有机溶剂和抑制剂对毕赤酵母表达TpmD酶活性影响

Effects of organic solvents and inhibitors on the decolorization activity of TpmD expressed in Pichia Pastoris

【目的】本文在利用毕赤酵母成功分泌表达高活性三苯基甲烷类染料降解酶TpmD的基础上,研究不同有机溶剂和常见酶抑制剂对纯化后的重组脱色酶TpmD活性的影响。【方法】将毕赤酵母分泌表达的重组酶TpmD经超滤浓缩及镍离子亲和层析柱纯化后得到纯化的重组TpmD酶。利用分光光度计法测定其对孔雀石绿脱色活性,并研究了各种有机溶剂和抑制剂对TpmD酶脱色活性的影响。通过脱色过程溶氧测定和全波长扫描分析反映了DTT与NADH作为TpmD辅因子时对孔雀石绿脱色反应的表观区别。【结果】甲醇对酶活性抑制作用较弱,重组酶在甲醇中能维持约90%的活性,乙醇和丙酮则使重组酶活性则迅速丧失。低浓度二甲基亚砜有利于重组酶活性的维持,30%的二甲基亚砜可抑制一半以上酶活性。L-半胱氨酸、叠氮化钠及低浓度的EDTA对重组酶活性影响较小,较高浓度的EDTA才显示较强抑制作用;表面活性剂SDS有强烈的抑制作用,极低的浓度也能完全抑制重组酶活性。抗氧化剂二硫苏糖醇(DTT)对重组脱色酶的作用十分独特,它可替代NADH辅助脱色反应并增加脱色速率,但在反应终点脱色总效率约为NADH作为辅酶时的92%。DTT辅助TpmD脱色孔雀石绿过程中的耗氧情况及脱色产物与NADH作为辅酶时明显不相同。【结论】发现不同有机溶剂和常见抑制剂对重组酶活性的影响存在较大差异,首次证实DTT能辅助TpmD脱色反应,这一脱色反应过程的作用机理与NADH为辅酶催化的反应原理完全不同。

[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 uhrafihration and Ni^2＋ 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 DTF or NADH. This is the first report about DTr 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 DTr was a novel reaction process. The mechanism of reaction with DTr as cofactor is completely different from that with NADH as coenzyme.