Enzymatic production of chitooligosaccharides has high value in medicine and other fields. However, low chitinase activity and yield of chitooligosaccharides limit the production and application. Herein, we used a series of molecular biology strategies to increase the expression of chitinase in Bacillus subtilis WB600. Upon addition of the signal peptide NprB, Chisb was successfully secreted to the outside of the cell and extracellular expression level reached 35.54 U/mL. Furthermore, optimizing Ribosome Binding Sites (RBSs) with spacer sequences, and combining molecular docking technology with site-directed mutagenesis, the expression level and the specific activity of Chisb was further increased to 51.67 U/mL and 249.62 U/mg, respectively. When colloidal chitin was used as the substrate, the chitooligosaccharides detected by ion chromatography were (GlcNAc)1-5, and the total yield of chitooligosaccharides was 14.4%. Our results indicate that strategies for increasing Chisb expression contribute to the study and application of chitinase and the production of chitooligosaccharides.
Keywords: Bacillus subtilis WB600; Chitinase; Chitooligosaccharides; RBS optimization; Signal peptides; Site-directed mutagenesis.
Copyright © 2019 Elsevier Inc. All rights reserved.