Cloning and characterization of Aspergillus niger genes encoding an alpha-galactosidase and a beta-mannosidase involved in galactomannan degradation.
Alpha-galactosidase (EC 3.2.1.22) and beta-mannosidase (EC 3.2.1.25) participate in the hydrolysis of complex plant saccharides such as galacto(gluco)mannans. Here we report on the cloning and characterization of genes encoding an alpha-galactosidase (AglC) and a beta-mannosidase (MndA) from Aspergillus niger. The aglC and mndA genes code for 747 and 931 amino acids, respectively, including the eukaryotic signal sequences. The predicted isoelectric points of AglC and MndA are 4.56 and 5.17, and the calculated molecular masses are 79.674 and 102.335 kDa, respectively. Both AglC and MndA contain several putative N-glycosylation sites. AglC was assigned to family 36 of the glycosyl hydrolases and MndA was assigned to family 2. The expression patterns of aglC and mndA and two other genes encoding A. niger alpha-galactosidases (aglA and aglB) during cultivation on galactomannan were studied by Northern analysis. A comparison of gene expression on monosaccharides in the A. niger wild-type and a CreA mutant strain showed that the carbon catabolite repressor protein CreA has a strong influence on aglA, but not on aglB, aglC or mndA. AglC and MndA were purified from constructed overexpression strains of A. niger, and the combined action of these enzymes degraded a galactomanno-oligosaccharide into galactose and mannose. The possible roles of AglC and MndA in galactomannan hydrolysis is discussed.
Ademark
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Department of Biochemistry, Center for Chemistry and Chemical Engineering, Lund University, Sweden.
de Vries
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Hägglund
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Stålbrand
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Visser
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001234:Aspergillus niger / Q000201:enzymology* / Q000235:genetics*; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002244:Carbon / Q000378:metabolism; D003001:Cloning, Molecular; D005690:Galactose / Q000031:analogs & derivatives; D006031:Glycosylation; D006868:Hydrolysis; D008351:Mannans / Q000737:chemistry / Q000378:metabolism*; D008361:Mannosidases / Q000737:chemistry* / Q000235:genetics*; D008969:Molecular Sequence Data; D009005:Monosaccharides / Q000737:chemistry; D016133:Polymerase Chain Reaction; D017434:Protein Structure, Tertiary; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D000519:alpha-Galactosidase / Q000737:chemistry* / Q000235:genetics*; D044902:beta-Mannosidase
D000596:Amino Acids; D008351:Mannans; D009005:Monosaccharides; C012990:galactomannan; D002244:Carbon; D008361:Mannosidases; D000519:alpha-Galactosidase; D044902:beta-Mannosidase; D005690:Galactose