蜡状芽孢杆菌CZ磷酸甘露糖异构酶基因的克隆表达及酶学性质研究

Gene cloning and expression of a phosphomannose isomerase from Bacillus cereus CZ and characterization of the recombinant enzyme

将蜡状芽孢杆菌CZ中的磷酸甘露糖异构酶基因(pmi)进行克隆,并在大肠杆菌中进行异源表达。将PCR扩增得到的pmi基因与p ET-22b(+)表达载体进行连接,转入大肠杆菌BL21(DE3)中,构建重组菌株BL21-p ET22b(+)-pmi,并成功表达了重组磷酸甘露糖异构酶。结果显示:克隆得到pmi基因序列全长为948 bp,编码315个氨基酸。通过镍柱His Trap HP亲和层析法纯化得到具有活性的重组酶,其蛋白分子大小约为40.8 ku。酶学性质结果显示:该酶的最适反应温度为35℃,在30~40℃酶活力相对稳定;最适p H为7.0,在弱碱性条件下保存12 h后仍存有50%以上酶活力;不同低浓度的金属离子Ni^(2+)、Ca^(2+)、Zn^(2+)、Cu^(2+)和Mg^(2+)均对该酶表现出不同程度的激活作用,其中Mn^(2+)对该酶激活作用最显著,当其浓度为1 mmol/L时,激活作用最大,而Co^(2+)对其有明显的抑制作用。

The Bacillus cereus CZ phosphomannose isomerase gene （pmi） were cloned and heterogeneously expressed in Escherichia coli.The pmi gene which was amplified by using PCR was connected with pET-22b（ ＋ ）expression vector,then the recombinant plasmid was transferred to E.coli BL21 （DE3）.The recombinant strain BI21-pET22b（ ＋ ）-pmi was constructed and successfully expressed recombinant enzyme.The results showed that pmigene（948 bp）was cloned and encoded 315 amino acids.The crude recombinant enzyme was purified by HisTrap HP Ni-affinity chromatography and the molecular mass of the purified recombinant enzyme was detected about 40.8 ku. The enzymatic characteristics analysis showed that the optimal temperature was estimated to be 35℃, and it retained relatively stable at 30 - 40℃. Meanwhile, the optimal pH of the recombinant enzyme was around 7.0, and relative enzyme activity was retained 50% in weak alkaline environment for 12 h. Various low concentrations of metal ion, such as Ni^2＋ , Ca^2＋, Zn^2＋ , Cu^2＋ , Mg^2＋ , could activate the recombinant enzyme in different degree.Mn^2＋ had most obviously of activation with a concentration of 1 mmol/L,whereas Co^2＋ had obviously inhibitory effect on the recombinant enzyme.