醋酸菌中乙醛脱氢酶的分离纯化及酶学性质

Isolation,purification and enzymatic properties of aldehyde dehydrogenase from Acetobacter pomorum

以实验室筛选得到的醋酸菌(Acetobacter pomorum)为实验菌株发酵产酶,通过细胞破壁,采用(NH_4)_2SO_4沉淀、透析、DEAE-Sepharose离子交换层析及Superdex G-75凝胶过滤层析分离纯化得到乙醛脱氢酶的酶液,并考察其酶学性质。该酶分子质量为221.60 k Da,单个亚基分子质量约为54.41 k Da,为四聚体结构;纯酶液比活力20.25 U/mg,纯化倍数为10.16倍,乙醛脱氢酶(aldehyde dehydrogenase,ALDH)的回收率为6.53%。酶学性质研究表明,ALDH促进乙醛分解的最适温度为50℃,40~50℃相对酶活力稳定性好;该酶的最适p H为7.0,当p H在5.5~7.5内酶活力表现稳定;金属离子对酶活性的影响实验表明,Na^+、K^+、Zn^(2+)、Ba^(2+)对该酶酶有不同程度抑制作用,而Mg^(2+)、Ca^(2+)、Al^(3+)、Li^+、Cu^(2+)具有促进作用;ALDH的最适底物为乙醛,相对偏好直链醛类。ALDH活性较大,为后期表达和深入研究其生物学功能提供理论和数据支持。

Acetaldehyde dehydrogenase（ALDH）was isolated and purified by the Acetobacter pomorum obtained from the selfscreening of the laboratory,cell disruption,ammonium sulfate fractionation,DEAE-Sepharose fast flow chromatography and Superdex G-75 prep grade chromatography separation and purification of enzyme liquid aldehyde dehydrogenase,and its enzymatic properties was studied.The molecular weight of the enzyme was 221.60 k Da,in which the subunit molecular mass was54.41 k Da,respectively.The crude enzyme was purified 10.16 times with 20.25 U/mg of enzyme activity and the recovery rate of ALDH was 6.53%.The enzyme properties showed that its optimal reaction temperature was 50℃,and the enzyme had a good stability between 40℃and 50℃;The optimal reaction p H of ALDH was 7.0,and there was a good stability between p H5.5and p H7.5.The effects of different metal ions on enzyme activity showed that the enzyme was strongly inhibited by Na＋,K＋,Zn^（2＋）,Ba^（2＋）,however,Mg^（2＋）、Ca^（2＋）、Al^（3＋）、Li＋、Cu^（2＋）activated the enzyme activity.The substrate specificity of ALDH showed that the enzyme had higher enzyme activity on acetaldehyde,which was on straight aldehydes.ALDH had high enzyme activity,which provided a foundation for further investigation of biological function.