细菌脱色酶TpmD的酶学特性研究

从嗜水气单胞菌DN322中分离纯化出能够对三苯基甲烷类染料结晶紫、碱性品红、灿烂绿及孔雀绿进行高效脱色的脱色酶,命名为TpmD。在测定TpmD分子量、等电点及对不同三苯基甲烷染料脱色的动力学参数、脱色过程对分子氧及NADH/NADPH具有依赖性的基础上,又进一步从黄素FAD/FMN对酶活力的影响、酶抑制剂、酶蛋白N-末端测序及酶溶液的特征吸收光谱等方面对TpmD的酶学本质进行了分析。结果表明,TpmD不含核黄素,其脱色活性也不因加入FAD或FMN而提高。TpmD的N-末端氨基酸序列与多种氧化还原酶具有同源性。甲吡酮及维生素C(Vc)对TpmD的脱色活性具有明显的抑制作用。TpmD酶蛋白的溶液在408nm处有一特征吸收峰,但在连二亚硫酸钠的还原条件下通入CO气体后,该酶却不具有P450酶在450nm处的特征吸收峰。上述结果显示脱色酶TpmD是一种新的氧化酶。

Properties of a triphenylmethane dyes decolorization enzyme (TpmD) from Aeromonas hydrophila strain DN322

A novel bacterial enzyme for decolorization of triphenylmethane dyes from Aeromonas hydrophila strain DN322 was purified and named TpmD.The basic properties of this enzyme including molecular weight,isoelectric point Km as well as the optimum temperature and pH were determined and the enzyme was identified as an NADH/NADPH-dependent oxygenase in previous research.Based on previous results,the effect of different inhibitor including Vc,metyrapone,rotenone,antimycin A and NaN-3 as well as the effect of FAD and FMN on the activity of TpmD were measured.The results indicated that the activity of the decolorization enzyme was inhibited by Vc and metyrapone in a concentration-dependent manner,but wasn't inhibited by rotenone,antimycin A and NaN-3.The activity of the decolorization enzyme was not enhanced by addition of FAD or FMN.The solution of the enzyme protein displayed only a single peak at 408nm in the Soret region,a characteristic peak of porphyrin,but did not show the characteristic peak of the cytochrome P450 proteins at 450nm in sodium dithionite(DTN)-reduced enzyme solution after treatment with carbon monoxide.The amino acid sequence of N-terminal of TpmD provided further evidence that the enzyme is an oxygenase.All these results suggest that decolorization enzyme TpmD is a new hemo-containing oxygenase.The decolorization enzyme would be a good material for further research of the enzymological mechanism of triphenylmethane dyes decolorization by bacteria.