谷氨酸棒杆菌海藻糖合成酶基因的分子进化及其酶学性质的研究
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摘要
海藻糖合成酶是酶法生产海藻糖的关键酶,通过分子内转糖基作用,将α,α-1,4糖苷键连接的麦芽糖转化为α,α-1,1糖苷键连接的海藻糖,由于海藻糖对生物大分子的非特异性保护作用,使其备受关注,为了降低生产成本,各国科学家纷纷对其生产和应用技术进行了大量的研究。现有的海藻糖合成酶存在一些缺点:如转化效率不够高、热稳定性较差、反应时间较长等。因此,有必要对已有的海藻糖合成酶进行改造,使其工业性能有所改善。
本论文采用半理性设计方法,以海藻酮糖合成酶(trehalulose synthaseMutB from Pseudomonas mesoacidophila MX-45)晶体结构为模板,在网站SWISS-MODEL上对来自谷氨酸棒杆菌(Corynebacterium glutamicum)海藻糖合成酶进行同源建模,并对其初始结构作了能量优化,结合分析不同来源的海藻糖合成酶基因的保守序列,选择了保守区的位点F244、D247、R245、E289进行定点(饱和)突变,并在保守区外的A288进行定点突变,结果表明:R245、E289分别饱和突变为其它的19个氨基酸后,共获得了38个突变子,但均无酶活;对D247定点突变成结构相似的氨基酸即Glu和Asn后也丧失酶活;突变体F244C、F244L、F244W、F244Y和A288G的比活力均大大降低,分别降低为野生型(WT)的38%、24%、62%、64%和35%,而A288变成T288后没有酶活。与野生型(WT)相比,突变酶F244C、F244W、A288G的Km值基本不变,突变酶F244L、F244Y对底物麦芽糖的亲和力降低了;突变酶F244Y的最适温度和野生型一样,均为27℃,而F244C、F244L、F244W和A288G比野生型提高了5℃;各突变体的最适pH均有所下降,其中F244C、F244Y和A288G最适反应pH为7.5,比野生型的8.0均下降了约0.5个单位,而F244L和F244W均为6.5,均下降了近1.5个单位;突变酶的热稳定性均有不同程度的提高,其中F244Y、F244W和A288G的Tm值比野生型的提高约1℃;F244L提高约2℃,而F244C热稳定性提高了近4℃。通过分析和比较这些突变酶的性质变化,可为今后研究海藻糖合成酶的结构和功能的关系提供有用的数据和信息,为进一步对海藻糖合成酶的分子改造提供理论依据。
Trehalose synthase is the key enzyme in enzymatic process to produce trehalose.It can catalyze maltose bonded in anα,α-1,4-glycosidic linkage to produce trehalose withα,α-1,4-glycosidic linkage.Recently,Trehalose has been widely attended because of it's non-special protecting function on big molecule of biology.The scientists from many countries have been engaging in studying on the technology of production and application to reduce it's cost of production. The existing trehalose synthase have some disadvantages:the transform efficiency is not enough high;The thermostability is low;The reaction time is long and so on.Therefore,it is necessary to perform mutagenesis on trehalose synthase to improve the industrial capability.
In this paper,trehalose synthase was evolved by half-rational design.The three-dimensional model of TreS from Corynebacterium glutamicum was constructed based on the crystal structure of trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45 on the SWISS-MODEL Internet.The energy of primal structure was optimized.Combined with analyzing the conserved region of trehalose synthase amio sequence from different origin.The conserved amio acid R245 and E289 were predicted likely as active site and were mutated.Further more,the position F244、D247and A288 near the two sites(R245 and E289)were performed site-mutagenesis.The results indicated that the activity was lost after R245 and E289 were converted to other 19 kinds of amio acids and that the mutants D247E and D247N were also shown no activities.The reason of activity lost probably was that these positions were important to maintain the structure stability of the protein,and they possibly were crucial residues or active sites of the enzyme.The specific activity of the mutants F244C、F244L、F244W、F244Y and A288G were decreased largely and reduced to 38%、24%、62%、64%and 35%of the wild-type respectively.After the position of A288 was changed to A288T,it was shown that the activity was disrupted.Compared with the wild-type,The Km of F244C、F244W、A288G were not significantly altered,but the affinity of F244L and F244Y enzyme for the maltose substrate were decreased;The optimum temperature of F244Y was 27℃which the same as the wild-type,and the mutants F244C、F244L、F244W and A288G were enhanced 5℃more than the wild-type;The optimum pH of all the mutants were decreased,The optimum pH of F244C、F244Y and A288G were 7.5 that dropped 0.5 pH units compared with those of the wild-type,and the F244L and F244W were reduced 1.5 units;The thermostability of all the mutants were increased in various degree,The mutant F244Y、244W and A288G increased about more one degree than that of the wild type;F244L increased two degree and F244C enhanced four degree approximately.It can not only provide useful datum and information in studying the relation of the configuration and function by analyzing and studying these changes about the properties of the mutants but also can afford the theoretic basis to perform molecule evolution on trehalose synthase for the future.
本论文采用半理性设计方法,以海藻酮糖合成酶(trehalulose synthaseMutB from Pseudomonas mesoacidophila MX-45)晶体结构为模板,在网站SWISS-MODEL上对来自谷氨酸棒杆菌(Corynebacterium glutamicum)海藻糖合成酶进行同源建模,并对其初始结构作了能量优化,结合分析不同来源的海藻糖合成酶基因的保守序列,选择了保守区的位点F244、D247、R245、E289进行定点(饱和)突变,并在保守区外的A288进行定点突变,结果表明:R245、E289分别饱和突变为其它的19个氨基酸后,共获得了38个突变子,但均无酶活;对D247定点突变成结构相似的氨基酸即Glu和Asn后也丧失酶活;突变体F244C、F244L、F244W、F244Y和A288G的比活力均大大降低,分别降低为野生型(WT)的38%、24%、62%、64%和35%,而A288变成T288后没有酶活。与野生型(WT)相比,突变酶F244C、F244W、A288G的Km值基本不变,突变酶F244L、F244Y对底物麦芽糖的亲和力降低了;突变酶F244Y的最适温度和野生型一样,均为27℃,而F244C、F244L、F244W和A288G比野生型提高了5℃;各突变体的最适pH均有所下降,其中F244C、F244Y和A288G最适反应pH为7.5,比野生型的8.0均下降了约0.5个单位,而F244L和F244W均为6.5,均下降了近1.5个单位;突变酶的热稳定性均有不同程度的提高,其中F244Y、F244W和A288G的Tm值比野生型的提高约1℃;F244L提高约2℃,而F244C热稳定性提高了近4℃。通过分析和比较这些突变酶的性质变化,可为今后研究海藻糖合成酶的结构和功能的关系提供有用的数据和信息,为进一步对海藻糖合成酶的分子改造提供理论依据。
Trehalose synthase is the key enzyme in enzymatic process to produce trehalose.It can catalyze maltose bonded in anα,α-1,4-glycosidic linkage to produce trehalose withα,α-1,4-glycosidic linkage.Recently,Trehalose has been widely attended because of it's non-special protecting function on big molecule of biology.The scientists from many countries have been engaging in studying on the technology of production and application to reduce it's cost of production. The existing trehalose synthase have some disadvantages:the transform efficiency is not enough high;The thermostability is low;The reaction time is long and so on.Therefore,it is necessary to perform mutagenesis on trehalose synthase to improve the industrial capability.
In this paper,trehalose synthase was evolved by half-rational design.The three-dimensional model of TreS from Corynebacterium glutamicum was constructed based on the crystal structure of trehalulose synthase MutB from Pseudomonas mesoacidophila MX-45 on the SWISS-MODEL Internet.The energy of primal structure was optimized.Combined with analyzing the conserved region of trehalose synthase amio sequence from different origin.The conserved amio acid R245 and E289 were predicted likely as active site and were mutated.Further more,the position F244、D247and A288 near the two sites(R245 and E289)were performed site-mutagenesis.The results indicated that the activity was lost after R245 and E289 were converted to other 19 kinds of amio acids and that the mutants D247E and D247N were also shown no activities.The reason of activity lost probably was that these positions were important to maintain the structure stability of the protein,and they possibly were crucial residues or active sites of the enzyme.The specific activity of the mutants F244C、F244L、F244W、F244Y and A288G were decreased largely and reduced to 38%、24%、62%、64%and 35%of the wild-type respectively.After the position of A288 was changed to A288T,it was shown that the activity was disrupted.Compared with the wild-type,The Km of F244C、F244W、A288G were not significantly altered,but the affinity of F244L and F244Y enzyme for the maltose substrate were decreased;The optimum temperature of F244Y was 27℃which the same as the wild-type,and the mutants F244C、F244L、F244W and A288G were enhanced 5℃more than the wild-type;The optimum pH of all the mutants were decreased,The optimum pH of F244C、F244Y and A288G were 7.5 that dropped 0.5 pH units compared with those of the wild-type,and the F244L and F244W were reduced 1.5 units;The thermostability of all the mutants were increased in various degree,The mutant F244Y、244W and A288G increased about more one degree than that of the wild type;F244L increased two degree and F244C enhanced four degree approximately.It can not only provide useful datum and information in studying the relation of the configuration and function by analyzing and studying these changes about the properties of the mutants but also can afford the theoretic basis to perform molecule evolution on trehalose synthase for the future.
引文
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