Improving thermostability of Bacillus amyloliquefaciens alpha-amylase by multipoint mutations

Susu Yuan; Renxiang Yan; Biyu Lin; Renkuan Li; Xiuyun Ye

doi:10.1016/j.bbrc.2023.02.064

Improving thermostability of Bacillus amyloliquefaciens alpha-amylase by multipoint mutations

Biochem Biophys Res Commun. 2023 Apr 23:653:69-75. doi: 10.1016/j.bbrc.2023.02.064. Epub 2023 Feb 24.

Authors

Susu Yuan¹, Renxiang Yan¹, Biyu Lin¹, Renkuan Li¹, Xiuyun Ye²

Affiliations

¹ National Engineering Laboratory for High-efficient Enzyme Expression, Fuzhou, Fujian, China; The Key Laboratory of Marine Enzyme Engineering of Fujian Province, Fuzhou University, Fuzhou, Fujian, China; College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian, China.
² National Engineering Laboratory for High-efficient Enzyme Expression, Fuzhou, Fujian, China; The Key Laboratory of Marine Enzyme Engineering of Fujian Province, Fuzhou University, Fuzhou, Fujian, China; College of Biological Science and Engineering, Fuzhou University, Fuzhou, Fujian, China. Electronic address: xiuyunye@fzu.edu.cn.

PMID: 36857902
DOI: 10.1016/j.bbrc.2023.02.064

Abstract

The medium-temperature alpha-amylase of Bacillus amyloliquefaciens is widely used in the food and washing process. Enhancing the thermostability of alpha-amylases and investigating the mechanism of stability are important for enzyme industry development. The optimal temperature and pH of the wild-type BAA and mutant MuBAA (D28E/V118A/S187D/K370 N) were all 60 °C and 6.0, respectively. The mutant MuBAA showed better thermostability at 50 °C and 60 °C, with a specific activity of 206.61 U/mg, which was 99.1% greater than that of the wild-type. By analyzing predicted structures, the improving thermostability of the mutant MuBAA was mainly related to enhanced stabilization of a loop region in domain B via more calcium-binding sites and intramolecular interactions around Asp187. Furthermore, additional intramolecular interactions around sites 28 and 370 in domain A were also beneficial for improving thermostability. Additionally, the decrease of steric hindrance at the active cavity increased the specific activity of the mutant MuBAA. Improving the thermostability of BAA has theoretical reference values for the modification of alpha-amylases.

Keywords: Alpha-amylase; Bacillus amyloliquefaciens; Thermostability; Three-dimensional model.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Amino Acid Sequence
Bacillus amyloliquefaciens* / genetics
Bacillus amyloliquefaciens* / metabolism
Bacillus*
Enzyme Stability
Mutation
Temperature
alpha-Amylases / chemistry
alpha-Amylases / genetics
alpha-Amylases / metabolism

Substances

alpha-Amylases