OVERPRODUCTION OF 1,2-ALPHA-MANNOSIDASE, A GLYCOCHAIN PROCESSING ENZYME, BY ASPERGILLUS-ORYZAE

A recombinant strain of Aspergillus oryzae has been constructed in whi ch 1,2-alpha-mannosidase, an intracellular glycochain professing enzym e with specificity toward 1,2-alpha-mannosidic linkages, has been over expressed. For the construction, the N-terminal signal-encoding sequen ce of the 1,2-alpha-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 ex pression plasmid pTAPM1. A transformant of A. oryzae (the strain PM-1) secreted a great deal of heterogeneous 1,2-alpha-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 c ulture. N-terminal amino acid analysis indicated that the signal pepti de was removed from the secreted enzyme. The Penicillium 1,2-alpha-man nosidase expressed in A. oryzae did not show any notable difference fr om the enzyme from P. citrinum in such properties as M-r, specific act ivity, CD spectra, or kinetic parameters. Man(7)GlcNAc(2) accumulated temporarily during the degradation of Man(9)GlcNAc(2) to Man(5)GlcNAc( 2) by fungal 1,2-alpha-mannosidase.