甲基营养菌MP688葡萄糖脱氢酶基因分离鉴定及性质研究

Isolation and Characterization of Glucose Dehydrogenase Gene in Methylovorus sp. MP688

目的:鉴定甲基营养菌MP688中的葡萄糖脱氢酶基因。方法:对甲基营养菌MP688基因组序列进行比对和分析,找到与已知细菌葡萄糖脱氢酶同源性最高的基因序列mpq_2164,且该基因所编码蛋白经分析具有跨膜结构域。设计引物扩增mpq_2164和缺失跨膜区域序列的s-mpq_2164,将PCR产物克隆到表达载体pET-15b上,在大肠杆菌BL21中完成异源重组表达,然后通过组氨酸标签镍柱亲和层析纯化,采用DCIP法测定葡萄糖脱氢酶的活力。结果:分离了甲基营养菌MP688中的葡糖糖脱氢酶基因,并实现了s-mpq_2164的高效异源重组表达;MPQ_2164的氨基酸序列与已知的葡萄糖脱氢酶相似性很低,但酶活测定结果表明S-MPQ_2164具有很高的葡糖糖脱氢酶活性。结论:MPQ_2164是一个依赖于吡咯喹啉醌的葡萄糖脱氢酶,去掉跨膜结构域有利于该蛋白的异源表达。

Objective： To isolate glucose dehydrogenase genes in Methylovorus sp. MP688 and to study the function of the enzyme. Methods： The protein sequence in Methylovorus sp. MP688 which shared the highest similarity with typical bacterial glucose dehydrogenase was isolated through BLAST program from NCBI, containing transmembrane domain at its amino terminal. The fragment of mpq_2164 and s-mpq_2164 （without the sequence of transmembrane domain） were first amplified fi＇om genome of MP688 and ligated with pET-15b. The resultant plasraids were then transformed into E.coli BL21 respectively to acquire the recombinant proteins. The 6×His-tagged proteins was induced by 1 mmol/L IPTG and purified by Ni^2＋ affinity chromatography. The glucose dehydrogenase activity was determined by using DCIP method. Results： A putative glucose dehydrogenase gene was isolated and the corresponding catalytic domain was successfully expressed in a soluble form in E.coli BL21. Although the protein sequence of MPQ_2164 shared very low similarity with reported glucose dehydrogenase, it could oxidise D-glucose in the presence of pyrroloquinoline quinone（PQQ）. Conclusion： MPQ_2164 served as a PQQ dependent glucose dehydrogenase in Methylovorus sp. MP688. Deletion of the transmembrane region promoted the protein expression in E.coli.