重组ZEN降解酶Oxa的纯化及特性研究

Purification and Enzymatic Characters of Recombinant Oxidase Responsible for Zearalenone Biodegradation

摘要:

以表达可高效降解玉米赤霉烯酮（Zearalenone，ZEN，ZEA）的氧化酶Oxa的毕赤酵母工程菌GS115/pPIC9K-Oxa为研究对象，将发酵液经乙醇沉淀、阴离子交换色谱和超滤纯化后，检测重组Oxa酶降解ZEN的活力，分析其二级结构，并对相关酶学特性进行研究。结果表明，氧化酶Oxa的纯化倍数达到20倍以上，纯化后的Oxa针对ZEN的降解率达到80%以上，其二级结构主要由无规卷曲和β-折叠组成，辅以少量的β-转角和α-螺旋，该酶的最适作用温度和pH分别为60 ℃和9.0，并且在50~70 ℃和pH 9.0~11.0时保持60%以上的ZEN降解率，具有典型的耐高温碱性酶的特征，反应体系中适量的Cu2+、Fe2+和Fe3+的加入能提高其降解活力。本论文为进一步研究氧化酶Oxa的分子结构及作用方式奠定了基础，对ZEN的生物降解具有显著的实际意义。

Abstract:

The recombinant Pichia pastoris strain GS115/pPIC9K-Oxa expressing oxidase Oxa derived from Acinetobacter sp.SM04 was studied, including purification of fermentation supernant, identification of secondary structure of recombinant Oxa, ZEN-degradating ability and enzymatic characteristics. After ethanol precipitation, anion-exchange chromatography and ultrafiltration, recombinant Oxa was puri?ed more than 20 folds and exhibited remarkable efficiency in ZEN removement by over 80% degradation rate. Its secondary structure is mainly composed of random coils and β-sheet, supplemented by less β-turn and α-helix. Oxa acted as a typical thermostable alkalase by which the ZEN degradation rate was maintained above 60% in pH 9.0-11.0 buffer from 50 to 70°C. In addition, the optimum temperature and pH were 60°C and 9.0, respectively. Some proper concentration of Cu2+, Fe2+ and Fe3+ individually in environment would accelerate degradation of Oxa. The results could be helpful to evaluate the molecular structure and mode of action of Oxa in future. It would make significant practical sense for the biodegradation of zearalanone.