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PCR cloning and heterologous expression of Scytalidium thermophilum laccase gene in Aspergillus sojae

Koçlar, Gülden
In this study, Scytalidium thermophilum laccase gene was first cloned into E. coli and then heterologously expressed in A. sojae. S. thermophilum is a thermophilic fungus with an important role in determining selectivity of compost produced for growing Agaricus bisporus. S. thermophilum laccase gene was first cloned by Novo Nordisk Bio Tech, Inc. in 1998. This laccase gene (lccS) has an open reading frame of 2092bp. It is composed of five exons punctuated by four small introns. The coding region, excluding intervening sequences is very GC-rich (60.8% G+C) and encodes a preproenzyme of 616 amino acids: a 21 amino acid signal peptide and a 24 amino acid predicted propeptide. lccS gene was amplified using specific primers to exclude the signal and pro-peptide coding regions and ligated to expression vector pAN52-4. The recombinant plasmid was used to transform Aspergillus sojae ATCC11906 (pyrG-). Heterologuos expression was observed in glucose-containing media, under the control of the glyceraldehydes 3-phosphate dehydnogenese promoter and the secretion signal of glucoamylase gene. Laccase gene is an important step towards the high level expression of this enzyme in a GRAS eucaryotic host and for further biotransformation and enzyme engineering studies. In this study also bioinformatic analysis of N-terminal and C-terminal propeptide cleavage sites of fungal proteins including laccases were studied.