褐色嗜热裂孢菌脱色过氧化物酶的表达及发酵条件优化

Expression and fermentation optimization of dye-decolorizing peroxidase from Thermobifida fusca

为了提高来源于褐色嗜热裂孢菌(Thermobifida fusca)的脱色过氧化物酶(TfuDyP)对蒽醌染料降解能力,将含有目的基因的重组质粒pET-28a(+)-TfuDyP,转化至E.coli BL21进行异源表达,并对重组TfuDyP的发酵条件进行优化,分析酶活与血红素饱和度之间的关系。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(sodium dodecyl sulfate-polyacrylamide gel electrophoresis,SDS-PAGE)检测到分子质量为46 kDa的重组TfuDyP蛋白条带。TfuDyP的最佳诱导条件为:诱导剂(IPTG)浓度0.3 mmol/L,诱导时间14 h,诱导温度30℃,在此条件下,TfuDyP比酶活达到27.9 U/g。氯高铁血红素、5-氨基乙酰丙酸(5-ALA)、谷氨酸(Glu)、FeCl2、MnCl2均可提高重组TfuDyP的催化活性,酶活的提高与血红素饱和度之间存在一定的正相关性。实验结果可为利用外源添加物提高血红素的饱和度,应用于染料脱色过氧化物酶的工业发酵提供理论依据。

This study aimed to improve the catalytic ability of Thermobifida fusca dye-decolorizing peroxidase(TfuDyP)to degrade anthraquinone dyes.The recombinant plasmid pET-28a(+)-TfuDyP was transformed into Escherichia coli BL 21 for heterologous expression,and the fermentation conditions of recombinant TfuDyP was optimized.Additionally,the relationship between enzyme activity and heme saturation was analyzed.SDS-PAGE showed that the molecular weight of recombinant TfuDyP was 46 kDa.Moreover,induction with 0.3 mmol/L IPTG at 30℃for 14 h was determined to be the optimal fermentation condition.Under this condition,the specific activity of TfuDyP was 27.9 U/g.Furthermore,hemin,5-aminolevulinic acid(5-ALA),glutamic acid(Glu),FeCl 2 and MnCl 2 promoted the enzyme activity.There was a positive correlation between the increase in enzyme activity and heme saturation.Therefore,this study provides a theoretical basis for improving heme saturation by adding exogenous promoters and applying this in industrial fermentation of dye-decolorizing peroxidase.