关键词: 毕赤酵母, 解淀粉芽孢杆菌, 壳聚糖酶, 壳寡糖, 基质辅助激光解吸电离飞行时间质谱, 核磁共振

Abstract: 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 (MALDITOF 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 nonreducing 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.

Key words: Bacillus amyloliquefaciens, chitosanase, Pichia pastoris, chitooligosaccharides, matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS), nuclear magnetic resonance (NMR)