毕赤酵母发酵表达金属硫蛋白的制备及活性

Preparation and Activity of Metallothionein from the Fermentated Yeast(Pichia pastoris)

以Cu Cl2诱导的巴斯德毕赤酵母发酵液为原料,比较高温沉降和等电点沉淀法对发酵液中杂蛋白去除效果,进而通过超滤膜分离、DEAD-FF琼脂糖凝胶层析及EDTA螯合脱除Cu2+,纯化制备MT,并评价其对自由基的清除活性。结果发现,相比于等电点沉淀法,采用80℃保温10 min,再以8000 r/min离心20 min的高温沉淀法,对发酵液中杂蛋白去除及MT保持效果较佳;发酵液再经50 ku平板膜超滤浓缩、DEAE-FF离子交换层析,有效保留了发酵液中MT并去除杂蛋白;采用EDTA螯合去除MT中结合的Cu2+,其脱除率达61.90%;纯化制备MT对Pb2+、Cr6+、Cd2+和Cu2+表现出一定再吸附能力,并具有较好的OH·,O2-·和DPPH自由基清除活性。研究结果可为酵母源MT的分离提取以及进一步活性开发与应用,提供一定的参考与基础。

In this study, the methods to prepare metallothionein（MT） from the fermentation of yeast（Pichia pastoris） using high temperature heating and isoelectric precipitation were compared and used ias a pre-treatment process to remove the miscellaneous proteins in the fermentation broth. Next, the ultrafiltration membrane, DEAD-FF chromatography, and the action of EDTA chelation were conducted to get high content of MT product from the yeast fermentation. The antioxidant activity in vitro of obtained MT was also evaluated. The results indicated that the high temperature heating（80 ℃ heating 10 min, then centrifugation at 8000 r/min for 20 min） showed better effects on the removal of heteroprotein and retention of MT, compared with the treatment of isoelectric precipitation. Additionally, the MT was significantly extracted and purified from the fermentation broth by the following processes of ultrafiltration concentration（50 ku） and DEAE-FF chromatography. The combined Cu2＋ by extracted MT were removed by the chelation of EDTA, the concentrations of which were decreased by 61.90%. Finally, the obtained MT exhibited the re-adsorption capacity of metal elements and good capacities of OH·, O2-·, and DPPH scavenging activity. These results can provide a reference and theoretical basis for the extration, the development, and the application of MT from yeast resources.