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Improving the Catalytic Activity and Thermostability of MAS1 Lipase by Alanine Substitution

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Abstract

MAS1 is a lipase isolated from Streptomyces sp. strain W007 with potential application in biotechnology. Structural analysis of MAS1 lipase showed that eight amino acids with bulkier side located in the substrate-binding pocket may be involved in affecting catalytic performance. Alanine substitutions of those residues were conducted to reduce steric clash of catalyzed pocket and probe their functional roles. The kcat/Km of mutants H108A, F153A, and V233A increased to 2.3-, 2.1-, and 1.4-fold, respectively. Interestingly, the half-life (60 °C) of F153A had shifted to 523 min after mutagenesis, which was fivefold enhancement toward that of MAS1 wide-type. Furthermore, higher hydrolysis ability of mutants H108A and F153A toward palm stearin of high melting temperature made them potentially applicable in oil/fat modification. Our work provided an example to obtain biocatalysts with desired catalytic behaviors by protein engineering.

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Abbreviations

WT:

Wild type

PUFA:

Polyunsaturated fatty acids

TAG:

Triacylglycerol

pNP:

p-Nitrophenol

PCR:

Polymerase chain reaction

SDS-PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

FAs:

Fatty acids

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Acknowledgements

This work was made possible with funding provided by the National Natural Science Foundation of China (21376098, 31471690, 31601398) and Science and Technology Planning project of Guangdong Province (2016B090920082).

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Correspondence to Dongming Lan or Yonghua Wang.

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Zhao, G., Wang, J., Tang, Q. et al. Improving the Catalytic Activity and Thermostability of MAS1 Lipase by Alanine Substitution. Mol Biotechnol 60, 319–328 (2018). https://doi.org/10.1007/s12033-018-0062-y

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  • DOI: https://doi.org/10.1007/s12033-018-0062-y

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