Molecular cloning of a laccase isozyme gene from Pleurotus sajor-caju and expression in the heterologous Pichia pastoris host.
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 degrees C and pH 3.5. The enzyme was strongly inhibited by sodium azide and thioglycolic acid but not by EDTA.
Soden
D M
DM
National Food Biotechnology Centre1 and Microbiology Department2, University College, National University of Ireland, Cork, Ireland.
O'Callaghan
J
J
National Food Biotechnology Centre1 and Microbiology Department2, University College, National University of Ireland, Cork, Ireland.
Dobson
A D W
ADW
National Food Biotechnology Centre1 and Microbiology Department2, University College, National University of Ireland, Cork, Ireland.
D003001:Cloning, Molecular; D015966:Gene Expression Regulation, Fungal; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D042845:Laccase; D000432:Methanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D020076:Pleurotus / Q000201:enzymology* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D007527:Isoenzymes; D011994:Recombinant Proteins; D010088:Oxidoreductases; D042845:Laccase; D000432:Methanol