AUTHOR=Zhao Ting , Li Yuan , Yuan Siqi , Ye Yang , Peng Zhifu , Zhou Rongqing , Liu Jun TITLE=Structure-Based Design of Acetolactate Synthase From Bacillus licheniformis Improved Protein Stability Under Acidic Conditions JOURNAL=Frontiers in Microbiology VOLUME=Volume 11 - 2020 YEAR=2020 URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.582909 DOI=10.3389/fmicb.2020.582909 ISSN=1664-302X ABSTRACT=

Catabolic acetolactate synthase (cALS) plays a crucial role in the quality of liquor because of its ability to catalyze the synthesis of the endogenous precursor product α-acetolactate of the aromatic compound tetramethylpyrazine (TTMP) and acetoin. However, the vulnerability of cALS to acidic conditions limits its application in the Chinese liquor brewing industry. Here we report the biochemical characterization of cALS from B. licheniformis T2 (BlALS) that was screened from Chinese liquor brewing microorganisms. BlALS showed optimal activity levels at pH 7.0, and the values of Km and Vmax were 27.26 mM and 6.9 mM⋅min–1, respectively. Through site-directed mutagenesis, we improved the stability of BlALS under acidic conditions. Replacing the two basic residues of BlALS with acidic mutations (N210D and H399D) significantly improved the acid tolerance of the enzyme with a prolonged half-life of 2.2 h (compared with wild-type BlALS of 0.8 h) at pH 4.0. Based on the analysis of homologous modeling, the positive charge area of the electrostatic potential on the protein surface and the number of hydrogen bonds near the active site increased, which helped BlALSN210D–H399D to withstand the acidic environment; this could extend its application in the food fermentation industry.