Rational engineering of phospholipase C from Bacillus cereus HSL3 for simultaneous thermostability and activity improvement

Yonghui Zhang; Peng Dai; Rongkai Liu; Wenlong Liu; Anfeng Xiao; Jian Li; Guiling Li; Jingwen Liu

doi:10.1016/j.jbiotec.2022.06.009

Rational engineering of phospholipase C from Bacillus cereus HSL3 for simultaneous thermostability and activity improvement

J Biotechnol. 2022 Aug 20:355:1-9. doi: 10.1016/j.jbiotec.2022.06.009. Epub 2022 Jun 28.

Authors

Yonghui Zhang¹, Peng Dai², Rongkai Liu², Wenlong Liu², Anfeng Xiao¹, Jian Li², Guiling Li³, Jingwen Liu⁴

Affiliations

¹ College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China; Fujian Provincial Engineering Technology Research Center of Marine Functional Food, Xiamen, Fujian Province 361021, China; Xiamen Key Laboratory of Marine Functional Food, Xiamen 361021, China.
² College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China.
³ College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China; Fujian Provincial Engineering Technology Research Center of Marine Functional Food, Xiamen, Fujian Province 361021, China; Xiamen Key Laboratory of Marine Functional Food, Xiamen 361021, China. Electronic address: glli2016@jmu.edu.cn.
⁴ College of Ocean Food and Biological Engineering, Jimei University, Xiamen 361021, China; Collaborative Innovation Center of Marine Food Deep Processing, Dalian Polytechnic University, Dalian 116034, China. Electronic address: ljwsbch@163.com.

PMID: 35772600
DOI: 10.1016/j.jbiotec.2022.06.009

Abstract

As an essential enzyme for phospholipid degradation, phospholipase C (PLC) has been used for enzymatic degumming of vegetable oils and production of valuable phospholipid derivatives. In this study, rational engineering based on B-factor analysis and molecular dynamic simulation analysis were employed to rationally identify mutation candidates and a PLC double mutant F96R/Q153P was designed from Bacillus cereus HSL3. Compared to the wild-type PLC, F96R/Q153P exhibited significantly improved thermal properties, including higher temperature optima and better thermal stability. It showed the highest optimal reaction temperature (90 °C) reported so far. F96R/Q153P displayed 4.94 times kcat and 2.37 times k_cat/K_m as much as the wild-type, as well as improved substrate adaptability. Structural insights revealed that the mutations caused reduced proportion of random coil and constraint of certain loop fluctuations. These results demonstrated the great potential of knowledge-based rational design for improving the catalytic characteristics of industrial enzymes in the enzymatic degumming process.

Keywords: Enzymatic degumming; Phospholipase C; Rational design.

MeSH terms

Bacillus cereus* / genetics
Catalysis
Enzyme Stability
Hot Temperature
Kinetics
Phospholipids
Temperature
Type C Phospholipases* / chemistry
Type C Phospholipases* / genetics

Substances

Phospholipids
Type C Phospholipases