海洋微生物Fulvimarina manganoxydans磷酸酶FM2382的克隆表达及酶学性质

Gene cloning, expression and characterization of a novel phosphatase obtained from a marine bacterium Fulvimarina manganoxydans

为促进海洋微生物及基因资源的开发应用,将海洋微生物Fulvimarina manganoxydans sp.nov.8047的磷酸酶基因fm2382在大肠杆菌(Escherichia coli)中异源表达、纯化,并研究磷酸酶FM2382的酶学性质.设计引物从F.manganoxydans sp.nov.8047基因组DNA中扩增出磷酸酶fm2382基因,克隆至pET28(a)表达载体中,构建重组菌株,表达纯化后对磷酸酶FM2382的酶学性质进行分析.结果表明:磷酸酶FM2382最适反应温度为45℃,最适反应pH 7.1,70-80℃高温处理1 h后仍具35%左右的活力,在pH 7.1-9.0范围内具有较好的稳定性;金属离子对磷酸酶活力有不同程度的影响,其中Mg^(2+)、Mn^(2+)具有强烈的激活作用;K_m=1.42×10^(-3)mol/L,V_m=2.5×10^(-7)mol/L.本研究表明F.manganoxydans sp.nov.中获得的fm2382基因可以在大肠杆菌中高效表达且FM2382是一种新的磷酸酶.

To characterize phosphatase FM2382, we cloned the fm2382 gene of Fulvimarina manganoxydans sp. nov. 8047 and expressed it in Escherichia coli. Based on the genomic DNA sequence of F. manganoxydans sp. nov.8047, we designed a pair of primers and amplified phosphatase fm2382 gene by PCR methods. Then fm2382 was cloned into the vector of pET-28 a and expressed in E. coli. The recombinant protein was purified to investigate enzymatic properties of phosphatase FM2382. The nucleotide sequencing result showed that the fm2382 gene had 890 base pairs and encoded 289 amino acid residues. The purified FM2382 exhibited phosphatase activity with optimum temperature of 45 °C and optimum p H of 7.1. Moreover, the FM2382 was stable between pH 7.1and pH 9.0 for 1 hour, and the relative activity held 40% after storing at 70–80 °C for 1 hour. Furthermore, FM2382 was activated by Mg（2＋）, Mn（2＋）, and inhibited by Zn（2＋）, EDTA. Lineweaver-Burk assay revealed that FM2382 had K_m of and 1.42 × 10（-3） mol/L and V_m of 2.5×10（-7） mol/L. This research first reported that gene fm2382 from F. manganoxydans sp. nov. 8047 could be highly expressed in E. coli, and the enzymatic characterization results suggested that FM2382 is a novel phosphatase.