Alkaline Protease from a Non-toxigenic Vibrio sp.

272 Pages·2010·3.11 MB·English

Alkaline Protease from a Non-toxigenic Vibrio sp. (V26)
and its Applications

Thesis submitted to
Cochin University of Science and Technology
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY
In
MICROBIOLOGY

UNDER THE FACULTY OF MARINE SCIENCES

By
MANJUSHA K.

DEPARTMENT OF MARINE BIOLOGY, MICROBIOLOGY AND
BIOCHEMISTRY
SCHOOL OF MARINE SCIENCES
COCHIN UNIVERSITY OF SCIENCE AND TECHNOLOGY
KOCHI-682016, INDIA

July 2011
Dedicated to the
Loving Memory of my
Grandfather
M.K Panikkar
Certificate

This is to certify that the thesis entitled “Alkaline Protease from a Non-
toxigenic Vibrio sp. (V26) and its Applications” is an authentic record of research
work carried out by Ms. Manjusha K. under my supervision and guidance in the
Department of Marine Biology, Microbiology and Biochemistry, School of Marine
Sciences, Cochin University of Science and Technology, in partial fulfillment of the
requirements for the degree of Doctor of Philosophy in Microbiology and no part
thereof has been presented before for the award of any degree, diploma, or
associateship in any University.

Dr. A.V. Saramma
(Supervising Teacher) Associate Professor
Dept. of Marine Biology, Microbiology and
Biochemistry, School of Marine Sciences
Cochin University of Science and Technology
Cochin-682016

Kochi-682016
July, 2011
Declaration

I hereby do declare that the thesis entitled “Alkaline Protease from
a Non-toxigenic Vibrio sp. (V26) and its Applications”, is a genuine
record of research work done by me under the supervision and guidance of
Dr. A.V. Saramma, Associate Professor, Department of Marine Biology,
Microbiology and Biochemistry, School of Marine Sciences, Cochin
University of Science and Technology and that no part of this work has
been presented for the award of any degree, diploma or associateship in any
University or Institution earlier.

Manjusha K.

Kochi-16
July, 2011
Acknowledgement
This thesis would not have been possible without the support and help of several
individuals who in one way or another have contributed and extended their valuable
assistance in the preparation and completion of this study. It is a pleasure to convey my
gratitude to all of them in my humble acknowledgement.
First and foremost, I wish to express my utmost and profound gratitude to my
supervising guide Dr. A. V. Saramma, Associate Professor, Department of Marine
Biology, Microbiology and Biochemistry, for her valuable guidance, support,
understanding and encouragement throughout my work; whilst allowing me the room to
work in my own way. I am grateful to her for the constructive comments and careful
evaluation of my thesis. It’s been a privilege to work under her guidance.
I would like to place on record my indebtedness to Prof (Dr.) I.S Bright Singh,
Coordinator, National Centre for Aquatic Animal Health, CUSAT, for giving me the
permission to work and use the facilities at the centre. I am extremely grateful to him, for
his timely guidance, interesting discussions; unflinching support that has helped me
overcome the obstacles encountered during my research work. His dedication and sincerity
has inspired me. It’s been an honour to work under him.
I gratefully acknowledge all the help and support extended to me by The Head,
Department of Marine Biology, Microbiology and Biochemistry. I would also like to thank
Dr H. S. Ram Mohan, Dean and Director, School of Marine Sciences for allowing me to
utilize the facilities of the School of Marine Sciences.
It is a pleasure to express my gratitude to Prof (Dr.) Babu Philip for his expert
advice, Dr. Rosamma Philip for her willingness to clear my silliest to the most serious of
doubts, Dr A. A Mohamed Hatha for all the valuable suggestions that helped me improve
my work. I would also like to thank Dr. Aneykutty Joseph, Dr. Bijoy Nandan and
Dr. C.K.Radhakrishnan, former Head of the Department for all their help and
encouragement.
I wholeheartedly thank Dr. Mohandas, Emeritus Professor, School of
Environmental Studies, Dr Valsamma Joseph and Dr. Sajeevan T.P, faculty members at
NCAAH for all their valuable suggestions. I would like to thank Mr. Angel, Department
of Statistics for the statistical advice.
I would like to express my sincere gratitude to my lecturers at St. Teresa’s College
Mrs Gladys Francis and Mrs Reema Kuriakose for all their encouragement. I would also
like to thank each and every teacher of mine right from my kinder garden to my post
graduation.
Words fail me to express my appreciation and gratitude to my dear friends
Dr. Sreedevi N Kutty and Dr. Simi Joseph. P for their unselfish and unfailing support
throughout my work. They have been a great company in this otherwise exhausting journey
of research. I thank Sreedevi for helping me with the references and Simi for her untiring
efforts in the proof reading of my thesis.
I would like to thank my friends Dr. Merina Varghese and Manjusha K.P my
former degree class mates for all the help extended to me. I wish to express my gratitude to
my former colleague and friend Dr. Latha Unnikrishnan for the advice, encouragement
and the sisterly affection rendered.
I gratefully acknowledge the untiring support and timely help rendered to me by
Dr. Sanil Kumar, Ms. Swapna P Antony, Mr. Anit M Thomas and Mr.Hari Shankar.
Collective and individual acknowledgements are also owed to my seniors
Dr. Newby Joseph, Dr. Meera Venugopal, Dr. Maya Paul, Dr. Sincy Joseph, Dr. Selvan.
S, Dr. Annies Joseph, Dr.Lakshmi G.Nair, Mr. Neil, Dr Rejish, Dr. Sreedharan,
Ms. Smitha S.L, Dr. Smitha V Banu, Dr. Anupama G. Nair, Mr. Abdul Jaleel, Dr. Venu,
Mr. Harikrishnan and Dr.Anila Devi Kunjamma for all their help and advice.
In my daily work I have been blessed with a friendly and cheerful group of fellow
colleagues. I owe a lot to Ms. Deepthi Augustine, Ms. Divya P.S, Ms. Nifty John,
Ms. Rosemine for proof reading of my thesis in the midst of all their activity. I
wholeheartedly thank my colleagues Mr.Abhilash KR, Ms. Jisha V.K,, Ms. Sini P.J,
Dr. Mujeeb, Ms. Remya Vardarajan, Ms. Prabha Pillai, Ms. Jisha Jose, Ms. Jimly C.J,
Ms. Remya K.D, Mr.Prajith, Mr. Manoj, Ms. Neetu, Ms. Deborah Alexander,
Mr. Shubhankar Ghosh, Mr Anil Kumar, Mr Navin, Ms. Divya T. Babu ,Ms. Neena and
all the other research scholars of the department, for always being there to lend a helping
hand.
I sincerely thank my senior colleagues at NCAAH Dr. Manju, Mr. Deepesh,
Dr. Somanath Pai, Dr.Jasmine and Dr. Anas for all their help and suggestions.
I would like to thank Ms. Divya Jose and Ms Sreelakshmi B. for being great co-
workers, for their understanding and support. I am immensely grateful to Ms. Sunitha
Poulose for sparing her valuable time and unfailing moral support. Dr. Seena Jose and
Mr Jayesh have helped me understand the complexities of animal cell culture and I greatly
acknowledge their help. Ms. Priyaja P., Ms Vrinda S. and Ms. Giji have been a great help
and I sincerely thank them all. I would also like to acknowledge Mr. Prem, Ms. Blessy,
Ms. Deepa, Ms. Surekha and all my other friends at NCAAH for their help and support.
I am grateful to the librarian and other staff of the School of Marine Sciences
library for their valuable cooperation. I would like to thank both the office and technical
staff of the Department of Marine Biology, Microbiology and Biochemistry and NCAAH
for all their help.
I thank the University Grants Commission and Department of Biotechnology,
Govt. of India for the financial assistance provided.
I would like to thank everybody who has played a part in the successful
completion of this thesis, as well as express my apology that I could not mention it
personally.
Where would I be without my family? My parents deserve special mention for
their unconditional love and sacrifice. I fall short of words, to express my gratitude to my
Father Mr.Mohana Dasan for being so supportive in all my endeavors and my Mother
Mrs Shobha Mohana Dasan for being my friend and mentor. It is my mother’s prayers
that has got me this far. I would like to thank my brother Mr. Mukund for all the love
and affection. My heart felt gratitude to my Grandparents Late M.K Panikkar, Late
Rugumini amma, Late K.G Kurup and Mrs Padmavathy amma, for all their love and
blessings. I would like to express my deep sense of gratitude to all my aunts, uncles and
cousins for their untiring support, love and encouragement.
Last, but not the least, I would like to thank the Almighty God for giving me the
strength to move on despite all odds. Thank you, God for giving me a great family,
excellent teachers and wonderful friends.

Manjusha. K
Abbreviations
A Absorbance at 280 nm
280
ºC Degree Celsius
% Percentage
ANOVA Analysis of variance
APV26 Alkaline protease from Vibrio sp.(V26)
BATH Bacterial adhesion to hydrocarbon test
bp Base pair
BLAST Basic Local Alignment Search Tool
BSA Bovine serum albumin
CaCo-2 Human colon carcinoma cells
CCD Central Composite Design
Da Dalton
DEAE Diethyl amino ethyl
DMSO Dimethyl sulphoxide
DNA Deoxyribonucleic acid
DOE Design of Experiments
EDTA Ethylene-diamine tetra acetic acid
FBS Fetal bovine serum
FCCCD Face Centered Central Composite Design
g Gram
HA/protease Hemagglutinin/protease
HeLa Henritta Lacks, human cervical carcinoma cell lines
HEp-2 Human larynx epithelial cell lines
hrs hours
IAA Iodo acetic acid
kDa kilo Dalton
LC Lethal concentration, 50%
50
L Litre
M molar
MEM Minimal essential media
mg milligram
ml Milli litre
mM milliMolar
mol/L Moles per litre
mOsm milliosmole
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyl tetrazolium bromide
nm nanometer
O.D Optical density
PBD Plackett-Burman Design
PBS Phosphate buffered saline
PCR Polymerase chain reaction
PMSF Phenylmethylsulphonyl fluoride
pmol Picomoles
PVP Poly vinyl pyrrolidone
RBCs Red Blood Cells
RNA Ribonucleic acid
rpm revolutions per minute
RSM Response surface methodology
RTG-2 Rainbow trout gonadial cell lines
SAT Salt aggregation test
SDS Sodium dodecyl sulphate
sec seconds
TCA Trichloroacetic acid
U Protease Unit
µ microgram
µl microlitre
Chapter 1 General Introduction ...............................................................1-14
Chapter 2 Phenotypic and Molecular Identification of the
Producer Strain ........................................................................15-59
2.1 Review of Literature 17
2.1.1. Identification 17
2.1.1.1 Phenotypic Identification 17
2.1.1.2 Phylogenetic identification and 16S rRNA 18
2.1.1.3 Genotypic identification 20
2.1.2 Putative virulence traits 21
2.1.2.1Adherence and hydrophobicity 22
2.1.2.2 Toxins 24
2.1.2.3 Regulation of virulence factors 26
2.1.3 Antibiotic susceptibility 27
2.2 Materials and Methods 28
2.2.1 Experimental organism 28
2.2.2 Identification of the selected mangrove isolate Vibrio sp.
(V26) 29
2.2.2.1 Morphology 29
2.2.2.2 Phenotypic characterization 29
2.2.2.3 Molecular characterization 34
2.2.2.4 Putative virulence traits 37
2.2.2.4.1Serogrouping 37
2.2.2.4.2 Virulence genes 37
2.2.2.4.3 Adherence Assay 39
2.2.2.4.4 Hydrophobicity 39
2.2.2.5 Antimicrobial susceptibility test 40
2.3 Results 41
2.3.1 Morphology 41
2.3.2 Phenotypic Identification 41
2.3.3 Molecular identification 43
2.3.4 Putative Virulence Traits 45
2.3.4.1 serogrouping 45