Ca(2+)-induced sporulation in the fungus Achlya : the biological roles of [Beta]-(1->3)-glucans and key enzymes

167 Pages·1998·13.5 MB·English

by  Du, Xin, 1962-

CA^^-INDUCED SPORULATION IN THE FUNGUS ACHLYA THE
:
BIOLOGICAL ROLES OF p-(l->3)-GLUCANS AND KEY ENZYMES
By
XINDU
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF
THE UNIVERISTY OF FLORIDA
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE
DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1998
InMemoryofMy Mother
and
To My Baby and My Wife
u
ACKNOWLEDGMENTS
I thank Drs. George Bowes, Ross Brown, Alice Harmon and James Preston for
serving as members ofmy advisory committee and fortheir generous time and expertise. I
NMR
also thank Dr. John Gander for providing his facility for study. Dr. Gregory Erdos and
Scott Whittker ofICBR EM core lab for their technical assistance.
I would like to express my sincerest appreciationto Dr. J. T. Mullins, my advisory
chairman, for his invaluable guidance throughout this study and his support ofthe research
with his time, monies, kindness, humor, patience and facilities.
I amtruly indebted to mywhole family, my father, sisters, brother and their
families, my parents-in-law and brother-in-law, for their love, encouragement and constant
support and inspiration.
Finally, my greatest debt ofgratitude goes to my wife, Lan Mu, for her love,
patience, support and understanding, especially, for her sacrifice during this specialtime.
Ill
TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS
iii
LIST OF TABLES
vi
LIST OF FIGURES
vii
ABSTRACT
x
Chapter 1. INTRODUCTION 1
Chapter 2. REVIEW OF LITERATURE 3
The Organism, Achlva hisexualis 3
Hydrolytic Enzymes andNutrient Limitation 5
Fungal p-glucans and Associated P-(l->3)-glucanases 8
Fugal Sporulation 12
Chapter 3. ACTIVITY OF THE ENZYMES PHOSPHODIESTERASE
AND PHOSPHOMONOESTERASE: RESPONSE TO NUTRIENT
LIMITATION
14
Introduction 14
Materials and Methods 16
Results 20
Discussion 43
Chapter 4. ACTIVITY OF THE ENZYMES p-GLUCANASE AND
p-GLUCOSIDASE: RESPONSE TO NUTRIENT LIMITATION. 48
..
Introduction 48
Materials and Methods 51
Results 52
Discussion 76
IV
Page
Chapter 5. PURIFICATION AND CHARACTERIZATION
OF A p-(l->3)-GLUCANASE FROM ACHLYA DURING
CA^^-INDUCED SPORULATION 81
Introduction 81
Materials and Methods 82
Results 87
Discussion 116
Chapter 6. UTILIZATION OF THE CYTOPLASMIC
P-(l->3)-GLUCANS OF ACHLYA DURING CA^^-INDUCED
SPORULATION
120
Introduction 120
Materials and Methods 121
Results 123
Discussion 140
REFERENCES
144
BIOGRAPHICAL SKETCH
154
V
LIST OF TABLES
Table Page
1 The composition ofmodified enriched medium (MEM) 19
2 The comparison ofthe highest total and specific activities of
PDEase and PMEase ofthe different cultural conditions 42
3 The comparison ofthe highest total and specific activities of
P-glucosidase and P-glucanase ofthe different cultural conditions. 75
4 Purification ofthe P-(l->3)-glucanase 91
5 The effect ofmetal ions and protease inhibitors onthe
purified P-(l->3)-glucanase 106
6 Relative rates ofhydrolysis ofP-glucans by the purified
P-(l->3)-glucanase 107
7 Effect ofsporulation on mycelial weight and glucan ofAchlva 127
8 Glucan and phosphate utilization ofAchlva during induced
sporulation 128
9 The ratios ofmono- and di-ester linkages ofphosphoglucan
during sporulation ofAchlva 139
VI
LIST OF FIGURES
Figures Page
1 Total mycelial activities ofphosphomonoesterase (PMEase) and
phosphodiesterase (PDEase) ofA. bisexualis on modified
enriched medium (MEM) 22
2 Estimated specific activities ofphosphomonoesterase (PMEase)
and phosphodiesterase (PDEase) ofA. bisexuahs on modified
enriched medium (MEM) 24
3 Total mycelial activities ofphosphomonoesterase (PMEase) and
phosphodiesterase (PDEase) ofA. bisexualis fi'om
MEM-minus-P culture 26
4 Estimated specific activities ofphosphomonoesterase (PMEase)
and phosphodiesterase (PDEase) ofA. bisexualis from
MEM-minus-P culture 28
5 Total mycelial activities ofphosphomonoesterase (PMEase) and
phosphodiesterase (PDEase) ofA. bisexualis using different
phosphate concentrations 30
6 Estimated specific activities ofphosphomonoesterase (PMEase)
and phosphodiesterase (PDEase) ofA. bisexualis using different
phosphate concentrations 32
7 Total mycelial activities ofphosphomonoesterase (PMEase) and
phosphodiesterase (PDEase) ofA. bisexualis fi-omthe lowest
phosphate culture 34
8 Estimated specific activities ofphosphomonoesterase (PMEase)
and phosphodiesterase (PDEase) ofA. bisexualis fi’omthe
lowest phosphate culture 36
Vll
2678
Figures Page
9 Total mycelial activities ofphosphomonoesterase (PMEase) and
phosphodiesterase (PDEase) ofA. hisexualis during
Ca2"t"-induced sporulation 38
10 Estimated specific activities ofphosphomonoesterase (PMEase)
and phosphodiesterase (PDEase) ofA. hisexualis during
Ca2_|_-^induced sporulation 40
11 Total mycelial activities ofp-glucosidase and p-glucanase of
A- hisexualis on modified enriched medium (MEM) 55
115 Estimated specific activities ofP-glucosidase and p-glucanase
ofA- hisexualis on modified enriched medium (MEM) 57
13 Total mycelial activities ofP-glucosidase and p-glucanase
ofA- hisexualis fi'om MEM-minus-P culture 59
14 Estimated specific activities ofP-glucosidase and p-glucanase
ofA- hisexualis fi'omMEM-minus-P culture 61
19
Total mycelial activities ofP-glucosidase and P-glucanase of
20 A- hisexualis using different phosphate concentrations 63
1 Estimated specific activities ofP-glucosidase and p-glucanase
ofA. hisexualis using different phosphate concentrations 65
1 Total mycelial activities ofP-glucosidase and P-glucanase of
A- hisexualis fromthe lowest phosphate culture 67
1 Estimated specific activities ofP-glucosidase and P-glucanase
ofA- hisexualis fromthe lowest phosphate culture 69
Total mycelial activities ofp-glucosidase and P-glucanase of
A- hisexualis during Ca -induced sporulation 71
Estimated specific activities ofP-glucosidase and P-glucanase
ofA. bisexuaUs during Ca -induced sporulation 73
21 Summary ofpurificationprocedures ofthe p-(1->3)-glucanase... 90
22 Separation ofP-(1->3)-glucanases fi*om 70% (NH4)2S04
fi'actionation onhydrophobic interaction column 92
Vlll
Figures Page
23 Cation exchange chromatography ofthe P-(l->3)-glucanase 94
24 Anion exchange chromatography ofthe P-(l->3)-glucanase 96
25 Gel filtration chromatography ofthe P-(l->3)-glucanase 98
26 SDS-PAGE ofproteins during purification ofthe P-glucanase.. 100
27 The effect ofpH onthe activity and stability ofthe p-glucanase. 102
28 The effect oftemperature onthe activity and stabihty
ofthe P-(l->3)-glucanase 104
29 Chromatogramofthe P-(1->3)-gluceinase hydrolysate following
various reactiontimes oflaminarin 108
30 Chromatogramofthe P-(1->3)-glucanase hydrolysate following
various reactiontimes ofneutral glucan 110
31 Chromatogram ofthe P-(1->3)-glucanase hydrolysate following
various reactiontimes ofphosphoglucan 112
32 The effect ofconcentration using several different substrates
onthe activity ofthe P -(l->3)-glucanase 114
33 Batch fi’actionation ofmycelial glucan fi’omAchlva 125
34 Gel filtration ofthe phosphoglucans ofAchlva fi’om 0, 4, 8
and 12 h sporulation onP-60 129
NMR
35 Proton-decoupled phosphorus-3 spectrum of
1
phosphoglucan fi'om 0 h sporulation 131
NMR
36 Proton-decoupled phosphorus-31 spectrumof
phosphoglucan from4 h sporulation 133
NMR
37 Proton-decoupled phosphorus-31 spectrum of
phosphoglucan fi'om 8 h sporulation 135
NMR
38 Proton-decoupled phosphorus-31 spectrum of
phosphoglucan fiom 12 h sporulation 137
39 Proposed diagramfor the synthesis and degradation ofP-glucans 143
.
ix
Abstract ofDissertationPresented to the Graduate School ofthe
University ofFlorida in Partial FulfiUment ofthe Requirements
for the Degree ofDoctor ofPhilosophy
CA^^- INDUCED SPORULATION IN THE FUNGUS ACHLYA THE
:
BIOLOGICAL ROLES OF p-(I->3)-GLUCANS AND KEY ENZYMES
By
XINDU
May, 1998
Chairman: J. T. Mullins
Major Department: Botany
This research utilized the fungus Achlva under growthconditions which caused the
development ofasexual reproductive structures termed sporangia. Achlva accumulates, under
optimal growth conditions, reserve P-(l->3)-glucans which containphosphate and glucose.
%
These reserves represent 14 ofthe dry weight ofthe fungal mycelium, and the phosphate is
linked to the glucose by both mono- and diphosphoester bonds.
The first series ofexperiments defined cultural conditions whichproduced the highest
activities ofthe several enzymes which are required to hydrolyze the reserve p-(l->3)-glucans
releasing both glucose and phosphate. It was found that the concentrations ofphosphate and
carbon supplied inthe cultural medium greatly influenced the activities ofthese enzymes. The
highest activities ofphosphodiesterase and phosphomonoesterase were found withthe lowest
concentration ofphosphate (0.015 mM). Whenthe glucose supply was limited, the highest
X