番木瓜几丁质酶在毕赤酵母中的表达、表征及水解产物分析  

Expression and characterization of chitinase from Carica papaya in Pichia Pastoris and analysis of its hydrolysates

作　　者：吴丽娜 程功[1] 焦思明 冯翠 李建军[1] 杜昱光[1] WU Lina;CHENG Gong;JIAO Siming;FENG Cui;LI Jianjun;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：《生物加工过程》2021年第6期626-633,共8页Chinese Journal of Bioprocess Engineering

基　　金：国家重点研发计划(2018YFC0311105);国家自然科学基金联合基金(U1608255);中科院重点部署项目(KFZD-SW-218)。

摘　　要：为了研究来源于番木瓜(Carica papaya)的几丁质酶在毕赤酵母中异源表达、表征及水解产物,笔者重新设计了来源于番木瓜的几丁质酶基因序列,命名为cpchi19。将cpchi19构建到毕赤酵母表达载体pGBG1中得到重组菌,通过质粒构建、发酵表达、酶学性质研究及对低脱乙酰度壳聚糖酶解产物进行分析。该几丁质酶在毕赤酵母中表达量为0.18 mg/mL,酶活为0.32 U/mL;最适pH和温度分别为5.6和75℃;该酶在70℃及以下较稳定。超高效液相色谱四极杆飞行时间质谱(UPLC QTOF MS)分析发现,水解主要产物为聚合度2~20、不同脱乙酰度的38个壳寡糖组分。核磁共振(NMR)分析显示,部分乙酰化壳寡糖的还原末端主要为N乙酰氨基葡萄糖,同时含少量氨基葡萄糖,而非还原末端则同时包含N乙酰氨基葡萄糖及氨基葡萄糖。来源于番木瓜的几丁质酶在毕赤酵母中实现表达并成功制备低脱乙酰度壳寡糖,为该酶及其水解产物的应用提供了理论支撑。To study the heterologous expression,characterization and hydrolysis product analysis of chitinase derived from Carica papaya in Pichia pastoris,we redesigned the chitinase gene sequence derived from papaya and named it cpchi19.Recombinant bacteria were constructed by constructing cpchi19 into the P.pastoris expression vector pGBG1,through plasmid construction,fermentation expression,enzyme characterization and analysis of partially acetylated chitooligosaccharide enzymatic hydrolysis products.The expression level of chitinase in recombinant P.pastoris was 0.18 mg/mL,and the enzyme activity was 0.32 U/mL.The optimal pH and temperature of the enzyme were 5.6 and 75℃respectively,and the enzyme was stable below 70℃.Ultra-high performance liquid chromatography-quadrupole time-of-flight mass spectrometry analysis showed that the main products of hydrolysis were 38 chitooligosaccharides with a degree of polymerization of 220 and different degrees of deacetylation.Nuclear magnetic resonance analysis showed that the reducing ends of most partially acetylated chitooligosaccharides was N-acetylglucosamine and those of few was glucosamine,while the non-reducing ends contained both N-acetylglucosamine and glucosamine.We successfully expressed the chitinase derived from Carica papaya and prepared partially acetylated chitooligosaccharide,which laid the foundation for application of this enzyme and its hydrolysates.

关 键 词：毕赤酵母 番木瓜 几丁质酶 壳寡糖 超高效液相色谱四极杆飞行时间质谱 核磁共振 

分 类 号：TS245.9[轻工技术与工程—制糖工程] Q814[轻工技术与工程—食品科学与工程]