黄翅大白蚁来源β-葡萄糖苷酶的分子改造  

Molecular modification of β-glucosidase from the midgut of Macrotermes barneyi

作　　者：姜淑喆 李净净[1] 曹春静 申玉龙[1] 倪金凤[1] Shuzhe Jiang;Jingjing Li;Chunjing Cao;Yulong Shen;and Jinfeng Ni(State Key Laboratory of Microbial Technology, Shandong University, Ji ＇nan 250100, Shandong, China)

机构地区：[1]山东大学微生物技术国家重点实验室,山东济南250100

出　　处：《生物工程学报》2018年第7期1081-1090,共10页Chinese Journal of Biotechnology

基　　金：国家重点基础研究发展计划(973计划)(No.2011CB707402);国家自然科学基金(Nos.31272370;30870085)资助~~

摘　　要：纤维素水解成为葡萄糖需要一系列纤维素酶的作用,其中β-葡萄糖苷酶(β-glucosidases)起着至关重要的作用。来自于培菌白蚁中肠的β-葡萄糖苷酶(MbmgBG1)具有较高的葡萄糖耐受性(1.5 mol/L的葡萄糖,保持60%以上的酶活力),但是,酶活力低和热稳定性差限制了β-葡萄糖苷酶(MbmgBG1)在食品以及工业领域中的应用。因此通过对保守氨基酸附近的非保守氨基酸定点突变,获得点突变体(F167L、T176C、E347I、R354K、N393G和V425M),其中突变体F167L、R354K的比活力(底物pNPG)比MbmgBG1分别高出约2倍和4倍。突变体的K_(cat)/K_m值比野生型大,反映了突变体对底物的亲和力以及催化能力比MbmgBG1强。当酶活力保留60%以上时,MbmgBG1所耐受的葡萄糖浓度为1.5 mol/L,而F167L为2.0 mol/L,R354K为3.0 mol/L。这些特性的增强表明,对活性中心附近保守区域内的非保守氨基酸突变,可以较大程度地影响活性,因此需要更深入地研究β-葡萄糖苷酶的活性中心位点,进行改造以提高催化效率。Cellulose hydrolysis to glucose requires a series of cellulase enzymes, of which β-glucosidases play a crucial role. β-glucosidase（MbmgBG1） derived from the midgut of Macrotermes barneyi has higher glucose tolerance（maintaining more than 60% enzyme activity at 1.5 mol/L glucose）. However, low enzyme activity and poor thermal stability limit the applications of β-glucosidase in food industries. Point mutants（F167L, T176C, E347I, R354K, N393G and V425M） were obtained by site-directed mutagenesis of non-conserved amino acids near conserved amino acids. Among them, the specific activities against to substrate p NPG of two mutants（F167L and R354K） were about 2-fold and 4-fold higher than that of MbmgBG1. K_（cat）/K_m values were also higher than that of the wild-type, reflecting stronger affinity to the substrate and higher catalytic ability of mutants than MbmgBG1. When the glucose concentration was 1.5 mol/L, the enzyme activity of MbmgBG1 was about 60% of the original activity. F167L and R354K kept 60% enzymatic activity when the glucose concentrations of was 2.0 mol/L and 3.0 mol/L, respectively. These results lay a foundation for further studies on the catalytic efficiency of β-glucosidase.

关 键 词：Β-葡萄糖苷酶 点突变 动力学常数 葡萄糖耐受性 

分 类 号：Q55[生物学—生物化学]