具有辅助降解纤维素功能的大斑刚毛座腔菌糖苷水解酶GH61的鉴定、异源表达及功能分析  

Identification,Heterologous Expression and Functional Analysis of a GH61 Family Glycoside Hydrolase from Setosphaeria turcica with the Assisting Function in Degrading Cellulose

作　　者：马玉倩 孙东辉 岳浩峰 辛佳瑜 刘宁[2] 曹志艳[2,3] MA Yu-qian;SUN Dong-hui;YUE Hao-feng;XIN Jia-yu;LIU Ning;CAO Zhi-yan(Collage of Life Science,Hebei Agricultural University,Baoding 071000;College of Plant Protection,Hebei Agricultural University,Baoding 071000;State Key Laboratory of North China Crop Improvement and Regulation,Baoding 071000)

机构地区：[1]河北农业大学生命科学学院,保定071000 [2]河北农业大学植物保护学院,保定071000 [3]华北作物改良与调控国家重点实验室,保定071000

出　　处：《生物技术通报》2023年第4期124-135,共12页Biotechnology Bulletin

基　　金：国家自然科学基金项目(31901827,32072370);河北省自然科学基金项目(C2020204039);国家现代农业产业技术体系(CARS-02-25);中央引导地方科技发展资金项目(226Z6502G)。

摘　　要：糖苷水解酶61家族(GH61)属于一类同时具有氧化作用和水解作用的纤维素降解酶类,既具有微弱的纤维素内切酶活性,也可通过氧化作用破坏纤维素晶体结构促进纤维素酶对木质纤维素的降解,在生物质资源的利用方面具有潜在的应用价值。对大斑刚毛座腔菌Setosphaeria turcica的GH61家族基因进行鉴定及生物信息学分析,通过转录组数据分析及荧光定量PCR验证,筛选出受玉米秸秆木质纤维素底物诱导表达的GH61家族基因StGH61-11。将StGH61-11进行重组表达,使用2,6-二甲氧基苯酚氧化反应测定其酶活力并优化诱导条件,表征其酶学性质并检测其对纤维素酶水解木质纤维素的促进作用。结果表明,在S.turcica基因组中存在21个GH61家族基因,且S.turcica在玉米秸秆的诱导下滤纸酶活明显增加,对其进行转录组分析发现,在以玉米秸秆为碳源时GH61家族基因中有11个基因的表达量增加。将其中StGH61-11基因在大肠杆菌中诱导表达,最佳诱导条件为25℃、1 mmol/L IPTG诱导9 h,此时获得的蛋白比活力可达到(54.08±1.67)U/g。重组蛋白StGH61-11的最佳催化条件为温度50℃、pH 5,在最佳催化条件下可以显著提高纤维素酶水解玉米秸秆的活性,协同度最高可达2.5,糖化率最高可达46.5%。Glycoside hydrolase 61 family(GH61)belongs to the category of both oxidation and hydrolysis of cellulose degradation enzymes,it has a weak cellulose endonuclease,also can promote cellulase by oxidative damage to cellulose crystal structure on the degradation of lignocellulose,in the use of biomass resources it has potential application value.The GH61 family gene of Setosphaeria turcica was identified and bioinformatics to it was analyzed.Through transcriptome data analysis and fluorescence quantitative PCR verification,the GH61 family gene StGH61-11,induced by the carbon source corn stalk,was screened out.StGH61-11 was heterologous expressed,and its enzyme activity was determined by oxidation reaction with 2,6-dimethoxy-phenol,and the induction conditions were optimized.Its enzymatic properties were explored and the effect of StGH61-11 on promoting cellulase hydrolysis of cellulose was studied.The results showed that there were 21 GH61 family genes in S.turcica genome,and the filter paper enzyme activity of S.turcica significantly increased under the induction of corn stover.Transcriptome analysis of S.turcica showed that the expression levels of 11 genes in GH61 family increased when corn stover was used as carbon source.StGH61-11 was heterologous expressed in Escherichia coli.The optimal induction condition was 1 mmol/L IPTG at 25℃for 9 h,and the specific activity reached(54.08±1.67)U/g.The optimal catalytic conditions were 50℃and pH 5 for the recombinant protein StGH61-11 and the recombinant protein significantly improved the activity of cellulase hydrolysis of corn stover,with the highest degree of synergy up to 2.5 and the highest glycosylation rate up to 46.5%.

关 键 词：GH61糖苷水解酶家族 多糖单加氧酶 纤维素酶 AA9 大斑刚毛座腔菌 协同降解 异源表达 

分 类 号：Q814[生物学—生物工程]