关键词: 枯草芽孢杆菌, 壳聚糖酶, 高效表达, 酶学性质, 壳寡糖

Abstract: In this study, the gene encoding chitosanase (BsCsn46) was cloned from Bacillus subtilis and expressed at high level in Pichia pastoris GS115. The enzymatic and hydrolytic properties of BsCsn46 were further investigated. The extracellular chitosanase activity and protein concentration was up to 50 370 U/mL and 15.7 mg/mL after the high cell-density fermentation of the recombinant P. pastoris in a 5 L fermentor, respectively. BsCsn46 was purified by ion-exchange chromatography using Q-Sepharose Fast Flow. The purified enzyme had a high specific activity of 4 065.7 U/mg towards chitosan and showed maximal activity at pH 6.0 and 55 ℃, and it was stable at temperature below 45 ℃. The hydrolysis of 3 g/100 mL chitosan by BsCsn46 yielded predominantly chitooligosaccharides with degree of polymerization (DP) 2–4. The degree of hydrolysis reached 92.8% and the yield of chitooligosaccharides was up to 90.9%. The high enzyme production level and the remarkable hydrolysis efficiency make BsCsn46 a promising candidate for application in the industrial preparation of chitosanase and chitooligosaccharides.

Key words: Bacillus subtilis, chitosanase, high-level expression, enzymatic properties, chitooligosaccharides