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.
作者
梁爱玲
刘文婷
武攀
李倩
高健
张洁
刘卫东
贾士儒
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)
出处
《中国生物工程杂志》
CAS
CSCD
北大核心
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。
