构巢曲霉内切纤维素酶A10在毕赤酵母中的表达及重组蛋白质的表征

Overexpression and characterization of endo-cellulase A10 from Aspergillus nidulans in Pichia pastoris

摘要:

内切纤维素酶是纤维素酶高效降解纤维素的一个关键因子，已广泛应用于工业生物技术领域。对来源于腐生真菌构巢曲霉的一个内切纤维素酶进行过表达及详细的酶学性质研究，研究结果表明：该内切纤维素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达到0.89 mg/mL；该酶的最适反应pH和温度分别为4.0和80 ℃，在pH 2.0–12.0之间表现出了很好的稳定性；在温度￡60 ℃时，该酶非常稳定，当温度370 ℃，酶的稳定性大大降低；Co2+、Mn2+和Fe2+促进了CMCase活性，而Pb2+、Ni2+和Cu2+等金属离子表现出了一定的抑制作用。因此，该构巢曲霉内切纤维素酶表现出了非常好的耐酸、耐碱以及一定的耐热性等性能，具有开发为商品酶的潜力，为深入开发构巢曲霉来源糖苷酶的应用奠定了基础。

关键词:构巢曲霉，内切纤维素酶，酶学性质，二价金属离子，发酵

Abstract:

Endo-cellulases are important to efficiently hydrolyze cellulose, and widely used in biotechnology. In this study, we overexpressed and characterized an endo-cellulase from Aspergillus nidulans. This endo-cellulase was successfully overexpressed in flasks and fermentor, and its concentration in fermentor reached 0.89 mg/mL. The optimal pH and temperature of the were 4.0 and 80 ℃ respectively, and it was very stable between pH 2.0 and 12.0. It was thermally stable below 60 ℃, whereas it was inactivated very quickly above 70 ℃. Its CMCase activity could be enhanced by Co2+, Mn2+ and Fe2+, whereas it was inhibited by Pb2+, Ni2+ and Cu2+. Therefore, this endo-cellulase exhibited good pH stability and thermostability below 60 ℃, and has the potential as commercial enzymes.

Key words:Aspergillus nidulans, endo-cellulase, enzymological properties, divalent metal ion, fermentation