毕赤酵母表达解淀粉芽孢杆菌壳聚糖酶及其水解制备可控结构壳寡糖

Expression of Chitosanase from Bacillus amyloliquefaciens in Pichia pastoris and Its Application in Preparation of Chitooligosaccharides with Controllable Structure

优化并全合成解淀粉芽孢杆菌(Bacillus amyloliquefaciens)壳聚糖酶编码基因并在毕赤酵母(Pichia pastoris)中实现分泌表达,表达产物的蛋白质量浓度达到0.23 mg/mL。壳聚糖水解酶的最适pH值为5.0,最适温度为45℃,比活力达52.2 U/mL。该酶在50℃以下较稳定。利用该酶水解低脱乙酰度壳聚糖并对产物进行了组成及结构分析。基质辅助激光解吸电离飞行时间质谱分析结果显示,酶解产物中包含聚合度3～15、不同脱乙酰度的壳寡糖。核磁共振鉴定结果显示,壳寡糖组分的还原末端及非还原末端均主要由氨基葡萄糖组成。综上,本研究高效表达了来源于解淀粉芽孢杆菌的壳聚糖酶,并制备了确定末端结构的壳寡糖,为壳寡糖的结构与功能关系研究提供理论支持。

The chitosanase-encoding gene of Bacillus amyloliquefaciens was optimized, synthesized and expressed in Pichia pastoris. The protein concentration of the expressed product was 0.23 mg/mL. The optimum pH and temperature of the enzyme were 5.0 and 45 ℃, respectively, and the specific activity reached 52.2 U/mL. The chitosanase was continuously thermo-stable at 50 ℃. Chitosan with low deacetylation was hydrolyzed by this enzyme and the composition and structure of the hydrolysate were analyzed. Matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS) analysis showed that this hydrolysate contained chitooligosaccharides with degree of polymerization of 3-15 and different degrees of deacetylation. Nuclear magnetic resonance (NMR) results indicated that both the reducing and non-reducing ends of oligosaccharide fractions were mainly composed of glucosamine. In summary, we efficiently expressed chitosanase from B. amyloliquefaciens and prepared chitooligosaccharides with determined terminal structures, which will lay a theoretical foundation for the study of the structure-function relationship of chitooligosaccharides.