RT - Journal Article T1 - Cloning and heterologous expression of Laccase in Pichia pastoris and determination of some biochemical properties JF - gebsj YR - 2019 JO - gebsj VO - 8 IS - 2 UR - http://gebsj.ir/article-1-309-en.html SP - 121 EP - 131 K1 - laccase K1 - Pichia pastoris K1 - heterologous expression K1 - bioremediation K1 - cloning AB - Laccase (EC 1.10.3.2) enzymes are multi-copper oxidase which catalyze the oxidation of aromatic and non- aromatic compounds with electron reduction of molecular oxygen to water. These enzymes catalyze the oxidation of a wide array of phenolic compound and have been used in different industrial field. In this study, Nucleotide sequence of laccase (accession number: AY081188.1) was optimized according to the codon preference of Pichia pastoris. Laccase gene was synthesized and cloned into pPICZalpha A. under control of AOX1 promoter then transformed to P. pastoris by electroporation. 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 by presence of 0.4 mM Cu+2, at 25°C culture temperature, by 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 3 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 that Pichia pastoris can be useful tool for heterologous laccase expression in order to use in industrial application such as wastewater treatment, biosensor & etc. LA eng UL http://gebsj.ir/article-1-309-en.html M3 ER -