里氏木霉几丁质酶表达及其水解产物组成与结构分析  

Expression of Chitinase from Trichoderma reesei and Analysis the Composition and Structure of its Hydrolysates

作　　者：焦思明 程功[1] 张毓宸 冯翠 任立世 李建军[1] 杜昱光[1] JIAO Si-ming;CHENG Gong;ZHANG Yu-chen;FENG Cui;REN Li-shi;LI Jian-jun;DU Yu-guang(State Key Laboratory of Biochemical Engineering,Institute of Process Engineering,Chinese Academy of Sciences,Beijing 100190,China)

机构地区：[1]中国科学院过程工程研究所生化工程国家重点实验室

出　　处：《中国生物工程杂志》2018年第10期137-144,共8页China Biotechnology

基　　金：国家重点研发计划(2017YFD0200902);国家自然科学基金联合基金(U1608255);中科院重点部署(KFZD-SW-218)资助项目

摘　　要：优化并全合成里氏木霉几丁质酶基因,在毕赤酵母中实现分泌表达。产物几丁质酶的蛋白浓度达0. 17mg/ml,最适pH为5. 6,最适温度为65℃,酶活为0. 52U/ml。该酶在50℃及以下较稳定。利用该酶水解低脱乙酰度壳聚糖并对产物的组成及结构进行分析。超高效液相色谱-四极杆飞行时间质谱（ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry,UPLC-QTOF MS）检测及分析结果显示,酶解产物中包含至少41种聚合度2-18,不同脱乙酰度的壳寡糖组分;核磁共振（nuclear magnetic resonance,NMR）检测及分析结果显示,产物壳寡糖的还原端主要为N-乙酰氨基葡萄糖,非还原端则同时含有N-乙酰氨基葡萄糖及氨基葡萄糖。相关结果可为壳寡糖的结构与功能关系研究提供参考。Chitinase encoding gene of Trichoderma reesei was optimized,synthesized and secretorily expressed in Pichia pastoris. The protein concentration of the expressed chitinase reached 0. 17 mg/ml. The optimum pH and temperature of the chitinase was 5. 6 and 65℃,respectively,and enzymatic activity reached0. 52 U/ml. The chitinase was continuously thermostable at 50℃. The low deacetylated chitosan was hydrolyzed by this enzyme and the composition and structure of these hydrolysates were analyzed. Ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry（ UPLC-QTOF MS） results showed that these hydrolysates contained at least 41 different kinds of chitooligosaccharides with degree of polymerization of 2 - 18 and different degree of deacetylation. Nuclear magnetic resonance（ NMR） results indicated that,the reducing end of these oligosaccharide fractions were mainly composed of N-Acetylglucosamine while both glucosamine and N-Acetylglucosamine were found in the non-reducing end. These results would provide a reference for study of the relationship between the structure and function of chitooligosaccharides.

关 键 词：里氏木霉 几丁质酶 毕赤酵母 壳寡糖 UPLC-QTOF MS NMR 

分 类 号：Q55[生物学—生物化学]