嗜热革节孢GH1 BGL1进口端位点对酶活性及葡萄糖耐受性的影响  

The Substrate Entrance Residues of GH1 BGL1 from Scytalidium thermophilium Affect Enzyme Activity and Glucose Tolerance

作　　者：刘玉姣 高金鹏 刘娇娇 李多川[1] LIU Yu-jiao, GAO Jin-peng, LIU Jiao-jiao, LI Duo-chuan(Department of Fungus, College of Plant Protection, Shandong Agricultural University, 271018, Chin)

机构地区：[1]山东农业大学植物保护学院菌物学系,泰安271018

出　　处：《菌物研究》2018年第1期36-42,共7页Journal of Fungal Research

基　　金：国家863计划课题(2012AA10180402);国家科技支撑计划课题(2015BAD15B05);国家自然科学基金项目(31571949)

摘　　要：糖苷水解酶第一家族(GH1)β-葡萄糖苷酶(BGL1)有葡萄糖耐受性,进口端位点对酶活性及葡萄糖耐受性有很大影响,但具体作用机制尚不清楚。对嗜热革节孢GH1 BGL1进口端的W168、L173、F348、W349、C169、F180、D237、Y179、A260、H307、N335和E437这12个氨基酸残基进行定点突变,将突变酶与野生酶(WT)在毕赤酵母中表达,表达产物纯化后进行酶活性和葡萄糖耐受性测定。与WT相比,所有突变酶活性均有所降低,其中W168H、N335F和W349G几乎丧失活性。突变F180H、D237S、A260N和H307Y的Km低于WT,所有突变的kcat都降低。除L173Q外,其余突变都保持葡萄糖耐受性,在高浓度(400 mmol/L)葡萄糖时,Y179F和D237S酶活受到显著抑制。本研究表明,进口端位点对酶活性及葡萄糖耐受性均具有一定影响,催化活性通道的结构特异性可能是葡萄糖耐受机制。Some β-glucosidase belonging to the GH1 family exhibit glucose tolerance. However,the molecular mechanism of glucose tolerance still keeps elusive. In this study,12 amino acid residues of W168,L173,F348,W349,C169,F180,D237,Y179,A260,H307,N335 and E437 in the substrate entrance region were mutated and expressed in Pichia pastoris. All mutants showed reduced enzyme activity,compared to wild type（ WT）. The enzyme activity of W168H,N335F and W349G almost were fully lost. Four mutants,F180H,D237 S,A260N and H307Y,had smaller Kmvalues than that of WT. Except L173 Q, other mutants still maintained glucose tolerance,whereas Y179F and D237S showed strong inhibition at high glucose concentrations（ 400 mmol/L）.This study suggested that the substrate entrance residues can affect enzyme activityand glucose tolerance,and the specificity of catalytic channel structural may be involved in glucose tolerance.

关 键 词：Β-葡萄糖苷酶 定点突变 进口端 葡萄糖耐受性 

分 类 号：Q556.2[生物学—生物化学]