Alkaline Proteases from Bacteria Isolated from Cochin Estuary

221 Pages·2014·3.57 MB·English

by  Meera Venugopal

ALKALINE PROTEASES FROM BACTERIA
ISOLATED FROM COCHIN ESTUARY
Thesis submitted to
Cochin University of Science and Technology
in partial fulfilment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
IN
MICROBIOLOGY
UNDER THE FACULTY OF MARINE SCIENCES
By
MEERA VENUGOPAL
DEPARTMENT OF MARINE BIOLOGY, MICROBIOLOGY AND BIOCHEMISTRY
COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
KOCHI-682016, INDIA
AUGUST 2004
CERTIFICATE
This is to certify that the thesis entitled ALKALINE PROTEASES
1-"ROM BACTERIA ISOLATED I-‘ROM COCHIN ESTLIARY is an authentic
record of the research work carried out by Ms. Meera Venugopal, under my
supervision and guidance in the Department of Marine Biology, Microbiology and
Biochemistry, Cochin University of science and Technology, in partial fulfilment of
the requirements for the degree of Doctor of Philosophy in Microbiology of Cochin
University of Science and Technology, and no part thereof lzas been presented for the
award of any other degree, diploma or associateship in any university.
/ D.’
/5
Dr. A. V. Saramma
Reader in Microbiology
Department of Marine
Biology, Microbiology and
Biochemistry
Cochin University of Science
and Technology
Kochi-682016
Kochi - 16
9 August 2004
DECLARATION
I hereby declare that the thesis entitled ALKALINE PROTEASES
FROM BACTERIA ISOLATED I-‘ROM COCHIN ESTLIARY is an authentic
record of the research work carried out by me under the supervision and guidance of
Dr. A. V. Saramma, Reader in Microbiology, Department of Marine Biology,
Microbiology and Biochemistry, Cochin University of Science and Technology, for
the degree of Doctor of Philosophy in Microbiology of Cochin University of Science
and Technology, and no part thereof has been presented for the award of any other
degree, diploma or associateship in any university.
Meera Venugopal
Kochi - 16
9 August 2004
ACIGVO WLEDGEMENTS
I wish to record my sincere thanks and deepest sense of gratitude to my guide
Dr. A. V. Saramma, Reader in Microbiology, Department of Marine Biology, Microbiology and
Biochemistry for her unfailing guidance, invaluable suggestions, critical assessment and the
careful scrutiny of the manuscript. I am extremely grateful to her for the constant
encouragement, support and kind understanding.
I thank Dr. C. K. Radhakrishnan, Head, Department of Marine Biology, Microbiology and
Biochemistry, for his encouragement and support.
My thanks are due to Dr. K. T. Damodaran, Director, School of Marine Sciences,
CUSAT.
I thank Prof. (Dr) N. R. Menon, Honorary Director, C - IMCOZ, School of Marine
Sciences, CUSAT. I wish to record my sincere thanks to Prof (Dr) R. Damodaran, former
Dean, Faculty of Marine sciences, for the encouragement and suggestions.
I gratefully acknowledge my indebtedness to Prof. (Dr.) Babu Philip, Department of
Marine Biology, Microbiology and Biochemistry, for his advice, valuable suggestions and
constant encouragement.
I express my heartfelt thanks to Dr. Rosamma Philip, Senior lecturer, Department of
Marine Biology, Microbiology and Biochemistry for her valuable advice, encouragement and kind
understanding.
I am thankful to all the teachers of the Department of Marine Biology, Microbiology and
Biochemistry, for all the encouragement and support. I am thankful to the non-teaching staff of
this department for their helps and support.
I am sincerely gratefixl to Dr. I. 5. Bright Singh, Reader, School of Environmental studies,
for his valuable suggestions and encouragement.
I record my gratitude to Dr. Krishna Iyer, Retd. Scientist, CIFT, for the guidance in the
statistical analysis of the data.
I extent my heartfelt gratitude to Dr. Beatrice Amar, Dr. Biji Mathew and Dr. Sreevalsam
Gopinath, for all their help, constant encouragement and benevolent support. I thank Ms.
Minimol. K.G, Ms. Sarlin P. ], Ms. Bindu Araham and Dr. Newby Ioseph for their support and
encouragement.
It will be difficult to find words to express my feelings of gratitude to my friends
Ms. Maya Paul, Mr. Harish R, Ms. Sincy Ioseph, Mr. Selven S, Mr. Sajeevan T. P, and
Mr. Neil Scholastine Correya without whose sincere efforts it would have been difficult for me to
complete the work in time. I am deeply indebted to them for their support and I extend my
heartfelt gratitude to them. I express my sincere thanks to Ms. Lakshmi G. Nair,
Ms. Annies Ioseph, Ms. Simi Joseph, Ms. Iaya Kuruvilla, and Ms. Smitha for their wholehearted
support and friendship.
I would like to place on record my gratitude and appreciation to Mr. K. P. Krishnan,
Research Fellow, National Institute of Oceanography, Goa, for his help and support. I express my
sincere thanks for the help and support extended by Ms. Sreedeoi and Ms. Vrinda.
I thank my friends Mr. Padmakumar, Mr. Harikrishnan, Mr. Anilkumar, Mr. Ialeel,
Mr. Noushar Khan and Mr. Manoj, for their friendship and encouragement. My sincere thanks
are due to Dr. Nandini Menon, Dr. Arun A.U., Dr. Vinu Chandran, Dr. Bindu P. C,
Ms. Shajna, Dr. Iayasree, Dr. Ioydas, and Dr. Suchitra Varior for their encouragement and
support. I thank Ms. Bindu Bhaskaran, Ms. Anupama Nair, Ms. Soja Louis, Ms. Sreeja,
Ms. Iisha and Ms. I-‘eebaRani for their support.
My thanks to all the research scholars and students of the Department Marine Biology,
Microbiology and Biochemistry for their support.
I am deeply indebted to Dr. Nisha N.R. for her kind understanding, support and care, and
I extend my heartfelt gratitude to her. My sincere thanks to Ms. Resmi T. R., for her friendship
and support.
I had been extremely privileged to have the love, encouragement, support and care, from
Dr. Valsamma Ioseph, Dr. Manjusha M., Ms. Priyaja P., and Ms. Seena ]ose to whom I am
deeply indebted. I extend my deepest feelings of gratitude to them.
I am extremely thankful to Mr. Iayaprakash and Mr. Somanath Pai for the kind help and
support throughout the period of the study. I extent my heartfelt gratitude to Mr. Arias Abdul
Aziz and Mrs. Jasmine Anas for their kind help and encouragement. My sincere thanks are due
to my friends Mr. Rajeesh, Mr.Sudheer, Mr. Sreedharan, Ms. Preetha, and Ms. Manju and Ms.
Ii Ii for their support.
I wish to express my sincere thanks to CSIR, New Delhi for the award of Iunior and
Senior Research Fellowship.
I wish to express my feelings of gratitude to my brother Mr. Praveen who had always been
my inspiration and I am much indebted to him for his invaluable suggestions at crucial junctures
of this work and I sincerely thank him for all the care, support and encouragement I received from
him. My deep feelings of gratitude are due to my brother Mr. Arun who had been always
supportive and caring. I specially thank him for the sincere efforts he has put in for the
completion of this thesis.
My deepest feelings for the love and care of my beloved parents, who had been the
motivation in all my endeavours, cannot be contained in words, and I am indebted to them for all
my achievements. I fondly cherish the love and support of my brother as my fortune and I extent
my sincere thanks to him for his contributions for the completion of this work.
I am much obliged to my parents in - law for their great love, care, support and
encouragement. I extent my heartfelt gratitude to their love.
I am grateful to the care, love and aflection I received from all my family members and all
my friends.
Finally, I fondly dedicate this work, to the love and sacrifice of my dearest husband.
Meera Venugopal
CHAPTER 1C OINnTEtNrTSoduction 1- 6
CHAPTER2 2 .Is1ola tiRon aendv Scrieeeniwng o f oProfte aslei Ptroedurciang tBuactreriea 7 -8 26
2.2 Materials and Methods
_L_L
2.2.1 Collection of samples and isolation of bacteria
_L_L
2.2.2 Sampling sites _L'9
2.2.3 Identification of cultures
2.2.4 Primary screening for protease production
2.2.5 Secondary screening for potent strains producing alkaline protease
2.3 Results
2.4 Discussion
2.4.1 Selected strains
2.4.2 Mangroves- a unique ecological niche to be conserved
fi)k)|\J_L_L_L_L-b(J—¥O)U'I-|>La-J
CHAPTER 3 Efofectf of CAulturle kConaditilonisn on eGro wPth arndo Protdeuctiaonses 27­
3.1 Review of literature
3.2 Materials and Methods
3.2.1 Selected strains
3.2.2 Growth media
3.2.3 lnoculum preparation
3.2.4 Measurement of growth
3.2.5 Preparation of crude enzyme
3.2.6 Assay of alkaline protease
3.2.7 Effect of different physico chemical parameters on growth and alkaline
protease production
3.3 Statistical analysis
3.4 Results
3.4.1 Effect of shaking speed on growth and protease production
3.4.2 Effect of period of incubation on growth and protease production
3.4.3 Effect of pH on growth and protease production
3.4.4 Effect of temperature on growth and protease production
3.4.5 Effect of NaCl concentration on growth and protease production
3.4.6 Effect of metallic salts on growth and protease production
3.4.7 Effect of carbon sources on growth and protease production
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3.4.8 Effect of Nitrogen sources on growth and protease production
3.5 Discussion
3.5.1 Effect of shaking speed on growth and protease production
3.5.2 Effect of period of incubation on growth and protease production
3.5.3 Effect of pH on growth and protease production
3.5.4 Effect of temperature on growth and protease production
3.5.5 Effect of NaCl on growth and protease production
3.5.6 Effect of metallic salts on growth and protease production
3.5.7 Effect of carbon sources on growth and protease production
3.5.8 Effect of Nitrogen sources on growth and protease production
CHAPTER 4 Purification and Characterisation of Proteases I
4.1 Review of literature
4.2 Materials and Methods
4.2.1 Detennination of protein
4.2.2 Determination of molecular weight by SDS PAGE
4.2.3 Zymography (Activity Gel)
4.2.4 Assay of alkaline protease
4.2.5 The enzyme kinetics
4.3 Statistical Analysis
4.4 Results
4.4.1 Purification of the alkaline proteases
4.4.2 Molecular weight detennination
4.4.3 Effect of pH on protease activity
4.4.4 Effect of pH on the stability of alkaline protease
4.4.5 Effect of temperature on enzyme activity
4.4.6 Effect of temperature on stability of the enzymes
4.4.7 Effect of substrate concentration on protease activity
4.4.8 Effect of various metallic salts on protease activity
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4.4.9 Effect of inhibitors on protease activity —L3(.0
4.5 Discussion :LoN
4.5.1 Purification of the alkaline proteases _L3N
4.5.2 SDS PAGE and molecular weight $1
—|
4.5.3 Zymogram analysis 3
—-L CO
4.5.4 Effect of pH on protease activity and stability 111
4.5.5 effect on temperature on protease activity and stability 113
4.5.6 Effect of substrate concentrations on enzyme activity- Kinetic constants 117
4.5.7 Effect of metal ions on the activity and stability of proteases 115
4.5.8 The mechanistic classes of proteases based on inhibitor studies 118
CHAPTER 5 Application of the Alkaline Proteases as Detergent Additives 123­ 147
5.1 Review of literature 126
5.2 materials and methods 128
5.2.1 Effect of pH on the activity and stability of proteases 128
5.2.2 Effect of temperature on the activity and stability of proteases 128
5.2.3 Activity of the proteases on different protein substrates 128
5.2.4 Stability of the proteases in different commercial detergents 129
5.2.5 Stability of the proteases in SDS, Triton X- 100 and H202 129
5.3 Results 130
5.3.1 Effect of pH on the activity and stability of proteases 130
5.3.2 Effect of temperature on the activity and stability of proteases 130
5.3.3 Activity of the proteases on different protein substrates 131
5.3.4 Stability of the proteases in different commercial detergents 131
5.3.5 Effect of H202 on the activity and stability of proteases 135
5.3.6 Effect of SDS on the activity and stability of proteases 136
5.3.7 Effect of Triton X- 100 on the activity and stability of proteases 138
5.4 Discussion 140
5.4.1 Effect of pH on the activity and stability of proteases 140
5.4.2 Effect of temperature on the activity and stability of proteases 141
5.4.3 Activity of the proteases on different protein substrates 143
5.4.4 Stability of the proteases in different commercial detergents 144
5.4.5 Effect of H202 on the activity and stability of proteases 146
5.4.6 Effect of SDS and Triton X- 100 on the activity and stability of proteases 147
CHAPTER 6 Summary 150-153
APPENDIX 154-167
REFERENCES 168-197
ABBREVIATIONS
Abs. Absorbance
rpm revolutions per minute
"19 Milligram
mM Milli moles
Mg Microgram
pl Microlitre
hr. Hour
Km Michelis Menten Constant.
Gram
nm nanometer
% Percentage
9 I“ gram per litre
Fig. Figure
et al. Co-authors
ASW Artificial Sea Water
°C Degree Celsius
TEMED NNN'N’ Tetramethyl ethylenediamine.
APS Ammonium persulphate
SDS Sodium Dodecyl Sulphate.
BSA Bovine Serum Albumin
PEG Poly ethylene glycol
EDTA Ethylenediaminetetraaceticacid
EGTA Ethyleneglycol-O, 0' bis, (2 amino ethyl) NNN'N’
tetraaceticacid
PMSF Phenyl methane sulphonyl fluoride
Iodo Acetic Acid
DTT Dithiothreitol
ANOVA Analysis of variance
LSD Least Significant Difference
DFP Diisopropyl fluorophosphates
E64 L-trans- epoxysucciny|- leucylamido (4-guanidino butane)
ONPG 0- Nitrophenyl-B-D Galactopyranoside
HPLC High Performance Liquid Chromatography
IEC lon Exchange Chromatography
O/129 2,4 - diamino-6,7 diisopropyl pteridine
CHAPTER 1
INTRODUCTION