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