Cloning and heterologous expression of Laccase in pichia pastoris and determination some of biochemical properties

Laccase (EC 1.10.3.2) are multi-copper oxidase which catalyze the oxidation aromatic and non- aromatic compounds with electron reduction of molecular oxygen to water. Nucleotide sequence of laccase (accession number : ) was optimized according codon preference of Pichia pastoris. Gene was synthesized and cloned into pPICZalpha A. laccase under control of AOX1 promoter was transformed to P.pastoris by electroporation and integrated to P.pastoris genome. Methanol concentration, copper salts concentration and temperature were varied in order to enhance laccase expression in the heterologous system. Optimal fermentation condition for laccase production in shake flask cultivation using BMGY medium were obtained: presence of 0.4 mM Cu+2, culture temperature 25°C, 0.8% methanol added into culture every 24 h. the volumetric activity was achieved 9600 U/L after 12 day culture in fernbach flask. The biochemical properties of recombinant laccase and kinetic parameters were studied. The laccase activity was optimal at pH 4 and 40°C. Its Km value was 0.089 mM For ABTS [2, 2´azino-bis (3-ethylbenzthiazoline-6-sulphoric acid)]. The result of this study was showed Pichia pastoris can be useful tool for heterologous laccase expression in order to use in industrial application such as: waste water treatment, biosensor & etc.