摘要
木聚糖酶是微生物半纤维素降解体系中的一种关键酶,被广泛地应用在工业中的多个领域。本研究为了提高木聚糖酶Umxyn10A (ABL73-883.1)的热稳定性,将Umxyn10A与GH 10家族4种耐热木聚糖酶进行多序列同源比对以及三维结构的同源建模分析,选定了Umxyn10A第31位氨基酸位点进行定点突变,将氨基酸Ala (A)突变为Phe (F)。分别将Umxyn10A和Umxyn10AA31F在大肠杆菌中进行重组表达,分析2种重组酶的酶学特性,结果发现,Umxyn10AA31F的最适反应温度为85℃,较野生重组酶提高了5℃;在65℃下的半衰期为105 min,较野生重组酶(15 min)提高了6倍;在70℃下突变重组酶的半衰期为15 min,较野生重组酶(5 min)提高了2倍;与此同时突变重组酶的pH耐受区间较原酶也有一定的增大。结果表明,将第31位氨基酸位点Ala突变为Phe能够显著的提高Umxyn10A的热稳定性。
Xyla nase is a key enzyme in microbial hemicellulose degradation system, which is widely used in many industrial fields. To improve the thermal stability of the xylanase Umxyn10 A(ABL73-883.1), the mutant enzyme Umxyn10AA31Fwas obtained by replacing 31 site Ala(A) with Phe(F) based on comparison of primary structure of four heat-resistant xylanases in GH 10 family and the homologous modeling of three-dimensional structures. The recombinant expression of Umxyn10A and Umxyn10AA31Fin E. coli and the enzymatic characteristics of the two recombinant enzymes were analyzed. The results show that: Umxyn10AA31Foptimum temperature is 85℃, 5℃ higher than that of the original enzyme;Under the 65℃ has a half-life of 105 min, a wild recombinant enzyme(15 min) raised six times;Mutation the half-life of recombinant enzymes under 70 ℃ for 15 min, a wild recombinant enzyme(5 min) increased two times;At the same time, the pH tolerance range of mutant recombinase was also increased. The results showed that the mutation of the 31 st amino acid site Ala to Phe could significantly improve the thermal stability of Umxyn10A.
作者
邓巧平
王龙
谭曼利
冯家勋
刘君梁
Deng Qiaoping;Wang Long;Tan Manli;Feng Jiaxun;Liu Junliang(State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources,College of Life Science and Technology,Guangxi University,Nanning,530004)
出处
《基因组学与应用生物学》
CAS
CSCD
北大核心
2020年第3期1137-1142,共6页
Genomics and Applied Biology
基金
广西自然科学基金创新研究团队项目(2012GXNSFGA060005)资助。
关键词
木聚糖酶
热稳定性
定点突变
Xylanase
Thermostability
Site-specific mutagenesis
