摘要
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.
作者
程功
焦思明
冯翠
贾培媛
任立世
杜昱光
CHENG Gong;JIAO Siming;FENG Cui;JIA Peiyuan;REN Lishi;DU Yuguang(State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences,Beijing 100190, China)
出处
《食品科学》
EI
CAS
CSCD
北大核心
2019年第8期73-78,共6页
Food Science
基金
"十三五"国家重点研发计划重点专项(2016YFB0301502)
国家自然科学基金联合基金项目(U1608255)
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
Bacillus amyloliquefaciens
chitosanase
Pichia pastoris
chitooligosaccharides
matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF MS)
nuclear magnetic resonance (NMR)