Expression of Pichia Etchellsii B-Glucosidase gene in Escherichia coli; regulation and characterization of th encoded product

EXPRESSION OF PICHIA ETCHELLSII

fi-GLUCOSIDASE GENE IN ESCHERICHIA COLI;

REGULATION AND CHARACTERIZATION OF THE ENCODED PRODUCT

by

MANJULA PANDEY

Department of Biochemical Engineering and Biotechnology

THESIS SUBMITTED

FOR THE AWARD OF THE DEGREE OF DOCTOR OF PHILOSOPHY

INDIAN INSTITUTE OF TECHNOLOGY, DELHI

HAUZ KAHS, NEW DELHI - 110 016, INDIA

JULY, 1996

CERTIFICATE

JILi9 is to certi/y that the theii9 titled ''x'uaSGieW ^0/ PicLia

.tchd%u a-

this

dy Ms. Manjula Pandey

the Indian institute o/ Jechnolog y, flew .,Delhi, /or the award o/ the Theree o/ `

Doctor of Philosophy'

con/ormity with rules and regulations o/ the Indian institute o/ Jechnology

.2

DR. SAROJ MISHRA

ACKNOWLEDGEMENT

It is my privilege to express deep gratitude and indebtedness towards my supervisor Dr. Saroj Mishra for her invaluable guidance and encouragement at every step of my work. She never failed to extend a helping hand even for experimental work whenever I was in a muddle. Her complete involvement with my work and optimism helped me to pursue the objective confidently.

I also acknowledge my gratitude towards the faculty of DBEB for their valuable suggestions and criticisms throughout this work.

Thanks are due to Mrs. S. Mathai, and Mr V.K. Ghosh for making everything available in the lab and for maintaining the cultures.

Thanks are also extended to Mrs. Sunita Abrol, Mrs. Neera Verma and Mr.

Subhash Anand for their cooperation.

I sincerely thank staff members Mr. Rana, Mr. Bhagwan Singh, Mr. Mukesh Anand and Negi to help me whenever requested and specially Kishan who always helped me to find crucial things like chemicals, glasswares, syringe, cotton etc. during the course of this work.

I would like to thank Mr. Hemant Deoliya and Mr. Gajendra Singh for their skilful help in word processing of this document.

Special thanks to my friends for their pleasant company and support. I would also like to thank all my colleagues for maintaining a coordial atmosphere in the lab.

There are others whose names although did not surface here yet they will be remembered for their contribution.

Last but not the least I thank my parents, brother and sisters for their constant support and affection which kept me going.

(MANJULA PANDEY)

CONTENTS

CHAPTER PAGE NO.

ABSTRACT (V)

I INTRODUCTION AND OBJECTIVES 1

2 LITERATURE REVIEW 12

3 MATERIALS AND METHODS 45

4 : RESULTS 77

5 DISCUSSION 130

6 CONCLUSIONS ]48

REFERENCES 153