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

146 Pages·1993·3 MB·English

by  Tuekam, Brigitte Albertine

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. CM. 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.
in
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
6-Glucanases 25
B-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 ofGlycopeptides 34
pPGM
Dilute Acid Hydrolysis of with Release
ofGalactose 34
Preparation of Oligosaccharides from pPGM 35
Isolation of Enzymes 35
Assay for Enzyme Activity 37
GalactofuranosidaseAssay 37
Assays for Hydrolases 38
GalactoseOxidase Assay 39
Chemical Assays 39
Total Carbohydrate 39
Reducing Carbohydrate 40
Total Phosphate 40
Protein 40
Formaldehyde 41
Chromatography 41
PaperChromatography 41
Thin-Layer Chromatography 42
Ion-Exchange Chromatography 42
Detection of Compounds on Paper and Thin-
LayerChromatography 42
Gel Filtration 43
Gel Electrophoresis 44
SDS-Polyacrylamide 44
Non-DenaturatingPolyacrylamide 44
Isoelectric Focusing (IEF) 45
NH
-Terminal Analysis 46
2
Determination of Kinetic Properties of Exo-fi-
Galactofuranosidase 46
Optimum pH for Activity 47
pH Stability Profile 47
Optimum Temperature for Activity 47
TemperatureStability 47
Km Vm
and Determination 48
Substrate Specificity of Exo-B-Galactofuranosidase 48
RESULTS 50
EnzymePurification 50
Anion Exchange Chromatography 1 51
Anion Exchange Chromatography II 51
Cation Exchange Chromatography 56
Superose-12 Gel Filtration 1 59
Superose-12 Gel Filtration II 59
Homogeneity ofthe Purified Exo-B-Galactofuranosidase 59
SDS-PAGE 64
Non-Denaturating PAGE 68
IsoelectricFocusing 68
Carbohydrate Content 72
AminoTerminalAminoAcid 73
Properties ofExo-B-Galactofuranosidase 73
pH Profile 73
pH Stability 73
Temperature Optimum 76
Temperature Stability 76
Enzymatic Products from pP,GM" and pP GM" 79
30
Km andVm : 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-6-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. Summary 97
7. Czapek-Dox Agar 109
8. Raulin-Thom Medium 110
9. Standard Growth Medium Ill
vn
. .
LIST OF FIGURES
Figure Page
1 Elution Profile of Exo-B-Galactofuranosidase from DE-52 Anion
ExchangeColumn 53
1
2. Fractionation of Partially Purified Exo-B-Galactofuranosidase on
DE-52 Anion Exchange Column II 55
DEAE
3. Fractionation of Galactofuranosidase Preparation Obtained from
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
onSuperose-12 FPLC Column 63
6. Photograph ofExo-B-Galactofuranosidase Following SDS-PAGE 67
7. Photograph of Exo-B-Galactofuranosidase Following IEF 70
8. Zymogram ofExo-B-Galactofuranosidase Following IEF 72
9. Optimum pH for Activity and pH Stability of
Exo-6-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
Vlll
GM"
12. Gel Filtration ofGalactofuranosidase Treated-pP30 83
13. Activity ofPurified Exo-B-Galactofuranosidaseon pPGM"s 85
14. Gel Filtration of Oligosaccharides Obtained by Mild Acid
Hydrolysis of pPjGM" 90
15. Plot ofthe Initial Reaction Rate vs Substrate Concentration 94
16. Cornish-Bowden Plots A and B for Determination of K^ and Vm 113
17. Cornish-Bowden Plots C and D for Determination of Km and Vm 114
IX
LIST OF ABBREVIATIONS
CH 0- Total carbohydrate
2
DEAE- Diethylaminoethyl
CM- Carboxymethyl Sepharose
ELISA- Enzyme-linked immunosorbent assay
Gal- D-Galactose
Galf- D-Galactofuranosyl residue
Rhap- L-Rhamnopyranose
Glc - D-Glucopyranose
p
Gal NAc- N-Acetyl-D-galactopyranose
p
Gal - D-Galactopyranose
p
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