Hexose oxidase from the red alga Chondrus crispus. Purification, molecular cloning, and expression in Pichia pastoris.
Hexose oxidase from Chondrus crispus catalyzes the oxidation of a variety of mono- and disaccharides including D-glucose, D-galactose, maltose, and lactose. The enzyme has previously been partially purified and was reported to be a highly glycosylated, copper-containing protein with a relative molecular mass of approximately 130,000 (Sullivan, J. D., and Ikawa, M. (1973) Biochim. Biophys. Acta 309, 11-22). We report here the purification to homogeneity of hexose oxidase from C. crispus. The purified enzyme was cleaved with cyanogen bromide and endoproteinase Lys-C and the peptide fragments were subjected to amino acid sequence analysis. Oligonucleotides were designed on the basis of the peptide sequences and a cDNA clone encoding C. crispus hexose oxidase was obtained using polymerase chain reaction on reverse transcribed cDNA. The nucleotide sequence of the hexose oxidase cDNA contained an open reading frame of 546 amino acid residues with a predicted relative molecular mass of 61,898. No significant sequence similarity was found between hexose oxidase and other protein sequences available in data bases. Expression of the hexose oxidase cDNA in Pichia pastoris as an active enzyme confirmed the identity of the DNA sequence. Native hexose oxidase from C. crispus was characterized and compared with purified, recombinant enzyme.
Hansen
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Biotechnological Institute, Koglevej 2, DK-2970 Horsholm, Denmark.
Stougaard
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D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D004187:Disaccharides / Q000378:metabolism; D017343:Genes, Plant; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000378:metabolism; D010843:Pichia / Q000235:genetics; D011994:Recombinant Proteins / Q000378:metabolism; D000461:Rhodophyta / Q000737:chemistry / Q000201:enzymology / Q000235:genetics*; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity
D018076:DNA, Complementary; D004187:Disaccharides; D009005:Monosaccharides; D011994:Recombinant Proteins; D000429:Alcohol Oxidoreductases; C021354:hexose oxidase