山羊溶酶体α-甘露糖苷酶不同结构域片段的制备及酶活性的鉴定  

Expression and characterization of different domain fragments from Capra hircus lysosomalα-mannosidase

作　　者：赵一宇 吴莹[1] 赵影 赖毕 李勤凡[1] ZHAO Yiyu;WU Ying;ZHAO Ying;LAI Bi;LI Qinfan(Biotorin Laboratory,College of Veterinary Medicine,Northwest A&F University,Yangling,Shaanxi 712100,China)

机构地区：[1]西北农林科技大学动物医学学院生物毒素实验室,陕西杨凌712100

出　　处：《中国兽医学报》2022年第12期2495-2499,共5页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(31860724)。

摘　　要：为分析溶酶体α-甘露糖苷酶(lysosomalα-mannosidase,LAM)亚基结构与酶活性的关系,分别截短、扩增含有山羊溶酶体α-甘露糖苷酶(Capra hircas LAM,ChLAM)高度保守活性位点的亚基片段ChA、ChC、ChD和ChT,将其作为目的基因构建酵母表达载体pPIC9K-ChA、pPIC9K-ChC、pPIC9K-ChD和pPIC9K-ChT,线性化后电转至酵母GS115感受态细胞,以1%甲醇诱导表达,SDS-PAGE和Western blot检测蛋白表达,并对表达产物进行LAM活性检测。结果显示,毕赤酵母中成功表达了有活性的ChLAMt蛋白,相对分子质量约为90 kDa,活性为35 U;而ChA、ChC、ChD表达产物不具有酶的活性。ChLAMt酶活性最适温度为45~55℃,最适pH5.5,Zn^(2+)和Ca^(2+)对ChLAMt酶活性有促进作用,ChLAMt蛋白与野生型全长编码的ChLAM蛋白有相似的酶活特性,说明A、C、D这3个亚基上高度保守的活性位点共同参与酶的催化反应,为ChLAM的研究提供了资料。In order to analyze the relationship between subunit structure and enzyme activity of lysosomalα-mannosidase(LAM),truncated and amplified ChA,ChC,ChD and ChT respectively which containing high conserved active sites of Capra hircas LAM(chLAM).The constructed plasmids pPIC9 K-ChA,pPIC9 K-ChC,pPIC9 K-ChD and pPIC9 K-ChT were linearized,then transformed into the Pichia yeast GS115,the expressed proteins were induced under 1%methanol and detected by SDS-PAGE and Western blot,detecting the LAM activity of the expressed proteins.The results showed that the protein of ChLAMt protein was about 90 kDa,and the activity of expressed product was 35 U.Whereas,there were no activity in ChA,ChC and ChD expressed proteins.The optimum catalytic temperature of ChLAMt proteins was 45-55℃,and the optimal catalytic pH was 5.5,Zn^(2+)and Ca^(2+)could promote the activity of ChLAMt proteins,indicating that the high conserved active sites have to interact with each other to participate in the catalytic reaction of the enzyme.This study provides data for ChLAM research.

关 键 词：山羊溶酶体α-甘露糖苷酶 亚基片段 毕赤酵母 苦马豆素 

分 类 号：S852.2[农业科学—基础兽医学]