基于宏基因策略的新颖木聚糖酶基因cbxynA克隆及其重组酶酶学性质  

Cloning of a Novel Xylanase Gene cbxynA Based on Macro Gene Strategy and the Characterization of Recombinant Enzyme

作　　者：熊科[1,2] 崔晓亭 王小艺[1,4] 赵峙尧 柳佳芸 裴鹏刚 邓蕾 熊苏玥 Xiong Ke;Cui Xiaoting;Wang Xiaoyi;Zhao Zhiyao;Liu Jiayun;Pei Penggang;Deng Lei;Xiong Suyue(Beijing Engineering and Technology Research Center of Food Additives,Beijing Technology & Business University,Beijing 100048;Beijing Advanced Innovation Center for Food Nutrition and Human Health,Beijing Technology & Business University,Beijing 100048;Beijing Laboratory for Food Quality and Safety,Beijing Technology & Business University,Beijing 100048;Beijing Key Laboratory of Flavor Chemistry,Beijing Technology & Business University,Beijing 100048)

机构地区：[1]北京工商大学北京市食品添加剂工程技术研究中心,北京100048 [2]北京工商大学北京食品营养与人类健康高精尖创新中心,北京100048 [3]北京工商大学北京市质量与安全重点实验室,北京100048 [4]北京工商大学北京市风味化学重点实验室,北京100048

出　　处：《中国食品学报》2019年第9期37-44,共8页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家研发计划项目(2017YFC1600605);北京自然科学基金资助项目(6172003);国家自然科学基金资助项目(31601408)

摘　　要：研究构建了库容量约为1.3×10^4个克隆子且外源片段的总长为30Mb的土壤宏基因组文库。基于功能策略,从文库中筛选到分解木聚糖底物的阳性克隆子。该克隆经序列分析发现一个822bp的潜在的编码木聚糖酶基因(cbxynA)的开放阅读框,其编码的氨基酸序列与来自古细菌属编码的木聚糖酶的相似度最高仅为45%,说明该序列为一条未知的新序列。cbxynA在大肠杆菌中的重组表达产物的分子质量为29ku,与预测结果一致。对其原核表达条件进行优化,结果表明:质粒在培养5h后加入0.7mmol/LIPTG,23℃下诱导25h后表达,木聚糖酶活力为52U/mL,较初始值提高4.3倍。而重组酶(CBXYNA)最适pH和最适温度分别为5.2和55℃。金属离子Na^+、K^+、Li^+、Ca^2+能提高该酶的酶活,而另外一些金属离子如Mg^2+、Mn^2+、Zn^2+、Fe^3+、Al^3+、Cu^2+能抑制其活性。该酶以水溶性玉米芯木聚糖、燕麦木聚糖、水不溶性木聚糖为底物时,其相对酶活力分别为162%,139%,74%。This research construct the soil metagenomic library,which have 1.3×10^4 storage capacity and 30 Mb length of the total gene.Screening the positive clone can hydrolyze xylan from the library based on the functional strategy.Sequence analysis revealed that cbxynA potential encoding xylanase gene with 822 bp length.Only 45% of its amino acid sequence similar with the encoding xylanase from Halobacteriaceae archaeon(Archaea),which indicated that it’s a novel unknown sequence.Molecular weight of cbxynA expressed in the E.coli was 29 ku consistented with predicted size.At the same time,the prokaryotic expression conditions were optimized.The results showed that the plasmid cultivated for 5 h,then added 0.7 mmol/L IPTG at 23 ℃ inducing for 25 h,and the xylanase activity was 52 U/mL.The optimum pH and temperature of the recombinant enzyme CBXYNA were 5.2 and 55 ℃,respectively.The metal ions of Na^+,K^+,Li^+ and Ca^2+ can enhance the enzyme activity.While Mg^2+,Mn^2+,Zn^2+,Fe^3+,Al^3+ and Cu^2+ can inhibit the activity of the enzyme.When CBXYNA taken water soluble corncob xylan and oat xylan,water-insoluble corncob xylan as substrate,the relative enzyme activity was 162%,139%,74%,respectively.

关 键 词：土壤微生物 宏基因组文库 木聚糖酶 酶学性质 

分 类 号：Q78[生物学—分子生物学]