Details

Autor(en) / Beteiligte

• Jiang, Zhu
• Niu, Tengfei
• Lv, Xueqin
• Liu, Yanfeng
• Li, Jianghua
• Lu, Wei
• Du, Guocheng
• Chen, Jian
• Liu, Long
• Zhou, Ning-Yi

Titel

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

Ist Teil von

• Applied and environmental microbiology, 2019-09, Vol.85 (17)

Ort / Verlag

United States: American Society for Microbiology

Erscheinungsjahr

2019

Link zum Volltext

Quelle

Alma/SFX Local Collection

Beschreibungen/Notizen

• 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 and perform molecular engineering to improve catalytic performance. First, we screened 12 signal peptides for diacetylchitobiose deacetylase secretion in , 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 decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the increased from 5.11 μM s in the wild type to 7.56 μM s in the mutant R157T. We successfully achieved efficient secretory production and improved the catalytic efficiency of diacetylchitobiose deacetylase in , and this provides a good foundation for the application of diacetylchitobiose deacetylase in the production of chitosan oligosaccharides and monosaccharides.