Kusumoto K, Hsieh D P
Department of Environmental Toxicology, University of California, Davis 95616, USA.
Can J Microbiol. 1996 Aug;42(8):804-10. doi: 10.1139/m96-101.
The esterases from the cell-free extracts (CFEs) of Aspergillus parasiticus ATCC15517, an aflatoxin-producing strain, catalyzing the hydrolytic conversion of versiconal hemiacetal acetate (VHA) to versiconal was biochemically studied. The specific activity of the enzymes increased 2.5-fold during incubation of mycelia through 40-55 h. No metal ions were required for enzyme stability, but EDTA at 1 mM and dithiothreitol at 0.5-5 mM increased its stability. Three peaks of VHA esterase activity were resolved when the proteins in the CFEs prepared from the mycelia of different ages were separated by anion-exchange column chromatography, suggesting that at least three VHA esterases were present in the eluate of this purification step. One of these esterases extracted from the mycelia of a 55-h culture was partially purified in five steps by means of preparative chromatography and fast protein liquid chromatography. The partially purified enzyme when reacted with [14C]diisopropylfluorophosphate followed by sodium dodecyl sulfate - polyacrylamide gel electrophoresis gave a single radiolabelled band, which corresponded to a protein of 32 kDa. The molecular mass of the partially purified VHA esterase determined with gel filtration was around 60 kDa. The results suggested that the enzyme consists of two isomeric subunits.
对产黄曲霉毒素的寄生曲霉ATCC15517的无细胞提取物（CFE）中的酯酶进行了生化研究，该酯酶催化乙酸Versiconal半缩醛（VHA）水解转化为Versiconal。在菌丝体培养40 - 55小时期间，酶的比活性增加了2.5倍。酶的稳定性不需要金属离子，但1 mM的EDTA和0.5 - 5 mM的二硫苏糖醇可提高其稳定性。当用阴离子交换柱色谱法分离不同年龄菌丝体制备的CFE中的蛋白质时，可分辨出三个VHA酯酶活性峰，这表明在该纯化步骤的洗脱液中至少存在三种VHA酯酶。从55小时培养的菌丝体中提取的其中一种酯酶通过制备色谱法和快速蛋白质液相色谱法分五步进行了部分纯化。将部分纯化的酶与[14C]二异丙基氟磷酸反应，然后进行十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳，得到一条单一的放射性标记带，其对应于一种32 kDa的蛋白质。用凝胶过滤法测定的部分纯化的VHA酯酶的分子量约为60 kDa。结果表明该酶由两个异构亚基组成。
