Enhancing the Thermostability of Rhizomucor miehei Lipase with a Limited Screening Library by Rational-Design Point Mutations and Disulfide Bonds

Applied and Environmental Microbiology
Guanlin LiYunjun Yan

Abstract

Rhizomucor miehei lipase (RML), as a kind of eukaryotic protein catalyst, plays an important role in the food, organic chemical, and biofuel industries. However, RML retains its catalytic activity below 50°C, which limits its industrial applications at higher temperatures. Soluble expression of this eukaryotic protein in Escherichia coli not only helps to screen for thermostable mutants quickly but also provides the opportunity to develop rapid and effective ways to enhance the thermal stability of eukaryotic proteins. Therefore, in this study, RML was engineered using multiple computational design methods, followed by filtration via conservation analysis and functional region assessment. We successfully obtained a limited screening library (only 36 candidates) to validate thermostable single point mutants, among which 24 of the candidates showed higher thermostability and 13 point mutations resulted in an apparent melting temperature ([Formula: see text]) of at least 1°C higher. Furthermore, both of the two disulfide bonds predicted from four rational-design algorithms were further introduced and found to stabilize RML. The most stable mutant, with T18K/T22I/E230I/S56C-N63C/V189C-D238C mutations, exhibited a 14.3°C-higher [For...Continue Reading

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Citations

Sep 16, 2018·Applied and Environmental Microbiology·Shalev GihazAyelet Fishman
Nov 5, 2019·Critical Reviews in Food Science and Nutrition·Qiuming ChenWanmeng Mu
Mar 25, 2019·The Journal of General and Applied Microbiology·Liutengzi CaiChenyan Zhou
Apr 10, 2021·Emerging Topics in Life Sciences·Nitu SinghKinshuk Raj Srivastava
Oct 29, 2019·Journal of Chemical Information and Modeling·Xingrong FangLi Xu
Aug 30, 2021·International Journal of Biological Macromolecules·Raphael Oliveira SenaEliane Pereira Cipolatti
Sep 7, 2021·Applied Microbiology and Biotechnology·Siti Hajar HamdanThean Chor Leow

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