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 k/K 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.
Engineering
of enzymes on the basis of protein structures are rational
and efficient approaches to acquire biocatalysts of desired performances.
In this study, we focused on a special mono- and diacylglycerol lipase
(MDGL) isolated from the lipolytic enzyme-enriched fungus Aspergillus oryzae and discovered improved variants
based on its crystal structure. We first solved the crystal structure
of Aspergillus oryzae lipase (AOL)
at 1.7 Å resolution. Structure analysis and sequence alignment
of AOL and other MDGLs revealed that the residue V269 is of vital
importance for catalysis. Replacement of the V269 in AOL with the
corresponding residues in other MDGLs has led to noticeable changes
in hydrolysis without sacrificing the thermostability and substrate
specificity. Among the investigated variants, V269D exhibited about
a six-fold higher hydrolysis activity compared to the wild type. Molecular
dynamics simulations and protein–ligand interaction frequency
analyses revealed that the Asp substitution enhanced the substrate
affinity of AOL. Our work sheds light on understanding the catalytic
process of AOL and helps tailoring MDGLs with desired catalytic performance
to fulfill the demand for biotechnological applications.
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