Chitosanase plays an important role in enzymatic production of chitosan oligosaccharides (COSs). The present study describes the gene cloning and high-level expression of a high-efficiency chitosanase from Bacillus mojavensis SY1 (CsnBm). The gene encoding CsnBm was obtained by homologous cloning, ligated to pPICZαA, and transformed into Pichia pastoris X33. A recombinant strain designated X33-C3 with the highest activity was isolated from 120 recombinant colonies. The maximum activity and total protein concentration of recombinant strain X33-C3 were 6,052 U/ml and 3.75 g/l, respectively, which were obtained in fed-batch cultivation in a 50-l bioreactor. The optimal temperature and pH of purified CsnBm were 55°C and 5.5, respectively. Meanwhile, CsnBm was stable from pH 4.0 to 9.0 and 40 to 55°C. The purified CsnBm exhibited high activity toward colloidal chitosan with degrees of deacetylation from 85 to 95%. Furthermore, CsnBm exhibited high efficiency to hydrolyze different concentration of colloidal chitosan to produce COSs. The result of this study not only identifies a high-efficiency chitosanase for preparation of COSs, but also casts some insight into the high-level production of chitosanase in heterologous systems.

Chitosan oligosaccharides (COSs) are widely applied in many areas due to its various biological activities. Controllable preparation of COSs with desired degree of polymerization (DP) via suitable chitosanase is of great value. Herein, a novel glycoside hydrolase (GH) family 46 chitosanase (SlCsn46) from marine Streptomyces lydicus S1 was prepared, characterized and used to controllably produce COSs with different DP. The specific activity of purified recombinant SlCsn46 was 1,008.5 U/mg. The optimal temperature and pH of purified SlCsn46 were 50°C and 6.0, respectively. Metal ions Mn2+ could improve the stability of SlCsn46. Additionally, SlCsn46 can efficiently hydrolyze 2% and 4% colloidal chitosan to prepare COSs with DP 2–4, 2–5, and 2–6 by adjusting the amount of SlCsn46 added. Moreover, COSs with DP 2–4, 2–5, and 2–6 exhibited potential application value for prolonging the shelf-life of pre-packaged Tofu. The water-holding capacity (WHC), sensorial properties, total viable count (TVC), pH and total volatile base nitrogen (TVB-N) of pre-packed tofu incorporated with 4 mg/mL COSs with DP 2–4, 2–5, and 2–6 were better than those of the control during 15 days of storage at 10°C. Thus, the controllable hydrolysis strategy provides an effective method to prepare COSs with desired DP and its potential application on preservation of pre-packed tofu.