Enzymatic production of diverse N-acetyl chitooligosaccharides employing a novel bifunctional chitinase and its engineered variants

Bioproduction of diverse N -acetyl chitooligosaccharides from chitin is of great value. In the study, a novel GH family 18 bifunctional chitinase gene ( PsChi82 ) from Paenibacillus shirakamiensis was identified, expressed and biochemically characterized. PsChi82 was most active at pH 5.0, and 55 °C, and displayed remarkable pH stability with the broad pH range of 3.0–12.0. It showed high chitosanase activity of 10.6 U mg −1 and diverse hydrolysis products of GlcNAc, (GlcNAc) 2 , GlcN-GlcNAc and (GlcN) 2 -GlcNAc, which may facilitate comprehensively understanding of structure-function relationships of N -acetyl COSs. Three engineered variants were then expressed and characterized. Among them, PsChi82-CBM26 possessed specific activity of 25.1 U mg −1 against colloidal chitin, which was 2.1 folds higher than that of PsChi82. The diverse N -acetyl COSs were subsequently produced by PsChi82-CBM26 with a sugar content of 23.2 g L −1 . These excellent properties may make PsChi82-CBM26 potentially useful for N -acetyl COSs production in the food and chemical industries. • A novel bifunctional chitinase gene ( PsChi82 ) was successfully expressed in E. coli . • The great pH stability and high chitosanase activity of PsChi82 were determined. • PsChi82 showed diverse hydrolysis products including two types of paCOSs. • Three engineered variants were designed, and PsChi82-CBM26 possessed high activity. • An efficient bioprocess for diverse N -acetyl COSs production was developed.