Overproduction of 1,2-α-Mannosidase, a Glycochain Processing Enzyme, by Aspergillus oryzae

Abstract

  A recombinant strain of Aspergillus oryzae has been constructed in which 1,2-α-mannosidase, an intracellular glycochain processing enzyme with specificity toward 1,2-α-mannosidic linkages, has been overexpressed. For the construction, the N-terminal signal-encoding sequence of the 1,2-α-mannosidase gene (msdC) from Penicillium citrinum was replaced with that of the aspergillopepsin I signal, and the fused gene was inserted between amyB promoter-terminator elements in the expression plasmid pTAPM1. A transformant of A. oryzae (the strain PM-1) secreted a great deal of heterogeneous 1,2-α-mannosidase into the culture media, which was purified by CM ion-exchange chromatography. Approximately 21 mg of the purified enzyme was obtained per liter of culture. N-terminal amino acid analysis indicated that the signal peptide was removed from the secreted enzyme. The Penicillium 1,2-α-mannosidase expressed in A. oryzae did not show any notable difference from the enzyme from P. citrinum in such properties as M_r, specific activity, CD spectra, or kinetic parameters. Man₇GlcNAc₂ accumulated temporarily during the degradation of Man₉GlcNAc₂ to Man₅GlcNAc₂ by fungal 1,2-α-mannosidase.