Studies on the enzymes and their applications

163 Pages·2015·3.61 MB·English

by  Dr Ashok Pandey

Novel esterases from microbes through
classical and metagenomics approach:
Studies on the enzymes and their
applications

Thesis submitted under the Faculty of Science of the
Cochin University of Science and Technology
For the award of the degree of

Doctor of Philosophy
in

Biotechnology

Deepthy Alex
Reg. No. 3453

Centre for Biofuels, Biotechnology Division
CSIR-National Institute for Interdisciplinary Science & Technology
Thiruvananthapuram 695019, INDIA
March 2014
National Institute for Interdisciplinary Science and Technology
Council of Scientific and Industrial Research, Government of India

Pappanamcode, Thiruvananthapuram 695019, Kerala, India
Phone : [phone], Fax: [phone]
Centre for Biofuels
Email : [email protected] / [email protected]
Biotechnology Division

Dr Rajeev Kumar Sukumaran, MSc, PhD, PGDBi
Senior Scientist
20 March 2014

DECLARATION

I hereby declare that the work presented in this thesis entitled “Novel esterases from microbes
through classical and metagenomics approach: Studies on the enzymes and their applications”
is a bona fide record of the research carried out by Ms Deepthy Alex (Reg No.3453), under my
guidance and supervision, at the CSIR-National Institute for Interdisciplinary Science and
Technology, Thiruvananthapuram, India. I declare that all suggestions made by the audience
during Pre-synopsis seminar and recommended by the doctoral committee have been
incorporated in this thesis. I also declare that this work or no part of this has been submitted for
the award of any degree, diploma, associateship or any other title or recognition.

Rajeev Kumar Sukumaran
Thiruvananthapuram
20 March 2014

DECLARATION

I hereby declare that the work presented in this thesis entitled “Novel esterases from microbes
through classical and metagenomics approach: Studies on the enzymes and their
applications” is based on the original work done by me under the guidance of Dr Rajeev Kumar
Sukumaran, Senior Scientist, Biotechnology Division, CSIR-National Institute for Interdisciplinary
Science and Technology, Thiruvananthapuram, India and the thesis or no part of it has been
submitted elsewhere for the award of any Degree, Diploma, Associateship or any other Title or
Recognition.

Deepthy Alex
Dedicated to my Parents
Acknowledgements

Pursuing a PhD is indeed an adventurous experience accompanied with hardships,
frustrations, encouragement and trust, a journey with so many kind and helpful hearts. As I reach the
end of this journey, I realize that it was in fact, a team work that got me here. Words will not suffice
to express my gratitude to all those who have helped me, yet I would like to leave a note of thanks to
all who have been with me on this journey and helped me see it through.

First and foremost, I am deeply grateful for the constant support, insight and patience of my
supervisor Dr Rajeev Kumar Sukumaran for his guidance without which this thesis would not have
been possible. You have been a true mentor to me dear Sir. I would like to thank you for the faith you
have had in me to accept me as your PhD student without any hesitation and also for all the
constructive criticism and extensive discussions about my work thereafter.

I express my sincere gratitude to Dr Sarita G Bhat, Head, Department of Biotechnology,
CUSAT and Expert Member of my Doctoral Committee for her valuable suggestions which helped in
improving my research studies.

I would like to thank Prof Dr.Ashok Pandey, Head of Biotechnology Division, NIIST for his
valuable suggestions and support throughout my research work. The regular quarterly reviews and
seminars conducted in our division under his command has indeed groomed us a lot in developing our
skills. I deeply appreciate your ways in managing our division.

I am thankful to Director, Dr. Suresh Das, Former Director, T.K Chandrasekhar, and Former
acting Director Dr. B.C Pai, NIIST, CSIR, Trivandrum for providing me with all the facilities for
completing my research.

My sincere and warm thanks to Dr.Sindhu for her valuable inputs and suggestions in my
work especially in the metagenomic work. I extend my thanks to Dr. Madhavan Nampoothiri, Dr.
Binod Parameswaran, Dr Ramesh, Dr. M Arumugham, Mr. Kiran Kumar and Dr. D Leena for their
critical suggestions and help during my research work.

I take this opportunity to thank the Doctoral Committee and Research Committee of
CUSAT, Academic Program Committee of NIIST and NIIST-CUSAT Research Council for all the
timely help during the entire course of my work and thesis submission. I also thank all the
administrative and supporting staff of CUSAT and NIIST for their support and help.

I am grateful to Dr. Vijaya Lakshmi Amma, Mr Sivan Kutty Nair, Mr. KM Prakash,
Ms Radhamani, Ms Sasikala and Ms Rajalakshmi for their assistance in my work. I also acknowledge
the supporting staff in NIIST especially the Library and Administration staff.

Team Biotech has been a wonderful community in which to ‘grow up’ as a research student:
the personal and professional relationships forged in these years will stay with me forever. I am
particularly grateful to Vidya, Ushasree, Arya and Neetha; my lab mates as well as roommates for
they have made my stay comfortable and memorable here. I deeply cherish the fun we had together for
the past five years.

I extend my thanks to Rajasree and Abraham for all their generous and timely help. I express
my gratitude towards my seniors Dr. Reeta, Dr Nisha, Dr Raji, Dr Sayed, Dr Aswathy, Dr Swetha,
Dr Dhanya, Dr Gincy, Dr Sai and Dr Shyam who kept me going in the beginning. With immense
pleasure I wish to extend my sincere thanks to my friends Vipin, Kiran, Aravind, Anusree, Deepa,
Kuttiraja, Preeti, Divya, Varsha, Nimisha, Sabeela, Vani, Sajna, Arjun, Lalitha, Leya, Nishant,
Soya, Ayman, Meena, Dilna, Vini, Sneha, Surya and all friends of my Division for their support,
encouragement and care.

My sincere thanks to students of other divisions especially Janu, Linsha, the photochemistry
section of CSIR-NIIST for providing me with DLS and AFM facilities and the AP & NP division
for FAME analyses.

Words cannot express my deepest thankfulness to my parents for the support, concern,
prayers and sacrifices they have offered without which it would not have been possible to reach where
I am today. I am dedicating my thesis to my parents. Care and support of my brother Deepu and sister
in law Elizabeth and my sister Deepa and brother- in- law Bino and their two wonderful kids Diya
and Isa. I thank them all for the unconditional love they have showered on me.

Above all, I express my gratefulness to the Almighty for guiding me and strengthening me
through all the difficulties and for making my paths straight, for I believe it’s Your plans and not
mine that has taken me through this wonderful journey.

Deepthy Alex
LIST OF PUBLICATIONS

Published in SCI journals

1. Deepthy Alex, Ashok Pandey, Rajeev K Sukumaran, 2014, Esterase active in polar organic
solvents from the yeast Pseudozyma sp NII 08165, Enzyme Res (In Press)
http://www.hindawi.com/journals/er/aip/494682/

2. Jajaja Vidya, Sweta Swaroop, Sudheer K Singh, Deepthy Alex, Rajeev K Sukumaran, Ashok
Pandey, 2011, Isolation and characterization of a novel α-amylase from a metagenomic
library of Western Ghats of Kerala, India, Jalaja Vidya, Sweta Swaroop, Biologia 66 (6):
939—944

Communicated

1. Deepthy Alex, Abraham Mathew, Rajeev K Sukumaran. 2014, Esterases immobilized on
aminosilane modified magnetic nanoparticles as a catalyst for biotransformation reactions
(Communicated to Bioresource Technology- Ref BITE-S-14-02007).

Book Chapters

1. Rajeev K Sukumaran, Lalith Devi Gottumukkala, Rajasree Kuniparambil, Deepthy Alex,
Ashok Pandey, 2011, Butanol from Biomass: Revisiting ABE fermentation, In : Pandey A,
Larroche C, Ricke SC, Dussap CG, Gnansounou E (eds) Biofuels: Alternative Feedstocks and
Conversion Processes, Academic Press, London, pp-571-586

Papers/Poster Presented in Conferences/Seminars

1. Deepthy Alex, Ashok Pandey, Rajeev K Sukumaran, 2011, Purification and characterization
of thermo and halotolerant esterase sfrom a metagenomic library constructed from soap
factory effluent, In International Conference on “New Horizons in Biotechnology” organized
by CSIR-NIIST and BRSI, Trivandrum Nov 21-24.
2. Deepthy Alex, Sindhu Raveendran, Ashok Pandey, Rajeev K Sukumaran, 2010, Esterase
from soil DNA through metagenomics, In International conference on Genomic Sciences,
Madurai Kamaraj University, Madurai, November 12 -14, 2010,
3. Deepthy Alex, Anju Shainu, Ashok Pandey, Rajeev K Sukumaran, 2009, Studies on
temperature and solvent tolerant esterase from an yeast like isolate NII 08165.
International Conference on Emerging Trends in Biotechnology and VI BRSI Convention,
Banaras Hindu University, Varanasi, India, December 4-6
CONTENTS

Chapter 1 Introduction and Review of Literature 1
1.1. Introduction 1
1.2. Review of Literature 3
1.2.1. Different reactions catalyzed by lipases 3
1.2.2. Lipases and Esterases 4
1.2.3. Sources of lipase 4
1.2.3.1. Microbial lipases 5
1.2.4. Specificity of Lipases 5
1.2.5. Active site of lipase and the mechanism of action 7
1.2.6. Lipases with important properties 8
1.2.6.1. Solvent tolerant lipases 8
1.2.6.2. Thermostable lipases 8
1.2.6.3. Lipases showing tolerance to NaCl (Halo tolerant lipases) 8
1.2.7. Applications of lipases 9
1.2.8. Fermentative production of lipases and optimization of production 10
1.2.9. Screening for lipases 12
1.2.10. Purification of lipases 13
1.2.11. Magnetic nanoparticle immobilized lipase for biodiesel production 14
1.2.12. Cloning of lipases from yeasts 15
1.2.13. Metagenomic approach for novel biocatalysts 16
1.3. Objectives of the work 19

Chapter 2 Materials and Methods 21
2.1. Microorganisms and culture conditions 21
2.2. Medium for Enzyme production 22
2.3. Preparation of inoculum 22
2.4. Production of Esterase 23
2.5. Analytical methods 23
2.5.1 Enzyme assay 23
2.5.2 Protein assay 23
2.6. Electrophoresis and Zymogram analyses 24
2.6.1 Poly Acrylamide Gel Electrophoresis (PAGE) and Zymogram 24
analyses
2.6.2 Agarose Gel Electrophoresis 24
2.7. Yeast chromosomal DNA isolation 25
2.8. Plasmid isolation from metagenomic clones 25
2.9. Production of electrocompetant cells 26
2.10 General Molecular Biology Techniques and Software 26
2.11. Experimental Data Analyses 26

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Chapter 3 Screening and selection of a novel microbe 27
for esterase production
3.1. Introduction 27
3.2. Materials and Methods 28
3.2.1. Sample collection and primary screening for Esterase producers 28
3.2.2. Esterase production 28
3.2.3. Native PAGE and Zymogram analysis 28
3.2.4. Identification of microbial strains 29
3.2.4.1. Morphological identification of NII08165 29
3.2.4.2. Molecular identification of NII08165 29
3.2.5. Enzyme Profile 29
3.2.5.1. Amylase 30
3.2.5.2. Xylanase 30
3.2.5.3. Cellulase 30
3.2.5.4. Pectinase 30
3.2.5.5. Protease 30
3.3 Results and Discussion 31
3.3.1. Screening and isolation of lipase producing cultures 31
3.3.2. Lipase production by BTL1 31
3.3.3. Zymogram analysis 32
3.3.4. Identification of the isolate BTL 1 33
3.3.4.1. Morphological identification 33
3.3.4.2. Molecular identification of isolate NII 08165 (BTL1) 34
3.3.5 Enzyme profile 36
3.4 Conclusion 37

Chapter 4 Fermentative production of esterase from 39
Pseudozyma sp NII 08165
4.1 Introduction 39
4.2 Materials and Methods 40
4.2.1 Strain and initial cultivation conditions 40
4.2.2. Enzyme Assay 40
4.2.3 Optimization of parameters affecting esterase production 41
4.2.3.1. Screening of parameters affecting esterase production by fractional 41
factorial design
4.2.3.2. Optimization of significant parameters using response surface 43
method
4.3 Results and discussion 45
4.3.1. Optimization of parameters affecting esterase production 45
4.3.1.1. Screening of parameters influencing esterase production by 45
Pseudozyma sp.
4.3.1.2. Optimization of critical parameters identified by factorial design 48
4.4 Conclusion 52

ii
Chapter 5 Purification and characterization of esterase from 53
Pseudozyma sp NII 08165
5.1 Introduction 53
5.2 Materials and methods 55
5.2.1. Studies on the properties of the partially purified esterase 55
5.2.1.1 Partial purification of Pseudozyma esterase 55
5.2.1.2 Temperature tolerance and Thermostability 55
5.2.1.3 pH tolerance and pH stability 55
5.2.1.4 Solvent Tolerance 55
5.2.1.5 Halo Tolerance 56
5.2.2. Purification of the LIP1 esterase of Pseudozyma sp NII 08165 56
5.2.2.1. Ultra filtration 56
5.2.2.2 Ammonium sulfate precipitation 56
5.2.2.3. Hydrophobic interaction chromatography (HIC). 56
5.2.3 Enzyme Characterization 57
5.2.3.1. Molecular weight determination 57
5.2.3.2. Enzyme kinetics 57
5.2.3.3 Properties of purified isoform LIP 1 57
5.3 Results and discussion 58
5.3.1. Studies on the properties of partially purified enzyme 58
5.3.1.1. Temperature tolerance and temperature stability 58
5.3.1.2. pH tolerance and pH stability 59
5.3.1.3. Solvent tolerance 59
5.3.1.4. Halo tolerance 60
5.3.2. Purification of LIP 1 enzyme 61
5.3.3. Characterization and Properties of LIP1 from 63
Pseudozyma SP NII 08165
5.3.3.1. Molecular weight determination 63
5.3.3.2. Enzyme kinetics 63
5.3.3.3. Solvent tolerance of LIP1 64
5.3.3.4. Halo tolerance of purified LIP 1 65
5.3.3.5 Thermo tolerance 65
5.4 Conclusion 66

Chapter 6 Construction and screening of metagenomic libraries for novel 67
lipases
6.1 Introduction 67
6.2. Materials and methods 70
6.2.1. Collection of environmental soil samples 70
6.2.2. Isolation and purification of environmental DNA 70
6.2.3. Construction of metagenomic libraries: Fosmid Library 71
6.2.3.1. Confirmation of fosmid clones 71
6.2.3.2. Insert size determination of fosmid clones 72
6.2.3.3. Screening of fosmid clones for esterases 72
6.2.4. Construction of metagenomic libraries: BAC library 72
6.2.4.1. Confirmation of BAC clones 72
6.2.4.2. Insert size determination of BAC clones 73
6.2.4.3. Screening of BAC clones for esterases 73
6.3. Results and Discussion 74
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