title: Cloning and expression of a tyrosinase from Aspergillus oryzae in Yarrowia lipolytica: application in L-DOPA biotransformation.
abstract: 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.
authors.affiliation: Institute of Bioinformatics and Biotechnology, University of Pune, Pune 411007, India.
authors.forename: Ashit
authors.initials: A
authors.lastname: Rao
authors.forename: Priya
authors.initials: P
authors.lastname: Pimprikar
authors.forename: Chandrika
authors.initials: C
authors.lastname: Bendigiri
authors.forename: Ameeta Ravi
authors.initials: AR
authors.lastname: Kumar
authors.forename: Smita
authors.initials: S
authors.lastname: Zinjarde
mesh_terms: D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D001711:Biotransformation; D003001:Cloning, Molecular; D005656:Fungal Proteins / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007980:Levodopa / Q000378:metabolism*; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics* / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism; D025062:Yarrowia / Q000235:genetics / Q000378:metabolism*
chemical_list: D005656:Fungal Proteins; D014443:Tyrosine; D007980:Levodopa; D014442:Monophenol Monooxygenase