Secretory Expression Fine-Tuning and Directed Evolution of Diacetylchitobiose Deacetylase by Bacillus subtilis

Diacetylchitobiose deacetylase has great application potential in the production of chitosan oligosaccharides and monosaccharides. This work aimed to achieve high-level secretory production of diacetylchitobiose deacetylase by Bacillus subtilis and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in B. subtilis, and the signal peptide YncM achieved the highest extracellular diacetylchitobiose deacetylase activity of 13.5 U/ml. Second, by replacing the HpaII promoter with a strong promoter, the P43 promoter, the activity was increased to 18.9 U/ml. An unexpected mutation occurred at the 5' untranslated region of plasmid, and the extracellular activity reached 1,548.1 U/ml, which is 82 times higher than that of the original strain. Finally, site-directed saturation mutagenesis was performed for the molecular engineering of diacetylchitobiose deacetylase to further improve the catalytic efficiency. The extracellular activity of mutant diacetylchitobiose deacetylase R157T reached 2,042.8 U/ml in shake flasks. Mutant R157T exhibited much higher specific activity (3,112.2 U/mg) than the wild type (2,047.3 U/mg). The K decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the V_max increased from 5.11 μM s^-1 in the wild type to 7.56 μM s^-1 in the mutant R157T.IMPORTANCE We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in Bacillus subtilis, and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.