Enhanced activity of Rhizomucor miehei lipase by directed evolution with simultaneous evolution of the propeptide

Applied Microbiology and Biotechnology
Jue WangHongwei Yu

Abstract

Propeptides are short sequences that facilitate the folding of their associated proteins. The present study found that the propeptide of Rhizomucor miehei lipase (RML) was not proteolytically removed in Escherichia coli. Moreover, RML was not expressed if the propeptide was removed artificially during the cloning process in E. coli. This behavior in E. coli permitted the application of directed evolution to full-length RML, which included both propeptide and catalytic domain, to explore the role played by the propeptide in governing enzyme activity. The catalytic rate constant, k (cat), of the most active mutant RML protein (Q5) was increased from 10.63 ± 0.80 to 71.44 ± 3.20 min(-1) after four rounds of screening. Sequence analysis of the mutant displayed three mutations in the propeptide (L57V, S65A, and V67A) and two mutations in the functional region (I111T and S168P). This result showed that improved activity was obtained with essential involvement by mutations in the propeptide, meaning that the majority of mutants with enhanced activity had simultaneous mutations in propeptide and catalytic domains. This observation leads to the hypothesis that directed evolution has simultaneous and synergistic effects on both functiona...Continue Reading

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Citations

Mar 1, 2016·The Journal of General and Applied Microbiology·Zhiyuan WangDong He
Aug 26, 2016·Applied Microbiology and Biotechnology·Chengcheng YangHongwei Yu
May 12, 2015·Biotechnology and Applied Biochemistry·Xiaoxue TongLene Lange
Aug 2, 2017·Letters in Applied Microbiology·H CaiF Feng
Sep 14, 2021·Frontiers in Bioengineering and Biotechnology·Ting MiaoWei Jiang

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