Engineering of a thermo-alkali-stable lipase from Rhizopus chinensis by rational design of a buried disulfide bond and c
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BIOCATALYSIS - ORIGINAL PAPER
Engineering of a thermo‑alkali‑stable lipase from Rhizopus chinensis by rational design of a buried disulfide bond and combinatorial mutagenesis Rui Wang1,2 · Shang Wang1 · Yan Xu1 · Xiaowei Yu1 Received: 17 June 2020 / Accepted: 9 October 2020 © Society for Industrial Microbiology and Biotechnology 2020
Abstract To improve the thermostability of the lipase (r27RCL) from Rhizopus chinensis through rational design, a newly introduced buried disulfide bond F223C/G247C was proved to be beneficial to thermostability. Interestingly, F223C/G247C was also found to improve the alkali tolerance of the lipase. Subsequently, six other thermostabilizing mutations from our previous work were integrated into the mutant F223C/G247C, leading to a thermo-alkali-stable mutant m32. Compared to the wildtype lipase, the associative effect of the beneficial mutations showed significant improvements on the thermostability of m32, 30 with a 74.7-fold increase in half-life at 60 °C, a 21.2 °C higher T50 value and a 10 °C elevation in optimum temperature. The mutated m32 was also found stable at pH 9.0–10.0. Furthermore, the molecular dynamics simulations of m32 indicated that its rigidity was enhanced due to the decreased solvent-accessible surface area, a newly formed salt bridge, and the increased ΔΔG values. Keywords Rhizopus chinensis · Thermo-alkali-stable lipase · Disulfide bond design · Molecular dynamics simulations
Introduction Lipases (triacylglycerol lipase EC.3.1.1.3) are a class of enzymes that can catalyze the long-chain triglyceride hydrolysis, ester synthesis, and ester exchange reactions. Owing to their high catalytic efficiency and environmental friendliness, lipases have found important applications in various industries including food, oil, pharmaceuticals, paper, leather, detergent, and cosmetics [1]. Rhizopus sp. is one of the most important lipase producers [2]. The high sn-1, 3-positional specificity, enantioselectivity, and activity in non-aqueous media of Rhizopus lipases are the most Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10295-020-02324-1) contains supplementary material, which is available to authorized users. * Xiaowei Yu [email protected] 1
Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China
School of Pharmaceutical Science, Jiangnan University, Wuxi 214122, Jiangsu, China
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desirable characters for the industrial applications [2]. However, most Rhizopus lipases are mesophilic and exhibit low thermostability [3]. Thus, endeavors have always been pursued to improve their thermostability, for even slight improvement can save huge costs during the manufacturing processes [4, 5]. The thermostability of the enzyme is mainly affected by salt bridge, hydrogen bond, hydrophobic stacking, and disulfide bond. Among these, disulfide bond is one of the most important interactions to stabilize the enzyme structure [6], which c
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