Molecular Cloning and Characteristic Features of a Novel Extracellular Tyrosinase from Aspergillus niger PA2.
Aspergillus niger PA2, a novel strain isolated from waste effluents of food industry, is a potential extracellular tyrosinase producer. Enzyme activity and L-DOPA production were maximum when glucose and peptone were employed as C source and nitrogen source respectively in the medium and enhanced notably when the copper was supplemented, thus depicting the significance of copper in tyrosinase activity. Tyrosinase-encoding gene from the fungus was cloned, and amplification of the tyrosinase gene yielded a 1127-bp DNA fragment and 374 amino acid residue long product that encoded for a predicted protein of 42.3 kDa with an isoelectric point of 4.8. Primary sequence analysis of A. niger PA2 tyrosinase had shown that it had approximately 99% identity with that of A. niger CBS 513.88, which was further confirmed by phylogenetic analysis. The inferred amino acid sequence of A. niger tyrosinase contained two putative copper-binding sites comprising of six histidines, a characteristic feature for type-3 copper proteins, which were highly conserved in all tyrosinases throughout the Aspergillus species. When superimposed onto the tertiary structure of A. oryzae tyrosinase, the conserved residues from both the organisms occupied same spatial positions to provide a di-copper-binding peptide groove.
Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, India.
Pragati
P
Agarwal
Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, India.
Jyoti
J
Singh
Department of Biotechnology, Indian Institute of Technology Roorkee, Roorkee, 247667, India. rpsbsfbs@iitr.ac.in.
R P
RP
Singh
D000595:Amino Acid Sequence; D001234:Aspergillus niger / Q000737:chemistry / Q000145:classification / Q000201:enzymology*; D001665:Binding Sites; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005285:Fermentation; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006639:Histidine / Q000737:chemistry* / Q000378:metabolism; D007526:Isoelectric Point; D007700:Kinetics; D007980:Levodopa / Q000096:biosynthesis*; D008958:Models, Molecular; D014442:Monophenol Monooxygenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010461:Peptones / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005656:Fungal Proteins; D010461:Peptones; D011994:Recombinant Proteins; D007980:Levodopa; D006639:Histidine; D003300:Copper; D014442:Monophenol Monooxygenase; D005947:Glucose
A. niger; Cloning; Extracellular; L-DOPA; Tyrosinase