脱色希瓦氏菌S12 非特异性偶氮还原酶基因表达及特性

Expression and characterization of the nonspecific azoreducase of Shewanella decolorationis S12

摘要:

摘要：【目的】对脱色希瓦氏菌S12 (Shewanella decolorationis S12)的acpD基因(登录号EF198254)及其表达活性进行研究。【方法】采用DNAMAN软件对该基因进行序列分析。利用PCR技术克隆含原有启动子的目的基因，与pGM-T载体连接后转化仅有微弱偶氮还原活性的大肠杆菌TOP10(Escherichia coli TOP10)中进行表达。通过分光光度法测定偶氮染料的还原活性。【结果】序列分析表明，该基因编码198个氨基酸残基组成的多肽，与希瓦氏菌ANA-3(Shewa

Abstract:

Abstract: [Objective] In order to identify the function of acpD in azoreduction, the gene was obtained from Shewanella decolorationis S12 and expressed in Escherichia coli TOP10 which showed little azoreduction activity. [Methods] Gene cloning and expression of acpD were performed by pGM-T vector. Sequences were analyzed by DNAMAN software and azoreduction activity was assayed spectrophotometrically. [Results] The deduced amino acid sequences from acpD of S. decolorationis S12 showed 61％ identity to FMN-dependent NADH-azoreductase from E. coli JM109 with extremely conservative amino acid sequences at FMN binding sites. Moreover, the resulting recombinant E. coli TOP10 (pGMT-N) exhibited high azoreduction activity for azo dyes of different polarity by intact cells and their extracts. On the other hand, NADH was used as electron donor to drive azoreduction and FMN could enhance the reduction activity. [Conclusion] The product of acpD from S. decolorationis S12 is a nonspecific FMN-dependent NADH-azoreductase, which transfers electrons from NADH via FMN to azo bond, and exhibits high azoreduction activity under in vivo or in vitro conditions.