Kumar Adepu Kiran, Goswami Pranab
Department of Biotechnology, Indian Institute of Technology Guwahati, Guwahati-781039, Assam, India.
Biochim Biophys Acta. 2008 Nov;1784(11):1552-9. doi: 10.1016/j.bbapap.2008.06.009. Epub 2008 Jun 24.
An alcohol oxidase was isolated from the microsome of n-hexadecane grown Aspergillus terreus and purified by ion exchange chromatography. The oxidase was found to act on short chain-, long chain-, secondary-, and aromatic-alcohol substrates with highest affinity for n-heptanol (K(M)=0.498 mM, K(cat)=2.7x10(2) s(-1)). The native protein molecular mass was 269+/-5 kDa and the subunit molecular masses were 85-, 63-, 43-, 27-, and 13-kDa. The isoelectric point of the proteins was within 8.3-8.5. High aggregating property of the protein was demonstrated by AFM, DLS and TEM analyses. Chemical analysis showed the presence of oleic acid and palmitic acid at a ratio of 2:1 in the purified protein. This lipoidic nature of the protein particles was correlated to the high aggregating property. In this flavoenzyme, flavin was non-covalently but avidly associated. Peptide mass fingerprinting studies showed the presence of two FAD binding domains in 63 kDa protein. Among these two FAD binding domain sequences only the YPVIDHEYDAVVVGAGGAGLR peptide shows 45-50% sequence homology with the reported N-terminal sequences of other known alcohol oxidases. Non-redundant database search of 63- and 43-kDa subunits peptide sequences showed no sequence similarity with the other alcohol oxidase protein reported till now.
从以正十六烷为碳源培养的土曲霉微粒体中分离出一种醇氧化酶，并通过离子交换色谱法进行纯化。发现该氧化酶作用于短链、长链、仲链和芳香醇底物，对正庚醇的亲和力最高（K(M)=0.498 mM，K(cat)=2.7x10(2) s(-1)）。天然蛋白质分子量为269±5 kDa，亚基分子量分别为85 kDa、63 kDa、43 kDa、27 kDa和13 kDa。蛋白质的等电点在8.3 - 8.5之间。原子力显微镜（AFM）、动态光散射（DLS）和透射电子显微镜（TEM）分析表明该蛋白质具有高度聚集特性。化学分析显示纯化后的蛋白质中油酸和棕榈酸的比例为2:1。蛋白质颗粒的这种类脂性质与高度聚集特性相关。在这种黄素酶中，黄素是非共价但紧密结合的。肽质量指纹图谱研究表明在63 kDa的蛋白质中存在两个FAD结合结构域。在这两个FAD结合结构域序列中，只有YPVIDHEYDAVVVGAGGAGLR肽与其他已知醇氧化酶报道的N端序列显示出45 - 50%的序列同源性。对63 kDa和43 kDa亚基肽序列进行非冗余数据库搜索，结果显示与迄今报道的其他醇氧化酶蛋白质没有序列相似性。
