CAJ | 학술논문

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。
목적：감정갑기영양균MP688중적포도당탈경매기인。방법：대갑기영양균MP688기인조서렬진행비대화분석，조도여이지세균포도당탈경매동원성최고적기인서렬mpq_2164，차해기인소편마단백경분석구유과막결구역。설계인물확증mpq_2164화결실과막구역서렬적s-mpq_2164，장PCR산물극륭도표체재체pET-15b상，재대장간균BL21중완성이원중조표체，연후통과조안산표첨얼주친화층석순화，채용DCIP법측정포도당탈경매적활력。결과：분리료갑기영양균MP688중적포당당탈경매기인，병실현료s-mpq_2164적고효이원중조표체；MPQ_2164적안기산서렬여이지적포도당탈경매상사성흔저，단매활측정결과표명S-MPQ_2164구유흔고적포당당탈경매활성。결론：MPQ_2164시일개의뢰우필각규람곤적포도당탈경매，거도과막결구역유리우해단백적이원표체。
Objective: To isolate glucose dehydrogenase genes in Methylovorus sp. MP688 and to study the func-tion of the enzyme. Methods: The protein sequence in Methylovorus sp. MP688 which shared the highest similari-ty with typical bacterial glucose dehydrogenase was isolated through BLAST program from NCBI, containing trans-membrane domain at its amino terminal. The fragment of mpq_2164 and s-mpq_2164(without the sequence of transmembrane domain) were first amplified from genome of MP688 and ligated with pET-15b. The resultant plas-mids 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 Ni2+ 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 pro-tein sequence of MPQ_2164 shared very low similarity with reported glucose dehydrogenase, it could oxidise D-glu-cose in the presence of pyrroloquinoline quinone(PQQ). Conclusion: MPQ_2164 served as a PQQ dependent glu-cose dehydrogenase in Methylovorus sp. MP688. Deletion of the transmembrane region promoted the protein expres-sion in E.coli.