Cloning and characterization of a tyrosinase gene from the white-rot fungus Pycnoporus sanguineus, and overproduction of the recombinant protein in Aspergillus niger.
A new tyrosinase-encoding gene (2,204 bp) and the corresponding cDNA (1,857 nucleotides) from the white-rot fungus Pycnoporus sanguineus BRFM49 were cloned. This gene consisted of seven exons and six introns and encoded a predicted protein of 68 kDa, exceeding the mature tyrosinase by 23 kDa. P. sanguineus tyrosinase cDNA was over-expressed in Aspergillus niger, a particularly suitable fungus for heterologous expression of proteins of biotechnological interest, under the control of the glyceraldehyde-3-phosphate-dehydrogenase promoter as strong and constitutive promoter. The glucoamylase preprosequence of A. niger was used to target the secretion. This construction enabled the production of recombinant tyrosinase in the extracellular medium of A. niger. The identity of the purified recombinant protein was confirmed by N-terminal amino acid sequencing. The maturation process was shown to be effective in A. niger, and the recombinant enzyme was fully active, with a molecular mass of 45 kDa. The best transformant obtained, A. niger D15#26-e, produced extracellular tyrosinase activities of 534 and 1,668 U l(-1) for monophenolase and diphenolase, respectively, which corresponded to a protein yield of ca. 20 mg l(-1).
Halaouli
Sonia
S
UMR 1163 INRA-Université de Provence de Biotechnologie des Champignons Filamenteux, IFR 86 de Biotechnologie Agro-Industrielle de Marseille, 163 Avenue de Luminy, Case 925, 13288 Marseille Cedex 09, France.
Record
Eric
E
Casalot
Laurence
L
Hamdi
Moktar
M
Sigoillot
Jean-Claude
JC
Asther
Marcel
M
Lomascolo
Anne
A
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics*; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics
D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase