嗜热β-葡萄糖苷酶的原核表达、纯化及其活性

Prokaryotic expression,purification and activity of thermophilic β-glucosidase

目的原核表达、纯化嗜热β-葡萄糖苷酶,并检测其活性。方法从嗜热细菌Fervidobacterium pennivorans基因组DNA中PCR扩增β-葡萄糖苷酶基因,插入原核表达载体p ET-28a中,构建重组表达质粒,转化E.coli BL21(DE3)Codon Plus,筛选阳性重组菌,IPTG诱导表达。采用镍柱亲和层析法纯化重组酶;紫外吸收法检测酶活力和底物选择性。结果 PCR退火温度为67℃时,可扩增得到单一的目的基因条带;测序结果显示,重组表达质粒中目的基因的核苷酸序列与细菌基因组中该基因序列同源性为100%,未发现终止密码子及氨基酸的改变;表达的重组酶相对分子质量约为54 000;纯化的目的蛋白纯度达95%,浓度为10 mg/L;该重组酶能够高效催化对硝基苯-β-D-葡萄糖苷(p NPG)的水解,60℃条件下的比活力达124 293.5 U/mg。结论成功在E.coli中表达了嗜热细菌Fervidobacterium pennivorans来源的具有较高催化活力和底物专一性的β-葡萄糖苷酶。

Objective To express thermophilic β-glucosidase in prokaryotic cells,purify the expressed product and determine its activity. Methods β-glucosidase gene was amplified by PCR from the genomic DNA of Fervidobacterium pennivorans and inserted into prokaryotic expression vector p ET-28 a. The constructed recombinant plasmid was transformed to competent E. coli BL21（DE3）Codon Plus,and positive recombinants were screened and induced with IPTG.The expressed recombinant protein was purified by Ni-NTA spin column affinity chromatography and determined for enzyme activity and the substrate specificity by UV absorption. Results A single target gene band was obtained at an annealing temperature of 67 ℃. DNA sequencing proved that the homology of nucleotide sequence of target gene in recombinant plasmid was 100% to that in bacterial genome. No change was observed in codon or amino acids. The expressed recombinant protein,with a relative molecular mass of about 54 000,reached a purity of 95% and a concentration of 10 mg / L after purification. This enzyme effectively catalyzed the hydrolysis of p-nitrophenylglucopyrinoside,of which the specific activity was 124 293. 5 U / mg at 60 ℃. Conclusion A novel thermophilic β-glucosidase from Fervidobacterium pennivorans was successfully expressed in E. coli,which showed high catalytic activity and substrate specificity.