粘细菌Myxococcus sp.CYD-1海藻糖合酶基因克隆及酶学特性分析

Cloning,expression and characterization of a trehalose synthase from Myxococcus sp.CYD-1

【目的】海藻糖合酶(trehalose synthase,EC.5.4.99.16)可通过转糖苷作用将麦芽糖转化成为海藻糖,该反应仅需一步,且所用原料低廉,在酶法生产海藻糖上显示出一定的应用潜力。本文以粘细菌Myxococcus sp.CYD-1为材料,挖掘新的海藻糖合酶资源。【方法】以菌株CYD-1基因组DNA为模板,使用加尾PCR的方法克隆编码海藻糖合成酶的基因MCTs,构建含有组氨酸标签的重组表达载体,并将重组质粒导入大肠杆菌BL21(DE3)中进行原核表达。重组蛋白经Ni^(2+)-NTA纯化,以麦芽糖为底物研究其酶学性质,包括最适温度、最适pH、温度和pH稳定性及金属离子和抑制剂的影响。【结果】从菌株CYD-1基因组中克隆到一个编码海藻糖合成酶的基因MCTs。MCTs全长1659 bp,编码一个552个氨基酸的蛋白,分子量大小为65 kU。氨基酸序列分析表明MCTs与来源于Myxococcus sp.V11的海藻糖合酶相似性为92.36%,与其他已报道的海藻糖合酶有较大差异。MCTs具有高度保守的基序202DAVPYL207,244EANQ247和307RNHDEL312,以及活性中心三联体Asp202-Glu244-Asp310,三维建模结果显示MCTs的催化结构域具有典型的(α/β)8桶状结构,表明MCTs归属于糖苷水解酶GH13家族。重组蛋白在E.coli BL21(DE3)中大量可溶性表达后,通过Ni^(2+)-NTA将目的蛋白纯化至电泳纯。重组酶rMCTs最适反应温度为40℃,最适反应pH为6.5,最大比活力为105.6 U/mg。重组酶rMCTs在30℃下处理4 h,残留酶活为50%,但在65℃下处理1.5 h活力丧失。重组酶rMCTs在pH 5.0~8.0范围内有活性,在pH 6.0~7.5处理12 h活力稳定。1 mmol/L Ca^(2+)对重组酶rMCTs有轻微的激活作用,Co^(2+)、Fe^(3+)、Cu^(2+)对重组酶rMCTs有抑制作用,其余金属离子对酶活力无影响。甲醇、乙醇、丙酮等有机溶剂和SDS对重组酶rMCTs有强烈的抑制作用,TritonX-100对rMCTs酶活力无显著影响。【结论】MCTs具有潜在的应用价值,丰富了海藻糖合成酶资源。

【Objective】Trehalose synthase(EC.5.4.99.16),which converts maltose to trehalose,is considered to be a potential biocatalyst for trehalose production.This enzymatic process has the advantage of a simple reaction and uses an inexpensive substrate.We aimed to discover and characterize a trehalose synthase from Myxococcus sp.CYD-1.【Method】Genomic DNA extracted from strain CYD-1 was used as a template for trehalose synthase gene cloning by PCR reaction.The PCR product was digested with restriction endonucleases Nde I and HindⅢand then ligated into pET-29a vector.After plasmid transformation into E.coli BL21(DE3)cells,the recombinant enzyme was expressed by IPTG induction and purified by nickel affinity chromatography(Ni^(2+)-NTA).Characterization of recombinant rMCTs,including optimal pH and temperature,metal ion dependence,and inhibitor,was performed using maltose as the substrate.The effect of pH and temperature on the enzyme activity was also measured.【Result】A trehalose synthase gene(MCTs)isolated from Myxococcus sp.CYD-1,encoding 552 amino acids with a putative molecular size of 65 kU,was expressed and characterized.The amino acid sequence of MCTs shared the highest identity(92.36%)with the trehalose synthase from Myxococcus sp.V11,but it is completely different from other experimentally characterized bacterial trehalose synthases.The results of homology modeling showed that MCTs possess the common trehalose synthase motifs such as 202DAVPYL207,244EANQ247 and 307RNHDEL312.A catalytic triad Asp202-Glu244-Asp330 was shown to be conserved in the MCTs.Threedimensional modeling results show that MCTs has a typical(α/β)8-barrel structure,indicating that MCTs belongs to the glycosyl hydrolase family 13.The purified recombinant enzyme had an optimum temperature of 40℃and a pH optimum around 6.5.For the target substrate maltose,the specific enzyme activity of rMCTs was calculated to be approximately 105.6 U/mg protein under the optimal reaction conditions.The rMCTs retained full activity after 4 h of incubation at below 30°C and retained about 50%,but it lost 100%of the original activity after 1.5 h of incubation at 65℃.In terms of pH,the rMCTs was active in a narrow range of pH(5.0~8.0)conditions,and the enzyme was stable in the range of pH 6.0~7.5 in treatments at 4℃for 12 h.The enzyme activity was slightly stimulated by Ca^(2+),and strongly inhibited by 1 mmol/L Co^(2+),Fe^(3+),Cu^(2+).Other metal ions had almost no effect.Recombinant enzyme activity was strongly inhibited by 5 mg/mL SDS,10%acetone,ethanol and acetonitrile and weakly inhibited by 20 mg/mL TritonX-100.【Conclusion】The results of the characterization showed that it could be a potential novel enzyme in industrial applications.