枯草芽孢杆菌壳聚糖酶在毕赤酵母中的高效表达及其酶解特性

研究枯草芽孢杆菌壳聚糖酶基因（BsCsn46）在巴斯德毕赤酵母（Pichia pastoris）GS115中的高效表达、重组酶性质及其酶解特性。重组菌在5 L发酵罐高密度发酵后胞外酶活力高达50 370 U/mL，蛋白质量浓度15.7 mg/mL。粗酶经强阴离子交换层析纯化，纯酶比活力为4 065.7 U/mg，最适pH 6.0，最适温度55 ℃，在45 ℃以下保持稳定。该酶水解3 g/100 mL壳聚糖得到主产物为二糖、三糖和四糖的壳寡糖，水解率为92.8%，壳寡糖得率为90.9%。本研究的重组壳聚糖酶产酶水平和水解效率高，为工业化制备壳聚糖酶及大规模制备壳寡糖的应用提供了理论支持。

High-Level Expression of Bacillus subtilis Chitosanase in Pichia pastoris and Its Hydrolytic Properties

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.