Improving the Catalytic Activity and Thermostability of MAS1 Lipase by Alanine Substitution
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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.
KeywordsMAS1 lipase Steric clash Alanine substitution Site-directed mutagenesis
Polyunsaturated fatty acids
Polymerase chain reaction
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
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).
Compliance with Ethical Standards
Conflict of interest
The authors have declared no conflict of interests.
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