吡咯喹啉醌产生菌筛选方法建立及菌种筛选

Establishment of the screening method and isolation of PQQ producing strains

摘要:

吡咯喹啉醌(PQQ)是一种氧化还原酶的辅酶,具有多种生理功能。扩增得到大肠杆菌葡萄糖脱氢酶(GDH)基因,并利用表达载体pET28a在E.coli BL21(DE3)中进行了表达。纯化了可溶性表达产物,并建立了基于GDH的重组酶法分析PQQ的方法。确定了甲基营养菌筛选模型,从2000余份土样中分离得到一株PQQ高产生菌MP606,在未经培养条件优化及诱变选育的条件下PQQ产量达113mg/L。从该菌培养液中制备得到了产物的结晶,HPLC分析、特征光谱分析以及酶法分析均证实该产物为PQQ。扩增并分析了MP606的16S rDNA序列,结果显示该菌16S rDNA序列与12种甲基营养菌都具有95%以上同源性,其中与食甲基菌属两菌株的16S rDNA序列同源性达99%。

Abstract:

Pyrroloquinoline quinone (PQQ) is a cofactor of some oxido-reductases with many important physiological effects and potential pharmaceutical applications. The glucose dehydrogenase of Escherichia coli，being a candidate for enzymic detection of PQQ，is known to be a quinoprotein which is obligately dependant on PQQ as cofactor. The gdh gene of E. coli was amplified and cloned into plasmid pET28a. The recombinant GDH was overexpressed in soluble form in E. coli BL21(DE3). A bioassay method was established for determination of PQQ by the purified GDH. A screening model was set up for the enrichment of methylotrophic bacteria. Together with the above bioassay method，over 2000 soil samples were screened for the isolation of high-yielding PQQ producing strains. A methylotrophic strain，named MP606，was thus isolated. The PQQ production of MP606 is determined to be 113mg/L without conditional optimization and genetic breeding. The PQQ crystal was obtained from the culture supernatant which has been identified by HPLC，absorption spectra assay，and enzymatic analysis. The 16S rDNA of MP606 was amplified and sequenced. According to the comparison of 16S rDNA sequences，overall similarity value between strain MP606 and 12 typical methylotrophic bacteria is above 95%. The highest value is with two strains of Methylovorus，which reached at 99%.