宇佐美曲霉酸性β-甘露聚糖酶的纯化及性质研究

Purification and Some Properties of an Acidic β-Mannanase from Aspergillus usamii

采用硫酸铵分部盐析、Phenyl Sepharose CL-4B疏水层析、Sephadex G-75凝胶过滤层析、DEAE Sepharosefast flow阴离子交换层析等分离技术,从宇佐美曲霉(Aspergillus usamii)YL-01-78菌株固态曲浸出液中分离出1种SDS-PAGE电泳纯和HPLC色谱纯的酸性β-甘露聚糖酶组分,其最终回收率为12.6%,纯化倍数为32.3。经SDS-PAGE和凝胶过滤层析测得该纯化酶的分子质量分别为42 ku和40 ku,表明该酶以单体形式存在;其等电点pI为4.2;含糖量为23.2%。以角豆胶半乳甘露聚糖为底物,测得该酶的Km值和Vmax值分别为6.3 mg/mL和1 667μmol/(min.mg)。

An acidic β- mannanase from solid-state fermentation culture of the strain Aspergillus usamii YL- 01-78 was purified by buffer extraction, ammonium sulfate precipitation, Phenyl Sepharose CL-4B hydrophobic interaction chromatography, Sephadex G-75 gel filtration and DEAE Sepharose Fast Flow ion-exchange chromatography. The purified hydrolase was homogeneous as examined by SDS-PAGE and HPLC. After these steps the enzyme was purified by 32.3-fold with a recovery of 12.6 %. The molecular mass of the purified enzyme was estimated to be 40 ku by Sephadex G-75 gel filtration and 42 ku by SDS-PAGE, which indicated that the β-mannanase was a monomer. The isoelectric point was estimated to be 4.2 by IEF-PAGE. Its carbohydrate content was 23.2 %. Kinetics coefficients Km and Vmax Of the β-mannanase were 6.3 mg/mL and 1 667 μmol. min^- 1. mg- 1 respectively with the galactomannan from locust bean gum as substrate.