Kolaríková Katerina, Galuszka Petr, Sedlárová Iva, Sebela Marek, Frébort Ivo
Department of Biochemistry, Palacký University, Slechtitelů 11, 783 71, Olomouc, Czech Republic.
Mol Biol Rep. 2009 Jan;36(1):13-20. doi: 10.1007/s11033-007-9146-7. Epub 2007 Sep 27.
The aim of this work was to prepare recombinant amine oxidase from Aspergillus niger after overexpressing in yeast. The yeast expression vector pDR197 that includes a constitutive PMA1 promoter was used for the expression in Saccharomyces cerevisiae. Recombinant amine oxidase was extracted from the growth medium of the yeast, purified to homogeneity and identified by activity assay and MALDI-TOF peptide mass fingerprinting. Similarity search in the newly published A. niger genome identified six genes coding for copper amine oxidase, two of them corresponding to the previously described enzymes AO-I a methylamine oxidase and three other genes coding for FAD amine oxidases. Thus, A. niger possesses an enormous metabolic gear to grow on amine compounds and thus support its saprophytic lifestyle.
这项工作的目的是在酵母中过表达后,从黑曲霉制备重组胺氧化酶。使用包含组成型PMA1启动子的酵母表达载体pDR197在酿酒酵母中进行表达。从酵母的生长培养基中提取重组胺氧化酶,纯化至同质,并通过活性测定和MALDI-TOF肽质量指纹图谱进行鉴定。在新发表的黑曲霉基因组中进行相似性搜索,鉴定出六个编码铜胺氧化酶的基因,其中两个与先前描述的酶AO-I(一种甲胺氧化酶)相对应,另外三个基因编码FAD胺氧化酶。因此,黑曲霉拥有巨大的代谢机制以在胺化合物上生长,从而支持其腐生生活方式。