摘要
为改造黑曲霉来源的β-甘露聚糖酶基因Man26A,获得耐温性重组β-甘露聚糖酶,本试验通过易错PCR技术(在PCR体系中加入不同水平的MnCl_(2)),将目的基因Man26A克隆至载体pGAPZαA上,构建重组质粒pGAPZαA-Man26A,将重组质粒电转化至毕赤酵母GS115中,在含有100μg/mL博来霉素的YPDS平板上筛选耐温性突变体,并测定重组酶的酶学性质。本试验筛选出2株产耐温性β-甘露聚糖酶的重组突变体3-228和4-59,两重组酶的最适pH和温度分别为5.0和45℃,均与野生型酶相同,但与野生型相比,两重组酶的工作温度范围更加宽泛,且温度稳定性显著提高;金属离子Fe^(2+)、Mg^(2+)、Mn^(2+)、K+、Ca^(2+)、Co^(2+)对β-甘露聚糖酶活力有不同程度的激活作用,SDS、EDTA、Cd^(2+)、Cu^(2+)、Ni^(2+)、Ag^(2+)、Fe^(3+)、Pb^(2+)等对酶活力有不同的抑制作用。2个耐温性重组β-甘露聚糖酶均具有工作温度范围广、耐温性强等特点,提高了其在饲料工业上的应用潜力,证明了易错PCR技术是提高酶温度稳定性的有效方法。
In order to modlify the β-mannanase gene Man26A derived from Aspergillus niger,and obtain temperature resistance recombinantβ-mannanase,using error-prone PCR technology(adding different levels of MnCl_(2) to the PCR system),the target gene Man26A was cloned into the vector plasmid pGAPZαA,and a recombinant plasmid pGAPZαA-Man26A expressingβ-mannanase was constructed.The recombinant plasmid was electroporated into Pichia pastoris GS115,the temperature resistance mutants were screened on YPDS plates containing 100μg/mL zeocin,and the enzymatic properties of the recombinant enzymes were determined.Two recombinant mutants 3-228 and 4-59 producing temperature resistanceβ-mannanase were screened in this experiment.The optimal pH and temperature of the two enzymes were the same as the wild type,which was 5.0 and 45℃respectively.However,compared with the wild type,the working temperature range of the two enzymes were wider,and their thermal stability were significantly improved;Metal ions Fe^(2+),Mg^(2+),Mn^(2+),K+,Ca^(2+),Co^(2+)could activateβ-mannanase activities with varying degrees,while SDS,EDTA,Cd^(2+),Cu^(2+),Ni^(2+),Ag^(2+),Fe^(3+),Pb^(2+)had different inhibitory effects on the enzymes activities.Two temperature resistant recombinantβ-mannanases were obtained in this experiment,both of which had the characteristics of wide working temperature range and strong temperature resistance compared with the wild type enzyme,which improved the potential application ofβ-mannanase in the feed industry,and proving that error-prone PCR technology was an effective method to improve enzyme temperature stability.
作者
陈晓飞
李珊珊
周伏忠
刘德海
王佰涛
刁文涛
CHEN Xiaofei;LI Shanshan;ZHOU Fuzhong;LIU Dehai;WANG Baitao;DIAO Wentao(Institute of Biology Co.,Ltd.,Henan Academy of Science,Zhengzhou,Henan Province 450008,China;Key Laboratory of Microbial Engineering of Henan Province,Zhengzhou,Henan Province 450008,China)
出处
《中国饲料》
北大核心
2025年第17期53-60,共8页
China Feed
基金
河南省科技厅联合基金重点项目(225200810041)
河南省科学院功能微生物绿色转化创新团队(20230114)
河南省微生物资源利用院士工作站建设提升项目(241005024)
河南省科技开发联合基金项目(225200810109)。
关键词
Β-甘露聚糖酶
易错PCR
耐温性
酶学性质
β-mannanase
error-prone PCR
temperature resistance
enzymatic property
