Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-alpha-D-mannosidase.
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.
Maras
M
M
Department of Molecular Biology, Flanders Interuniversity Institute for Biotechnology, Ghent, Belgium.
Callewaert
N
N
Piens
K
K
Claeyssens
M
M
Martinet
W
W
Dewaele
S
S
Contreras
H
H
Dewerte
I
I
Penttilä
M
M
Contreras
R
R
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*
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