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Cloning and heterologous expression of Laccase in Pichia pastoris and determination of some biochemical properties
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Elham Soleimani   , GhorbanAli Nematzadeh * , Rouha Kasra kermanshahi , Susana Rodriguez-Couto |
| Genetic & Agricultural Biotechnology Institute of Tabarestan(GABIT). Sari Agricultural Sciences & Natural Resources University (SANRU), Sari, Iran , Gh.nematzadeh@gmail.com |
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Abstract: (4949 Views) |
| 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. |
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| Keywords: laccase, Pichia pastoris, heterologous expression, bioremediation, cloning |
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Full-Text [PDF 898 kb]
(1698 Downloads)
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Type of Study: Research |
Subject:
Microrganisms and Viruses
Received: 2019/07/10 | Accepted: 2019/08/26 | Published: 2019/11/11
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