Overproduction of 1,2-alpha-mannosidase, a glycochain processing enzyme, by Aspergillus oryzae.
A recombinant strain of Aspergillus oryzae has been constructed in which 1,2-alpha-mannosidase, an intracellular glycochain processing enzyme with specificity toward 1,2-alpha-mannosidic linkages, has been overexpressed. For the construction, the N-terminal signal-encoding sequence 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 expression 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 culture. N-terminal amino acid analysis indicated that the signal peptide was removed from the secreted enzyme. The Penicillium 1,2-alpha-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-alpha-mannosidase.
Yoshida
T
T
Department of Applied Biological Chemistry, Faculty of Agriculture, Tohoku University, Sendai, Japan.
Nakajima
T
T
Ichishima
E
E
D000595:Amino Acid Sequence; D001236:Aspergillus oryzae / Q000201:enzymology* / Q000235:genetics; D015153:Blotting, Western; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D007700:Kinetics; D008361:Mannosidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics; D010957:Plasmids / Q000737:chemistry; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics; D013050:Spectrometry, Fluorescence; D014170:Transformation, Genetic
D011994:Recombinant Proteins; D008361:Mannosidases; C046090:mannosyl-oligosaccharide 1,2-alpha-mannosidase