Enhancement of chitosanase secretion by Bacillus subtilis for production of chitosan oligosaccharides

Chitosan oligosaccharides (COSs) are produced by hydrolysis of chitosan (the deacetylation product of chitin) by chitosanases. COSs are widely used in food, textile, pharmaceutical, and medical applications. The chitosanase (Csn) encoded by the Bacillus subtilis 168 csn gene, which belongs to the glycosyl hydrolase family 46, was recombinantly expressed in B. subtilis PT5. Csn fused with various signal peptides was secreted into culture supernatants and its production evaluated. The highest specific activity achieved was 156 ± 4.68 U/ml using the aprE signal peptide rather than the original csn signal peptide. The optimal temperature and pH for enzyme activity were determined as 47 °C and 5.4, respectively, using central composite design (CCD). Recombinant Csn could efficiently hydrolyze both α and β type chitosan to dimer, trimer, and tetramer COSs. Further, the optimal medium for chitosanase production was predicted and successfully determined using Box-Behnken experimental designs. The maximum Csn protein production level that could be achieved using the optimal fermentation medium (0.76% lactose, 1.63% yeast extract, and 2.31% glutamate) was 208.23 ± 5.19 U/ml in a 5-l fermenter. The present study demonstrates that recombinant B. subtilis Csn has potential for use as a biocatalyst to develop industrial-scale production of COSs.