Threonine 22 (Thr22) located in catalytic center near the catalytic amino acid Glu19 was non-conserved in Bacillus species chitosanase. In order to study the function of Thr22, saturation mutagenesis was carried out towards P121N, a mutant previously constructed in our laboratory. Compared with P121N, which was designated as the wild type (WT) in this research, the specific enzyme activity of all mutants was decreased, and that of the T22P mutant was decreased by 91.6 %. Among these mutants, the optimum temperature decreased from 55 °C to 50 °C for 10 mutants and 45 °C for 4 mutants, respectively. The optimum temperature of mutant T22P was 40 °C. In order to analyze the reasons for the changes in enzymatic properties of the mutants, molecular docking analysis of WT and its mutants with substrate were performed. The hydrogen bond analysis around position 22 also conducted. The substitution of Thr22 was found to significantly affect the enzyme-substrate complex interaction. In addition, the hydrogen network near position 22 has undergone obvious changes. These changes may be the main reasons for the changes in enzymatic properties of the mutants. Altogether, this study is valuable for the future research on Bacillus chitosanase.
Keywords: Bacillus subtilis; Chitooligosaccharide; GH46 family chitosanase; Molecular dock; Molecular dynamics; Site-saturation mutagenesis; Thermostability.
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