Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411007, India.
Appl Microbiol Biotechnol. 2011 Dec;92(5):951-9. doi: 10.1007/s00253-011-3400-6. Epub 2011 Jun 18.
Amplification of the tyrosinase gene (melO) from the genomic DNA of Aspergillus oryzae NCIM 1212 yielded a 1.6-kb product. This gene was cloned into pYLEX1, and the resulting pTyro-YLEX1 vector was transformed in Yarrowia lipolytica strain Po 1 g. A clone displaying the highest specific activity for tyrosinase (10.94 U/mg) was used for obtaining the complementary DNA (cDNA) and for protein expression studies. cDNA sequence analysis indicated the splicing of an intron present in the melO gene by Po 1 g. Native polyacrylamide gel electrophoresis, acidification at pH 3.0 followed by activity staining with L-DOPA indicated the expression of an active tyrosinase. The clone over-expressing the tyrosinase transformed L-tyrosine to L-DOPA. On optimization of conditions for the biotransformation (pH 4.0, temperature 60° C and with 3.5 mg of biomass), 0.4 mg/ml of L-DOPA was obtained.
从米曲霉 NCIM 1212 的基因组 DNA 中扩增得到酪氨酸酶基因（melO），得到一个 1.6kb 的产物。该基因被克隆到 pYLEX1 中，所得的 pTyro-YLEX1 载体被转化到解脂耶氏酵母 Po 1 g 中。一个显示出最高酪氨酸酶比活性（10.94 U/mg）的克隆被用于获得 cDNA 和进行蛋白质表达研究。cDNA 序列分析表明，Po 1 g 对 melO 基因中的内含子进行了拼接。天然聚丙烯酰胺凝胶电泳、pH 3.0 酸化后用 L-DOPA 进行活性染色表明表达了一种活性的酪氨酸酶。过表达酪氨酸酶的克隆将 L-酪氨酸转化为 L-DOPA。在优化生物转化条件（pH 4.0、温度 60°C 和 3.5mg 生物质）后，获得了 0.4mg/ml 的 L-DOPA。
