A strain producing chitinase, isolated from potato stem tissue, was identified as Bacillus licheniformis by biochemical properties and 16S RNA sequence analysis. Statistical experimental designs were used to optimize nine independent variables for chitinase production by B. licheniformis AT6 strain in submerged fermentation. Using Plackett-Burman design, (NH4)2SO4, MgSO4.7H2O, colloidal chitin, MnCl2 2H2O, and temperature were found to influence chitinase production significantly. According to Box-Behnken response surface methodology, the optimal fermentation conditions allowing maximum chitinase production were (in gram per liter): (NH4)2SO4, 7; K2HPO4, 1; NaCl, 1; MgSO4.7H2O, 0.1; yeast extract, 0.5; colloidal chitin, 7.5; MnCl2.2H2O, 0.2; temperature 35 °C; pH medium 7. The optimization strategy led to a 10-fold increase in chitinase activity (505.26 ± 22.223 mU/mL versus 50.35 ± 19.62 mU/mL for control basal medium). A major protein band with a molecular weight of 61.9 kDa corresponding to chitinase activity was clearly detected under optimized conditions. Chitinase activity produced in optimized medium mainly releases N-acetyl glucosamine (GlcNAc) monomer from colloidal chitin. This enzyme also acts as an exochitinase with β-N-acetylglucosaminidase. These results suggest that B. licheniformis AT6 secreting exochitinase is highly efficient in GlcNAc production which could in turn be envisaged as a therapeutic agent or as a conservator against the alteration of several ailments.
Keywords: Bacillus licheniformis; Box–Behnken design; Chitinase; N-acetyl glucosamine; Plackett–Burman design.