Cloning and expression of fungal Trametes Laccase gene in yeast host and characterization of recombinant enzyme biochemical properties and kinetic parameters
Subject Areas : Food Microbial Contamination
Ardeshir Hesampour
1
(Department of Biology, Tehran Central Branch, Islamic Azad University)
Nooshin Mohandesi
2
(Department of Biology, Islamic Azad University Central Tehran Branch, Tehran, Iran)
Keywords: Yeast, Pichia Pastoris, Laccase, Trametes,
Abstract :
Laccases have received much attention from researchers in last decades due to their ability to oxidase both phenolic and non-phenolic lignin related compounds as well as highly recalcitrant environmental pollutants, which makes them very useful for their application to several biotechnological processes. Despite of Trametes Laccase wildly application as detoxification of industrial effluents, its native low expression, makes it unsuitable for industrial application. In this study, heterologous expression of Trametes Laccase gene in methylotrophic yeast, P.pastoris with αMF as signal peptide and PpinkαHC expression vector under methatol induction in BMGY and BMMY media was investigated and expressed active recombinant laccase biochemical and biophysical properties in compare with native Trametes Laccase was studied. According to codon performance of P.pastoris, Trametes Laccase gene sequence designed and synthetic genes under control of inducible AOX1 promoter was successfully high expressed as active form in yeast host, SDS-PAGE analysis showed the recombinant laccase in size of 65KDa with Km=140µM.Analysis of Laccase biochemical and biophysical properties demonstrated that recombinant Laccase has wide pH profile with pH optima 4.8 and optimum temperature of 60 °C with high activity in P.pastoris host. We conclude that recombinant P.pastoris Laccase could fulfil a serried of predefined industrial quality criteria to be used in industry like, broad pH optimum ,substrate specify and proper thermal stability. Therefore studied methylotrophic yeast could be an appropriate host for expression and production of recombinant Laccase, with industrial application.
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