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铅黄肠球菌(Enterococcus casseliflavus)在诱导剂甘油存在的条件下,经发酵培养可合成和分泌α-磷酸甘油氧化酶。该菌的无细胞抽提液可通过聚乙二醇(PEG)/(NH4)2SO4双水相萃取法有效进行α-磷酸甘油氧化酶的分离和纯化。研究结果表明,PEG分子大小、PEG浓度、pH、(NH4)2SO4浓度和无机盐及粗酶液的加入量是影响该萃取系统的主要因素,最终确定的最适双水相系统为16.5%(m/m)PEG2000、13.2%(m/m)(NH4)2SO4、pH7.5和30%(m/m)的粗酶加入量。无机盐NaCl的加入对萃取α-磷酸甘油氧化酶会产生负面影响,导致低的纯化倍数和活性回收率。此双水相萃取体系用于放大提取α-磷酸甘油氧化酶,其酶活性回收率可达89%、分配率为1.2、纯化倍数为7.0。
Enterococcus casseliflavus can be synthesized and secreted α-phosphoglycerol oxidase by fermentation in the presence of an inducer of glycerol. The bacterial cell-free extract of the bacterium can effectively separate and purify the α-phosphoglycerol oxidase by a polyethylene glycol (PEG) / (NH 4) 2 SO 4 aqueous two-phase extraction method. The results showed that PEG molecular size, PEG concentration, pH, (NH4) 2SO4 concentration and inorganic salts and crude enzyme solution were the main factors affecting the extraction system. The final optimum aqueous system was 16.5% ( m / m) PEG2000, 13.2% (m / m) (NH4) 2SO4, pH 7.5 and 30% (m / m) The addition of inorganic salts NaCl negatively affects the extraction of alpha-glycerophosphate oxidase resulting in low purification fold and activity recovery. This aqueous two-phase extraction system is used to amplify and extract α-phosphoglycerol oxidase with an average recovery rate of 89%, a partition ratio of 1.2 and a purification fold of 7.0.