Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host

• Published in: Microbiology (Society for General Microbiology) Vol. 148; no. 12; pp. 4003 - 4014
• Main Authors: Soden, D. M, O'Callaghan, J, Dobson, A. D. W
• Format: Journal Article
• Language: English
• Published: Reading Soc General Microbiol 01.12.2002; Society for General Microbiology
• Subjects: AOX1 gene; Biological and medical sciences; Cloning, Molecular; Fundamental and applied biological sciences. Psychology; Gene Expression Regulation, Fungal; Growth, nutrition, metabolism, transports, enzymes. Molecular biology; Isoenzymes - antagonists & inhibitors; Isoenzymes - chemistry; Isoenzymes - genetics; Isoenzymes - metabolism; lac4 gene; Laccase; methanol; Methanol - pharmacology; Microbiology; Molecular Sequence Data; Mycology; Oxidoreductases - antagonists & inhibitors; Oxidoreductases - chemistry; Oxidoreductases - genetics; Oxidoreductases - metabolism; Pichia - enzymology; Pichia - genetics; Pichia pastoris; Pleurotus - enzymology; Pleurotus - genetics; Pleurotus sajor-caju; Promoter Regions, Genetic; Recombinant Proteins - metabolism; Sequence Analysis, DNA; Substrate Specificity; thioglycolic acid; Enzyme; Isozyme; Basidiomycetes; Gene expression; Pichia; Fungi; Pleurotus; Ascomycetes; Carbohydrate; Oxidoreductases; Molecular cloning; Heterologous; Thallophyta; Laccase
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-148-12-4003

National Food Biotechnology Centre 1 and Microbiology Department 2 , University College, National University of Ireland, Cork, Ireland Author for correspondence: A. D. W. Dobson. Tel: +353 21 4902743. Fax: +353 21 4903101. e-mail: a.dobson{at}ucc.ie The Psc lac4 gene from Pleurotus sajor-caju has been cloned and expressed in the heterologous host Pichia pastoris , under the control of the AOX1 methanol inducible promoter. The native Ple. sajor-caju laccase signal sequence was effective in directing the secretion of lac4 expressed in Pic. pastoris . The control of media pH and temperature was found to be important in obtaining sufficient quantities of the protein to allow purification and subsequent biochemical characterization. The recombinant Psc Lac4 was purified to electrophoretic homogeneity and was shown to be immunologically related to Pleurotus eryngii Lac1. The purified laccase was estimated to have a molecular mass of around 59 kDa, to have a carbohydrate content of approximately 7% and a calculated pI of 4·38. The enzyme oxidized the substrates 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol, syringaldazine and guaiacol, exhibiting optimal pHs of 3·3, 6, 6·5 and 7 respectively. With ABTS as substrate the enzyme displayed optimal activity at 35 °C and pH 3·5. The enzyme was strongly inhibited by sodium azide and thioglycolic acid but not by EDTA. Keywords: white-rot fungus, heterologous expression, laccase, biochemical characterization Abbreviations: ABTS, 2,2-azinobis(3-ethylbenzthiazoline-6-sulphonate); DMP, 2,6-dimethoxyphenol; GRAS, generally regarded as safe The GenBank accession number for the sequence of the Pleurotus sajor-caju laccase 4 gene ( lac4 ) reported in this paper is AF297228 .