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

发布时间：2018-05-28 11:32

  本文选题：磷酸甘露糖异构酶(PMI) + 蜡状芽孢杆菌　； 参考：《食品工业科技》2017年06期

【摘要】：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。
[Abstract]:The phosphomannose isomerase gene of Bacillus cereus CZ was cloned and expressed in E. coli. The pmi gene amplified by PCR was ligated with pET-22b () expression vector and transferred into E. coli BL21DE3. The recombinant strain BL21-p ET22b (-pmib) was constructed, and the recombinant phosphate mannose isomerase was successfully expressed. The results showed that the total length of pmi gene was 948 BP, encoding 315 amino acids. The recombinant enzyme was purified by nickel column His Trap HP affinity chromatography and its protein molecular size was about 40. 8 ku. The results of enzymatic properties showed that the optimum reaction temperature was 35 鈩，

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