摘要
内酯水解酶ZHD101对玉米赤霉烯酮降解效果显著,是目前研究最广泛的玉米赤霉烯酮降解酶,但由于热稳定性较低,无法满足饲料生产制粒过程中的温度要求。作者利用理性设计和分子改造在其分子中引入二硫键,以提高ZHD101的热稳定性。通过在分子引入七对半胱氨酸双突变(A110C/P196C、S136C/R189C、D143C/P181C、S147C/P181C、D199C/A202C、L200C/A231C、R204C/G205C)以形成潜在的二硫键,研究其对热稳定性的影响。结果表明,突变体S136C/R189C和D143C/P181C在50℃加热处理2 min后的残余活性高于野生型,其中突变体D143C/P181C的残余活性是野生型的2倍,且室温下活力损失小于10%。在此基础上设计四突变(S136C/D143C/P181C/R189C),热稳定性和活力并不优于双突变D143C/P181C。本研究结果为提高ZHD101在饲料工业上的应用潜力打下了基础。
Lactonase ZHD101 which can degrade ZEN effectively is being widely studied.However,its application in feed industry was limited by low thermostability.In this study,we attempted to improve the thermostability of ZHD101 through structure-based rational design and molecular engineering of disulfide bridges.Specifically,7 mutants(A110C/P196C,S136C/R189C,D143C/P181C,S147C/P181C,D199C/A202C,L200C/A231C,and R204C/G205C)were constructed for disulfide bridge formation.Two mutants,S136C/R189C and D143C/P181C,showed enhanced thermostability.Notably,D143C/P181C mutant,which exhibits similar specific activity as wild type enzyme,showed two-fold increase in thermostability after heated at 50℃for 2 min.A quadruplet mutant(S136C/D143C/P181C/R189C)was subsequently constructed and examined,but it showed lower thermostability comparing to D143C/P181C mutant.This study provides significant benefit for the application of ZHD101 in further commercial utilizations.
作者
许中霞
刘桂智
刘卫东
郭瑞庭
李华钟
郑迎迎
XU Zhongxia;LIU Guizhi;LIU Weidong;GUO Ruiting;LI Huazhong;ZHENG Yingying(Key Laboratory of Industrial Biotechnology,Ministry of Education,Jiangnan University,Wuxi 214122,China;Tianjin Institute of Industrial Biotechnology,Chinese Academy of Sciences,Tianjin 300308,China)
出处
《食品与生物技术学报》
CAS
CSCD
北大核心
2019年第7期71-77,共7页
Journal of Food Science and Biotechnology
基金
国家自然科学基金项目（31400678）
国家863计划项目（2012AA022209）
江苏省优势学科建设工程资助项目
