Abstract
BACKGROUND: Fungal peroxidases are oxidoreductases that utilize hydrogen peroxide to catalyze lignin biodegradation. RESULTS: PER-K (peroxidase synthesis codon gene) was transformed from Aspergillus niger strain AN512 deposited in the National Center for Biotechnology Information with the accession number OK323140 to Escherichia coli strain (BL21-T7 with YEp356R recombinant plasmid) via calcium chloride heat-shock method. The impact of four parameters (CaCl(2) concentrations, centrifugation time, shaking speed, growth intensity) on the efficacy of the transformation process was evaluated. Furthermore, peroxidase production after optimization was assessed both qualitatively and quantitatively, as well as SDS-PAGE analysis. The optimum conditions for a successful transformation process were as follows: CaCl(2) concentrations (50 mM), centrifugation time (20 min), shaking speed (200 rpm), and growth optical density (0.45). PCR and gel electrophoresis detect DNA bands with lengths 175, 179, and 211 bps corresponding to UA3, AmpR, and PER-K genes respectively besides partially sequencing the PER-K gene. Pyrogallol/hydrogen peroxide assay confirmed peroxidase production, and the activity of the enzyme was determined to be 3924 U/L. SDS-PAGE analysis also confirms peroxidase production illustrated by the appearance of a single peroxidase protein band after staining with Coomassie blue R-250. CONCLUSION: A successful peroxidase-gene (PER-K) transformation from fungi to bacteria was performed correctly. The enzyme activity was screened, and partial sequencing of PER-K gene was analyzed successively. The protein 3D structure was generated via in silico homology modeling, and determination of binding sites and biological annotations of the constructed protein were carried out via COACH and COFACTOR based on the I-TASSER structure prediction.