谷氨酸棒杆菌(Corynebacterium glutamicum)海藻糖合成酶的定点突变及其酶学性质研究

Study on Site-directed Mutagenesis and the Enzyme Properties of Trehalose Synthase from Corynebacterium glutamicum

以已知晶体结构的Pseudomonas mesoacidophila MX-45菌株海藻酮糖合成酶(MutB)的晶体结构为模板,在SWISS-MODEL模建立谷氨酸棒杆菌(Corynebacterium glutamicum)海藻糖合成酶的立体结构,并对初始结构作能量优化,通过氨基酸序列比对,选择TreS-glu保守区内的氨基酸R245、D247、E289、F244和保守区外的氨基酸A288进行定点突变,并对突变酶F244C、F244L、F244W、F244Y、A288G、R245X、E289X、D247N、D247E进行纯化和酶学性质研究,比较突变子对酶活性和热稳定性的影响。结果表明,R245、E289突变为其它的19个氨基酸后酶活力全部丧失,D247E和D247N也丧失酶活,F244C、F244L、F244W、F244Y和A288G的比活力分别降低到TreS-glu的38%、24%、62%、64%和35%,A288突变成T288后没有酶活。与TreS-glu相比,F244C、F244W、A288G的Km值基本不变,F244L、F244Y对底物麦芽糖的亲和力降低,F244Y的最适反应温度和TreS-glu相同,均为27℃,而F244C、F244L、F244W和A288G的最适温度提高到32℃。与TreS-glu相比,突变酶的最适反应pH值均有所下降,其中F244C、F244Y和A288G的为7.5,比TreS-glu的8.0均下降了约0.5个单位,而F244L和F244W的为6.5,比TreS-glu的8.0均下降了近1.5个单位。与TreS-glu相比,突变酶的热稳定性均有不同程度提高,其中F244Y、F244W和A288G的Tm值比TreS-glu的提高约1℃,F244L提高约2℃,F244C提高了近4℃。

A three-dimensional model of trehalose synthase from Corynebacterium glutamicum was constructed based on the crystal structure of trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45 on the SWISS-MODEL and the energy of its primal structure was optimized.Combined with analyzing the conserved region of trehalose synthase genes from different origin,mutagenesis was performed on the amino acids in TreS-gluhis conserved region（R245,D247,E289,F244） and out of the conserved region（A288）.The mutants F244C,F244L,F244W,F244Y,A288G,R245X,E289X,D247N and D247E were purified and their impacts on enzyme activity and thermal stability were analyzed.The results showed that the activity was lost after R245 and E289 were converted to other 19 kinds of amino acids.The mutants D247E and D247N revealed also no activity.The specific activity of the mutants F244C,F244L,F244W,F244Y and A288G were decreased to 38%,24%,62%,64% and 35% of the wild-type（TreS-gluhis）,respectively.The mutant A288T showed its activity to be disrupted completely.Compared with the wild-type,the km of F244C,F244W,A288G was not significantly altered,but the affinity of F244L and F244Y enzymes was decreased for the maltose substrate.The optimum temperature of the mutant F244Y was 27℃ that was the same as the TreS-glu,while the mutants F244C,F244L,F244W and A288G enhanced their optimum temperature to 32℃.The optimum pH value of all the mutants was decreased in comparison with that of the wild type.The optimum pH of F244C,F244Y and A288G dropped 0.5 pH value units and reached to 7.5,and that of F244L and F244W decreased 1.5 units.The thermostability of all the mutants increased in various degree,about one degree in the F244Y,F244W and A288G,two degrees in the F244L and four degrees in the F244C than that of the wild type.