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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| Format: | Thesis |
| Language: | English |
| Published: |
2017
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/69536/1/fbsb%202017%2039%20ir.pdf |
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| Summary: | 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. |
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