Isolation, purification and characterization of Penicillium charlesii G. Smith exo-beta-galactofuranosidase

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Title:
Isolation, purification and characterization of Penicillium charlesii G. Smith exo-beta-galactofuranosidase
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xiii, 130 leaves : ill. ; 29 cm.
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Tuekam, Brigitte Albertine, 1961-
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Thesis (Ph. D.)--University of Florida, 1993.
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Includes bibliographical references (leaves 115-129).
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by Brigitte Albertine Tuekam.
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Vita.

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ISOLATION, PURIFICATION AND CHARACTERIZATION OF Penicillium
charlesii G. SMITH EXO-B-GALACTOFURANOSIDASE











BY

BRIGITTE ALBERTINE TUEKAM


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1993
















I would like to dedicate this work to the memory of my father, Gabriel

Tamdem, who passed away before its completion. Thank you, Daddy, for

teaching me how to believe in myself; I love you.













ACKNOWLEDGEMENTS

I am deeply grateful to Dr. John Edward Gander, chairman of my

committee, for his encouragement, continued support, patience and constant

effort to assist me throughout the course of these studies.

I would also like to express my sincere thanks to Dr. E.M. Hoffmann,

Dr. J.F. Preston III, Dr. K.T. Shanmugam and Dr. C.M. Allen, members of

my committee, for their time, assistance and encouragement.

Special thanks go to my husband, Dr. Tuekam and our children,

Rosine, Sandrine and Steve, whose love, support and sacrifice contributed in

achieving this work.

I also thank my mother Julienne Tamdem and my sister Horthense D.

Tamdem.













TABLE OF CONTENTS

Pages

ACKNOWLEDGEMENTS...........................................................iii

LIST OF TABLES...................................................................vii

LIST OF FIGURES...................................................................viii

LIST OF ABBREVIATIONS.........................................................xx

ABSTRACT..............................................................................xxii

INTRODUCTION.......................................................................1..

OBJECTIVE..............................................................................5

REVIEW OF LITERATURE..................................................... 6

Galactofuranosyl-Containing Extracellular or Cell
Envelope/Wall Glycoproteins and Polysaccharides.......................6

Bacteria.....................................................................6
Protozoa.................................................................9
Fungi......................................................................10

Glycohydrolases.................................................................19

Cellulases................................................................19
a-Glucanases............................................................. 22
B-Glucanases................................................................25
8-Mannanases.........................................................26
Galactanases............................................................28

MATERIALS AND METHODS......................................................32

General Procedure..............................................................32
Organism.........................................................................33

iv












Culture Condition and Growth Media........................................33
Isolation of Glycopeptides..................................... .... ......... 34
Chemical Modification of Glycopeptides.................................... 34
Dilute Acid Hydrolysis of pPGM with Release
of Galactose...........................................................34
Preparation of Oligosaccharides from pPGM....................35
Isolation of Enzymes......................................................... 35
Assay for Enzyme Activity..................................................37
Galactofuranosidase Assay.............................................37
Assays for Hydrolases ..... ........................................38
Galactose Oxidase Assay........................................... 39
Chemical Assays.............................................. ............... 39
Total Carbohydrate ...............................................39
Reducing Carbohydrate................................................40
Total Phosphate... .. ... ........................................... ......... 40
Protein ...................................... ......... ...............40
Formaldehyde.........................................................41
Chromatography ............................... .................................41
Paper Chromatography................................... ..............41
Thin-Layer Chromatography.....................................42
Ion-Exchange Chromatography.................................... 42
Detection of Compounds on Paper and Thin-
Layer Chromatography............................................. 42
G el Filtration....................... .................................43
Gel Electrophoresis.......................................................... 44
SDS-Polyacrylamide................................................. 44
Non-DenaturatingPolyacrylamide................................... 44
Isoelectric Focusing (IEF) ............................................. 45
NH2-Terminal Analysis .............................. .... ........ ......... 46
Determination of Kinetic Properties of Exo-B-
Galactofuranosidase...........................................................46
Optimum pH for Activity.........................................47
pH Stability Profile...................................................47
Optimum Temperature for Activity................................. 47
Temperature Stability................................................. 47
Km and Vm Determination..........................................48












Substrate Specificity of Exo-B-Galactofuranosidase.............48

RESULTS........................................................... ....................50

Enzyme Purification.................................................. .......50
Anion Exchange Chromatography I................................ 51
Anion Exchange Chromatography II................................51
Cation Exchange Chromatography....... .............................56
Superose-12 Gel Filtration I........................................59
Superose-12 Gel Filtration II ....................................... 59
Homogeneity of the Purified Exo-B-Galactofuranosidase................ 59
SDS-PAGE .................................................................64
Non-Denaturating PAGE...........................................68
Isoelectric Focusing....................................................68
Carbohydrate Content ............................................................ 72
Amino Terminal Amino Acid ................................................. 73
Properties of Exo-B-Galactofuranosidase.......................... ........73
pH Profile............................................................ 73
pH Stability..............................................................73
Temperature Optimum............................................. 76
Temperature Stability...... ........................................ 76
Enzymatic Products from pP2GM"i and pP30GM" .................79
Km and Vm ............................................................ 86

DISCUSSION.................................................................... ....96

CONCLUSION.......................................................... .............107

SUMMARY................................................................ ....... 108

APPENDIX............................................................................. ...109

REFERENCES.................................................................................115

BIOGRAPHICAL SKETCH............................. ........................... 130



vi

















LIST OF TABLES

Table Page

1. Purification of Exo-B-Galactofuranosidase....................................65

2. Action of Exo-B-Galactofuranosidase on Various
Oligosaccharides.................................................................87

3. Analysis of pPGMs Before and After
Treatment with Exo-B-Galactofuranosidase.................................. 88

4. Kinetic Constants for Hydrolysis of Various Substrates
by Exo-B-galactofuranosidase.......................................... ......... 92

5. Activities of Purified Exo-B-Galactofuranosidase Preparation
Toward Substrates for Phospho mono- and diesterases,
and Other Glycosidases.......................................................... 95

6. Sum m ary............................................................................ 97

7. Czapek-Dox Agar................................................................109

8. Raulin-Thom Medium........... .......................................... 110

9. Standard Growth Medium.......................................................111
















LIST OF FIGURES


Figure Page

1. Elution Profile of Exo-B-Galactofuranosidase from DE-52 Anion
Exchange Column I......................................... ............ .......... 53

2. Fractionation of Partially Purified Exo-B-Galactofuranosidase on
DE-52 Anion Exchange Column II...............................................55

3. Fractionation of Galactofuranosidase Preparation Obtained from DEAE
Cellulose Chromatography on CM-Sepharose Cation Exchange .............58

4. Elution Profile of Exo-B-Galactofuranosidase Activity on
Superose-12 FPLC Gel Filtration Column.......................................61

5. Second Gel Filtration of Exo-B-Galactofuranosidase Activity
on Superose-12 FPLC Column...................................................63

6. Photograph of Exo-B-Galactofuranosidase Following SDS-PAGE............67

7. Photograph of Exo-B-Galactofuranosidase Following IEF......... ....... 70

8. Zymogram of Exo-B-Galactofuranosidase Following IEF....................72

9. Optimum pH for Activity and pH Stability of
Exo-B-Galactofuranosidase.............................................. ...75

10. Optimum Temperature and Temperature Stability for Activity of
Exo-B-Galactofuranosidase.................................. .................... 78

11. Photograph of Paper Chromatogram of pPGM"s after Treatment
with Exo-B-Galactofuranosidase................................. ............. 81












12. Gel Filtration of Galactofuranosidase Treated-pPo0GM"......................83

13. Activity of Purified Exo-B-Galactofuranosidase on pPGM"s.................85

14. Gel Filtration of Oligosaccharides Obtained by Mild Acid
Hydrolysis of pP2GM"............................................................90

15. Plot of the Initial Reaction Rate vs Substrate Concentration................94

16. Cornish-Bowden Plots A and B for Determination of Km and Vm,.........113

17. Cornish-Bowden Plots C and D for Determination of Km and Vm .........114













LIST OF ABBREVIATIONS


CHO2- Total carbohydrate

DEAE- Diethylaminoethyl

CM- Carboxymethyl Sepharose

ELISA- Enzyme-linked immunosorbent assay

Gal- D-Galactose

Galf- D-Galactofuranosyl residue

Rhap- L-Rhamnopyranose

Glc,- D-Glucopyranose

GalpNAc- N-Acetyl-D-galactopyranose

Galp- D-Galactopyranose

IEF- Isoelectric focusing

kd- Kilo-dalton

Man- D-Mannose

Manp- D-Mannopyranosyl

NMR- Nuclear Magnetic Resonance

pPGM- Peptidophosphogalactomannan

pP2GM"- pPGM fraction II containing 2 phosphate residues












pP3oGM"- pPGM fraction II containing 30 phosphate residues

ppm- part per million

SDS-PAGE- Sodium dodecyl sulfate polyacrylamide gel electrophoresis












Abstract of Dissertation Presented to the Graduate School of
the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


ISOLATION, PURIFICATION AND CHARACTERIZATION OF Penicillium
charlesii G. SMITH EXO-8-GALACTOFURANOSIDASE

By

Brigitte Albertine Tuekam

May, 1993

Chairman: Dr. John Edward Gander
Major Department: Microbiology and Cell Science

Fungi of the genera Penicillium, Aspergillus, Talomyces and others produce soluble

extracellular polysaccharides, glycopeptides and glycoproteins. The major

extracellular polymers of Penicillium charlesii are glycopeptides:

peptidophosphogalactomannans (pPGMs). They are composed of B-D-(1-- > 5)-

galactofuranosyl, mannosyl, amino acyl and variable numbers of phophodiester

residues.

Penicillium charlesii produces extracellular enzymes such as phosphatases,

proteases and glycohydrolases. The percentage of galactofuranosyl residues in pPGM

decreases with increasing age of the culture; prior to this study, the enzyme that

catalyzes galactofuranosyl hydrolysis had not been purified to homogeneity nor its

properties investigated.









The focus of this research was to purify and characterize extracellular B-(1--

> 5)-galactofuranosidase from culture filtrates of P. charlesii. It was purified 100-

fold by ion-exchange and gel filtration chromatography; it contains 15% carbohydrate

as mannose. Mobility of the enzyme in nondenaturating PAGE indicates 2 forms of

apparent mass of 70 and 150 kd; it moves with an apparent mass of 75 kd, as a single

band in SDS PAGE. The pi determined by isoelectric focusing PAGE is 4.35; the

optimum pH of catalysis is 4.5. B-Galactofuranosidase is catalytically active between

250 -600C with maximum activity at 400C. The enzyme retains 94% of the original

activity after freezing and thawing in buffer. The enzyme catalyzes galactose release

from i) 1-O-B-methyl-D-galactofuranoside, ii) 5-O-8-galactofuranosyl-containing

peptidophosphogalactomannan and iii) 5-O-B-oligogalactofuranosides. Galactose was

not released from lactose or from p-nitrophenyl-B-D-galactopyranoside.

The specific activity of exo-B-galactofuranosidase on i) 1-O-8-methyl-D-

galactofuranoside; ii) pP2GM"; and iii) pP30GM" were 857, 417, and 134 IM hr'

mg-' protein, respectively. The apparent Km of the enzyme with these substrates is

2.6, 0.8 and 1.6 mM, respectively. Exo-B-galactofuranosidase did not release

phosphorylated galactofuranosyl residues from pPGM"s nor was it inhibited by D-

galactose-6-phosphate, D-mannose-6-phosphate or peptidophosphomannan. The

results provide the first indirect evidence showing that phosphodiesters in pPGMs have

a role in protecting the polymer from exo-B-galactofuranosidase catalyzed

depolymerization.












INTRODUCTION

Microorganisms such as bacteria and fungi are noted for the production

of extracellular and mural polysaccharides, glycoproteins and glycopeptides.

The cell surface glycoproteins may act as receptor molecules which are

involved in lectin-mediated coaggregation, or they may be involved in the

process of parasite attachment and internalization ( De Arrura et al., 1989;

McConville and Bacic, 1990; and Abeyguwardana et al., 1990, 1991a, 1991b).

Some of the extracellular polysaccharides are allergenic or toxic to humans

(Daley and Strobel, 1983; Sward-Nordmo et al., 1988). Many extracellular

glycoproteins are enzymatically active. Most extracellular enzymes have no

cellular substrate and they appear to have evolved as "scavenger" enzymes;

extracellular enzymes which have cellular substrates have been implicated in

several cellular processes such as differentiation, cell wall turnover and

growth.

Penicillium charlesii releases into the growth medium various

extracellular saccharide-containing polymers. The most well characterized

class of glycopeptides of this organism is composed of galactosyl, mannosyl,

amino acyl and phophodiesters residues (Preston et al., 1969a, 1969b; Gander

et al., 1974; Rick et al., 1974; Unkefer et al., 1982; Unkefer and Gander,

1990).









2

This class includes a heterogeneous group referred to as peptidophospho-

galactomannans (pPGMs). The percentage of galactose in pPGMs decreases as

the culture ages; this suggests the presence in the growth medium of enzymes

hydrolyzing B-D-(1-->5) linked galactofuranosyl residues.

Many extracellular glycoproteins produced by this fungus are hydrolytic

enzymes such phosphatases, proteases and glycohydrolases. Although several

investigators have studied the chemical composition and structures of

extracellular glycopeptides from Penicilliwn and Aspergillus, (Bardalaye and

Nordin, 1977, Barreto-Bergter et al., 1981; Unkefer et al., 1982; Bonetti et

al., 1990 and 1991), the function of these glycopeptides is still not understood.

However, a likely, but yet unproven role is that of a temporary storage form of

phosphate, carbohydrate, amino acid and phosphatide components (Gander and

Laybourn, 1981). Salt (1983), in his studies on extracellular glycoprotein

enzymes from Penicillium charlesii, proposed that these glycopeptides may

represent cleavage or degradation products of exocellular glycoproteins. This

hypothesis is consistent with the role for the glycopeptide in the post-

translational translocation of glycoprotein enzymes across the cell membrane

and cell wall of P. charlesii. While some evidence was presented in support of

this hypothesis, further testing is needed to demonstrate the relationship

between the glycopeptides and the extracellular glycoproteins. Isolating an










3

extracellular glycoprotein to apparent homogeneity and characterizing it should

provide clues to the relationship, if any, of glycopeptide and extracellular

glycoproteins. Also, understanding the role of enzymes that catalyze

depolymerization of these extracellular glycopeptides is fundamental in

determining the roles of the abundant phospho-glycopeptides; this may also

help in determining how they participate in the competitive process which

allows fungi to thrive in diverse environments.

In an attempt to determine the role of enzymes that catalyze the depoly-

merization of pPGM, we have recently focused our research on the purification

and characterization of exo-B-galactofuranosidase. Exo-B-Galactofuranoside-

containing polymers are immunodominant for some Penicillium and Aspergillus

spp (Preston et al., 1970; Bennett et al., 1985; Notermans et al., 1988;

Tuekam, 1991). Toxin produced by Helminthosporium sacchari is a B-D-(1--

> 5)-linked galactofuranoside-containing sesquiterpenoid (Livingston and

Scheffer, 1981). Understanding the extent of the activity of B-D-

galactofuranosidase in the depolymerization of B-D-galactofuranoside

containing-polymers is necessary in order to prevent any false results in assays

intended to detect these polymers.

An endo B(1-- >5) D-galactofuranase from Penicillium oxalicum which

specifically hydrolyzes (1-- > 5) linked B-D-galactofuranosyl residues in homo-

and heterogalactan has been reported (Reyes et al., 1992). An exo B-D-










4

galactofuranosidase isolated from a crude commercial preparation of

Trichoderma harzaniwn was purified and characterized by Van Bruggen-Van

Der Lugt et al. (1992). Penicillium charlesii also produces a wide variety of

enzymatically active extracellular glycoproteins, including acid

phosphomonoesterase, alkaline phosphomonoesterase, choline specific

phosphodiesterase, bis-(phenyl)-phosphate phosphodiesterase, N-acetyl-B-

glucoaminidase and a and 8 glucosidase (Salt, 1983; Abbas, 1987). Salt

(1983) has proposed that pPGM may be derived from these and other

extracellular enzymes by proteolysis. This research provides some insight into

this hypothesis.












OBJECTIVE

The purpose of this research is to determine the role of Penicillium

charlesii exo-B-galactofuranosidase in the depolymerization of pPGMs. The

purified galactofuranosidase will also be used to determine if there is any

similarity between this enzyme and pPGM.

Specific objectives are to

1-Isolate, purify and characterize exo-B-galactofuranosidase

2-Determine general properties of exo-l-galactofuranosidase such as pH

and temperature for activity, pi substrate specificity and kinetic

constants.

3-Determine the ability of the purified enzyme to catalyze hydrolysis of

phosphorylated glycopeptide.

4-Determine if exo-B-galactofuranosidase will cleave phosphorylated

galactofuranosyl residues.












REVIEW OF LITERATURE

Microorganisms such as bacteria and fungi often release a large variety

of polysaccharides, glycopeptides and glycoproteins into the culture medium.

These polymers often contain more than one species of sugar; mannose and

galactose are common major component sugars. Frequently one or more of

these sugars are derivatized with a phosphoryl or an acyl component. Some of

the glycoproteins are enzymatically active. The first section of this review of

literature will focus on the distribution and composition of extracellular and/or

cell wall polysaccharides and glycoproteins containing galactofuranosyl

residues and the second section will focus on glycohydrolases.

Galactofuranosyl-Containing Extracellular or Cell Envelope/Wall
Glycoproteins and Polysaccharides

Bacteria

Carbohydrate components of bacterial polysaccharides, glycoproteins,

glycolipids and proteoglycans associated with cell envelopes have a complex

structure. They are usually highly branched and feature a variety of chemical

modifications including acyl, ketal, phosphoryl and sulfuryl groups attached to

hexosyl, pentosyl or polyol sugars.









7

One of the first bacterial polysaccharides shown to contain

galactofuranosyl residues was obtained from Pneumococcus Type 34(41)

(Roberts, et al., 1963). They isolated a phosphorus-free pentasaccharide by

alkaline hydrolysis; the pentasaccharide was characterized as O-D-galacto-

furanosyl-(1-- > 3)-O-a-D-glucopyranosyl-(1-- > 2)-O-D-galactofuranosyl-(1--

> 3)-O-a-D-galactopyranosyl-(l-- > 2)-ribitol. Plackett and Buttery (1964)

isolated 6-O-8-D-galactofuranosyl-D-galactose from Mycoplasma mycoides

following treatment of a galactan obtained from that organism with dilute

mineral acid. Since these early investigations, galactofuranosyl-containing

saccharides have been isolated from several genera of bacteria.

Cell wall polysaccharides of certain oral Streptococcus spp. are

postulated to be receptor molecules for galactose and N-acetylgalactosamine

reactive fimbrial lectins of Actinomyces spp. Abeyguwardana et al. (1990,

1991a, 1991b) have demonstrated by selective chemical degradation,

methylation analysis, mass spectrometry and 'H- and 31P-NMR spectroscopy

that Streptococcus spp. cell wall polysaccharides consist of linear polymers of

hexasaccharide repeating units joined by phosphodiester bonds. The repeating

unit varies from one species to another.

S. oralis ATCC 10557 hexasaccharide repeating unit consists of [--> 6)

Galp (a-1-- > 3) Rhap(B-l-- > 4) Glcp(-l-- > 6) Galf(B--- > 6) Galp(B-1-- > 3)

GalpNAc(al--> P04] (Abeygunawarda et al., 1991a). S. oralis C104









8

hexasaccharide repeating unit is composed of [-->6) Galf(B-1--> 3) Galp(B-1--

> 6) Galf(B-l-- > 6) GalpNAc(B-1-- > 3) Galp(a-1-- > 1) Ribitol(5-- > P04]

(Abeygunawarda et al., 1991b).

S. sanguis J22 repeating unit is a heptasaccharide containing :[--> 6) a-

D-GalNAp (1-- > 3)-B-L-Rhap (1-- > 4)-B-D-Glcp (1-- > 6)-B-D-Galf (1-- > 6)-B-

D-GalpNAc (1--3) a-D-Galp (1--> P04], with a-Rhap attached (1-- > 2) to B-

L-Rhap (Abeygunawarda et al., 1990). Comparison of these structures

suggests that the similar lectin receptor activities may depend on the internal

galactofuranosyl residue linked B-(1--> 6) to Gal (B-I--> 3)GalNAc(a) or

GalNAc (B--- > 3) Gal(a).

Lipopolysaccharide of Actinobacillus pleuropneumonae, causative agent

of contagious pleuropneumonia in swine, was shown by chemical and NMR

methods to be a unique linear unbranched homopolymer containing exclusively

1--> 2 linked B-D-galactofuranosyl units (Perry, 1989).

Capsular polysaccharides of Klebsiella serotype K12 and K41 were

shown to contain, respectively, two and one galactofuranosyl residues (Beurret

et al., 1989). 0-Antigen side chain polysaccharides in the lipopolysaccharides

of Klebsiella serotype 02 are identified as a repeating unit structure which

consist of a disaccharide of 3-B-D-galactofuranosyl-(l--> 3)-a-galactopyrano-

syl. This repeating unit is identical to that of D-galactan present in a lipo-

polysaccharide of Klebsiella pneumoniae serotype 01 (Whitfield et al., 1992).









9

Mycobacterium spp cell wall arabinogalactan is covalently linked to

both peptidoglycan and high molecular weight branched mycolic acids. This

arabinogalactan constitutes approximately 35% of the cell wall mass (McNeil et

al., 1987). It has been demonstrated that arabinogalactan contains an

octasaccharide repeating unit which consists of six arabinofuranosyl residues

predominantly linked (1-->5) and two galactofuranosyl residues linked (1--

>5) or (1-->6), by a complex series of reactions involving methylation,

partial hydrolysis, sodium borodeuteride reduction, ethylation, total acid

hydrolysis, acetylation gas chromatography and mass spectrometry (McNeil et

al.,1987; Gruber and Gray, 1990; and Daffe et al., 1990).

Protozoa

Several galactofuranosyl containing high mannose type oligosaccharides

present in glycoprotein of many protozoa have been described (Gonzales

Clemente et al., 1990, Mendelson and Parodi, 1986). In eucaryotes,

oligosaccharides are processed as protein-linked glycans. Protein glycosylation

in most eucaryotes are initiated by the transfer of Glc3-Man,-GlcNAc2 from a

dolichol diphosphate derivative to asparagine residues on the polypetide chains.

Protein N-glycosylation in trypanosomid protozoa differs by the fact that, the

transferred oligosaccharides are nonglucosylated and contain galactosyl residues

(Gonzales Clemente et al., 1990). In trypanosomatid (parasitic protozoa)









10

Leptomonas samueli and Herpetomonas samuel pessoai, the carbohydrate

components of glycopeptides appeared to consist of Galf, Man, N-acetyl

glucosamine in various ratios; Galf is linked to mannose at nonreducing ends

of the transferred oligosaccharides.

Glycoproteins of Crithidia fasciculata and Crithidia harmosa also

contain galactofuranosyl residues added to the nonreducing end of mannosyl

residues. These galactofuranosides are added after removal of a single

mannose unit from one of the two mannoses originally present in the

transferred oligosaccharide (Mendelson and Parodi, 1986).

Cell surface glycoproteins of trypomastiges of Trypanosoma cruzi

contain B-D-galactofuranosyl residues which are involved in the attachment

and/or internalization of the parasite (De Arruda et al., 1989).

Lipophosphoglycan (LPG) of Leishmania spp. has a tripartite structure,

consisting of a phosphoglycan, a variable phosphorylated hexasaccharide glycan

core, and a lysoalkyl-phosphoinositol (lysoalkyl-Pi) lipid anchor. One

galactose of the glycan core is in the furanosyl configuration (McConville and

Bacic, 1990, McConville et al., 1990).

Fungi

Fungi are noted for their ability to produce extracellular and mural

glycoproteins and glycopeptides. The carbohydrate components consist primar-

ily of mannans, phosphomannans, galactans, galactomannans and glucans.










11

Extracellular glycopeptides of Ascobolus furfuraceus, with a molecular

mass of about 20 kd, contain mannose, galactose, glucose and glucosamine;

the molar ratio of each of these sugars varies with the media composition. The

variability in the amount of galactose, present as terminal furanosyl unit, may

reflect the action of an exo-B-galactofuranosidase (Groisman and Lederkremer,

1987). Evidence of two types of linkage between the carbohydrates and the

peptide moieties was provided by cleavage of the sugar chain by endo B-N-

acetyl-glucosaminidase and by alkaline treatment (Groisman and Lederkremer,

1987). Alkaline treatment of these extracellular glycopeptides results in an

increase in absorbance at 240 nm with concomitant decrease in threonine and

serine content.

B-Galactofuranosyl residues have also been described in the

proteogalactomannan from Neurospora crassa. These B-D-galactofuranosyl

units are linked to the C2 of a mannosyl residue in O-glycosidic and N-

glycosidic linkages of the peptides (Nakagima et al., 1984).

Helminthosporium sacchari, the causative agent of eyespot disease of

sugar cane, produces helminthosporoside which is a toxin. This toxin, purified

by thin layer, ion exchange and gel chromatography, was shown to contain an

oligosaccharide composed of B-(1-- > 5)-galactofuranoside units. This

oligosaccharide is linked to a sesquiterpenoid C15H22 (Livingston and Scheffer,

1981). Regulation of toxin production in the culture medium containing the










12

fungus is in part controlled by 8-galactofuranosidase produced by the fungus

(Daley and Strobel, 1983).

Cladosporium herbarun produces a glycopetide allergen Ag-54. The

carbohydrate moiety of this glycoprotein is similar to the one from Penicillium

charlesii described by Gander and coworkers (Preston et al., 1970). The

carbohydrate component of the Cladosporium polymer is a highly branched

galactoglucomannan with a molecular mass of 19 kd. Mannosyl residues are

linked a-(l-- > 2) and a-(l-- > 6), while glucosyl residues are linked (1-- > 4)

and (1--> 6). The (1--> 6) linked galactofuranosyl side chains are linked to

the C2 of the (1-- >6) linked mannosyl units (Sward-Nordmo et al., 1988).

The reducing end of mannosyl residues are O-linked to threonyl residues of a

peptide.

The yeast form of Cladosporium wernecki also produces a similar

variety of mannosyl rich, galactofuranosyl containing peptidophospho-

galactomannan (Lloyd, 1972). Trichophyton mentagrophytes elaborates a

variety of glycopeptides of molecular mass 30-40 Kd. These glycopeptides

contain primarily mannosyl residues (65-75%) with nonreducing terminal

galactofuranosyl residues (9-20%). The carbohydrate components are linked to

a peptide rich in threonine, serine, proline and glutamine residues (Barker et

al., 1967).










13

Cladosporium flavum extracellular glycopeptides consist of a

phosphorylated glucogalactomannan which is O-glycosidically linked to a

peptide rich in serine, threonine, asparagine, glutamine and proline residues.

Galactose, in these glycopeptides, is in the furanosyl configuration (Dow and

Callou, 1979; DeWit and Kodde, 1981).

Species of Cryptococcus are the only fungi with capsular

polysaccharides (Bhattacharjee et al., 1984). Cryptococcus polysaccharides are

composed of xylose, mannose, galactose and uronic acids. Cryptococcus

neoformans cell wall polysaccharides are characterized as glucurono-

xylomannan, galactoxylomannan and mannoprotein. Galactoxylomannan is

composed of mannose, galactose and xylose. Galactofuranosyl units occur

only in some types and are always present as non-reducing termini (James and

Cherniak 1992).

The major cell wall polysaccharides of Aspergillus fumigatus are

characterized as peptidogalactomannans (Azuma et al., 1968) and

galactomannans (Sakaguchi et al., 1969; Bennett et al.,1985). The

peptidogalactomannan has a mannan backbone composed of a-(l-- > 6)-linked

mannopyranosyl residues to which are attached a-(l--> 2) and a-(1--> 6)

linked mannopyranosyl residues, D-galactofuranosyl residues and glucosamine

(Azuma et al., 1971). The galactomannan consists of a branched core










14

containing a(1-->2)- and a-(1-->6)-linked mannose units with linear side

chains of B-(1-- >5)-galactofuranosyl and/or B-(1-- >4)-galactopyranosyl units

which are terminated by galactofuranosyl nonreducing end units (Bennett et al.,

1985; Latge et al., 1991; Stynen et al., 1992).

Aspergillus niger peptidogalactomannans consist of a core with a-(l--

> 6)-linked mannopyranosyl residues substituted at C2 with oligomannosides

and at C6 with (1-- >5)-linked B-D-galactofuranosyl units; the average length

of the galactofuran chains is 4 residues (Barreto-Bergter et al., 1981). Cell

wall galactomannans of Aspergillus niger consist of repeating units, with 2 to 5

mannopyranosyl residues joined by a-(l--> 2)-glycosidic linkages, connected

by a-1-->6 linkages between each manno-oligosaccharide unit. The manno-

pyranosyl residues are substituted at C2 with a tri- or tetra-saccharide of

galactose with the general structure B-D-galactofuranosyl-(1-- > 4)-(galacto-

pyranosyl),_2-(l-- > 4)-galactopyranosyl-1-- > (Bardalaye and Nordin, 1977).

Clutterbuck and coworkers (1934) first demonstrated that culture

filtrates of Penicillium charlesii G. Smith contain two extracellular

carbohydrate polymers, galactocarolose and mannocarolose, separable by

fractional precipitation with ethanol. Galactocarolose, a homopolymer of

galactosyl residues, is exceptionally labile to mild acid hydrolysis. Haworth et

al., 1937, showed by methylation and other chemical analysis that










15

galactocarolose contains 9 to 10 5-O-B-D-galactofuranosyl residues and that

mannocarolose contains four 2-O-a-D-mannopyranosyl residues attached

through a (1--> 6) linkage to another four 2-O-a-D-mannopyranosyl unit.

Investigations of the structure and biosynthesis of mannocarolose and

galactocarolose by Gander and coworkers in the 1960s revealed that both are

degradation products of a large phosphorylated extracellular precursor polymer

(Gander, 1960; Preston and Gander, 1968; Preston et al., 1969a, 1969b).

This polymer was later shown to be a peptidophosphogalactomannan (Gander

et al., 1974). These extracellular polysaccharides and glycopeptides are

believed to be primarily derived from glycoproteins, cell walls and/or

cytoplasmic membrane-bound lipo-glycoproteins.

Glycopeptides of P.charlesii, characterized as peptidophosphogalacto-

mannans (pPGMs), are protein-carbohydrate complexes with a molecular

weight in the range of 25 to 70 kd (Preston et al., 1969a, 1969b; Gander et

al., 1974; Rick et al., 1974; Rietschel-Berst et al., 1977). These pPGMs are

heterogeneous and are isolated from culture filtrates by filtration, precipitation

and anion exchange chromatography. The two major fractions, pPGM" and

pPGM'" (Salt and Gander, 1985), are composed of a mannan backbone with

approximately 80 mannopyranosyl residues linked to one another primarily

through a-(l-- >2) and a-(l--> 6) glycosidic bonds (Gander et al., 1974;










16

Rietschel-Berst et al., 1977; Unkefer and Gander, 1990). Reducing terminal

mannosyl residues of the mannan are O-linked to seryl or threonyl residues of

a peptide with 30-32 amino acyl units.

The peptide is rich in serine, threonine, alanine, glycine and proline

with almost no aromatic or sulfur containing amino acids (Rick et al., 1974).

These pPGMs also contain 12 manno-oligosaccharides, each with 1-3 residues,

attached to the peptide, 10-20 galactofuran chains with 2-10 5-O-B-D-

galactofuranosyl residues attached to the mannan backbone and a variable

number of phosphodiesters. Salt and Gander (1988) showed that the phosphate

content of the major pPGM species varies with the growth medium; pP30GM

has 30 phosphodiesters per molecule whereas pPIoGM and pP2GM have only

10 and 2, respectively. Both the mannan and galactan of pP30GM are highly

substituted with phosphodiesters bridged to the C-6 hydroxyl group of the

mannosyl or galactofuranosyl residue (Bonetti et al., 1990, 1991).

Approximately 10 phosphodiesters, primarily N-methyl-phosphoryl

ethanolamine, phosphoryl ethanolamine, phosphoryl choline are attached to the

mannan backbone (Salt and Gander, 1985). Galactan chains of pP30GM

contain approximately 20 phosphodiester residues. These phosphodiesters

consist of N-peptidyl-ethanolamine of various sizes and phosphoryl choline

(Bonetti et al., 1990).










17

Beachy (1977) isolated a galactofuranosyl-containing glycopeptide

associated with the vacuolar or cytoplasmic membrane of P. charlesii. This

glycopeptide had a molecular weight of 105,000 as determined by SDS-PAGE.

Sixty percent of the carbohydrate portion consisted of galactose in the

furanosyl configuration and the remaining was mannose in the pyranosyl

configuration. The amino acid composition showed that serine and threonine

accounted for approximately 38% of the amino acid content of the peptide.

This peptide is rich in alanine, aspartic acid/asparagine, glutamic

acid/glutamate, glycine and valine, and has no aromatic or sulfur containing

amino acids. Furthermore, the glycopeptide contained ethanolamine and

phosphate residues. The glycopeptide was deoxycholate-soluble and its

lipophilic character was attributed to a covalently linked sphingolipid base

tentatively identified as dihydrosphingosine.

Galactose content in pPGM decreases as the age of the culture increases

(Preston et al., 1969a). The organism also secretes an exo-B-galactofurano-

sidase which hydrolyses B(1-- > 5)-linked galactofuranosyl residues of pPGM

(Rietschel-Berst et al., 1977). The activity of the enzyme in the medium

accounts for the decrease in the galactose content of pPGM as the culture ages.

It was shown that 2-deoxy glucose inhibits secretion of the enzyme but not the

secretion of pPGM (Gander and Fang, 1974). These data suggest that the

enzyme is a glycoprotein containing mannosyl or glucosyl residues.










18

Rietschel-Berst et al. (1977) reported the isolation and the partial

purification of an exo-B-D-galactofuranosidase from Penicillium charlesii. This

purification was achieved by affinity and gel permeation chromatography,

using B-D-galactofuranosyl residues of pPGM as affinity ligand. The enzyme

catalyzes the release of galactose from methyl- and ethyl-B-D-galactofurano-

side, from 6-O-B-galactofuranosyl-D-galactose and from 5-O-B-D-galactofur-

anosyl containing peptidophospho-galactomannan. This enzyme was not active

on methyl-a-D-galactofuranosides or on the methyl-D-galactopyranosides. It

was later shown that the enzyme was contaminated with other glycohydrolases

and phosphodiesterases.

Pletcher et al. (1981) studied factors affecting the appearance of

exocellular exo-B-D-galactofuranosidase in culture medium of P. charlesii and

they concluded that the galactofuranosidase activity appeared in the medium

only after the medium is depleted of glucose and when the pH of the medium

is above 4. They also suggested that galactofuranosidase activity was destroyed

by an acid protease. It was shown later that when plates containing P.

charlesii have been stored at room temperature for longer than 2-4 weeks,

inoculum from these cultures showed acid protease release into the growth

medium (Abbas, unpublished).









19

Van Bruggen-Van Der Lugt et al. (1992) have demonstrated, in

addition to terminal and internal B-(1-->5)-linked galactofuranosides, B-(1--

>6) and B-(1,5,6)-linked galactofuranosides in Penicillium and Aspergillus

species using a purified exo-B-galactofuranosidase from Trichoderma

harzianum.

Glycohydrolases

Many microorganisms produce extracellular enzymes. The majority of

extracellular enzymes are depolymerases acting on polysaccharides, proteins

and nucleic acids; hydrolases constitute the majority of these enzymes,

although, the action of hexuronosyl lyases are well known also. These

extracellular enzymes have no cellular substrate and seem to have evolved as

scavenger enzymes which degrade polymeric material in the environment to

provide the organism with assimilable nutrients. Polysaccharides degrading

enzymes have a long history of commercial application in food processing,

horticulture, agriculture and protein research.

Among bacteria, Bacillus and Clostridium spp. are prolific producers of

extracellular glycohydrolase enzymes. Filamentous fungi and yeasts also secrete

a variety of these extracellular enzymes.












Cellulases

Cellulases are enzymes which hydrolyze B-(1--> 4) glycosidic bonds in

crystalline and/or amorphous cellulose, and in water soluble cellulose

derivatives such as carboxy-methyl or hydroxy-methyl celluloses. Cellulases

are inherently interesting. The study of cellulase initially focused on the means

of inhibiting their degradative action on cellulolytic material. More recently,

cellulases have gained an economic interest as they are prime agents of decay

and also the potential key to conversion of waste biomass into fermentation

feedstocks.

Three major types of enzymes are found in cellulase systems that can

degrade crystalline cellulose. They are: endo--(l-- > 4)-glucanase which

hydrolyzes cellulose at random to produce oligosaccharides with different

degrees of polymerization, exo-B-(-- > 4)-glucanase which acts by removing

glucose or cellobiose from the nonreducing end of the chain and B-glucosidase

which hydrolyzes cellooligosaccharides to release glucose (Wood and McCrae,

1982; Reese et al., 1976; Wood et al., 1980; Bhat and Wood, 1992).

Extracellular cellulases have been purified from a number of bacterial

species such as Clostridium spp. (Thomas and Zeikus, 1988; Lamed et al.,

1983; Lamed and Bayer, 1988), Cellumonas uda (Nakamura and Kitomura,

1988) and Streptomyces spp (Stutzenberger, 1991). Cellulolytic bacteria which

produced only cell bound cellulase such as Cytophoga have been described










21

(Chang and Thayer, 1977). All cellulotic bacteria produce an endo-B-(1-->4)-

glucanase and either a B-glucosidase or an exo-B-(1-- > 4)-glucanase or a

combination of both (Gong et al., 1977; Ohmiya et al., 1987). Cellulase

systems of Cellumonas uda consist of a cellobiohydrolase, exo-B-(l-- > 4)-

glucanase and a B-(1--> 4)-endo-glucanase. The former releases cellobiose and

a trace of cellotriose (Nakamura and Kitamura, 1988). Cellulose degrading

enzymes of Clostridium thermocellum are arranged into a multisubunit complex

called cellulosome, which can be extracellular or cell bound (Lamed and

Bayer, 1988; Ljungdahl et al., 1988).

Some fungal cellulase systems have been investigated in detail; the most

studied systems are those from Trichoderma spp. (McCleary and Shameer,

1987; Voragen et al., 1988; Shulein, 1988), Aspergillus spp. and Penicillium

spp. (Wood et al., 1980, Stewart and Parry, 1981; McCleary and Shameer,

1987; Teunissen et al., 1992; Jun et al., 1992). Most cellulolytic fungi

synthesize exo-B-(1-- > 4)-glucanase, endo-B-(1-- > 4)-glucanase and B-

glucosidase. It is proposed that the cellulase complex requires synergetic

action of at least two components for effective hydrolysis of cellulose (Voragen

et al., 1988). It is suggested that endo B-(1-- > 4)-glucanase first cleaves in a

random manner to produce chain ends for subsequent attacks by exo-enzymes

(Reese, 1977; Wood et al. 1980).










22
Cellulase activity is regulated through the synthesis of one or more

enzyme components, by induction and/or by catabolite repression (Yamane and

Suzuki, 1988), or by end-product inhibition of the activity of a single

component of the complex. Gong et al.(1977) have proposed a unified model

for regulation of cellulase synthesis. This mechanism of regulation takes into

account the importance of glucosidase in the regulation of glucose and

cellobiose levels in cells. In this model, cellulose and glucose represent the

limiting cellulotic products. Cellulose serves as a carbon source and a potential

inducer of the cellulase system whereas glucose and glycerol are repressor

(Steward and Leatherwood, 1976 ; Nisizawa et al., 1972; Canevascini et al.,

1979). B-Glucosidase is generally responsible for regulation of the whole

cellulotic process and is often the rate limiting enzyme (Kadam and Demain,

1989).

Stutzenberger (1985) proposed a model of regulation of cellulase

systems similar to the well studied ara operon of Escherichia coli. In this

model, the cellulose operon is an activator-controlled inducible system,

requiring the cooperative binding of two regulatory proteins to the control

region for maximal transcription.












a-Glucanases

Amylolytic enzymes are an important group of industrial enzymes.

They are conveniently divided into a and B glucanases depending on their

ability to hydrolyze a or B glucans respectively.

Various a-glucanases from a variety of sources have different

physicochemical properties and product patterns. a-Glucanases are widely

distributed in microorganisms and they fall into three major categories (Fogarty

and Kelly, 1980): i) exo-a-(1-- >4)-amylases, ii) endo-a-(l-- >4)-amylases and

iii) debranching enzymes.

Exo-a-(1--> 4)-amylases can be further classified as glucoamylases or

amyloglucosidases, B-amylases and a-glucosidases. Glucoamylases are exo-

acting amylases that catalyze hydrolysis of a-(l-->4) and a-(l-->6) linkages

of glucans to produce glucose. The relative rate of hydrolysis depends on the

enzyme source and on the linkages in the vicinity of the bond being

hydrolyzed. Glucoamylases occur primarily in fungi and are less frequent in

bacteria and yeasts (Singh and Agrawal, 1981). Aspergillus amylasee" was

shown by Ueda and Saha (1983) to be a glucoamylase. Fungal glucoamylases

can be divided into two groups depending on their efficiency of hydrolyzing

starch. The first type of fungal glucoamylase hydrolyzes starch completely to

glucose such as Rhizopus delemar glucoamylase. Hiromi and coworkers

(1973) have shown that this enzyme which catalyzes the depolymerization of










24

maltooligasaccharides, displays an intrinsic rate of hydrolysis which is constant

over a range of substrate containing 2 to 7 4-O-a-D-glucosyl residues, and that

the subsite affinities increase in an additive manner as the degree of

polymerization increases from 2 to 5. The other type of fungal glucoamylases

hydrolyzes only 80% of the the starch; glucoamylase from Aspergillus niger

falls in this category.

B-Amylases hydrolyzes a-(l-- >4)-glucans by sequentially removing

maltosyl units from nonreducing terminal ends. Such enzymes have been

isolated from Bacillus spp. (Fogarty and Griffin, 1975; Murao et al., 1979).

a-Glucosidases catalyze the hydrolysis of nonreducing end a-(l--> 4)

glucosyl residues; glucose is released. Major producers of a-glucosidases are

Aspergillus spp. and Bacillus spp.

Endo a-(1-->4)-amylases are enzymes that randomly hydrolyze a-(l--

>4) bonds in an endo-manner, but bypass a-(l--> 6)-linkages in amylopectin

and glycogen to produce a variety of oligosaccharides (maltose, maltotriose and

some glucose). The rate of hydrolysis of a-(1--> 4)-linkages depends on the

degree of branching. Endo a-(l-- >4)-amylases are produced by a wide range

of microorganisms including Bacillus spp., Aspergillus spp. and Penicillum

spp. Debranching enzymes--isomaltases and pullulanase--are enzymes whose

primary specificity is to hydrolyze a-(l--> 6)-linked glucose residues.

Isoamylases are produced by a wide variety of organisms such as Cytophaga










25

spp. and Streptomyces spp. (Gunja-Smith et al., 1970; Yagisawa et al., 1972).

Pullulanases from Bacillus spp. and Streptomyces spp. cleave a-1-->6 linked

glucose units of starch to produce linear dextrins (Yagisawa et al., 1972;

Urlaub and Wober, 1975).

B-Glucanases

The B-glucanases may be classified as exo- or endo-B-glucanases. In

addition, endo-B-glucanases may be further divided into two groups depending

on whether they produce large or small oligosaccharides (Mori et al., 1977;

Rey et al., 1982). The B-(1-- > 2)-glucanases, B-(1--> 3)-glucanases and B-

(1--> 6)-glucanases from fungi and/or bacteria have been described (Reese et

al., 1961, 1962; Bacon et al., 1970; Fleet and Manner, 1977; Bielecki and

Gallas, 1991; Cutfield et al., 1992). The B-(-- > 6)-glucanases from Bacillus

subtilis catalyze release of mannan containing 3% of glucose from the alkali

soluble polysaccharide fraction of yeast cell wall (Fleet and Manner, 1977).

Recent studies have focused on the B-(1--> 3)-glucanases from bacteria which

are important in enzymatic lysis of cell wall components of yeasts and fungi.

Streptomyces spp. 1228 produces 4 extracellular B-(1--> 3)-glucanases. It has

been shown that four B-(1-- > 3)-glucanases act synergistically to lyse yeast cell

walls (Bielecki and Galas, 1991). Bacillus circulans also produces

extracellular B-(1--> 3)-glucanase that lyses fungal cell walls. The enzyme is










26

an endo-8-(1--> 3)-glucanase and has a random cleavage pattern (Aano et al.,

1992). Exo-B-(1--> 3)-glucanase from Candida albicans has been crystallized

(Cutfield et al., 1992).

Penicillium italicum produces at least 3 types of B-(1-- > 3)-glucanases

with different modes of action in addition to a B-(1-- > 6)-glucanase. Type I

has an endo-glucanase activity, type II has an exo-glucanase activity, whereas

type III has both activities. Santos et al. (1978) have shown that type II and

type III are repressed by excess glucose and that the absence of glucose not

only results in an increase in synthesis of both types II and III, but also

triggers the synthesis of type I. Cooperativity in cell wall depolymerization by

fungal endo- and exo-glucanases has been reported by Jones et al. (1974).

Glucanases, as well as several other enzymes secreted by yeasts and

fungi, are glycoproteins. Their carbohydrate moieties consist of mannose,

glucose, galactose and N-acetylglucosamine. N-acetyl glucosamine is bound to

asparagine of the polypeptide chain (Villa et al., 1978; Sanchez et al., 1982;

Rosa et al., 1984).

B-Mannanases

The B-D-mannanases are enzymes that randomly hydrolyze B-(1-- >4)-

mannan chain of galactomannans, glucomannans, galactoglucomannans or

mannans from microorganisms and/or plants. The production of 8-










27

mannanases and mannosidases has been reported in fungi and bacteria

(Horikoshi, 1991; Araujo and Ward 1990).

Bacteria, such as, Bacillus spp., Aeromonas spp. and Streptomyces spp.,

produce endo-8-mannanases and/or exo-B-mannanases. An exo-B-mannanase

(B-(-- > 4)-D-mannan mannobiohydrolase), isolated from culture fluids of

Aeromonas spp, hydrolyzes B-(1-->4)-D-mannosyl unit of mannan with three

or more mannosyl residues and release mannobiose from the nonreducing end

of these oligosaccharides. This enzyme does not act on mannan substituted

with D-galactosyl (galactomannan) or with D-glucosyl (glucomannan) units.

These organisms also secrete endo-B-(1--> 4) mannanase (Araki and

Kitamikado, 1981).

Endo-8-mannanase from Bacillus spp. hydrolyzes mannooligosacchar-

ides larger than mannotriose and produces di-, tri- and tetra-saccharides.

Dekker and Richards (1976) reported constitutive and inducible bacterial

mannanases. Productions of higher mannanases activities in Bacillus spp. have

been shown to be induced by either the carbon source or the nitrogen source

(Araujo and Ward, 1990), or under alkaline conditions (Horikoshi, 1991).

Inducibles B- mannanases have also been described in Aeromonas spp. (Araki

and Kitamikado, 1982). Fungal mannanases have been reported by several

authors (Reese and Shibata, 1965; Erikksson and Winell, 1968). Aspergillus










28

niger B- mannanases have pH optima at 3.0 and 3.8 and a temperature

optimum of 650C (Erikksson and Winnell, 1968). Aspergillus tamarii

produced an inducible B-D-mannanase. This enzyme is a glycoprotein

containing N-acetylglucosamine, mannose and galactose and is secreted into

those growth media containing galactomannan. Hydrolysis of galactomannan,

by Apergillus niger B-mannanase, produces mannobiose and mannotriose. The

extent of hydrolysis depends on the galactose content (Civas et al., 1984); the

greater the percentage of galactose, the less the percentage of total

mannooligosaccharides released.

Galactanases

Galactanases, the enzymes hydrolyzing galactan components of cell wall

and extracellular polysaccharides of microorganisms and/or plants, are

produced by bacteria, fungi and plants (Dekkers and Richards, 1976; Nakano

et al., 1985, 1990; Tsumura et al., 1991). Three types of endo-B-galactanases

have been described so far: endo-B-(1-- > 3)-galactanases, endo-B- (1-- > 4)-

galactanases and endo-B-(l-- > 5)-galactanase.

Endo-B-(1-- > 3)-galactanase from Rhizopus niveus has been

characterized; this enzyme hydrolyzes 1,3-type arabinogalactans and releases

D-galactose, several 1,3- and 1,6-linked B-D-galactose oligosaccharides. Some

of these oligosaccharides contain arabinose (Hashimoto, 1971).










29

A B-D-galactanase specific for 1-(1--> 5)-linked galactofuranosyl

residues has been purified and characterized from culture filtrates of

Penicilliwnum oxalicum (Reyes et al., 1992). This enzyme is a basic

glycoprotein, which hydrolyzes B-(1-- > 5)-D-galactofuranosyl linkages in

homo- and heterogalactans with production of mono-, di- and trisaccharides.

Endo 8-(1-- > 4)-galactanases are the most characterized of the galactan

degrading enzymes. Galactotriose and galactotetraose are the main products of

the hydrolysis of galactan by Bacillus spp S2 endo galactanase. Bacillus spp

39 endo-B-(1--> 4)-galactanase primarily releases galactobiose and

galactotriose. This enzyme shows a unique property with two pH optima at

4.0 and 9.0. Bacillus subtilis K-50 endo-(8-1-- > 4)-galactanase yielded

galactotriose as the major end product. The end products of the activity of the

enzyme from B. subtilis var amylosacchariticus are similar to those from

Bacillus spp. 39 enzyme, while the products of the activity of the enzyme from

B. subtilis (wild type) are primarily galactotetraose (Labavitch et al., 1976;

Araujo and Ward, 1990). Two endo B-(l-->4)-galactanases from P. citrium

were found to hydrolyze B-(1--> 4)-galactoside polymers to release galactose,

galactobiose and galactotetraose (Nakano et al., 1985). These two enzymes

have similar physical and enzymatic properties although they are separable by

affinity chromatography and by polyacrylamide gel electrophoresis.










30

Bacillus subtilis produced an exo-B-(1--> 4)-galactanase that was

shown to hydrolyze B-(1--> 4)-galactosidic linkages with major production of

galactobiose and a minor production of galactotetraose. This enzyme also has

a transferase activity (Nakano et al., 1990). Araujo and Ward (1990) have

reported the production of inducible galactanases in Bacillus spp.; galactose

was the inducer.

Aspergillus oryzae, Bacillus spp. and Kluyveromyces spp. produce B-

galactosidases which hydrolyze B-D-galactobiose,-galactotriose and galacto-

oligosaccharides with release of D-galactose from the nonreducing end of the

galactan chain. An exo-B-D-galactofuranosidase was partially purified from the

culture filtrates of P. charlesii by Rietschel-Berst et al. (1977). This enzyme

catalyzes the release of galactose from methyl and ethyl B-D-galactofurano-

sides, from 6-O-B-galactofuranosyl D-galactose and from 5-O-B-D-galacto-

furanosyl-containing peptidophosphogalactomannan and is not active on either

1-O-a-methyl-D-galactofuranosides or on methyl-D-galactopyranosides.

Studies of factors affecting the appearance of this exo-B-D-galactofuranosidase

in the medium by Pletcher et al. (1981) showed that the enzyme is repressed

by glucose and at low pH.










31

An exo B-D-galactofuranosidase isolated from crude commercial

preparations of Trichoderma harzianum was partially purified and characterized

by Van Bruggen-Van der Lugt et al. (1992). This enzyme releases galactose

from oligosaccharides of 8-(l-->5)-linked galactofuranosides with four or

more residues and from heterogalactans.

It is apparent that galactofuranosyl-containing saccharides are

widespread in nature; however, they are not considered as common

constituents of oligo- or polysaccharides. Exo-B-galactofuranosidases and

galactofuranases have been isolated from a few species. Although the

galactofuranase was purified to homogeneity, the exo-B-galactofuranosidases

have not been purified to homogeneity. There have been no reports of either

exo-a-D-galactofuranase or exo-a-D-galactofuranosidase. Penicillium charlesii

was selected as the source of exo-B-galactofuranosidase production because of

its ability to produce and secrete relatively high activities of this enzyme

(Rietschel-Berst et al., 1977).












MATERIALS AND METHODS

All chemicals used were reagent grade. These chemicals were purchased

from Sigma Chemical Company and Fisher Scientific. Commercial enzyme

prepa-rations were purchased from Sigma Chemical Company and from

Worthington Biochemical Corporation. Filter papers, membrane dialysis

apparatus and filters were purchased from several sources such as Whatman,

Amicon and MSI. Columns and column chromatography supplies were

obtained from Pharmacia, Bio-Rad Laboratories, and Amicon. Electrophoresis

equipment and reagents were purchased from Bio-Rad and Pharmacia.

General Procedures

Spectrophotometric measurements at wavelengths between 280 nm and

820 nm were made using 1 cm path length quartz cuvettes in a Hewlett

Packard Model A Diode Array Spectrophotometer. Colorimetric assays for

total carbohydrate were determined at 480 nm using a Coleman II Jr. (Perkin-

Elmer) spectrophotometer. Samples of 1.3 ml were examined in 10 x 75 mm

borosilicate culture tubes. The pH of the culture filtrates and buffers was

determined using an Accumet pH meter 900 (Fisher Scientific) standardized

with appropriate buffers. The pH was also monitored using pH paper.

Filter papers (#4, Whatman) were used in the filtration harvest of P.

charlesii cultures. Dialysis was performed using Spectrapor membrane tubing

32










33
with standard cellulose acetate dialysis tubing of 1,000, 3,500 and 14,000 to

15,000 molecular weight nominal cut-off (Union carbide).

Enzyme preparations and buffers were filter-sterilized using a Millipore

filter or MSI apparatus with a filter membrane of 0.22 micron pore size.

Organism

Penicillium charlesii G. Smith 1887 was grown on Czapek Dox (CD)

agar plates containing 5% glucose and 2% agar for 2 to 3 weeks at room

temperature and stored at 40 C until use. Transfers were made every 3 to 4

months by streaking conidiospores on fresh plates.

Culture Conditions and Growth Media

Conidiospore suspensions in sterile solution containing 0.9% NaCl

(w/v) and 0.125% Tween 20 (w/v) were used for inoculation of liquid shake

cultures according to the following procedure: 2 ml of spore suspension

(- 106 conidiospores), were diluted in 150-200 ml sterile growth medium in a

500 ml notched wide mouth Erlenmeyer flask. Each flask was plugged with a

styrofoam disc and agitated on a New Brunswick Model G-10 Gyrotory shaker

at a setting of 10, (40 rpm). Liquid shake cultures were grown under constant

light at 200C. Media composition is listed in table 8 and 9 (Appendix).










34

Isolation of Glycopeptides

Six days after inoculation, the cultures were filtered through Whatman #

4 filter paper. The filtrate was dialyzed at 40 C against several changes of

distilled water. Peptidophosphogalactomannans (pPGMs) were isolated

according to the procedure described by Salt (1983). The procedure followed

is outlined in scheme I, (Appendix). After fractionation of pPGM on DEAE

cellulose, fraction 2 and 3 (pPGM" and pPGM"'), were recovered and stored

separately as lyophilized powders.

Chemical Modifications of Glycopeptides

Dilute acid hydrolysis of pPGM with release of galactose

This procedure involved the release of galactofuranosyl residues of

pPGM using mild acid conditions. The ratio of galactose concentration to that

of HC1 is critical depending on whether total or partial hydrolysis is desired.

For the conversion of galactan to galactose, a sample of pPGM containing

approximately 2 jpmol of hexosyl residues was treated with 10 ml of 0.01 N

HC1 for 90 min at 1000C. The reaction was terminated by neutralization with

1 N NaOH. Following this procedure, the low molecular weight substances

were removed by dialysis in cellulose acetate membranes (3500 MW cut-off).

The retentates were assayed for carbohydrate, protein, and phosphate.










35

Preparation of oligosaccharides from pPGMs

Oligosaccharides were obtained from pPGMs by mild acid hydrolysis.

Four hundred micrograms of pP2GM" (2.5 Mmoles of hexosyl residues) was

treated for 20 minutes at 1000 C with 1 ml of 0.01 N HC1 Dialysates (less

than 3500 MW) were concentrated and applied to a DE 52 anion exchange

column. The unbound material (washed through with water) was concentrated

and applied to a Bio Gel P4 column.

Isolation of Enzymes

Exo-B-galactofuranosidase from 18-21 day old Raulin-Thom liquid-

shake cultures of Penicillium charlesii was isolated in a 5-step procedure.

Medium, from 18-20-day cultures to which phenylmethylsulfonylfluoride

(PMSF) was added at a concentration of 104 M 24 hours before harvest, was

filtered through a Whatman # 4 paper.

Step 1. Culture filtrates, previously dialyzed at 40C against several

changes of 50 mM sodium citrate buffer, pH 5.0, were concentrated 10-fold in

an Amicon ultrafiltration cell with a YM 30 ultrafilter (30,000 MW cut-off)

and stored at 40 C.

Step 2. DEAE-cellulose chromatography I. A 50 ml sample of

concentrated proteins equivalent to 500 ml of culture medium, from step 1,

was applied at room temperature to a column of DEAE cellulose (3 X 28 cm)










36

equilibrated with 50 mM sodium citrate buffer, pH 5.0. The column was

washed with 500 ml of the above buffer. Exo-8-galactofuranosidase activity

eluted with unbound proteins. Fractions with B-D-galactofuranosidase activity

were collected and concentrated as before to approximately 2 ml and then

resuspended in up to 20 ml with 50 mM MOPS buffer, pH 7.5.

Step 3. DEAE-cellulose chromatography II. The pooled DEAE I

fraction containing galactofuranosidase activity from step 2 was applied at

room temperature to a column (2 X 25 cm) of DEAE cellulose, previously

equilibrated in 50 mM MOPS buffer (pH 7.5). The column was washed with

the 50 mM MOPS buffer (pH 5.0) to remove unbound proteins; washing

continued until no absorbance was detected at 280 nm. Elution was performed

with a stepwise gradient using washes of 0, 0.12, 0.25 and 0.5 M NaCl in 50

mM sodium citrate buffer, pH 5.0. Galactofuranosidase activity eluted with

0.25 M NaCI. Active fractions were pooled and dialyzed at 40C against 12.5

mM sodium tartrate buffer, pH 3.0.

Step 4. Carboxymethyl sepharose chromatography. Dialyzed samples

from step 3 were loaded onto a CM-sepharose (2 x 26 cm) column preequili-

brated with 12.5 mM sodium tartrate buffer, pH 3.0, and the column was

washed with 300 ml of the above buffer. Elution was performed again with a

stepwise gradient of 0 to 0.5 M NaCI in 50 mM sodium acetate buffer, pH










37

4.0. Exo-B-galactofuranosidase activity eluted with 0.25 M NaC1. Fractions

which showed galactofuranosidase activity were dialyzed at 40C against 10 mM

acetate buffer, pH4.0, containing 10 mM NaC1. The dialyzed samples were

filtered through a 0.22 im filter unit (Micron Separation Inc.) and the filtrate

was concentrated in a Centricon 30 microconcentrators (30,000 MW cut-off;

Amicon).

Step 5. FPLC Gel filtration. A concentrated sample ( 200 p1) from

step 4 was applied to a Superose-12 Fast Performance Liquid Chromatography

column, previously equilibrated with 10 mM sodium citrate at pH 4.0 and

containing 10 mM NaCl buffer. Elution was performed with the same buffer.

Galactofuranosidase positive fractions were pooled, concentrated and reapplied

to the same column.

Assays of Enzyme Activities

Galactofuranosidase assay

Exo-B-galactofuranosidase activity was assayed by two different

methods. Galactofuranosidase activity was determined as described by

Rietschel-Berst et al., (1977). Essentially, a reaction mixture containing 200

pl of 5 mM 1-O-8-methyl-D-galactofuranosidase, 20 to 200 pt of enzyme

preparation and 66 mM sodium acetate buffer at pH 4.0 in a total volume of

500 ul was incubated at 400 C for various lengths of time. Similar reaction










38

mixtures without either substrate or enzyme were used as controls. Substrates

were diluted in 66 mM acetate buffer unless stated otherwise. Galactose

released by the action of the enzyme was estimated either by Nelson test for

reducing sugars (Nelson, 1949) or by the coupled oxidation of galactose and o-

cresol catalyzed by galactose oxidase and peroxidase, respectively, (see below).

One enzyme unit is defined as the activity which releases 1.0 imol of galac-

tose at 400 C in a minute. The substrate, 1-O-B-methyl-D-galactofuranoside,

was synthesized and purified according to the method of Augestad and Berner

(1954) with a slight modification (Rietschel-Berst et al. 1977). Alternatively,

the activity of exo-B-galactofuranosidase was determined by monitoring the

change in optical rotation of the substrate using a Jasco DIP Digital

polarimeter. For this purpose, a reaction mixture containing 5 to 10 Mmol of

substrate (in 400 pl volume), 200 ,l of enzyme preparation and 1.5 ml of 66

mM acetate buffer, was incubated in a one decimeter cuvette. The optical

rotation was monitored periodically.

Assays for Other Hydrolases

Activities of other hydrolase were determined by spectophotometric

measurement at 410 nm of p-nitrophenol released by cleavage of p-nitrophenyl

esters or glycosides. Enzyme activities determined by this method were: a-D-

galactopyranosidase, B-D-galactopyranosidase, N acetyl B-D-glucosaminidase,










39

acid phosphatase, and phosphodiesterases ( bis-p-nitrophenyl phosphate and p-

nitrophenyl phosphoryl choline as substrates). Reaction mixtures contained 5

gmol of substrate in 200 jl volume, 200 pl of 66 mM acetate buffer at pH 4.0

and 100 p of enzyme preparation. After incubating for two hours at 400C the

reactions were stopped by the addition of 1.0 ml of 0.2 N NaOH, the

absorbance at 410 nm was measured. An extinction coefficient of 18.3 mM'

cm-' was used.

Galactose Oxidase Assay

Galactose released by the enzyme was routinely quantitated by the

method described in the Worthington Manual. Two mg of horseradish

peroxidase (Sigma) was dissolved in 10 ml of 0.1 M potassium phosphate

buffer, pH 8.0 with 5 j1 of o-cresol and designated solution A. Two hundred

p1 of solution A and 50 pt of galactose oxidase ( Worthington) were added to a

sample volume of 0.6 ml. The above solution was incubated at 370C for 30

minutes. The values obtained at 410 nm were compared with a reference

solution of 1 M/mol galactose.

Chemical Assays

Total Carbohydrate

Total carbohydrate was determined by the phenol-sulfuric acid method

of Dubois and coworkers (1956). In this procedure, samples in a total volume

of 0.3 ml aqueous solution were mixed throughly after addition of 20 Al of










40

80% phenol and 1.0 ml of concentrated H2SO4. The absorbances of the

preparations were determined at 490 nm and the values obtained compared

with a reference solution of 0.9 mM galactose and 0.3 mM mannose.

Reducing Carbohydrate

Reducing sugars were determined by the Nelson's test (1944) for

reducing sugars. Absorbance of the blue complex was determined at 600 nm.

Total Phosphate

Total phosphate was determined using the ashing technique of Ames

and Dubin (1960) and the color reagent of Ames (1966). In this assay, 20-200

pl4 aliquots of samples were taken to dryness over an open flame after addition

of 60 pl of 10% Mg(NO3)2. The above dried preparations were then

hydrolyzed by adding 0.6 ml of 0.5 N HCI and placing the reaction mixture in

a boiling water bath for 15 minutes. Molybdenum-ascorbate reagent (5:1, v/v)

in the amount of 1.4 ml was added to the cooled preparations and the final

solution was incubated at 450C for 20 minutes. Absorbances were then

determined at 820 nm and the values obtained were compared with a reference

solution of 0.4 /mol KH2PO4.

Protein

Protein was estimated by the micro procedure of BCA protein assay

(Pierce Biochemicals). In this assay, 0.1 ml of the sample was mixed with 2.0

ml of BCA reagent and incubated for 30 minutes at 600 C. Bovine serum










41

albumin (Pierce Biochemicals) at a concentration of 0.2 mg/ml was used as

reference.

Formaldehyde

Formaldehyde was estimated using the chromotropic acid assay of

McFadyen (1945). A solution of oligosaccharides or glycopeptides (4 to 7

moles anhydrohexose/ml in H20) was treated with approximately a 5-fold

molar excess of sodium metaperiodate for 18 hours in the dark at 40C. After

this period, the excess periodate was destroyed by addition of sodium arsenite.

The oxidized sample in a total volume of 0.1 ml was then added to 2 ml of

chromotropic acid solution (10 mg/ml)-12.5 N H2S04 reagent (1:4, v/v), and

incubated in 1000C water bath for 30 minutes. The samples were blanked

against a control lacking saccharides, but otherwise treated similarly. Their

absorbances were determined at 570 nm and the values obtained were

compared with a reference solution of 1.3 Amol of formaldehyde.

Chromatography

Paper Chromatography

Descending paper chromatography of monosaccharides was carried out

on 56 x 22 cm sheets of Whatman No. 3 paper. Chromatography was

conducted at room temperature in glass tanks lined with Whatman No. 3 paper

previously saturated with the solvent employed. Development of

chromatogram was carried out for 20 hours using Solvent A consisting of n-










42

butanol:pyridine:water, (6:4:3), (v/v/v). The bottom edge of the

chromatogram was serrated, and the solvent was allowed to drip off the

chromatogram.

Thin-Layer Chromatography

The dansyl derivatives of amino acids were separated and identified by

two-dimensional thin-layer chromatography on polyamide layer sheets (Chen

Chin Trading Co., Ltd.). Solvent A, consisting of 1.5% formic acid in water

was used for development in the first direction. Solvent B, consisting of

benzene:acetic acid, 9:1 (v/v), was used for development in the second

direction.

Detection of Compounds on Paper and Thin-Layer Chromatography

Reducing sugars were detected on paper chromatograms with alkaline

silver nitrate (Trevelyan et al., 1950).

Dansyl derivatives were detected on thin-layer chromatograms by

illumination of the chromatogram with ultra violet light.

Ion-Exchange Chromatography

Whatman DEAE cellulose (DE-52) and CM sepharose (fast flow)

obtained from Pharmacia were employed for the routine purification of the

enzyme. Precycling of the ion exchanger was carried out as prescribed in the

technical literature published by the Manufacturers. All ion exchangers were










43

washed, swollen, and/or fines removed as recommended by Manufacturers.

Column packing were pre-equilibrated in buffer when appropriate. Columns

were packed by filling their lower third with buffer or other appropriate

solution. A deaerated slurry of packing material was added and allowed to

settle to the desired height.

DEAE cellulose (DE-52) for glycopeptide isolation was equilibrated

with 0.05 M borate in situ. DEAE cellulose (DE-52) for enzyme isolation was

preequilibrated with 0.05 M sodium citrate (pH 5.0) or 0.05 M MOPS buffer

(pH 7.5). CM sepharose for enzyme isolation was preequilibrated with 12 mM

sodium tartrate (pH 3.0).

Gel Filtration

A Superose 12 gel filtration column (Pharmacia) was used in the final

step of enzyme purification. This column has a void volume of 7.5 ml

(determined with blue dextran MW = 2.0 x106). Enzyme preparations

containing 20 to 100 gg of protein in a total volume of 200 p1 of buffer (0.01

M acetate/ 0.01 M NaCl pH 4.0) were injected The column was washed

with the same buffer at a flow rate of 0.12 ml/min.










44

Gel Electrophoresis

SDS Polyacrylamide Gel

Polyacrylamide gel electrophoresis of exo-B-galactofuranosidase

preparation was performed using a Bio-Rad mini slab gel apparatus (Model

Mini 2-D) attached to a power supply unit (Fisher biotech Electrophoresis

Systems). Specialized reagents were obtained from Bio-Rad. Gels were cast

in 7 x 10 cm glass plates. Buffer systems were used as described by Laemmli

(1970). Separating and stacking gels contained 12% and 4% acrylamide,

respectively. Samples were applied to gels in SDS sample buffer (pH 6.8, 50

mM Tris HC1 containing 10% glycerol, 2% SDS, 5% mercaptoethanol, and

0.002% bromophenol blue), heated at 1000 C for 5 minutes. The separation

was achieved using constant current at 200 volts for approximately 45 minutes.

The gels were removed from plates, fixed and stained with Coomassie Brilliant

Blue R-250, silver stain followed by Coomassie Brilliant Blue R-250. Exo-B-

galactofuranosidase, a glycoprotein, stains poorly when stained with Coomassie

or silver stain alone. The staining procedure which requires the sequential

staining as indicated above gave visible bands.

Non-Denaturating Polyacrylamide Gel

Non-denaturating gel electrophoresis of exo-B-galactofuranosidase was

performed using the same apparatus as above. Buffer systems were used as










45

described in the Sigma technical bulletin No. MKR-137. Separating gels

contained 7% to 10% acrylamide solution; stacking gel contained 4%

acrylamide solution. Samples were applied to the gel in sample buffer

consisting of pH 6.7, 50 mM Tris-HCl:glycerol:water:bromphenol blue,

1:1:1:250 (v/v/v/w) expressed as ml and gg. The gels were removed from the

plates, fixed and stained as above. The molecular weights were determined by

the procedure described in the manufacturer technical bulletin (Ferguson, 1964;

Hendrick and Smith, 1968). This consists of determining the relative mobility

(Rf) of the protein in each gel relative to the tracking dye. Hundred times

logarithm of (Rf x 100) is plotted against the percent gel concentration for each

protein. The slope of such a plot is the retardation coefficient (K). From

these plots individual Krs are determined for each protein and the logarithm of

the negative slope is plotted against the logarithm of the molecular weight of

each protein. This graph produces a linear plot from which the molecular

weight of exo-B-galactofuranosidase was determined.

Isoelectric Focusing

Isoelectric focusing of exo-B-galactofuranosidase was performed using

the PhastSystem (Pharmacia). PhastGel IEF media precast homogeneous

(5%T, 3%C) polyacrylamide, pH 4.0 to 6.5, was used for the determination of

the isoelectric point. The procedure employed was that described in the

technical literature published by the Manufacturer of the apparatus.










46

NH_-Terminal Analysis

The dansylation technique of Woods and Wang (1967) as described by

Gray (1972) was used for the determination of the NH2-terminus of the enzyme

and bovine serum albumin as a reference protein. To a sample volume of 20

pl containing 75 gg of protein, was added 50 Al of 1% SDS. The mixture

was heated in a boiling water bath for 2-5 minutes. After cooling, 70 tl of N-

ethylmorpholine was added and mixed thoroughly. Dansyl chloride was then

added (105 pl; 25 mg/ml). Reaction was allowed to proceed for 1 hour at

room temperature. The labelled protein was precipitated by addition of 0.5 ml

of acetone to the reaction mixture. The precipitate was washed once with 80%

acetone, dissolved in 6 N HCI and held at 1000C overnight in capped "reacti"

vials. The hydrolysate was reduced to dryness by passing a stream of dry

nitrogen over it. The residue was dissolved in 10 pl of acetone and was

subjected to thin-layer chromatography.

Determination of the Kinetic Properties of Exo-B-galactofuranosidase

Optimum pH for activity. Reaction mixture containing i) 400 pl of

buffer (50 mM sodium citrate, pH 2 to 7.5 and 50 mM Tris-HC1, pH 8 to 9),

ii) 50 Al of 1-O-6-methyl-D-galactofuranoside (750 Ag) and 30 pl of the

purified galactofuranosidase preparation (60 ng of protein) was incubated for

90 minutes at 400C. The release of galactose after incubation with the enzyme

was determined by the galactose oxidase.










47

pH stability profile. Sixty nanograms of enzyme preparation in 200 1l

of buffer (50 mM sodium citrate, pH 2 to 7.5 and 50 mM Tris-HC1, pH 8 to

9) was incubated at 4-80C for 24 hours. After that period of time, the pH was

adjusted to 4 with 0.1 N HC1 or 0.1 N NaOH. Substrate (50 pl of 1-0-B-

methyl-D-galactofuranoside, 750 p/g), was then added and the reaction mixture

was then incubated for 120 minutes at 400C. Galactose released was

determined by the galactose oxidase test.

Optimum temperature for activity. A volume of 400 pl of buffer (66

mM sodium acetate at pH 4.0) was brought to the indicated temperature. The

purified enzyme was then added (30 l1, 0.12 4g of protein) with 50 Ml

(500pg) of 1-O-B-methyl-D-galactofuranoside. The reaction was allowed to

proceed for one hour. Galactose released was measured by the galactose

oxidase test.

Temperature stability. Purified enzyme ( 30 pl, 0.12 Mg of protein)

was added to 400 pl of buffer (66 mM sodium acetate, pH 4.0) adjusted to the

indicated temperature. The mixture was incubated for 45 minutes. After

readjusting the temperature to 400C, 50 pl (500tpg) of 1-O-B-methyl-D-

galactofuranoside was added and the reaction mixture incubated for one hour at

400C. Purified exo-B-galactofuranosidase preparation ( 200 pl, 60 ng of

protein) was incubate at -200C for 24 hours to determine the stability of the

enzyme to freezing and thawing. Then, the preparation was thawed; 300 pl of










48

66 mM acetate buffer, pH 4.0, containing 500 jig of 1-O-B-methyl-D-

galactofuranoside was added and the reaction allowed to proceed for 60

minutes.

Kand V.m determination. Km and Vm were determined on pP2GM"s

and pP30GM"s, 1-O-B-methyl-D-galactofuranoside, and galactofurano-

oligosaccharides enriched in galactofuranotetraose or galactofuranohexaose.

For this purpose, 820 /l of buffer (66 mM acetate, pH 4.0) containing

substrate concentrations ranging from 0.025 to 11 moles of nonreducing

terminal galactofuranosyl residues were added to 80 p of purified enzyme

preparations (0.7 to 3 Mg of protein) and the reaction mixture incubated at

400C. Sample sizes of 50 p1 were taken out at 15, 30, 60, 120, 180 and 240

minutes, and the reaction stopped by adding 550 ,1 of 0.1 M phosphate buffer,

pH 8.0. The release of galactose was then determined by the galactose oxidase

test. Km and Vm were determined from Cornish-Bowden plots.

Substrate specificity of the exo-B-galactofuranosidase. 1-O-B-Methyl-D-

galactofuranoside, Galf34 and Galfs-6 linked 8-D-(1--> 5), pP2GM" and

pP30GM" were used to test the specificity of the exo-B-galactofuranosidase.

The amount of galactose released was determined by the galactose oxidase test.

The reaction mixtures were the same as described above.

Activities of purified exo-B-galactofuranosidase preparation toward

substrates for phospho-monoesterases, phosphodiesterases and other










49

glycosidases were determined using p-nitrophenyl-a-galactopyranoside, p-

nitrophenyl-B-galactopyranoside, p-nitrophenyl-N-acetyl-B-glucosamine, p-

nitrophenyl-phosphocholine, p-nitrophenyl phosphate and bis(p-nitrophenyl)

phosphate. 200 jl1 of substrate (6 Amole) was added to 300 1/l of buffer (66

mM acetate pH 4.0) containing 1.2 tig of enzyme preparation. The reaction

was allowed to proceed at 400C for 150 minutes and then stopped by addition

of 1 ml of 0.2 N NaOH. The amount of product released was compared to

that of 1-O-B-methyl-D-galactofuranosidase in the same conditions galactosee

released was measured by galactose oxidase test).













RESULTS

Enzyme Purification

Rietschel-Berst et al. (1977) have reported the purification of

extracellular exo-B-galactofuranosidase to a nearly homogeneous state in one

step using a peptidophospho-galactomannan affinity column. Discontinuous gel

electrophoresis of a sample eluted from the affinity column with sodium acetate

buffer, pH 4.0, showed three major protein bands; while a sample eluted with

0.1 M phosphate buffer, pH 7.0, showed two major protein bands.

Pletcher et al. (1979) also failed to purify galactofuranosidase; at least

one of the contaminants was an endogeneous protease which inactivates galac-

tofuranosidase. She attempted to circumvent this problem by the addition of

phenylmethylsulfonylfluoride (PMSF) to inactivate serine proteases. However,

this treatment only partially inhibited proteases in the culture filtrates.

Another protocol for protein purification was developed. Medium,

from 18-20-day old cultures to which PMSF was added at a final concentration

of 10-4 M ( 3.4 mg/200 ml flask) 24 hours before harvest, was filtered and

concentrated in an Amicon ultrafiltration apparatus (MW cut off 30,000).

There was no detectable galactofuranosidase activity in the filtrate, all the

activity remaining in the retentate.

This indicates that galactofuranosidase MW is > 30,000.

50












Anion Exchange Chromatography I

Aliquots from Amicon-concentrated 18-21 day culture filtrates were

subjected to ion exchange chromatography on a DEAE-cellulose column in 50

mM sodium acetate buffer, pH 5.0. The distribution of protein and

galactofuranosidase activity is shown in Figure 1. The fractions collected from

the column were measured at 280 nm and assayed for exo-B-

galactofuranosidase activity. The bulk of the protein (60%) did not bind to the

column and it is in this effluent that exo-B-galactofuranosidase activity was

found. Total recovery of the exo-B-galactofuranosidase activity from this first

anion exchange column was 74%. Even though exo-B-galactofuranosidase does

not bind to the column at pH 5.0, 40% of the total protein bound and was

removed. Based on the increase in specific activity, this step represents about

a 1.2-fold purification of exo-B-galactofuranosidase from the culture filtrate.

On that basis, it was determined that anion exchange chromatography at pH

5.0 was a good first step to resolve exo-B-galactofuranosidase activity from

other proteins present in the culture filtrate that may interfere in later steps.

Anion Exchange Chromatography II

Exo-B-galactofuranosidase activity recovered from the first anion

exchange was applied to a second anion exchange column (DEAE-cellulose)

previously equilibrated with 50 mM MOPS buffer, pH 7.5 (Figure 2).





















Figure 1. Elution Profile of Exo-B-Galactofuranosidase from DE-52
Anion Exchange Column I

Culture filtrates were concentrated and applied to a DEAE-cellulose column
(3 x 28 cm) as described in Materials and Methods. The column was pre-equilibrated
with 50 mM sodium citrate, pH 5.0 and washed with the same buffer. The protein
was eluted with a stepwise gradient of NaC1. Fractions of 7.5 ml were collected.
Protein, (_ ), was monitored by absorbance at 280 nm. Exo-B-galactofuranosidase
activity, (---), was determined on 200 Cl samples of every other fraction; galactose
released was estimated from the increase in absorbance at 410 nm resulting from the
coupled oxidation of galactose and o-cresol by galactose oxidase and peroxidase,
respectively. Column fractions containing galactofuranosidase activity as indicated by
the bar above the galactofuranosidase peak of activity were pooled.
On this figure, as well as on all following figures, exo-B-galactofuranosidase
activity (----) represents a curve drawn by a computer program using all the gathered
data.






















3 1.20

/\
I
E E
I 0.90 c
0 '
0o 2 I 0.25 M NaCI 0.5 M NaCI


4 4 0.60

oC
0 0
Q 1 -
O O
I0.30 i




0 0.00
0 40 80 120 160


Fraction numbers






















Figure 2. Fractionation of Partially Purified Exo-B-Galactofuranosidase on DE-52
Anion Exchange Column I

Galactofuranosidase preparation (10 ml, 7 units) obtained from DE-52 Anion
Exchange Column I, was applied to a second DEAE cellulose column previously
equilibrated with 50 mM MOPS buffer, pH 7.5. Fractions size of 2.5 ml were
collected. Protein was eluted with a stepwise gradient of NaCl. Protein, (__), was
monitored by absorbance at 280 nm. Galactofuranosidase activity, (----), was
determined on 100 /l samples of every other fraction; galactose released was
estimated by an increase in absorbance at 600 nm resulting from the increase in
reducing sugars assayed by Nelson's test for reducing sugars. Fractions containing
galactofuranosidase activity as indicated by the bar were pooled.























1.00 2.50

I

E 2.00 E

0 0
O 0.12 M N~e 0.25 M NCO (O
e4 j- 1.50 c
'0.50 II 1.50

0a50







0.00 0.00
0 10 20 30 40 50

0 10 20 30 40 50


Fractions numbers










56
Protein eluted in two major and two minor peaks. Half of the protein, but

almost none of the exo-B-galactofuranosidase activity came through in the

effluent (first peak). The two minor protein peaks have no galactofuranosidase

activity. Exo-B-galactofuranosidase activity is eluted in the last protein peak

with 25 mM sodium citrate containing 0.25 M NaC1. Protein peaks without

galactofuranosidase activity were discarded without further analysis.

Approximately 73% of the galactofuranosidase activity applied to the column

was recovered in that protein peak.

Cation Exchange Chromatography

The protein fraction containing galactofuranosidase activity obtained

from DEAE-cellulose chromatography II was applied to a CM-sepharose

column in 12 mM sodium tartrate buffer, pH 3.0 (Figure 3). None of the

protein came through in the effluent. One minor peak of protein without exo-

8-galactofuranosidase activity was eluted with 0.12 M NaC1.

Galactofuranosidase activity, which was found as a shoulder on the protein

peak, eluted with 25 mM sodium citrate containing 0.25 M NaCl, pH 5.0. A

second peak of protein eluted with the same salt concentration but did not have

any galactofuranosidase activity.





















Figure 3. Fractionation of the Galactofuranosidase Preparation Obtained from DEAE-
Cellulose Chromatography on CM-Sepharose by Cation Exchange

Fractions containing galactofuranosidase activity ( 10 units) obtained from
DEAE-cellulose column II as described in "Materials and Methods", was loaded onto
a CM-sepharose column (2 x 25 cm) pre-equilibrated with 12.5 mM sodium tartrate
buffer, pH 3.0. Protein was eluted with a stepwise gradient of NaCl. Fractions of
2ml were collected. Protein, (_), was monitored by absorbance at 280 nm.
Galactofuranosidase activity, (----), was determined on 100 p~ samples of every other
fraction; galactose released was estimated by an increase in absorbance at 600 nm
resulting from the increase in reducing sugars assayed by Nelson's test for reducing
sugars. Fractions with galactofuranosidase activity, as indicated by the bar, were
pooled.














































Fraction numbers


0.50



0.40



0.30



0.20



0.10



0.00


3



E
C-

2 0
(0



U)
0-
(0


O(
0
n




0


0 10 20 30 40 50 60 70 80 90











Superose-12 Gel Filtration I

Fractions containing exo-8-galactofuranosidase activity from CM-

sepharose chromatography was loaded onto a gel filtration column (Superose

12) equilibrated with 10 mM sodium acetate buffer, pH 4.0 containing 10 mM

NaCl (Figure 4). Two distinct peaks of protein eluted as the column was

washed with the same buffer. Galactofuranosidase activity was associated with

the first peak of protein. Fractions with exo-B-galactofuranosidase activity

were then concentrated as indicated in "Materials and Methods" and reapplied

to the same column.

Superose 12 Gel Filtration II

Proteins with galactofuranosidase activity recovered from several gel

filtration I (Superose 12) steps were pooled, concentrated and reapplied to the

same gel filtration column (Figure 5). Protein eluted as a single peak which

contained exo-galactofuranosidase activity.

Homogeneity of the Purified Exo-B-Galactofuranosidase

The protein content of intermediate and final exo-B-galactofuranosidase

preparations were determined by BCA protein assay (Pierce). For comparative

purposes, samples from each step and with various protein concentrations were

assayed for exo-B-galactofuranosidase activity using 1-O-B-methyl-D-galacto-

furanoside as substrate.























Figure 4. Elution Profile of Exo-B-D-Galactofuranosidase Activity on Superose-12
FPLC Gel Filtration Column I

A galactofuranosidase preparation (200l), obtained from CM-sepharose cation
exchange column and concentrated as described in the Materials and Methods, was
applied to a Superose-12 FPLC gel filtration column (1.5 x 31 cm) previously
equilibrated with 10 mM acetate buffer, pH 4.0, containing 10 mM NaC1. Protein
elution was done with the same buffer at a flow rate of 0.12 ml/minute. Fractions of
250 Al were collected. Protein, (_), was monitored by absorbance at 280 nm.
Galactofuranosidase activity, (----), was determined on 20 l samples of every other
fraction; galactose released was estimated by an increase at 600 nm resulting from the
increase of reducing sugars as assayed by Nelson's test for reducing sugars.












61







0.30 3

I\
E E
0i
J I o
co 0.20 2 O
.4 I l l .4




S 0.10 1 -
U O
SI I

I I -
I \

O "
0.00 -- '' 0
0 10 20 30 40 50 60


Fraction numbers






















Figure 5. Second Gel Filtration of Exo-l-Galactofuranosidase Activity on Superose-12
FPLC Column

Galactofuranosidase positive fractions from the first gel filtration were
collected, concentrated 10-fold, filtered through a 0.2 MSI filter apparatus and 200 pl
loaded onto the same Superose-12 FPLC gel filtration column. Fractions of 250 p/
were collected. Protein, (__), was monitored by the absorbance at 280 nm.
Galactofuranosidase activity, (----), was assayed on 20 Al samples of every other
fraction and galactose released was estimated as described in figure 4.




















0.20


0.10


0.00


10 20 30


Fraction numbers


3



E

o
20

0



U

.Q




-0
40










64

The activity of exo-B-galactofuranosidase and total protein present at

each step are shown in Table 1. The crude preparation contained 20 units of

activity and 77 mg of protein per 1 L of medium. The 5-step procedure

resulted in a 100-fold purification of exo-B-galactofuranosidase with a 45%

recovery of activity. Thus, approximately 1% of the protein in the crude

preparation was exo-B-galactofuranosidase. Aliquots of exo-B-galacto-

furanosidase from gel filtration chromatography I and/or II were analyzed by

SDS-PAGE, by non-denaturating PAGE (not shown) and by IEF.

SDSPAGE. Aliquots of gel filtration I and II were analyzed by SDS-

PAGE (Figure 6). Gels, stained with 0.1% Coomassie Brillant Blue R-250

following Bio-Rad's silver staining procedure, showed a major band with an

approximate molecular weight of 75 kd in lane 2 corresponding to gel filtration

I. Lane 3 represents protein after gel filtration II.

The above results suggest that exo-B-galactofuranosidase has an apparent

molecular weight of 75 kd. Molecular weight was calculated from a

calibration curve plotted on a semi-log paper of the molecular weight vs

relative mobility in SDS-PAGE of low molecular weight protein standards

(Lanes 1 and 5).










65

Table 1. Purification of Exo-B-galactofuranosidase


Step protein activity Specific act. Recovery fold
mg Units Units/mg % purification





Crude 76.8 19.2 0.25 100

DEAE-I 47.6 14.3 0.3 74 1.2

DEAE-II 13.0 10.4 0.8 54 3.2

CM-Sepharose 1.2 5.8 4.8 30 19.2

FPLC-II 0.3 8.6 25.2 45 100






















Figure 6. Photograph of Exo-B-Galactofuranosidase Following SDS-PAGE

Samples were diluted 1:5 (v/v) in SDS sample buffer and loaded into wells of
gels cast in 7 x 10 cm glass plates (0.5 mm). Electrophoresis was carried out at
constant current of 8 mA until the dye front reached the bottom of the gel (30-45 min)
at a constant current of 8 mA. Following electrophoresis, the gels were stained with
Coomassie Brillant Blue R-250 in 40% methanol, 10% acetic acid. Both molecular
weight standard and samples were heated at 950C for 5 minutes.
The standard mixture contained (a) phosphorylase b (97.4 kd), (b) bovine
serum albumin (66.2 kd), (c) ovalbumin (45.0 kd), (d) carbonic anhydrase (31.0 kd),
(e) soybean trypsin inhibitor (21.5 kd), (f) lysozyme (14.0kd).
Lanes 1 & 5 .....Molecular weight standard mixture as shown above
Lane 2...........Exo-B-galactofuranosidase preparation after first gel filtration
Lane 3..........Exo-B-galactofuranosidase preparation after second gel filtration
The band in Lane 4 represents a spillover from Lane 3.












67









A










68

Non-Denaturating PAGE. Exo-B-galactofuranosidase preparations from

gel filtration II were subjected to non-denaturating PAGE using gels with

different polyacrylamide concentrations ( 7, 8, 9 and 10%). After staining

with 0.1% Coomassie Brilliant Blue R-250 following Bio-Rad silver staining

procedure, two bands were detected. Proteins in these bands had an

approximate molecular weight of 150 kd and 70 kd. The molecular weights

were determined by the procedure described in the Sigma technical bulletin

No. MKR-137 as discussed in "Material and Methods".

Isoelectric Focusing (IEF). Exo-B-galactofuranosidase preparations

from gel filtration II were isoelectrically focused on Phast Gel IEF ( 4-6.5 pi)

medium (Pharmacia). After staining the gel with Bio-Rad silver stain, a single

band (Figure 7) with a pi of 4.35 was detected in the lane containing enzyme

fraction from gel filtration II (Lane 2). The pi of exo-B-galactofuranosidase

was calculated from a calibration curve plotted as a function of pi calibration

standards vs distance from the cathode (Lanes 1 and 3). Isoelectric focusing

followed by slicing of the gel and assaying for galactofuranosidase activity in

the sliced bands revealed one major band with activity around pH 4.0 as shown

of Figure 8.





















Figure 7. Photograph of Exo-B-Galactofuranosidase Following IEF

Isoelectric focusing of galactofuranosidase preparation was performed using the
PhastSystem (Pharmacia). PhastGel IEF media precast polyacrylamide (5%T, 3%C),
pH 4.0 to 6.5 was used. Following IEF, gels were stained with silver stain.
pi markers were (a) glucose oxidase (pI-4.15), (b) soybean trypsin inhibitor (pl-4.55),
(c) B-lactalbumin (pi-5.20), (d) bovine carbonic anhydrase (pi-5.85)
Lanes 1 & 3.... pi calibration mixture
Lane 2 ........ Exo-B-galactofuranosidase preparation, containing 60 ng of protein.












1


a --
U~^



.*.a-


6>**-*
9~1


- S.
40
d iu


em

AU





















Figure 8. Zymogram of Exo-B-Galactofuranosidase Following IEF


Isoelectric focusing of galactofuranosidase preparation was performed using the
PhastSystem (Pharmacia). PhastGel IEF media precast polyacrylamide (5%T, 3%C),
pH 4.0 to 6.5 was used. Following IEF, gels were stained according to manufacturer
protocol (Bio-Rad) with silver stain.
pi markers were (a) glucose oxidase (pl-4.15), (b) soybean trypsin inhibitor (pl-4.55),
(c) B-lactalbumin (pl-5.20), (d) bovine carbonic anhydrase (pi-5.85)
Lane 1........ p calibration mixture
Lane 2 ........ Exo-B-galactofuranosidase preparation, containing 60 ng of protein.
To test for exo-B-galactofuranosidase activity, half of the gel was cut out and
incubated in 66 mM acetate buffer and the other half was stained with silver stain.
Bands of the unstained half were then sliced out as indicated. Gel pieces were passed
through a 5 cc syringe and made up to 1 ml in 66 mM sodium acetate buffer pH 4.0.
Three hundred microliters of that solution were added to 200 t1 of susbtrate (1-0-8-
D-methyl galactofuranoside, 500 pg). The reaction was allowed to proceed for 20
hours at 400 C. Galactose released was measured by the Nelson's test for reducing
sugars at 600 nm.














Absorbance at 600 nm

... o. p p oo p


















1 2









73

Carbohydrate Content

Carbohydrate content of exo-B-galactofuranosidase was determined on

16 pg of protein in 100 pl of 10 mM, pH 4.0 sodium acetate buffer. The

phenol-sulfuric acid assay was used. The results showed that the reaction

contained 2.7 jg (15% carbohydrate) by weight.

A sample containing 50 jg of protein was treated with 2 N HCI for 3

hours at 1000 C. The reaction mixture was analyzed by paper chromatography

and showed to contain mannose; no galactose was detected (data not shown).

N-Terminal Amino Acid Analysis

A sample containing about 2 tg of galactofuranosidase did not react

with DANSYL.C1; a control reaction with bovine serum albumin reacted. This

suggests that exo-B-galactofuranosidase amino-terminal group is blocked.

Properties of Exo-B-Galactofuranosidase

pH Activity Profile

When assayed over the pH values 2 to 9, exo-B-galactofuranosidase

appeared to be active over a defined pH range (3.5 to 6.0) (Figure 9A). The

optimal pH for exo-B-galactofuranosidase activity was at pH 4.0-4.5. Less than

50% of the activity was observed at pH 6 and above.

pH Stability

After incubating equal aliquots of enzyme in buffers of various pH





















Figure 9. Optimum pH for Activity and pH Stability of Exo-B-galactofuranosidase

Panel A: optimum pH for activity. Reaction mixture containing i) 400 p. of
buffer (50 mM sodium citrate, pH 2 to 7.5 or 50 mM Tris-HC1, pH 8 to 9), ii) 50 J1
of 1-O-B-methyl-D-galactofuranoside ( 750 gg) and iii) 30 Al of purified exo-B-
galactofuranosidase preparation (60 ng of protein) was incubated at 400C for 90
minutes. Galactose released was measured by the increase in absorbance at 410 nm
following the coupled oxidation of galactose and o-cresol by galactose oxidase and
peroxidase, respectively. Galactose released (0.138 Amoles), at pH 4.5, was given
the relative value of 100% activity.

Panel B: pH stability profile. Sixty nanograms of enzyme preparation in 400
~1 of buffer (50 mM sodium citrate, pH 2 to 7.5 or 50 mM Tris HC1, pH 8 to 9) was
incubated at 40C for 24 hours. After that period of time, the pH was adjusted to 4
with 0.1 N HCI or 0.1 N NaOH. Substrate, 50 jl of 1-O-B-methyl-D-
galactofuranoside (750 ,g) was then added. The reaction mixture was incubated at
400C for 120 minutes. Galactose released was measured by the increase at 410 nm
following the coupled oxidation of galactose and o-cresol by galactose oxidase and
peroxidase, respectively. Galactose released (0.158 .moles0, at pH 4.0, was given the
relative value of 100% activity.

















120

A
100


t 80
S- s\

S60


( 40
rr

20
O \,
0 I I

0 1 2 3 4 5 6 7 8 9 10

pH

120


100 B


- 80 -


60


r 40




0 I I
20 -



0 1 2 3 4 5 6 7 8 9 10

pH










76

values at room temperature for 24 hours, the residual galactofuranosidase

activity in the preparations was determined at pH 4.0. As shown in Figure 9B,

exo-B-galactofuranosidase is stable over a narrow pH range (3.5 to 5.5) in the

conditions used. The enzyme was most stable at pH 4-5 which is also the

optimal pH for activity of exo-B-galactofuranosidase.

Temperature Optimum

The optimal temperature for exo-B-galactofuranosidase activity was

determined by assaying purified enzyme preparations over a wide range of

temperatures as described in "Materials and Methods". As shown in Figure

10A the optimal temperature for exo-B-galactofuranosidase activity was 400C.

The rapid decline of activity after 600C is probably due to protein denaturation.

Temperature Stability

The thermal stability of exo-B-galactofuranosidase determined following

45 minutes incubation at indicated temperatures (Figure 10B) suggests that exo-

B-galactofuranosidase was stable up to 45 minutes between 40C to 500C. The

enzyme looses nearly all of its activity at 600C. About 94% of the enzyme

activity was recovered following freezing and thawing of the enzyme

preparation. The loss of enzyme activity at 600 C in the presence of substrate

(top panel) compared with that in the absence of substrate (lower panel)

showed decreases of 40% and 90%, respectively. This suggests that substrate






















Figure 10. Optimum Temperature and Temperature Stability for Activity of Exo-8-
galactofuranosidase

Panel A: Optimum temperature for activity. Four hundred microliters of
buffer (66 mM acetate at pH 4.0) was brought to the indicated temperature. The
enzyme was then added (30 pl, 0.12 Ag of protein) with 50 1 (500 Mg) of 1-0-B-
methyl-D-galactofuranoside. The reaction was allowed to proceed for 60 minutes.
Galactose released was measured by the increase at 410 nm following the coupled
oxidation of galactose and o-cresol by galactose oxidase and peroxidase respectively.
The release of 0.146 Mmoles of galactose at 400C was given the relative value of
100%.

Panel B: Temperature stability. Thirty microliters of enzyme preparation (0.12
Mg of protein) was added to 400 p of buffer (66 mM acetate, pH 4.0) adjusted to the
indicated temperature. The reaction mixture was incubated for 45 minutes. After
readjusting the temperature to 400C, 50 pl (500 tg) of 1-O-B-methyl-D-galacto-
furanoside was added and the reaction mixture incubated for one hour at 400C.
Activity at 80C in which 0.135 Mmoles of galactose were released was given the
relative value of 100%.














120


100 A


0 80


60


S40\
> 0 I
20 -

2O -



0 10 20 30 40 50 60 70 80 90 100

Temperature (degree C)
120


100 B-


S80
U
S60


) 40


20


0
0 10 20 30 40 50 60 70 80 90 100

Temperature (degree C)









79

provides protection from thermal inactivation.

Products Derived from pP2GM and pP0oGM Incubated with exo-B-
Galactofuranosidase

Exo-B-galactofuranosidase generates only one type of product after

limited or extensive hydrolysis of pPGMs. As depicted in Figures 11 and 12,

the only product formed from the action of exo-B-galactofuranosidase on

pPGMs and detected by paper chromatography and gel filtration on Bio-Gel P4

is galactose. The data in Figure 11 confirmed that the enzyme is an exo-acting

enzyme which sequentially catalyzes the removal of 8-(1-- > 5)-galacto-

furanoside from the nonreducing terminal end of the galactofuran chains.

Peptidophosphogalactomannan (pP30GM), with an observed optical

rotation of -0.214 (10.70 moles of galactofuranoside/ml), was treated with

purified exo-galactofuranosidase. Galactose released by the action of the

enzyme was detected as a change in the observed optical rotation (Figure 13A).

The activity of exo-B-galactofuranosidase resulted in a change of 0.1

degree ( 3.2 moles of galactose/ml) over a period of 110 hours. This

indicated that 29.7% of total galactose has been released.

Peptidophosphogalactomannan (pP2GM) was incubated with purified

exo-B-galactofuranosidase and the product formed was detected as a change in

the observed rotation (Figure 13B). Starting with an observed optical rotation

of -0.445 degree ( 22.75 /moles of galactofuranoside/ml) for pP2GM", release






















Figure 11. Photograph of Paper Chromatograms of pPGMs after Treatment with Exo-
B-galactofuranosidase.

Sample of pPGM"s (10 mg) was treated with exo-B-galactofuranosidase as
described under "Materials and Methods". Samples were taken out after 15 and 30
minutes and spotted on Whatman No. 3 paper. Development of chromatogram was
carried out for 20 hours using solvent consisting of n-butanol:pyridine:water, (6:4:3),
(v/v/v). Reducing sugars were detected on paper chromatogram with alkaline siver
nitrate.

Lanes 1 & 2.....pP30GM" after 15 and 30 minutes, respectively

Lane 3..........Galactose (Gal) and Mannose (Man)

Lanes 4 & 5.....pP2GM" after 15 and 30 minutes respectively













81

















'. "Man








.... .. .
Gal















.. g- .
*.tl- 1

*
.4,.,.


r
3 4


r 2.





















Figure 12. Gel Filtration of Galactofuranosidase Treated-pP30GM"

A reaction mixture containing i) 7 Mmoles of non-reducing terminal
galactofuranosyl residues in pP30GM", ii) 40 ptl of enzyme preparation (1.5 gg of
protein) and iii) 500 pl of 66 mM sodium acetate buffer at pH 4.0 was incubated at
400C for 24 hours. At the end of that period of time, the mixture was loaded onto a
Bio Rad P2 gel filtration column previously equilibrated with distilled deionized
water. Elution was performed with distilled deionized water. Fractions of 2 ml were
collected. Every other fraction was assayed for total carbohydrate, protein, and total
phosphate. Total carbohydrate (__) was determined by the phenol-sulfuric acid
method. Protein (_ _)was determined by the BCA protein assay from Pierce. Total
phosphate (----) was determined by the ashing technique of Ames.


















100 40

Celloblose galactose


75 30
IO

0
I 50 20
o i
0)I
I 0O

25 10 g




0 -- 0
10 20 30 40 50 60


Fraction numbers






















Figure 13. Activity of Purified Exo-B-Galactofuranosidase on pPGM"s

A reaction mixture containing 2 to 4 jumoles of non-reducing terminal
galactofuranosyl residues, 200 l1 of enzyme preparation (6 Ag protein) and 1.5 ml of
66 mM acetate buffer at pH 4.0 was incubated in a 1 dm cuvette in a Jasco DIP-360
polarimeter. Observed optical rotation was recorded every 60 minutes and the
reaction was allowed to proceed for 120 or 200 hours at room temperature as
indicated.
Panel A. pP30GM"
Panel B. pP2GM"















0.00

A

-0.06


i-----
a -0.13



-0.19 -



-0.25 L ,
0 24 48 72 96 120

hours
0.05

B
-0.04


-0.13


a -0.22


-0.32


-0.41


-0.50 4 1
0 40 80 120 160 200


hours










86

of galactose catalyzed by the action of exo-B-galactofuranosidase, resulted in a

change of 0.483 degree (15.33 moles of galactose/ml) over a period of 200

hours. These data indicate that 67.4% percent of total galactose has been

released by the enzyme.

These data agree with those obtained by measuring the galactose

released using the galactose oxidase assay (Table 2). Quantitative analysis of

pPGMs before and after treatment with purified exo-B-galactofuranosidase is

presented in Table 3. The relatively linear relationship between the change in

observed optical rotation versus time is an indication that there is negligible

product inhibition. This result was confirmed by using various concentrations

of galactose as inhibitor of exo-B-galactofuranosidase catalyzed hydrolysis of 1-

O-B-methyl-D-galactofuranoside. There was little inhibition by galactose over

the range of 1.38 mM to 4.16 mM (data not shown).

Km and Vm

In order to obtain oligosaccharides, pPGM from 2 mM phosphate

medium was hydrolyzed with 0.01 N HCI (Galf:H+, 10:1, M:M) at 1000C for

20 minutes. Anionic substances were removed by anion exchange

chromatography, and neutral substances fractionated on a gel filtration column.

Figure 14 shows the elution profile of the neutral substances from a Bio Gel P4

filtration column. The average size of oligomers was determined











Table 2. Action of Exo-B-Galactofuranosidase on Various Oligosaccharides



substrate Total CHO2 NRT galactose released



mM %

pP2GM" 30 6.5 73

pP30GM" 40 7.3 30

l-O-B-M-D-Galf 50 50 100

B-(1-- > 5)Galf-oligosaccharide

(Galf)6-5 5 0.9 100

(Galf)4.3 5.5 1.7 100

p-Nitrophenyl-a-galactopyranoside 42 42 <0.01

p-Nitrophenyl-B-galactopyranoside 43 43 <0.01



Substrates at indicated concentrations in an assay volume of 1 ml of buffer
(66 mM acetate, pH 4.0) containing 0.7 to 6 pjg of enzyme preparation were
incubated at 400C for various times. Galactose released was determined by
galactose oxidase assay and total carbohydrate was determined by the phenol-
sulfuric acid method.
Abbreviations: -NRT: non reducing terminal galactofuranosyl
residues; -CHO2: total carbohydrate; -Galf6-s: mixture of hexamer and
pentamer of galactofurano-oligosaccharides; -Galf4_3: mixture of tetramer and
trimer of galactofurano-oligosaccharides; l-O-B-M-D-Galf: 1-O-B-Methyl-D-
galactofuranoside.