强烈炽热球菌几丁质酶水解制备低脱乙酰度壳寡糖及其组成与结构分析  

Preparation of Chitooligosaccharides with Low Degree of Deacetylation by Recombinant Chitinase from Pyrococcus furiosus, Its Compositions and Structure Analysis

作　　者：程功[1] 焦思明 任立世 冯翠 康跻耀[1] 杜昱光[1] CHENG Gong;JIAO Siming;REN Lishi;FENG Cui;KANG Jiyao;DU Yuguang(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,北京100190

出　　处：《食品科学》2018年第18期115-119,共5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)

摘　　要：利用全基因合成方法合成了强烈炽热球菌(Pyrococcus furiosus)的几丁质酶编码基因并在大肠杆菌(Escherichia coli)中实现了可溶表达。利用该酶对低脱乙酰度壳聚糖进行水解并对获得的壳寡糖产物进行组成及结构分析。分子排阻高效液相色谱结果显示,水解产物相对分子质量分布范围为1 000~5 000。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2~9、不同脱乙酰度的壳寡糖。核磁共振对酶解产物壳寡糖的结构鉴定结果显示,所有寡糖组分的还原端均主要由两个连续的N-乙酰氨基葡萄糖组成。综上,本研究利用来源于强烈炽热球菌的几丁质酶制备了还原末端结构确定的低脱乙酰度壳寡糖,为复杂结构壳寡糖结构与功能关系研究提供了理论支持。The chitinase-encoding gene of Pyrococcus furiosus was synthesized and the recombinant enzyme solubly expressed in Escherichia coli.Chitosan with low degree of deacetylation was hydrolyzed by this enzyme and the composition and structure of the resulting chitooligosaccharides were analyzed.Size exclusion high performance liquid chromatography(SE-HPLC)results showed that the relative molecular masses of the hydrolysis products ranged from 1 000 to 5 000.Matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF MS)analysis revealed that the hydrolysates contained chitooligosaccharides with a degree of polymerization of 2?9 and different degrees of deacetylation.Structural characterization by nuclear magnetic resonance(NMR)indicated that the reducing end of all oligosaccharide fractions was mainly composed of two successive N-acetylglucosamines.In summary,we have prepared chitooligosaccharides with low degree deacetylation having determined reducing end structure using recombinant chitinase from P.furiosus,which can lay the foundation for the study of the structural-activity relationship of chitooligosaccharides with complex structures.

关 键 词：强烈炽热球菌 几丁质酶 壳寡糖 分子排阻高效液相色谱 基质辅助激光解吸电离飞行时间质谱 核磁共振 

分 类 号：TS245.9[轻工技术与工程—制糖工程]