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
投稿时间:2017-12-25  
DOI:  10.13345/j.cjb.170511
中文关键词:β-葡萄糖苷酶,点突变,动力学常数,葡萄糖耐受性
英文关键词:β-glucosidase, site-directed mutagenesis, kinetic constant, glucose tolerance
基金项目: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。
作者单位E-mail
姜淑喆 山东大学 微生物技术国家重点实验室山东 济南 250100  
李净净 山东大学 微生物技术国家重点实验室山东 济南 250100  
曹春静 山东大学 微生物技术国家重点实验室山东 济南 250100  
申玉龙 山东大学 微生物技术国家重点实验室山东 济南 250100  
倪金凤 山东大学 微生物技术国家重点实验室山东 济南 250100 jinfgni@sdu.edu.cn 
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中文摘要:
      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。
英文摘要:
      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.
姜淑喆,李净净,曹春静,申玉龙,倪金凤.黄翅大白蚁来源β-葡萄糖苷酶的分子改造[J].生物工程学报,2018,34(7):1081~1090
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