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BIOPROCESS DEVELOPMENT FOR ASYMMETRIC REDUCTIONS BY
Sachharomyces cerevisiae ENZYMES
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
PARAG ANIL PAREKH
This document is dedicated to my Family.
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
ACKNOWLEDGMENT S .............. .................... iv
LI ST OF FIGURE S .............. .................... vii
AB S TRAC T ......_ ................. ............_........x
1 BACKGROUND ................. ...............1....._.._ ......
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
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
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
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
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
Parag Anil Parekh
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
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.
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.
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.
RR1 ordiooxygenase RR1OH orOH
Figure 1-2. Enzyme catalyzed oxidations
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
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
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 NHfae re face CONH2
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
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 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)
Auxiliary J Auxillary
Substrate Substrate H2
Figure 1-5. Cofactor recycling by the coupled-substrate method 1
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
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
Glucose 6-Phosphate Dehydrogenase -
HO YO Glucose Dehydrogenase H~O ~O
HOH 1 HO tneuO
R =Phosphate NAD(P) NAD(P)H
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
Compound Chiral Product
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.
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
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
S Lstrai -2 p tre
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
-4 11.5mM/hr +, 8.5mM/hr 1.6mM/hr
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).
a 40 _1 50mM EHBA @1.25hrs
0 2 4 6 8
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.
0 5 10 15 20 25
-M Total -* Before centrifugation -A After centrifugation
Figure 2-5. Product removal by centrifugation. Whole cells resuspended in fresh aqueous
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
Figure 2-6. Experimental setup for in situ product removal
0 5 10 15 20
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
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.
Figure 2-8. Biphasic reactor (aqueous two phase system)
10 15 20 25 30
-C Organic +e Total
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.
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.
pik-8 pik-8 pik-9 pik-9 pik-28 pik-28 pik-29 pik29 pik-3 pik-3 paks- paks-
MOD 18 OOD 20 HOD 25
Figure 2-13. Reduction of ECAA by E. coli whole cells expressing different Bakers'Yeast
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.
0 10 20 30 40 50 60
-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
H OR O OHO OR O
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.
10 15 20
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
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.
0 5 10 15 20 25 30
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
Type of system for reduction
reductions in the aqueous medium. 200 mM substrate was added to the shake flask
-* 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.
acts pH extracts
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
Glucorse~ Glucose is Gluconate
PEP ATP glk1 ATP gt
Pyrurat GI case-6-P nL G Blcniate-8-P
pg? NADP NADrPHIc~l&
2-Ke o-3-deox y-6-
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:
fbp, fructose-1 ,6-bisphosphatase:
fba, fructose-1,6-bisphosphate aldolase:
tpi, triosephosphate isomerase:
gap, glyceraldehyde 3-phosphate
pgk, phosphoglycerate kinase:
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
gnd, 6-phosphogluconate dehydrogenase:
rpe, ribulose-5-phosphate epimerase:
rpi, ribose-5-phosphateisomerase transketolase;
rpi,ribo se -5-pho sphateisomerase
PI, NA~D IJ
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.
* 1.5 o
5 10 15 20 25
-* WT ~ pgi--*- pgi- P/G -m WT P/G
0 5 10 15 20 25 30 35 40
Spgi WT --pgi P/G -- -WT P/G
S14 -1 ---;C~ 2
a. 0.5 a.
0 5 10 15 20 25 30 35
~pgi -m-WT -- -pgi P/G -- -WT P/G
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
RO-Si--OR + 4 H20 Si(OH)4 + 4 ROH
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.
E +, ~30 E
0 2 4 6 8
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
301 -. -iC+ 500,
E 20 -1
," i.300 .
5 --- .... 10
0 2 4 6 8
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.
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
20 -1 ..2
0 2 4 6 8 10 12 14
+- -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
---*--- control 3:01
Figure 2-29. 2nd Reuse of immobilized cells with different Si: Buffer ratio
0 2 4 6 8 10 12 14 16
+--control 7t3:01 2:01 -M01:0
Figure 2-30. Productivity of the immobilized cells after 2nd TOUSe with different Si: Buffer
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.
10 8~~- ;
E 15 E
E 10 E
t; 4- / S
o 2 -1 o
0 24 6 810 12
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.
8 10 12
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.
- +- -control ~1:01 1t:03 ~1:04 ~1:05
Figure 2-33. Reuse of immobilized cells for the biotransformation with different
j 40- .
0 2 4 6 8 10 12 14 16
+- -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
0 5 10 15 20 25
+- -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.
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
Figure 2-37. Plug -flow column reactor
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
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
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
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
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.
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
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.
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.
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
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
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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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.