Affiliation:
1. Institute of Biology, College of Basic Medical Science, Shanxi University of Chinese Medicine, Jin Zhong 030600,
P.R. China
Abstract
Background:
A series of mutants of Rhizomucor miehei lipase (RML) screened through
four rounds of directed evolution were studied. Mutants' triglyceride hydrolysis activity was
assessed, and their genes were sequenced. Results showed that mutations in the propeptide can
improve the activity of RML during evolution. Two parts of propeptide (wild-type and mutant) and
mature region were connected by molecular simulation technology.
Methods:
The spatial structure of the most positive mutants containing the mutations in the
propeptide was mainly characterized by the increase in the opening angle of the lid structure in the
mature region of RML, the enhancement of the hydrophobicity of the active center, and the triad of
the active center shifted outward.
Results:
The three indexes above explain the mechanism of propeptide mutations on the activity
change of the target protein. In addition, statistical analysis of all the mutants screened in directed
evolution showed that: (1) most of the mutants with increased activity contained mutations of the
propeptide, (2) in the later stage of directed evolution, the number of active mutants decreased
gradually, and the mutations of inactivated protein mainly occurred in the mature region, and (3) in
the last round of directed evolution, the mutations distributed in the propeptide improved the mutant
activity further. The results showed that the propeptide reduced RML's evolutionary pressure and
delayed the emergence of the evolutionary platform.
Conclusion:
These findings reveal the role of propeptide in the evolution of RML and provide
strategies for the molecular transformation of other lipases.
Funder
general Natural Science fund project of Shanxi Natural Science Foundation
scientific and technological innovation project of colleges and universities in Shanxi Province
cultivation plan of scientific and technological innovation ability of Shanxi University of Chinese Medicine
Publisher
Bentham Science Publishers Ltd.
Subject
Biochemistry,General Medicine,Structural Biology
Cited by
1 articles.
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