The cDNA encoding long-chain fatty-acid alcohol oxidase(FAO) was isolated by using RT-PCR method from Candida cloacae grown in a medium containing oleic acid. The cDNA of fatty-acid alcohol oxidase was cloned into exp...The cDNA encoding long-chain fatty-acid alcohol oxidase(FAO) was isolated by using RT-PCR method from Candida cloacae grown in a medium containing oleic acid. The cDNA of fatty-acid alcohol oxidase was cloned into expression plasmid pET17b under T7 promoter and then transformed into E.coli BL21/DE3. The molecular weight of the expressed protein was estimated to be approximately 64 kD by SDS-PAGE. The expressed protein had a specific catalytic activity of fatty-acid alcohol oxidase and its activity was 1090±116 U/ml medium. Northern blotting revealed that there was high expression of fao mRNA in Candida cloacae yeast grown in a medium containing oleic acid.展开更多
分离纯化刺芹侧耳Pleurotus eryngii芳基醇氧化酶,并探究其酶学性质。通过硫酸铵盐沉、DEAE-Sepharose Fast Flow弱阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析和Source 15Q强阴离子交换层析,得到纯化的单一酶。经肽...分离纯化刺芹侧耳Pleurotus eryngii芳基醇氧化酶,并探究其酶学性质。通过硫酸铵盐沉、DEAE-Sepharose Fast Flow弱阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析和Source 15Q强阴离子交换层析,得到纯化的单一酶。经肽指纹图谱鉴定,确定其为芳基醇氧化酶,酶活回收率25.5%,纯化倍数38.2。结合SDS-PAGE和IEF-PAGE分析,确定其分子量和等电点分别为70k Da和4.2。以藜芦醇为底物,该酶最适反应p H为6.0,最适反应温度为70℃,金属离子Zn^(2+)、Fe^(2+)和Cu^(2+)对芳基醇氧化酶的活性抑制作用明显,Km和Vmax分别为0.921mmol/L和80U/mg。展开更多
文摘The cDNA encoding long-chain fatty-acid alcohol oxidase(FAO) was isolated by using RT-PCR method from Candida cloacae grown in a medium containing oleic acid. The cDNA of fatty-acid alcohol oxidase was cloned into expression plasmid pET17b under T7 promoter and then transformed into E.coli BL21/DE3. The molecular weight of the expressed protein was estimated to be approximately 64 kD by SDS-PAGE. The expressed protein had a specific catalytic activity of fatty-acid alcohol oxidase and its activity was 1090±116 U/ml medium. Northern blotting revealed that there was high expression of fao mRNA in Candida cloacae yeast grown in a medium containing oleic acid.
文摘分离纯化刺芹侧耳Pleurotus eryngii芳基醇氧化酶,并探究其酶学性质。通过硫酸铵盐沉、DEAE-Sepharose Fast Flow弱阴离子交换层析、Sephacryl S-200 High Resolution凝胶过滤层析和Source 15Q强阴离子交换层析,得到纯化的单一酶。经肽指纹图谱鉴定,确定其为芳基醇氧化酶,酶活回收率25.5%,纯化倍数38.2。结合SDS-PAGE和IEF-PAGE分析,确定其分子量和等电点分别为70k Da和4.2。以藜芦醇为底物,该酶最适反应p H为6.0,最适反应温度为70℃,金属离子Zn^(2+)、Fe^(2+)和Cu^(2+)对芳基醇氧化酶的活性抑制作用明显,Km和Vmax分别为0.921mmol/L和80U/mg。
