Isolation and characterization of a novel tyrosinase enzyme from native bacterial straine, production of the enzyme by cloning, characterization of the recombinant enzyme and analysis of its biotechnological applications such as production of l-dopa and melanin

Tyrosinase is a type 3 copper-containing enzyme that catalyzes the conversion of L-tyrosine to L-DOPA and finally to melanin. Melanin has a broad spectrum of biotechnological and biological functions and it is widely used in pharmacology, cosmetics and other industrial fields. L-DOPA is the preferred drug for treatment of Parkinson's diseases. In this study a Bacillus sp. having tyrosinase enzyme was isolated from native and the strain was identified by morphological, biochemical and molecular analysis. Production of the enzyme by the native isolate was optimized using classical and statistical (Box- Behnken design) methods and the enzyme was characterized. After that the tyrosinase coding gene was cloned in two expression system (E.coli BL21 (DE3) pLysS and Rosetta-gami 2 (DE3) pLysS) using pET22b as an expression vector and expression of the gene in the two expression system was compared. The gene fragment was sequenced using with Sanger sequencing method. The sequence was subjected to insilico analysis using different bioinformatics programs and websites. Conversion of l-tyrosine to L-DOPA and melanin was evaluated by TLC and HPLC methods. The isolated strain showed up to 99% homology with Bacillus megaterium. Production of the enzyme by the native M36 Bacillus sp. was determined as 0.05IU/ml, after optimization of culture condition it was reached to 0.38 IU/ml; also this product was reached to 31 IU/ml after optimization of expression of recombinant M36 tyrosinase enzyme. The optimum temperature for native and recombinant M36 tyrosinase activity was 40 °C and 45-50°C also; optimum pH for native and recombinant enzyme activity was 7.0 and 7.5 respectively. In SDS-PAGE analysis the native and recombinant M36 tyrosinase enzyme determined as 34kDa and 35 kDa, respectively. M36 melanin produced in this research, showed structural similarity with standard synthetic melanin from sigma according to the solubility, FT-IR and EPR analysis. Also it showed notable anti-bacterial, anti-UV and ant-oxidant effects. MTT assay showed the M36 melanin to have anti-cancer effect.