Directed evolution of AMS8 lipase towards enhanced activity and stability at low temperature

Cold active lipases have huge biotechnological prospects due to their high catalytic activity at low temperature. Generally, cold active lipases demonstrates high specific activity at low temperature and rapidly denatured in moderate range of temperature due to their thermosensitive nature. Ho...

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Main Author:	Arulu, Pireya Tharaseni
Format:	Thesis
Language:	English
Published:	2017
Subjects:	Lipase - Research Immobilized enzymes
Online Access:	http://psasir.upm.edu.my/id/eprint/69536/1/fbsb%202017%2039%20ir.pdf
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id	my-upm-ir.69536
record_format	uketd_dc
spelling	my-upm-ir.695362019-11-11T02:30:03Z Directed evolution of AMS8 lipase towards enhanced activity and stability at low temperature 2017-02 Arulu, Pireya Tharaseni Cold active lipases have huge biotechnological prospects due to their high catalytic activity at low temperature. Generally, cold active lipases demonstrates high specific activity at low temperature and rapidly denatured in moderate range of temperature due to their thermosensitive nature. However, the factor that contributes to this cold adaptation properties are still vague. AMS8 lipase is a Family 1.3 lipase produced by Antarctic Pseudomonas sp exhibits minimum activity at low temperature. The aim of this study is to evolve AMS8 lipase with enhanced activity and stability at low temperature and to study the effect of the amino acid substitution on the biochemical features of this lipase. The mutant library of AMS8 lipase was generated by error-prone PCR. Mutant M15 lipase was selected as it has the highest lipolytic activity at 20°C. M15 lipase was sequenced and two mutation points were identified which are R259C and V342E. In silico studies of this mutant has predicted that M15 lipase has increased structural flexibility compared to the native enzyme. Mutant M15 lipase was purified using gel filtration chromatography method. Biochemical characterization has revealed that M15 lipase has an optimum temperature at 20°C and highly stable at 10°C. M15 lipase was optimally active at pH 6 and stable within a small range of pH, 6-10. The catalytic activity of the mutant was boosted in the presence of Ca2+ and Na+. Moreover, M15 lipase was found to be tolerant towards hydrophobic organic solvents and demonstrated great specificity towards long-chain pNP esters and optimum activity was observed in pNP-laurate. Secondary structure analysis of M15 lipase revealed that the enzyme has attained more structural flexibility compared to the wild type. In conclusion, AMS8 lipase was successfully mutated via directed evolution strategy and the findings will be useful insight on the understanding of the cold active lipases properties. Lipase - Research Immobilized enzymes 2017-02 Thesis http://psasir.upm.edu.my/id/eprint/69536/ http://psasir.upm.edu.my/id/eprint/69536/1/fbsb%202017%2039%20ir.pdf text en public masters Universiti Putra Malaysia Lipase - Research Immobilized enzymes
institution	Universiti Putra Malaysia
collection	PSAS Institutional Repository
language	English
topic	Lipase - Research Immobilized enzymes
spellingShingle	Lipase - Research Immobilized enzymes Arulu, Pireya Tharaseni Directed evolution of AMS8 lipase towards enhanced activity and stability at low temperature
description	Cold active lipases have huge biotechnological prospects due to their high catalytic activity at low temperature. Generally, cold active lipases demonstrates high specific activity at low temperature and rapidly denatured in moderate range of temperature due to their thermosensitive nature. However, the factor that contributes to this cold adaptation properties are still vague. AMS8 lipase is a Family 1.3 lipase produced by Antarctic Pseudomonas sp exhibits minimum activity at low temperature. The aim of this study is to evolve AMS8 lipase with enhanced activity and stability at low temperature and to study the effect of the amino acid substitution on the biochemical features of this lipase. The mutant library of AMS8 lipase was generated by error-prone PCR. Mutant M15 lipase was selected as it has the highest lipolytic activity at 20°C. M15 lipase was sequenced and two mutation points were identified which are R259C and V342E. In silico studies of this mutant has predicted that M15 lipase has increased structural flexibility compared to the native enzyme. Mutant M15 lipase was purified using gel filtration chromatography method. Biochemical characterization has revealed that M15 lipase has an optimum temperature at 20°C and highly stable at 10°C. M15 lipase was optimally active at pH 6 and stable within a small range of pH, 6-10. The catalytic activity of the mutant was boosted in the presence of Ca2+ and Na+. Moreover, M15 lipase was found to be tolerant towards hydrophobic organic solvents and demonstrated great specificity towards long-chain pNP esters and optimum activity was observed in pNP-laurate. Secondary structure analysis of M15 lipase revealed that the enzyme has attained more structural flexibility compared to the wild type. In conclusion, AMS8 lipase was successfully mutated via directed evolution strategy and the findings will be useful insight on the understanding of the cold active lipases properties.
format	Thesis
qualification_level	Master's degree
author	Arulu, Pireya Tharaseni
author_facet	Arulu, Pireya Tharaseni
author_sort	Arulu, Pireya Tharaseni
title	Directed evolution of AMS8 lipase towards enhanced activity and stability at low temperature
title_short	Directed evolution of AMS8 lipase towards enhanced activity and stability at low temperature
title_full	Directed evolution of AMS8 lipase towards enhanced activity and stability at low temperature
title_fullStr	Directed evolution of AMS8 lipase towards enhanced activity and stability at low temperature
title_full_unstemmed	Directed evolution of AMS8 lipase towards enhanced activity and stability at low temperature
title_sort	directed evolution of ams8 lipase towards enhanced activity and stability at low temperature
granting_institution	Universiti Putra Malaysia
publishDate	2017
url	http://psasir.upm.edu.my/id/eprint/69536/1/fbsb%202017%2039%20ir.pdf
_version_	1747812712067366912

Directed evolution of AMS8 lipase towards enhanced activity and stability at low temperature

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