel glass containing the biocatalyst was

resuspended in non-growing minimal M-9 medium without NH4GI for the










biotransformation. The reduction of ethyl acetoacetate was used as the model reaction

and E. coli BL21(DE3) expressing the enzyme GRE2p harboring the plasmid pAA3 was

used for the bioconversions. Different experiments were performed to improve the

productivity of the biotransformation using immobilized cells. 50 mM substrate was

added in all the reactions. Also, in all the experiments the reusability of the immobilized

cells was checked. The immobilized cells were centrifuged and then resuspended in fresh

buffer for repeated use.

Optimal Total Si: Buffer Ratio for Better Product Formation.

The ratio of the total silica content to the buffer added with a fixed amount of cell

loading yielding gels with different properties was studied .The volume ratios used were

3:1, 2:1i, 1:1 respectively. The gelation occurred within one minute.


10 35

E +, ~30 E


6 20

I ~15





0 0
0 2 4 6 8
Time days

3:01 2:01 -a- :01---*--- Control

Figure 2-25. Product mM / gm catalyst for the reduction of ethyl acetoacetate using
immobilized whole cells in silica gel glass with different Si:Buffer ratio.
Secondary axis corresponds to control











35 600

301 -. -iC+ 500,
S25 -1
400
E 20 -1
," i.300 .
15
~200
S10

5 --- .... 10

0 0
0 2 4 6 8
Time days

3::01 2:01 1t :01 -+- -control

Figure 2-26. Productivity for the reduction of ethyl acetoacetate using immobilized whole
cells in silica gel glass with different Si:Buffer ratio. Secondary axis
corresponds to control.

The immobilized cells were checked for their reusability. The free and immobilized

cells were centrifuged and resuspended in a fresh medium and glucose and substrate was

added to the system for a new round of bioconversion.


18-
E 16-
S14-



S2-

i 0 21 4 8 10 12 1
H ~ ~ ~ ~ ~~~Tm days------~ ..~~.
o tol 30 :110
Fiur 2-7 es fimblzd el o h itasorainwt ifrni
Bufr ai













120

> 100






40

20 -1 ..2



0 2 4 6 8 10 12 14

Time days

+- -control 3:01 2:01 -M- 1:01


Figure 2-28. Productivity of the reused immobilized cells with different Si:Buffer ratio


These cells were again centrifuged and reused for another round of


biotransformation.


_I
__
I


Time days


---*--- control 3:01


2:01 1:01


Figure 2-29. 2nd Reuse of immobilized cells with different Si: Buffer ratio












120-
C 100-
80-
S60-
40-


LL0
0 2 4 6 8 10 12 14 16
Time days

+--control 7t3:01 2:01 -M01:0


Figure 2-30. Productivity of the immobilized cells after 2nd TOUSe with different Si: Buffer
ratio

For all the above experiments Si: Buffer ratio of 1:1 gave the best results and was


kept constant for the next experiments.

Different Composition of the Silica Gel Matrix for Better Product Formation

Once we determined the ratio of total Si:Buffer, we decided to optimize the silica

matrix by varying the Si:Ludox ratio. This ratio would the change the composition of the


gel and thus affect the characteristics of the matrix affecting the biotransformation.


121 25


10 8~~- ;
E 15 E

E 10 E
t; 4- / S
o 2 -1 o

0 0
0 24 6 810 12
Time days
1:01 1:02 1t:03 ~1:04 -11:05 +--control



Figure 2-31. Product mM / gm catalyst for the reduction of ethyl acetoacetate using
Immobilized whole cells in silica gel glass with different NaSi:Ludox ratio.
Secondary axis corresponds to control.











1300

250

200

150

S100

50
0

8 10 12


Time days


~1:01


1:02 1:030 ~1:04 -11:05 +- -Control


Figure 2-32. Productivity for the reduction of ethyl acetoacetate using immobilized whole
cells in silica gel glass with different NaSi:Ludox ratio. Secondary axis
corresponds to control.


Time days

- +- -control ~1:01 1t:03 ~1:04 ~1:05


Figure 2-33. Reuse of immobilized cells for the biotransformation with different
NaSi:Ludox ratio












100-
90-
S80-
70-
E 60-

j 40- .
S30-
2 20-
10

0 2 4 6 8 10 12 14 16
Time days

+- -control -A-1:01 -a- :03 -*-1:04 ~1:05


Figure 2-34. Productivity of the reused immobilized cells different NaSi:Ludox ratio

NaSi:Ludox with a ratio 1:3 gave the best results. The gels used for further tests

had the Si:Buffer ratio 1:1 and NaSi:Ludox ratio 1:3.


Effect of Amount of Cell Loading on the Product Formation

We studied the effect of different amounts of cell loading in the silica-gel matrix.

This was an important parameter affecting the bioconversions. There definitely is an


optimum loading of cells beyond which results in lower productivity. The loading

amount varied from 0.24 gm to 12 gm of E.coli BL2 1 (DE3) pAA3 (wet weight) in

200ml total volume of the system.




















































5 10 15 20


30

25

20

15



10


-70

-60


-40
-30





0 5 10 15 20 25


Time days

+- -control 2.4 ~-5 1.2 -A-2.4 4.8 -M-12 ~-50.24


Figure 2-35. Product mM / gm catalyst for the reduction of ethyl acetoacetate using
immobilized whole cells in silica gel glass with different amount of cell
loading. Secondary axis corresponds to sample with 0.24 g biocatalyst.


40
S35
30
S25
E20
S15
10
o
5
0


Time days

4.8 -M- 2 +- -control 2.4


0.22 4 ~- 1- 1.2 .4


Figure 2-36. Productivity for the reduction of ethyl acetoacetate using immobilized whole
cells in silica gel glass with different amount of cell loading.

We also studied the stability of immobilized cells. The immobilized cells stored at

room temperature for three weeks showed no deterioration and were perfectly normal.


The product formation was exactly the same as in the case of the immobilized cells used










immediately without storing. The free cells cannot be stored at room temperature for

more than a day.

All previously mentioned reactions were carried out in shake flask reactors. We

tried to use a plug-flow column reactor and were able to get the product, but the product

formation was low. We suspect that due to diffusion barrier created by the silica gel glass

around the biocatalyst.

Further studies are required to make the silica gel glasses with the right porosity to

enhance diffusion and increase the product formation rate. Also, this would enable us to

use the immobilized cells in plug-flow column type reactors further increasing the

productivity of the system

Substrate






Biocatalyst










Product

Figure 2-37. Plug -flow column reactor

Conclusion

In conclusion, we have tried to overcome the disadvantages of biocatalysis such as

the scale up of reactions, very low space-time yields and product or substrate inhibition,

etc. using different methods for each problem.









Production of optically active alcohols using either whole cells or enzymes by the

enantioselective reduction of ketones is a very important activity in biocatalysis. We

studied the reduction of ethyl butyryl acetate, ethyl 4-chloro acetoacetate and ethyl

acetoacetate. Each reduction was accompanied by its own unique set of problems that

were overcome using simple bioprocess solutions.

Ethyl butyrylacetate reduction yielding ethyl 3-hydroxy butyrylacetate was limited

due to the product inhibition of the biocatalyst. We applied simple techniques like in situ

product removal using solid resins and enhanced the productivity of the system.

Additional increases in the productivity can be achieved by using the in situ SFPR

technique i.e. the in-situ substrate feeding and product removal techniques wherein the

substrate feed is adjusted so that all the substrate added is converted to product which is

removed promptly from the reactor. Thus a steady concentration of the product and

substrate is maintained at all times, and longer reaction times can be achieved resulting in

higher productivities.

Another method that was explored to avoid product inhibition was the use of a

Biphasic reactor also known as an Aqueous Two Phase System (ATPS). In this method,

we added an organic solvent to the reactor and the product and substrate would partition

in the two phases. The total concentration of the inhibitory substance, the product in our

case, around the biocatalyst is reduced enabling more product formation. We used Bis-

Ethyl-Hexyl Phthalate (BEHP) as the organic phase because it is inexpensive and

increased the final product concentration of the product. BEHP also made product

recovery easier because most of the product can be removed from the organic phase by










simple distillation as the boiling point of BEHP is very high, allowing for the separation

of organic phase and the aqueous phase by centrifugation

Using solvents with better partition rates can optimize the biphasic approach. Ionic

liquid solvents are a good example because they are environmentally friendly, have

essentially no vapor pressure and can be reused, making them very attractive for

biocatalysis.

Reduction of ethyl-4 chloro acetoacetate presented different problems, such as

toxicity of the substrate to the whole cells. The amount of substrate tolerated by the

whole cells was very low, thus making the use of whole cells unattractive as it slowed the

reaction time substantially. The reactions with cell free extracts with a cofactor

regenerating system were tried with moderate success. The final product concentration

increased from 18 mM to 100 mM. pH control of this reaction system further increased

the product formation to 165 mM. Some basic studies regarding biphasic reactions using

benzene and ethyl acetate as organic solvents with the cell-free extracts helped increase

the rate of reaction. More studies with these biphasic organic reactions with pH and

temperature control are required to increase the final product titer and the productivity.

Application of metabolic engineering to further reduce the costs of the bioprocess

was successfully achieved. The glucose metabolism of the E coli cells was routed through

the pentose phosphate pathway rather than the Krebs cycle, thus increasing the amount of

NADPH/glucose consumed. This was achieved by knocking out the

phosphoglucoi somerase (pgi) gene from the E coli. The engineered E. coli consumed less

glucose per mol NADPH produced and thus reducing the operating costs of the

biotransformation.









Reduction of ethyl acetoacetate was used to confirm the validity of the knockout.

The wild type produced 1.25mol NADPH per mole of glucose consumed and the

engineered pgi knockout produced 2.25mol NADPH per mole of glucose consumed.

Immobilized cell systems wherein the whole cells were encapsulated in silica

matrix were studied. This system needs further studies to be developed by altering the

porosity of the silica gel glasses formed to enhance the diffusion of the substrate and the

product. This system has its advantages regarding the reuse and storage of the biocatalyst.

We have thus improved the bioprocess efficiency for the asymmetric reduction of P-keto

esters viz. ethyl butyryl acetate, ethyl 4-chloroacetoacetate, and ethyl acetoacetate using

various techniques.















CHAPTER 3
EXPERIMENTAL

Materials

Ethyl acetoacetate, ethyl butyrylacetate, ethyl 4-chloro acetoacetate were purchased

from Sigma. (R, S) Ethyl 3-hydroxy butyrate, (R, S) ethyl 3-hydroxy 4-chloro

acetoacetate, ethyl 3-hydroxy butyrylacetate purchased from Aldrich.

Resins Amberlite XAD 4, 8, 16 purchased from Aldrich and bis-ethyl hexyl

phthalate (BEHP) from TCI America.

Gas Chromatography

GC analysis was done on Hewlett-Packard model 5890 GC/FID with a Chirasil-Val

and DBl7 columns.

Ethyl butyrylacetate was analyzed on a 25m Chirasil-Val column. The temperature

program consisted of an initial time and temperature of 2 minutes and 80 oC, a gradient of

lo C/min until the temperature reached 1 10o C, and final time of 2 minutes at 180 oC

attained with a rate of 10 oC / min.

Ethyl 4-chloro acetoacetate was analyzed on a 25m Chirasil-Val column. The

temperature program consisted of an initial time and temperature of 2 minutes and 60 oC,

a gradient of 1 oC/min until a temperature of 90 oC was reached and then with a gradient

of 10 oC/min final temperature of 180 oC with a final time 2 minutes.

Ethyl acetoacetate was analyzed on a 30m DB-17 column. The temperature

program consisted of an initial time and temperature of 2 minutes and 60 oC, a gradient of

10 oC /min until the temperature reached 180 oC, and a final time of 2 minutes.










Sample Preparation for GC

200 Cl~ of aqueous reaction media was extracted with 600 Cl~ of ethyl acetate

containing 1 mM methyl benzoate as the internal standard. After one more extraction

with 600-Cl ethyl acetate and the two organic phases were combined. Samples were

mixed using a vortex mixer. Standard curves for ethyl 3-hydroxy butyrate, ethyl 3-

hydroxy 4-chloro acetoacetate, and ethyl 3-hydroxy butyrylacetate were prepared by

making aqueous solutions of different concentrations and extracted identically. The

starting materials, i.e. the P-keto esters ethyl acetoacetate, ethyl-4chloroacetoacetate and

ethyl butyryl acetate decomposed during the GC analysis and were therefore not

quantified.

Glucose Assay

The glucose concentration in the reactor was determined offline using a Trinder

assay kit commercially available from Diagnostic Chemicals Limited, Canada. The

absorbance of the reaction, which consists of 5 Cl1 of the reaction media and Iml of the

Trinder reagent mixed by inversion and incubated at 37 oC for 15 minutes, was measured

at 505 nm. The concentration of the glucose in the reactor was measure by comparing it

to the standard reference containing 0.4g/L glucose.

Media Preparation

Cell growth was always in Luria-Bertani (LB) medium. Composition of LB

medium: 10g/L tryptone, 5g/L Yeast Extract, 10g/L NaC1. For LB plates 15g/L of agar

was used for solidification. The medium was sterilized by autoclave. This medium was

supplemented with 4g/L glucose for cell growth. The glucose addition was done with 20

% glucose solution that is autoclaved separately.









Non-growing medium for biotransformation was always M-9 minimal medium that

was autoclaved before use. It consisted of di-sodium hydrogen phosphate 12.8 g/L,

Potassium di-hydrogen phosphate 3 g/L, NaCl 0.5g/L and NH4Cl that is omitted to

maintain non-growing conditions. 2 mM of CaCl2 and 0. 1 mM of MgSO4 and 4g/L

glucose are separately sterilized and added to the total volume.

Cell Culturing

The different E.coli BL21 (DE3) strain expressing a ketoreductase used for the

biotransformation were streaked fresh weekly from a 15% glycerol stock stored at -80 oC,

onto LB plates with 40 CLM kanamycin, incubated overnight until colonies formed. Single

colonies were picked to prepare inocula for cell growth. Fresh stock of the appropriate

strain was prepared whenever necessary using the correct plasmid via electroporation.

Biotransformations with Non-growing Cells

50ml of LB was inoculated with a single colony of E.coli expressing the required

ketoreductase with kanamycin (40 mg/L) and shaken overnight. The preculture was

added to 4L of LB media with 4g/L glucose, 40 mg/L kanamycin and 0.25ml of antifoam

AF-204 (Sigma) in New Brunswick fermentor. The cells were grown at 37 oC until the

OD600 reached ~1 .The temperature was then lowered to 30 oC and the cells were induced

with 0.1mM isopropyl thio galactoside (IPTG). The cells were harvested after

6.00hrs.During cell growth the agitation speed was 800 rpm and the aeration rate 1

lit/min was kept constant.

The cells were centrifuged at 6000 rpm for 30minutes and then resuspended in

100ml of M-9 medium without NH4C1.For biotransformation, the cell slurry was added to

1L of medium to a final OD600 Of about 18.









Biotransformation with In situ Product Removal External Column

The external in situ product removal column contained 200gms of XAD-4 resin

packed in a glass column attached to the fermentor. The aqueous reaction media was

passed through it when the product neared inhibitory levels. The product free medium

was subj ected to another cycle of biotransformation.

Biotransformation in Biphasic Reactor

The biotransformation is carried out as usual but with 50% of the aqueous reaction

phase is replaced by an organic phase. Hexadecane and Bis-ethyl hexyl phthalate were

used as the organic phase. BEHP yielded better results and was used for further studies.

Biotransformation with Cell-free Extracts

The biotransformation carried out with cell free extracts involved cell growth as

usual. The cell were lysed using an ultrasonicator or a French press. The results obtained

using with either method were similar. The lysed cell extract suspension was centrifuged

at 600 rpm for 15 minutes and the supernatant used for bioconversion. The total volume

of the shake flask reactor was kept constant at 50 ml.

Biotransformation in Biphasic Medium with Cell-free Extracts

The supernatant obtained from the above-described method was then resuspended

with organic solvents forming 50% of the total reactor volume. The organic solvents used

were benzene, ethyl acetate. The total volume of the system was constant at 50 ml.

Biotransformation with Immobilized Cells

Cells grown were harvested and washed twice with minimal media M-9 without

NH4C1. To maintain non-growing conditions they were resuspended in the M-9 minimal

media without NH4Cl and 10 % (by volume) glycerol. An aqueous sol using molar

solutions of sodium silicate silica (27 wt % SiO2 with 14 wt % NaOH from Sigma) and









colloidal silica (Ludox HS 40 from Aldrich) was synthesized. Phosphoric acid was used

to maintain a pH of 7.00.The slurry containing E.coli (Buffer) was immediately added to

the sol. Gelation time varied from instantaneous to 1 minute. The total volume for sol-gel

was kept constant at 200ml. This silica gel was air-dried for 75 hrs. The resultant silica

gel glass encapsulating the whole E.coli cells was crushed with a pestle and mortar and

resuspended in minimal media M-9 without NH4C1. Different ratios of total Si content

and the buffer (Si:Buffer) from 3:1, 2:1i, 1:1 were used for the reaction. Once this

parameter was fixed then the composition of the gel was varied by varying the silica ratio

of sodium silicate silica with the colloidal silica Ludox (HS 40) viz. 1:1, 1:2, 1:3, 1:4 and

1:5 yielding gels with different characteristics. The immobilised cells with Si:Buffer ratio

1:1 and the composition of gel with Si:Ludox ratio 1:3 gave best results. The effect of

loading of cells onto the silica matrices was studied by loading different amount of cells

in the system. The wet weight of the cells loaded on the silica matrix was 0.24 gm,

1.2gm, 2.4gm, 4.8gm, and 12 gm. The biotransformation was carried out in a shake flask

reactor by resuspending the crushed silica gel glass in 50 ml M-9 media without NH4C

and adding 50mM of ethyl acetoacetate as a substrate and glucose for recycling the

cofactors. Centrifuging the reaction mixture and resuspending the catalyst in a fresh

medium tested reusability of the immobilized cells for undergoing an additional round of

biotranformations.

Product Recovery

In case of whole cell reactions the substrate was converted to product completely

and then the aqueous reaction media was continuously extracted with ethyl acetate in a

continuous extractor. The arrangement was as in the figure.





























Figure 3-1. Experimental set-up for continuous extraction for product recovery

The ethyl acetate was concentrated and removed over a rotary evaporator leaving

the pure product behind. Distillation was used to separate product and substrate if

present. In the case of the ISPR column, passing acetone through the column and the

acetone evaporating the acetone over a rotary evaporator washed out the product

adsorbed on the column. In case of a biphasic system involving BEHP, the aqueous and

the organic phase were separated using centrifugation and the product from BEHP was

extracted by distillation. Product from the aqueous phase was recovered using the usual

method.

In the case of product recovery from cell-free extract systems, (NH4)2SO4 WAS

added to the medium to precipitate the proteins and the product then extracted using

chloroform from the reaction mixture. In the case of biphasic reactions with cell-free

extracts, the organic phase was removed using a separatory funnel and concentrated

giving the product, which was pooled with the product obtained from the aqueous

medium using the above-described method.
















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BIOGRAPHICAL SKETCH

Parag Anil Parekh is from Pune, India. He is a graduate of the Fergusson College,

Pune, with a Master of Science in organic chemistry. He joined the National Chemical

Laboratory as a Research Assistant. He was assigned to a proj ect sponsored by

Department of Biotechnology, Government of India, investigating the oxidation of

polyunsaturated fatty acids. He j oined the Department of Chemistry at the University of

Florida to advance his career gaining expertise in the field of bioprocess chemistry.