title: Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-alpha-D-mannosidase.
abstract: A cDNA encoding 1,2-alpha-D-mannosidase mds 1 from Trichoderma reesei was cloned. The largest open reading frame occupied 1571 bp. The predicted sequence contains 523 amino acid residues for a calculated molecular mass of 56,266 Da and shows high similarity to the amino acid sequences of 1,2-alpha-D-mannosidases from Aspergillus saitoi and Penicillium citrinum (51.6 and 51.0% identity, respectively). T. reesei mannosidase was produced as a recombinant enzyme in the yeast Pichia pastoris. Replacement of the N-terminal part with the prepro-signal peptide of the Saccharomyces cerevisiae alpha-mating factor resulted in high amounts of secreted enzyme. A three-step purification protocol was designed and the enzymatic properties were analyzed. The enzyme was characterized as a class-I mannosidase.
authors.affiliation: Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, Ghent, Belgium.
authors.forename: M
authors.initials: M
authors.lastname: Maras
authors.forename: N
authors.initials: N
authors.lastname: Callewaert
authors.forename: K
authors.initials: K
authors.lastname: Piens
authors.forename: M
authors.initials: M
authors.lastname: Claeyssens
authors.forename: W
authors.initials: W
authors.lastname: Martinet
authors.forename: S
authors.initials: S
authors.lastname: Dewaele
authors.forename: H
authors.initials: H
authors.lastname: Contreras
authors.forename: I
authors.initials: I
authors.lastname: Dewerte
authors.forename: M
authors.initials: M
authors.lastname: Penttilä
authors.forename: R
authors.initials: R
authors.lastname: Contreras
mesh_terms: D000595:Amino Acid Sequence; D001230:Aspergillus / Q000201:enzymology; D003001:Cloning, Molecular; D018076:DNA, Complementary; D008361:Mannosidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000072235:Mating Factor; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology; D010455:Peptides / Q000235:genetics; D010843:Pichia / Q000201:enzymology / Q000235:genetics; D021382:Protein Sorting Signals / Q000235:genetics; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D018076:DNA, Complementary; D010455:Peptides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D000072235:Mating Factor; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase