摘要
研究旨在改造株链球菌来源的α-半乳糖苷酶基因Gal_(2)7A,获得耐热性高的突变体。在常规PCR体系中加入不同水平的MnCl_(2),将基因克隆至载体pGAPZαA并成功构建表达酶产物的重组质粒pGAPZαA-Gal_(2)7A。将线性化的重组质粒电转至毕赤酵母GS115,在含有100 g/L博来霉素的YPDS平板上筛选耐热性提高的突变体,通过pNPG的方法测定重组酶的酶学性质为最适温度65℃,最适pH值6.5,Ag^(2+)、Hg^(2+)对重组酶的酶学活性具有明显的抑制作用,Cu^(2+)对重组酶的酶活性具有促进作用。研究表明,试验成功构建耐热α-半乳糖苷酶高效表达菌株29和54,在55℃保温10 min与同等条件下的野生型进行对比,加热前后的保留酶活比的增幅大于45%。
The aim of the study was to modifyα-galactosidase gene Gal_(2)7A from Streptococcus,a mutant with high heat tolerance was obtained.Different concentrations of MnCl_(2) were added into conventional PCR system,the gene was cloned into vector pGAPZαA,and the recombinant plasmid pGAPZαA-GAL27A expressing the enzyme product was successfully constructed.The linearized recombinant plasmid was electrically transferred to Pichia pastoris GS115,and the mutant with being improved heat tolerance was screened on YPDS plate containing 100 g/L zeocin.The activity and enzymatic properties of recombinant enzyme were determined by pNPG method.The enzymatic properties showed that the optimal temperature was 65℃,the optimal pH value was 6.5,Ag^(2+),Hg^(2+)had obvious inhibitory effect on enzymatic activity,Cu^(2+)can promote the enzyme activity.The study indicates that the heat-resistantα-galactosidase strains 29 and 54 are successfully constructed,compared with wild-type strains under the same conditions at 55℃for 10 min,the retention-enzyme activity ratio is increased by more than 45%after being heated.
作者
马焕
张秀江
胡虹
冯菲
解复红
向凌云
武艳芳
刁文涛
MA Huan;ZHANG Xiu-jiang;HU Hong;FENG Fei;XIE Fu-hong;XIANG Ling-yun;WU Yan-fang;DIAO Wen-tao
出处
《饲料研究》
CAS
北大核心
2023年第9期80-84,共5页
Feed Research
基金
河南省科学院基本科研业务费项目(项目编号:220605074)
河南省科学院杰出人才培养项目(项目编号:220405001)
河南省科学院基本科研业务费项目(项目编号:220605037、220605100、220605056、220605075)。
