黑曲霉β-甘露聚糖酶ManA耐热突变体的筛选  

Screening of thermostable mutants of β-mannanase ManA from Aspergillus niger

作　　者：李珊珊[1,2] 刘德海[1,2] 刁文涛[1,2] 陈晓飞[1,2] 权淑静[1,2] LI Shanshan;LIU Dehai;DIAO Wentao;CHEN Xiaofei;QUAN Shujing(Institute of Biology Co.,Ltd.,Henan Academy of Sciences,Zhengzhou 450008,China;Key Laboratory of Microbial Engineering of Henan Province,Zhengzhou 450008,China)

机构地区：[1]河南省科学院生物研究所有限责任公司,河南郑州450008 [2]河南省微生物工程重点实验室,河南郑州450008

出　　处：《中国酿造》2022年第3期62-68,共7页China Brewing

基　　金：中原科技创新领军人才项目(214200510011);河南省科学院杰青人才培养项目(201405005)。

摘　　要：为了改造黑曲霉(Aspergillus niger)来源的β-甘露聚糖酶ManA,获得耐热性高的突变体。利用易错聚合酶链式反应(PCR)技术对构建的表达质粒pGAPZαA-manA进行随机突变,将突变文库转化至毕赤酵母(Pichia pastoris)GS115,表达筛选耐热性高的突变体,并进一步对突变位点进行定点突变,筛选其他耐热性高的突变体。结果表明,ManA的H283R与H283K突变体相对于野生型在耐热性方面有显著提升,75℃加热30 min其相对酶活由26.66%分别提升至76.95%和83.57%;在pH 3.0~9.0的条件下,50℃保存24 h,H283K突变体与野生型的相对酶活均在90%以上,H283R突变体在pH 3.0时的相对酶活下降到了84.72%,两个突变体的最适温度、pH及比酶活与野生型没有明显差异。说明β-甘露聚糖酶ManA的283位组氨酸(H283)对其耐热性较为关键,将该位点突变为精氨酸(R)和赖氨酸(K)时,ManA的耐热性得到显著提升。ManA, a β-mannanase from Aspergillus niger, was modified to obtain thermostable mutants. The recombinant plasmid pGAPZα-manA was randomly mutated by error-prone polymerase chain reaction(PCR), the mutant library was transformed into Pichia pastoris GS115, and the thermostable mutant was screened. Further site-directed mutation was performed on the mutant site, to screen other thermostable mutants. The results showed that the heat resistant of mutants H283R and H283K of ManA were higher than the wild type, after heated for 30 min at 75 ℃, the relative enzyme activity of H283R and H283K mutants of ManA increased from 26.66% to 76.95% and 83.57%, respectively. Under the condition of pH3.0-9.0 and 50 ℃ for 24 h, the relative enzyme activity of the H283K and wild type retained more than 90%, while the relative enzyme activity of the H283R mutant decreased to 84.72% at pH 3.0. And there were no significant differences between the two mutants and wild type in terms of optimal reaction temperature, optimal reaction pH and specific activity. These results showed that histidine 283(H283) of β-mannanase ManA from A. niger was key to its heat tolerance, when the site was mutated to arginine(R) or lysine(K), the heat tolerance of ManA was significantly improved.

关 键 词：甘露聚糖酶 耐热突变体 易错PCR 定点突变 

分 类 号：Q789[生物学—分子生物学]