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

Abstract

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 Km decreased from 7.04 mM in the wild type to 5.19 mM in the mutant R157T, and the Vmax 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.

Chitin deacetylase: from molecular structure to practical applications

“…Additionally, the cell controlling system of Gram-positive bacteria can avoid protein misfolding and the formation of inclusion bodies [57]. Hence, to further improve the soluble expression level of PhDac for the catalytic production of GlcN, Jiang et al [40] heterologously expressed PhDac in B. subtilis WB600, whose six extracellular proteases were deleted from the chromosome of B. subtilis 168. With the optimized signal peptide and a stronger promoter, they achieved a 3112.2 U/mg extracellular expression of PhDac.…”

“…First, the low expression level of CDAs and purification difficulties of CODs are the primary limiting factors. Many studies have tried to improve the heterogeneous expression of CDAs by Escherichia coli, Bacillus subtilis, Pichia pastoris or other hosts [39][40][41]. On the other hand, considering the cost and environmental problems, softer reacting conditions are required in industrial production, which has distance with the reaction characteristics of CDAs [38].…”

“…Therefore, CDAs are necessary to be modified by protein engineering to meet the industrial requirements. Currently, researches on constructing expression systems and protein engineering of CDAs are still at the beginning stage and develop rapidly [37,38,40]. However, the work about these two fields has not been covered in the previous reviews.…”

Random mutagenesis by insertion of error-prone PCR products to the chromosome of Bacillus subtilis

“…However, proteins are usually expressed in cytoplasm in E. coli , making the screening of library more difficult if the substrate of the protein cannot be transported into cell. Therefore, the organisms such asBacillus subtilis and Pichia pastoris that can secret proteins into medium have been developed as alternative hosts for library generation (Jiang et al 2019;Liu et al 2017;Reetz et al 2007;Wang et al 2012).…”

Random Mutagenesis by Insertion of Error-Prone PCR Products to the Chromosome of Bacillus subtilis

“…However, proteins are usually expressed in cytoplasm in E. coli, making the screening of the library more difficult if the substrate of the protein cannot be transported into the cell. Therefore, the organisms such as Bacillus subtilis and Pichia pastoris that can secret proteins into medium have been developed as alternative hosts for library generation (Reetz and Carballeira, 2007;Wang et al, 2012;Liu et al, 2017;Jiang et al, 2019).…”