构巢曲霉内切甘露聚糖酶在毕赤酵母中的表达及重组蛋白质的表征

Overexpression and characterization of endo-β-1,4-mannanase from Aspergillus nidulans in Pichia pastoris

【目的】内切甘露聚糖酶是一类重要的半纤维素酶,能够有效水解半纤维素的第二大组分甘露聚糖,已广泛应用于工业生物技术领域。【方法】本文对来源于腐生真菌构巢曲霉(Aspergillus nidulans)的一个内切甘露聚糖酶在毕赤酵母中进行过表达及详细的酶学性质研究。【结果】该甘露聚糖酶在摇瓶和发酵罐条件下都成功获得表达,发酵罐条件下的蛋白质表达量高达3.9 mg/mL;该酶的最适pH和温度分别为4.0和60°C,在pH 5.0–9.0之间表现出了很好的稳定性;在温度≤40°C时,该酶非常稳定,当温度≥60°C,该酶的稳定性大大降低;Co^(2+)和Zn^(2+)促进了该酶的活性,而Pb^(2+)、Cu^(2+)、Mn^(2+)等金属离子表现出了一定的抑制作用。【结论】该构巢曲霉来源的内切甘露聚糖酶能在毕赤酵母中高效表达,表现出了一定的耐酸、耐碱及耐热等性能,具有开发为商品酶的潜力,为深入开发构巢曲霉来源的其他糖苷酶奠定了基础。

[Objective] Endo β-1,4-mannanases play important roles in hydrolysis of the β-1,4 glycosidic linkages in mannans and heteromannans, the second most abundant hemicellulosic polysaccharides in nature. [Methodsl In this study, we overexpressed and characterized an endo β-1,4-mannanase from Aspergillus nidulans in Pichia pastoris. [Results] A β-1,4-mannanase was successfully overexpressed in flasks and fermentor, and the yield of overexpressed protein in fermentor reached 3.90 mg/mL. The optimal pH and temperature of the enzyme were 4.0 and 60 ℃ respectively, and it was very stable over the pH ranges from 5.0 to 9.0. It was thermally stable below 40 ℃, whereas it was inactivated very quickly above 60 ℃. Its enzyme activities could be enhanced by Co2＋ and Zn2＋, whereas it was inhibited by Pb2＋, Mn2＋ and Cu2＋. [Conclusion] This endo-β-1,4-mannanase could be well produced by Pichiapastoris, and has a potential as commercial enzymes for application.