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

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Kcat/Km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。

Abstract:

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 pNPG of two mutants (F167L and R354K) were about 2-fold and 4-fold higher than that of MbmgBG1. Kcat/Km 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.