[Effects of organic solvents and inhibitors on the decolorization activity of TpmD expressed in Pichia pastoris].

Objective: We studied the effects of different organic solvents and inhibitors on the decolorization of recombinant triphenylmethane dyes decolorization enzyme (TpmD), expressed extracellularly from Pichia pastoris.
Methods: The recombinant TpmD was purified by ultrafiltration and Ni2+ affinity chromatography. The decolorization activity for malachite green was determined by UV-visible spectrophotometer. We studied the effects of some organic solvents and inhibitors on TpmD activity through the rate of decolorization decreased using malachite green as the substance. We also compared the difference between dithicthreitol (DTT) and NADH as the cofactor in assisting the decolorization by monitoring the dissolved oxygen consumption and the end products.
Results: The effect of methanol on the enzymatic activity was weak since TpmD still retained its high activity in the reaction environment containing 10% - 20% methanols. The ethanol and acetone made the enzymatic activity fade away quickly. Dimethylsulfoxide in low concentration was propitious to keep the TpmD activity, although 30% dimethylsulfoxide inhibited the enzymatic activity lost in a half. Ethylenediaminetetraacetic acid (EDTA) of low concentration, L-cysteine and NaN3 all exhibited only weakly inhibitory effects; higher concentrated (25 mmol/L) EDTA could strongly inhibit enzymatic activity and sodium dodecylsulfonate (SDS) inhibited the activity completely. The effect of DTT on TpmD activity was beyond the expectation. It could substitute of coenzyme NADH to assist and accelerate the enzyme in decolorizing reaction. We found that the dissolved oxygen consumption behaviors and reaction end products measuring by UV-Visible full wave-scan were completely different between the reactions assisted with DTT or NADH. This is the first report about DTT which can act as a cofactor for a decolorization enzyme.
Conclusion: The effects of different organic solvents and inhibitors on the enzymatic activity of TpmD are very different. By the dissolved oxygen assay and the end products analysis, we concluded that the decolorizing reaction assisted with DTT was a novel reaction process. The mechanism of reaction with DTT as cofactor is completely different from that with NADH as coenzyme.