Molecular cloning and enzymatic characterization of a Trichoderma reesei 1,2-alpha-D-mannosidase

A cDNA encoding 1,2-alpha-D-mannosidase mds1 from Trichoderma reesei was cl oned. The largest open reading frame occupied 1571 bp. The predicted sequen ce contains 523 amino acid residues for a calculated molecular mass of 56 2 66 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 recombi nant enzyme in the yeast Pichia pastoris. Replacement of the N-terminal par t with the prepro-signal peptide of the Saccharomyces cerevisiae alpha-mati ng factor resulted in high amounts of secreted enzyme. A three-step purific ation protocol was designed and the enzymatic properties were analysed. The enzyme was characterized as a class-I mannosidase. (C) 2000 Elsevier Scien ce B.V. All rights reserved.