Molecular cloning and characterization of a laccase gene from the basidiomycete Fome lignosus and expression in Pichia pastoris.
A cDNA encoding for a laccase was isolated from the white-rot fungus Fome lignosus by RT-PCR. It contained an open reading frame of 1,557 bp. The deduced mature protein consisted of 497 amino acids and was preceded by a signal peptide of 21 amino acids. The genomic DNA of the laccase, containing 11 introns, was cloned by PCR. The cDNA was cloned into the vectors pGAPZalphaA and pGAPZA, and expressed in the Pichia pastoris GS115. Laccase-secreting transformants were selected by their ability to oxidize the substrate 2'2-azinobis-(3-ethylbenzthiaoline-6-sufonic acid) (ABTS). The laccase activity obtained with the native signal peptide was found to be fivefold higher than that obtained with the alpha-factor secretion signal peptide. The presence of 0.4 mM copper was necessary for optimal activity of the enzyme. The highest activity value reached 9.03 U ml(-1), and the optimal secreting time was 2~3 days at 20 degrees C. The crude laccase was stable in a pH range from 6.0 to 10.0 and at temperatures lower than 30 degrees C in pH 4.5 for 24 h. The molecular mass of the enzyme was estimated to be 66.5 kDa by SDS-PAGE. The optimum pH and temperature were 2.4 and 55 degrees C. The Km and Vmax values for ABTS were 177 microM and 23.54 micromol min(-1) respectively. The extent of glycosylation of the purified enzyme was 58.6%.
Liu
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W
Department of Enzymology, Institute of Microbiology, Chinese Academy of Sciences, PO Box 2714, 100080 Beijing, PR China.
Chao
Y
Y
Liu
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S
Bao
H
H
Qian
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001487:Basidiomycota / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary; D042845:Laccase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000201:enzymology* / Q000235:genetics; D017422:Sequence Analysis, DNA
D018076:DNA, Complementary; D042845:Laccase