Molecular cloning and expression in S. cerevisiae of two exochitinases from Trichoderma harzianum.
The synthetic exochitinase substrate 4-methylumbelliferyl N-acetylglucosamine was used to identify seven full-length exochitinase-encoding cDNAs from a Trichoderma harzianum cDNA library by expression in yeast. The cDNA clones represented transcripts of two exochitinase genes, designated as exc1 and exc2, which cross-hybridized under moderate stringency conditions in genomic Southern blots. The exc1 cDNA encodes a 578 amino acid polypeptide showing 72% similarity to the exc2-encoded 602-residue polypeptide. The deduced exochitinase amino acid sequences were found to be homologous with mammalian and fungal hexosaminidases as well as a bacterial chitobiosidase. The substrate specificity of the recombinant enzymes expressed in S. cerevisiae indicates that the enzymes are N-acetylglucosaminidases releasing single N-acetylglucosamine residues from the non-reducing end of the chitin substrate.
Draborg
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H
GeneExpress, Novo Nordisk A/S, Copenhagen O, Denmark.
Kauppinen
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S
Dalbøge
H
H
Christgau
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary; D004023:Dictyostelium / Q000201:enzymology; D005656:Fungal Proteins; D015870:Gene Expression; D015723:Gene Library; D005800:Genes, Fungal; D006596:Hexosaminidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D012441:Saccharomyces cerevisiae; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic; D014242:Trichoderma / Q000201:enzymology* / Q000235:genetics
D018076:DNA, Complementary; D005656:Fungal Proteins; D011994:Recombinant Proteins; C097190:Exc1 protein, Trichoderma harzianum; C097191:Exc2 protein, Trichoderma harzianum; D006596:Hexosaminidases