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Volume 148, Issue 12

Molecular cloning of a laccase isozyme gene from and expression in the heterologous host

The GenBank accession number for the sequence of the laccase 4 gene () reported in this paper is AF297228.

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Abstract

The Psc gene from has been cloned and expressed in the heterologous host , under the control of the methanol inducible promoter. The native laccase signal sequence was effective in directing the secretion of expressed in . 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 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 438. The enzyme oxidized the substrates 2,2-azinobis-(3-ethylbenzthiazoline-6-sulphonate) (ABTS), 2,6-dimethoxyphenol, syringaldazine and guaiacol, exhibiting optimal pHs of 33, 6, 65 and 7 respectively. With ABTS as substrate the enzyme displayed optimal activity at 35 °C and pH 35. The enzyme was strongly inhibited by sodium azide and thioglycolic acid but not by EDTA.

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  • Published Online:
Keyword(s): ABTS, 2,2-azinobis(3-ethylbenzthiazoline-6-sulphonate) , biochemical characterization , DMP, 2,6-dimethoxyphenol , GRAS, generally regarded as safe , heterologous expression , laccase and white-rot fungus
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2002-12-01
2024-04-27
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Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris hostThe GenBank accession number for the sequence of the Pleurotus sajor-caju laccase 4 gene (lac4) reported in this paper is AF297228.
Microbiology 148, 4003 (2002); https://doi.org/10.1099/00221287-148-12-4003
/content/journal/micro/10.1099/00221287-148-12-4003
/content/journal/micro/10.1099/00221287-148-12-4003
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