Chen Xingjuan, Xu Meiying, Li Guangfei, Sun Guoping
South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510301, China.
Wei Sheng Wu Xue Bao. 2009 Oct;49(10):1323-31.
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.
Gene cloning and expression of acpD were performed by pGM-T vector. Sequences were analyzed by DNAMAN software and azoreduction activity was assayed spectrophotometrically.
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.
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.
为了鉴定acpD在偶氮还原中的功能，从脱色希瓦氏菌S12中获得该基因，并在偶氮还原活性很低的大肠杆菌TOP10中进行表达。
采用pGM-T载体进行acpD的基因克隆和表达。利用DNAMAN软件对序列进行分析，并用分光光度法测定偶氮还原活性。
脱色希瓦氏菌S12的acpD推导氨基酸序列与大肠杆菌JM109的黄素单核苷酸（FMN）依赖的烟酰胺腺嘌呤二核苷酸（NADH）偶氮还原酶有61%的一致性，在FMN结合位点氨基酸序列极其保守。此外，所得重组大肠杆菌TOP10（pGMT-N）的完整细胞及其提取物对不同极性的偶氮染料均表现出高偶氮还原活性。另一方面，以NADH作为电子供体驱动偶氮还原，FMN可增强还原活性。
脱色希瓦氏菌S12的acpD产物是一种非特异性的FMN依赖的NADH偶氮还原酶，它通过FMN将电子从NADH转移至偶氮键，并在体内或体外条件下均表现出高偶氮还原活性。
