脱色酶TpmD在大肠杆菌中的高效表达及纯化

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

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.

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.