纤维素堆囊菌源果糖激酶的克隆表达及其酶学性质研究

Gene cloning,expression,and enzymatic characterization of fructokinases isolated from Sorangium cellulosum

为促进果糖-6-磷酸的绿色生物制备,该研究对纤维素堆囊菌(Sorangium cellulosum)中的果糖激酶基因进行克隆、表达,探究重组酶酶学性质和底物特异性。以纤维素堆囊菌基因组DNA为模板,设计引物克隆果糖激酶基因SoCeFruK 1852和SoCeFruK 7579,构建重组表达质粒pET30a-SoCeFruK 1852和pET30a-SoCeFruK 7579,并在Escherichia coli(DE3)中诱导表达,纯化重组果糖激酶,研究反应产物和酶学性质。重组果糖激酶SoCeFruK1852和SoCeFruK7579的分子质量大小分别为31.2、32.8 kDa,蛋白质质量浓度为2.04、1.8 mg/mL,比活力分别为35.8、18.3 U/mg。SoCeFruK1852的最适温度和pH分别为37℃和4.5。金属离子Co^(2+)、Fe^(3+)、Zn^(2+)和化学试剂SDS、EDTA、Tween 20对酶活力有抑制作用,而Mg^(2+)和Mn^(2+)对酶活力有促进作用。以果糖为底物时,SoCeFruK1852的K_(m)值为0.54 mmol/L,V_(max)值为1.28μmol/s。SoCeFruK1852能够特异性催化果糖,但也能催化部分D-塔格糖。获得细菌来源的重组果糖激酶SoCeFruK1852和SoCeFruK7579,且SoCeFruK1852显示良好的酶活力和酶学性质,底物特异性较强,为后续相关研究奠定基础。

To promote the biocatalytic production of fructose-6-phosphate by recombinant of fructokinases,novel fructokinases from Sorangium cellulosum were isolated and well analyzed.The fructokinase genes SoCeFruK 1852 and SoCeFruK 7579 were cloned by designing primers from the genomic DNA of S.cellulosum.The recombinant plasmids of pET30a-SoCeFruK 1852 and pET30a-SoCeFruK 7579 were constructed and transformed into Escherichia coli(DE3)for overexpression.The recombinant fructokinases were purified.The activity and biochemical characterization of fructokinases were determined.The molecular weights of recombinant fructokinase SoCeFruK1852 and SoCeFruK7579 were about 31.2 kDa and 32.8 kDa.The protein concentrations were 2.04 and 1.8 mg/mL.The specific activities were 35.8 and 18.3 U/mg,respectively.The optimal temperature and pH of recombinant SoCeFruK1852 were 37℃and 4.5.The activity of SoCeFruK1852 was inhibited by Co^(2+),Fe^(3+),Zn^(2+),SDS,EDTA and Tween 20.However,Mg^(2+) and Mn^(2+) could enhance its activity.The K_(m) and V_(max) values of SoCeFruK1852 were 0.54 mmol/L and 1.28μmol/s when fructose was used as the substrate.SoCeFruK1852 could specifically catalyze fructose,whereas it also showed slight activity to D-tagatose.The recombinant fructokinases SoCeFruK1852 and SoCeFruK7579 isolated from bacteria S.cellulosum were successfully obtained.SoCeFruK1852 showed good enzymatic activity,characterization and highly substrate specificity,laying a foundation for the research related to recombinant fructokinases.