studies of an alkaline protease inhibitor from a streptomyces sp.

188 Pages·2002·0.98 MB·English

STUDIES OF AN ALKALINE PROTEASE
INHIBITOR FROM A STREPTOMYCES SP.

THESIS SUBMITTED TO
THE UNIVERSITY OF PUNE
FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
IN
BIOCHEMISTRY

BY
JUI PANDHARE

DIVISION OF BIOCHEMICAL SCIENCES
NATIONAL CHEMICAL LABORATORY
PUNE-411 008
INDIA

JUNE 2002
Acknowledgement

I am indeed fortunate to work under the competent guidance of Dr (Mrs)
Vasanti Deshpande whose continuous interest and motivation, and the genuine
insight into the problems have helped me to gain the deepest thoughts in
research. Her receptive outlook, unrelenting enthusiasm and positive approach
will forever remain a source of inspiration.

I express my deepest gratitude towards Dr (Mrs) Mala Rao for invaluable
discussions, timely advice, continuous encouragement and support throughout
my work.

I am highly indebted to Dr. Mohini Ghatge for her help and critical
analysis of the work. I thank her for the valuable discussions and her
inspiration and encouragement during the course of the work.

I thank my ex-labmates Sneha and Aparna for their help and co-
operation.

I am grateful to Dr Sushma for her timely help and inspirations.

My special thanks to my labmates Pankaj, Kavita, Swati, Sharmili,
Prashant, Ajit and Aarohi for the cordial and friendly atmosphere in the lab and
for their timely help.

Thanks are also due to my friends at the Division of Biochemical Sciences
Rajesh, Mohini Pathak, Rajeshree, KK Singh, Atul T, Raman, Nitin, Anil,
Feroz, Atul S, and Dheeraj for their support and cooperation.

I take this opportunity to thank my friends Sudeep, Anish, Nilesh, Vinod,
Devyani, Lata, Ramchander and Maneesha with whom I have shared many
memorable moments and for their help, co-operation, and inspiration
throughout this work. A special thanks to Sudipa for her understanding and
support.

I am thankful to our office staffs Usha, Indira, Satyali, and Mari for their
timely help rendered during the course of this work.
I also express my thanks to our efficient instrumentation staffs, Mr.
Kamthe, Mr. Karanjkar, Mr. Trehan for the help and maintenance of the
instruments.

I am thankful to our lab attendant Mr. R. Lambate for his untiring help
in routine chores.

I am thankful to Dr. Aditi Pant, Head, Division of Biochemical Sciences
for allowing me to use the facilities of the Department.

I am thankful to Dr. Paul Ratnasamy, Director, National Chemical
Laboratory, for granting me permission to submit this work in the form of
thesis.

The fellowship awarded by the Council of Scientific and Industrial
Research is duly acknowledged.

Words fail me, to express my feelings towards my all time friend CV for
constantly encouraging and motivating me to excel. His enduring support,
untiring help and inspiration will always be cherished. He has always been
there all the time with me in my ups and downs .I would also like to thank my
dear friend Swaroop for always being there for me.

I owe my deepest gratitude to my parents, my father for his inspiration
and encouragement without which it would not have been possible to embark
upon this journey in life, my mother for her endurance, teaching me to sustain
and work untiringly, my brother and sister-in-law for all the support and
encouragement and finally my nephew Ameya and my niece Ojasi for all the
delightful moments.

Jui Pandhare
Dedicated to My Family…………..
TABLE OF CONTENTS

DECLARATION i
CANDIDATE’S DECLARATION ii
ABBREVIATIONS iii
ABSTRACT iv-viii
PUBLICATIONS ix
PATENTS/POSTERS/PRESENTATIONS x

CHAPTER 1. GENERAL INTRODUCTION 1-36
Classification of proteases 2
Serine proteases 2
Serine alkaline proteases 3
Subtilisins 3
Mechanism of action 3
Cysteine proteases 6
Aspartic proteases 6
Metallo proteases 7
Protease inhibitors 7
Low-molecular weight inhibitors 8
Proteinaceous inhibitors 8
Serine protease inhibitors 9
Serine protease inhibitors from plants 9
Serine protease inhibitors from animals 10
Mechanism of action of serine protease inhibitors 10
Canonical inhibitors 15
Non canonical inhibitors 15
Serpins 15
Microbial serine protease inhibitors 16
Streptomyces subtilisin inhibitor family 17
Biochemical properties of SSI 17
Reactive site and Chemical modification 18
Protein-protein interactions 19
Denaturation studies 19
X-ray crystallography 20
Genetic engineering 22
Protein engineering 22
Applications of serine protease inhibitors 23
Defense tools for plant protection 23
Therapeutic agents 24
Miscellaneous applications 24
Future Prospects 25-26
References 28-36

CHAPTER 2. SCREENING AND ISOLATION OF ALKALINE PROTEASE
INHIBITOR PRODUCING MICROORGANISMS 37-49

Summary 37
Introduction 38-39
Materials and methods 40-42
Results 43-47
Production of APIs 43
Properties of APIs 44
Potency against different proteolytic enzymes 44
Molecular nature of APIs 44
Stability of APIs 45

pH stability 45
Temperature stability 46
Effect of additives on the thermostability 47
Discussion 48

References 49
CHAPTER 3. PURIFICATION AND BIOCHEMICAL CHARACTERIZATION
OF AN ALKALINE PROTEASE INHIBITOR (API)
FROM A STREPTOMYCES SP NCIM 5127. 50-65

Summary 50
Introduction 51
Materials and methods 52-54
Results 55-61
Characterization of the actinomycete strain producing API 55
Purification of API 55
Biochemical properties of API 57
Stoichiometry of binding of API with alkaline protease from
Conidiobolus sp. 60
Discussion 62-63
References 64-65

CHAPTER 4. API AS A NOVEL ANTIFUNGAL PROTEIN: PROTEASE
INHIBITORY ACTIVITY AS THE BIOCHEMICAL BASIS
OF ANTIFUNGAL ACTIVITY 66-85
Summary 66
Introduction 67-71
Host pathogen interaction 67
Antimicrobial proteins and peptides 67
Strategies developed for the control of fungal diseases 69
Protease inhibitors as defense tools against plant pathogens 70
Materials and Methods 72-73
Results 74-79
Antifungal activity of API 74
Co-purification of antiproteolytic and antifungal activities 75
Simultaneous loss of antiproteolytic and antifungal activities upon
heat inactivation of API 76
Tryptophan is essential for antiproteolytic and antifungal
activities 77
Functional role of disulfide linkages for the antiproteolytic
and antifungal activity of API 78
Discussion 80-82
References 83-85

CHAPTER 5. PROTEIN DISULFIDE ISOMERASE ACCELERATED
REFOLDING OF API: EFFECT OF MACROMOLECULAR
CROWDING ON REFOLDING KINETICS 86-108
Summary 86
Introduction 87-89
Materials and Methods 90-91
Results 92-100
Reactivation yield as a function of API concentration 92
Propensity of rd-API for aggregation 93
PDI-accelerated refolding of rd-API 94
Interaction of fluorescent labeled PDI with rd-API 96
Kinetics of reactivation of rd-API under crowded conditions in
the presence of PDI 98
Discussion 101-106
References 107-108

CHAPTER 6. INTERACTION OF API WITH ALKALINE PROTEASES:
KINETIC PARAMETERS INVOLVED IN THE
INACTIVATION OF THE PROTEASES 109-128
Summary 109
Introduction 110-111
Materials and Methods 112-115
Results 116-123
Kinetic Analysis of the Inhibition of Proteinase K 116
Fluorescence changes of Proteinase K due to binding of
API and the dependence of emission fluorescence on time
dependent binding of inhibitor. 121
Discussion 124-127
References 128

CHAPTER 7. INHIBITOR INDUCED THERMAL STABILITY OF
PROTEINASE K 129-142
Summary 129
Introduction 130-132
Strategies for improving protein thermostabilization 130
Use of additives 130
Chemical modification 131
Chemical cross linking 131
Enzyme immobilization 132
Protein engineering 132
Materials and Methods 133-134
Results 135-138
Temperature stability of Proteinase K 135
Time–dependent regain in activity of Proteinase K
in presence of API 135
Fluorometric analysis of Proteinase K at higher temperature 136
Secondary structural analysis of Proteinase K at higher temperature 138
Discussion 139-140
References 141-142
i

DECLARATION

This is to certify that the work incorporated in the thesis entitled “STUDIES OF AN
ALKALINE PROTEASE INHIBITOR FROM STREPTOMYCES SP.” submitted by Ms.
Jui Pandhare was carried out under my supervision at the Division of Biochemical Sciences,
National Chemical Laboratory, Pune, India. Materials obtained from other sources have been
duly acknowledged.

June 2002 Dr. (Mrs) Vasanti V.
Deshpande
Research Guide
Division of Biochemical Sciences