来源于Exophiala aquamarina的新型玉米赤霉烯酮水解酶的性质及底物结合中心关键氨基酸的功能研究  

Characterization and Function of Key Amino Acids in Substrate Bingding Center of a Novel Zearalenone Hydrolase from Exophiala aquamarina

作　　者：梁爱玲 刘文婷 武攀 李倩[2] 高健 张洁[2] 刘卫东[2] 贾士儒[1] 郑迎迎 LIANG Ai-ling;LIU Wen-ting;WU Pan;LI QianGAO Jian;ZHANG Jie;LIU Wei-dong;JIA Shi-ru;ZHENG Ying-ying(Key Lab of Industrial Microbiology,College of Biotechnology,Tianjin University of Science&Technology,Tianjin 300457,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China)

机构地区：[1]天津科技大学生物工程学院工业微生物教育部重点实验室,天津300457 [2]中国科学院天津工业生物技术研究所,天津300308

出　　处：《中国生物工程杂志》2021年第10期19-27,共9页China Biotechnology

基　　金：国家重点研发计划(2019YFA0905100、2018YFA0901201);天津市科学基金(18JCYBJC24300);天津市合成生物技术创新能力提升行动(TSBICIP-KJGG-002-06、TSBICIP-KJGG-001)资助项目。

摘　　要：目的:开发一种对α-玉米赤霉烯醇(α-zearalenol,α-ZOL)催化活性高的水解酶,以促进玉米赤霉烯酮的酶法完全脱毒,推动玉米赤霉烯酮降解酶在饲料业和畜牧业的应用。方法:在NCBI数据库中通过基因挖掘获得一个来源于Exophiala aquamarina CBS 119918的新型玉米赤霉烯酮水解酶EaZHD,基因全长792 bp。构建重组质粒pET46-Eazhd,并在大肠杆菌中成功表达。经NiNTA亲和层析和DEAE阴离子交换柱纯化后,利用HPLC测定残余底物浓度的方法来表征其酶学性质。结果:EaZHD对玉米赤霉烯酮(zearalenone,ZEN)的酶活力为0.764 U/mg,而对α-ZOL的酶活力为1.529 U/mg,是对底物ZEN降解活力的2倍。Ea ZHD在p H 8.6和温度40℃条件下的活性最高,并具有较好的热稳定性和碱性条件下较高的p H稳定性。通过对Ea ZHD活性中心附近的氨基酸进行突变和活性分析,确定了对催化活性起关键作用的三个氨基酸,以及对底物特异性有影响的两个关键氨基酸。结论:为推动饲料业和畜牧业中玉米赤霉烯酮的酶法脱毒奠定了基础。Objective:In order to promote the enzymatic complete detoxification of zearalenone and the application in feed industry,we proposed to develop a new hydrolase exhibiting higher activity toα-ZOL.Methods:A novel zearalenone hydrolase EaZHD sequence from Exophiala aquamarina CBS 119918 with a total length of 263 amino acids was obtained.The recombinant plasmid p ET46-Eazhd was constructed and expressed in E.coli.Then EaZHD was purified with Ni-NTA affinity chromatography and DEAE ion exchange column.The enzymatic properties and activity analysis were evaluated with HPLC.Results:The results showed that the activity of EaZHD was 0.764 U/mg against ZEN and 1.529 U/mg againstα-ZOL,which is 2-fold higher.The optimal pH of EaZHD was 8.6 and the optimal temperature was 40°C.It had a better stability at alkaline p H.The thermal stability is better than ZHD101.Three amino acid residues around active site were identified to play a key role in the catalysis activity and the other two residues were found to affect the substrate specificity.Conclusion:The study provides the basis for the enzymatic detoxification of zearalenone in the feed and stock raising industries.

关 键 词：新型玉米赤霉烯酮水解酶 α-玉米赤霉烯醇 酶学性质 酶法脱毒 

分 类 号：Q819[生物学—生物工程]