Expression and characterization of tyrosinase from Agaricus bisporus in Saccharomyces cerevisiae

Abstract:

[Objective] The aim of this research was to express the tyrosinase-coding PPO2 gene from Agaricus bisporus in Saccharomyces cerevisiae and to investigate the characteristics of tyrosinase in vitro and in vivo.[Methods] We cloned PPO2 gene by RT-PCR using the total RNA extracted from Agaricus bisporus. The expression vector pSP-G1-PPO2 was constructed and transformed into yeast. The recombinant protein was purified with Ni-NTA and the tyrosinase enzyme properties were evaluated. [Results] The optimum temperature of tyrosinase in vitro was 45℃. And the optimum pH was 7.0 and 8.0 using tyrosine and L-3,4-dihydroxyphenylalanine (L-DOPA) as substrate, respectively. In Saccharomyces cerevisiae, the yield of melanin increased with the rise in substrate concentration within 2.5 mg/mL. [Conclusion] We achieved heterologous expression of tyrosinase-coding gene PPO2 from Agaricus bisporus in Saccharomyces cerevisiae and characterized the enzyme properties. The melanin production positively correlates with the concentration of substrate tyrosine which indicates that tyrosinase could be a biosensor to report the content of tyrosine in yeast and could be used for the high throughput screening of high-yield tyrosine strains.

Key words:tyrosinase;heterologous expression;Agaricus bisporus;Saccharomyces cerevisiae;enzyme properties