应用易错PCR定向进化甘油脱氢酶  

Directed Evolution of Glycerol Dehydrogenase by Error Prone PCR

作　　者：李梓君[1] 方柏山[1] 杨仲丽[1] 刘嘉[1] 

机构地区：[1]华侨大学工业生物技术福建省高等学校重点实验室,福建泉州362021

出　　处：《华侨大学学报（自然科学版）》2010年第6期661-666,共6页Journal of Huaqiao University(Natural Science)

基　　金：国家重点基础研究发展(973)计划项目(2006AA020103);国家自然科学基金资助项目(20676048)

摘　　要：经过连续易错聚合酶链式反应(Error-Prone PCR)向克雷伯杆菌(Klebsiella pneumoniae)甘油脱氢酶基因中引入突变,并构建突变文库.依据突变体催化番红O显色的特性,采用琼脂平板法和96微孔板法相结合的高通量筛选方法,成功获得突变体gldA-74,其酶活力是亲本重组酶的2.73倍.通过软件对突变体gldA-74酶基因和亲本重组酶基因进行分析对比,发现突变体gldA-74酶基因发生了一处点突变(A964T),该突变使一处氨基酸被取代.将亲本重组酶和进化酶的高效表达产物经Ni柱纯化后,酶学性质的测定结果表明,甘油的Km值由0.58 mmol.L-1升高至0.63 mmol.L-1,而NAD的Km值由0.74 mmol.L-1升高至0.77mmol.L-1,并且酶的最适pH值由原来的11.5升高至12.5,而最适温度由65℃降至55℃.The GDH enzyme gene from Klebsiella pneumoniae was repeatedly amplified by two sequential error-prone polymerase chain reaction（PCR）,and the gene library was built.The mutant can catalyze safranin O to show colors,and on the base of this property,the gldA-74 mutant was obtained by high throughput screening method using active agar plates in combination with 96-well microplates.The activity of the gldA-74 mutant was showed 2.73 fold of that of the parent recombinase.Gene analysis of the gldA-74 mutant indicated that the mutant enzyme had a point mutation（A964T） which resulted in an amino acid being replaced.Then,the highly expressed productions of recombinase GDH and evolved GDH were purified by Ni-NTA and the enzymatic properties were determined.The results showed that the Km of GDH increased from 0.58 mmol·L-1 to 0.63 mmol·L-1,and the Km of NAD increased as well,from 0.71 mmol·L-1 to 0.77 mmol·L-1.The optimum pH of mutant enzyme also increased from 11.5 to 12.5 while the optimum temperature decreased from 65 ℃ to 55 ℃.

关 键 词：甘油脱氢酶 克雷伯杆菌 定向进化 易错聚合酶链式反应 

分 类 号：Q554.903[生物学—生物化学]