不同酿酒酵母菌株来源异麦芽糖酶IMA1的克隆、表达及表征

Cloning, overexpression and characterization of isomaltases IMA1 from different strains of Saccharomyces cerevisiae

摘要:

[目的]异麦芽糖酶IMA1在充分利用含有α-1，6-O-糖苷键的低聚糖中起着关键作用。[方法]在本研究中，对来自4株酿酒酵母菌株（包括3株嗜酸性菌株）来源的异麦芽糖酶IMA1进行克隆、表达、纯化和表征。[结果]研究发现，4种异麦芽糖酶IMA1表现出类似的pH和温度依赖性，但表现出不同的动力学参数和热稳定性。IMA1-A对α-MG（α-甲基葡糖苷）表现出最高的结合亲和力、转换数、催化效率和热稳定性。结构和序列分析表明，2个远离活性位点和底物结合位点的氨基酸的差异对异麦芽糖酶IMA1的动力学参数和热稳定性有重要影响。[结论]本研究结果对进一步研究异麦芽糖酶IMA1的结构-功能关系奠定了基础。

关键词:异麦芽糖酶IMA1;酿酒酵母;动力学参数;pH值-活性曲线;热稳定性

Abstract:

[Objective] Isomaltases IMA1 play key roles in full utilization of oligosaccharides containing α-1,6-O-glucosidic bonds. [Methods] We cloned, overexpressed, purified and characterized four isomaltases IMA1 from four strains of S. cerevisiae including three acidophilic ones. [Results] They showed similar pH and temperature dependence, but different kinetic parameters and thermostability. IMA1-A exhibited the highest binding affinity for α-MG (α-methylglucoside), turnover number, catalytic efficiency, and thermostability. Structure and sequence analysis revealed that even variation in two remote amino acids from the active residues and the substrate binding site could also lead to significantly different kinetic behavior and thermostability of isomaltases IMA1. [Conclusion] Our results will be useful for further investigation into the structure-function relationship of isomaltases IMA1.

Key words:isomaltase IMA1;Saccharomyces cerevisiae;kinetic parameters;pH-rate profile;thermostability