Cloning and extracellular expression of laccase enzyme from bacillus of Iranian hot spring into yeast cell Pichia pastoris

Laccase (EC 1. 10. 3. 2) can use to broad range oxidation of phenolic and non-phenolic substrate. Yeast host cell is highly acceptable for the production because of advantages in promoterstrength، secretion efficiency، or ease of growth to high cell density and non-toxicity than other host cell resources. The laccase gene from bacillus sp HR30 was synthesized with codon usage of pichia pastoris. Then، it was sub-cloned in expression vector (Ppink-α) and transferred in Pichia pastoris yeast host cell via electroporation method. For certification of cloning، the colony PCR and restriction enzyme digestion was done and the 1542 bp fragment was observed. Then، the laccase gene was expressed under different condition of temperature and copper sulphate concentration. The results showed that at these conditions، the enzyme activation on the syringaldazine substrate was variable. Also، reducing of temperature up to 20°C and 2 mM copper sulphate was caused improving of refolding and activity of enzyme.