脱色酶TpmD在大肠杆菌中的高效表达及纯化

用PCR方法从嗜水气单胞菌DN322基因组中扩增出编码三苯基甲烷类染料脱色酶TpmD的基因,与表达载体pET-22b(+)连接构建成重组质粒pET22-tpmD,转化大肠杆菌BL21(DE3)得到重组工程菌株.结果表明,经IPTG诱导,脱色酶基因可高效表达,粗酶液降解结晶紫、孔雀石绿、碱性品红、灿烂绿的比活力达到569.5,386.9,516.1,273.0U/g.表达产物经Ni-NTA亲和层析法一步纯化,蛋白纯度达94.05%.对4种染料的比活力分别达到1075.3,1042.8,903.9,484.3U/g,重组质粒稳定存在于工程菌中,便于规模化发酵生产.

Triphenylmethane dyes decolorization enzyme over expression and purification in E.coli

The gene coding triphenylmethane dyes degradation enzyme (TpmD) was amplified from genomic DNA of Aeromonas hydrophila strain DN322 by PCR and cloned into the expression vector pET22b (+). After being confirmed by sequencing, the recombinant vector pET22-tpmD was transformed into Escherichia coli BL21(DE3), and then a clone with high yeild of TpmD was screened. The expression of TpmD was induced with IPTG and the specific activity of TpmD from pET22-tpmD/BL21 (DE3)’s cell-free extract, were 569.5,386.9,516.1 and 273.0U/g with crystal violet, malachite green, fuchsin basic and brilliant green as substrates, respectively. After one step purification by Ni-NTA agarose column, the specific activities of TpmD were increased to 1075.3, 1042.8, 903.9 and 484.3U/g, the purity of this enzyme reached up to 94.05%. The stability of the recombinant plasmid makes it easy to produce the protein on a large scale fermention.