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Saturation mutagenesis and self‐inducible expression of trehalose synthase in Bacillus subtilis
Author(s) -
Liu Hongling,
Wang Xihui,
Yang Shaojie,
Wang Ruiming,
Wang Tengfei
Publication year - 2019
Publication title -
biotechnology progress
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.572
H-Index - 129
eISSN - 1520-6033
pISSN - 8756-7938
DOI - 10.1002/btpr.2826
Subject(s) - trehalose , bacillus subtilis , saturated mutagenesis , maltose , disaccharide , biochemistry , mutant , enzyme kinetics , recombinant dna , chemistry , enzyme , biology , bacteria , gene , active site , genetics
Trehalose is a nonreducing disaccharide synthesized by trehalose synthase (TreS), which catalyzes the reversible interconversion of maltose and trehalose. We aimed to enhance the catalytic conversion of maltose to trehalose by saturation mutagenesis, and constructed a self‐inducible TreS expression system by generating a robust Bacillus subtilis recombinant. We found that the conversion yield and enzymatic activity of TreS was enhanced by saturation mutations, especially by the combination of V407M and K490L mutations. At the same time, these saturation mutations were contributing to reducing by‐products in the reaction. Compared to WT TreS, the conversion yield of maltose to trehalose was increased by 11.9%, and the k cat / K m toward trehalose was 1.33 times higher in the reaction catalyzed by treS V407M‐K490L . treS V407M‐K490L expression was further observed in the recombinant B. subtilis W800N(Δσ F ) under the influence of P srfA , P cry3Aa , and P srfA‐cry3Aa promoters without an inducer. It was shown that P srfA‐cry3Aa was evidently a stronger promoter for treS V407M‐K490L expression, with the intracellular enzymatic activity of recombinant treS V407M‐K490L being over 5,800 U/g at 35 hr in TB medium. These results suggested the combination of two mutations, V407M and K490L, was conducive for the production of trehalose. In addition, the self‐inducible TreS V407M/K490L mutant in the B. subtilis host provides a low‐cost choice for the industrial production of endotoxin‐free trehalose with high yields.