Structure-Based Design of Acetolactate Synthase From Bacillus licheniformis Improved Protein Stability Under Acidic Conditions.

Ting Zhao,Rongqing Zhou,Zhifu Peng,Jun Liu,Siqi Yuan,Yuan Li,Yang Ye

doi:10.3389/fmicb.2020.582909

Ting Zhao, Rongqing Zhou + Show 5 more

Open Access

https://doi.org/10.3389/fmicb.2020.582909

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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.

Highlights

Tetramethylpyrazine (TTMP), which is a compound commonly found in sauce-flavored liquor and sesame-flavor liquor, is an important compound related to the flavor of Chinese liquor, and a health-care factor, that endows Chinese liquor with healthy functions (Kao et al, 2006)
ALS consists of Flavin adenine dinucleotide (FAD)dependent anabolic acetohydroxyacid synthase (AHAS) and FAD independent catabolic acetolactate synthase, but cALS is only found in some bacteria (Renna et al, 1993; Lee et al, 2015)
The specific activity of BlALSN210D−H399D was similar to that of WT BlALS, and there was no significant difference between the specific activity measured in this study and that reported by Huo et al (2018)

Summary

Introduction

Tetramethylpyrazine (TTMP), which is a compound commonly found in sauce-flavored liquor and sesame-flavor liquor, is an important compound related to the flavor of Chinese liquor, and a health-care factor, that endows Chinese liquor with healthy functions (Kao et al, 2006). The main pathway for the production of TTMP in fermentation processes of Chinese liquor has been confirmed, which was a breakthrough for the improvement of liquor quality (Zhu et al, 2010). Bacillus licheniformis produces TTMP with glucose as a raw material by its Abbreviations: cALS, catabolic acetolactate synthase; MTD, medium-temperature Daqu;ThDP, thiamine diphosphate; TTMP, tetramethylpyrazine; WT, wild-type. Protein’s Acid Stability Improving own multi-step enzymatic reaction (Figure 1; Fan et al, 2007; Zhu et al, 2010), and the biosynthesis is in need of a key enzyme, acetolactate synthase (ALS, EC 2.2.1.6) (Jia et al, 2017). Acetolactate synthase belongs to the family of thiamine diphosphate (ThDP)-dependent enzymes, which is involved in α-acetolactate biosynthesis. ALS consists of Flavin adenine dinucleotide (FAD)dependent anabolic acetohydroxyacid synthase (AHAS) and FAD independent catabolic acetolactate synthase (cALS), but cALS is only found in some bacteria (Renna et al, 1993; Lee et al, 2015)

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