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Chitosan oligosaccharides are depolymerization products of chitosan, which have a wide range of functional properties such as antimicrobial and antitumor effects.1) Chitooligosaccharides can be produced by chemical and enzymatic methods. The enzymatic production of chitooligosaccharides has become popular because chemical synthesis such as acid hydrolysis typically results in a large quantity of short-chain oligosaccharides and low yields.2,3) The enzymatic degradation of chitosan is generally conducted with chitosanase (EC 3.2.1.132), which catalyzes the endohydrolysis of β-1,4-linkages between D-glucosamine residues in a partially deacetylated chitosan.This study aims to optimize the production of chitosanase from Bacillus subtilis RKY3 through statistical experimental tools. The parameters significantly influencing both chitosanase production and cell growth were identified through Plackett-Burman design, by which maltose, beef extract, MgSO4, and incubation time were selected as the most significant variables. The optimum levels of the screened variables and their mutual interactions were determined through the steepest ascent method and
Box-Behnken design. Under the optimized conditions, 62.30 U/mL of chitosanase activity was predicted, which was in good accordance with the validation experiments, The optimized conditions resulted in a maximum chitosanase activity of 63.53±1.22 U/mL. This result is 11.3-fold increased chitosanase activity compared to that before optimization.