a-Galactosidase (EC 3.2.1.22) and b-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 a-galactosidase (AglC) and a b-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 a-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.Keywords: Aspergillus niger; a-galactosidase; b-mannosidase; cloning; expression.One of the most common polysaccharides in nature is O-acetylgalactoglucomannan, a branched heteroglycan that comprises up to 25% of the dry weight of softwoods [1]. It has a backbone of b-1,4-linked mannose and glucose residues in a ratio of 3 : 1, to which O-acetyl groups and a-1,6-linked galactose side groups are attached [1]. A similar, but less complex structure is found in galactomannan, a major storage polysaccharide in seeds of leguminous plants [2]. The complete enzymatic degradation of galactomannan into monosaccharides involves endo-1,4-b-mannanase (b-mannanase, EC 3.2.1.78), b-mannosidase (EC 3.2.1.25) and a-galactosidase (EC 3.2.1.22); in addition to these enzymes, degradation of O-acetylgalactoglucomannan also requires b-glucosidase and acetylesterase activities.b-Mannanase cleaves randomly within the main chain of galacto(gluco)mannans and mannans, and thereby produces oligosaccharides that can be further degraded by other enzymes. b-Mannosidase is an exoglycosidase that hydrolyses b-1,4-linked mannose residues from the nonreducing end of various oligosaccharides. The b-mannosidase from Aspergillus niger releases mannose from oligomeric as well as polymeric compounds, including mannan and galactomannan [3]. Genes encoding b-mannosidases have been isolated from mammals [4±6], and from a number of microorganisms [7±10]. All of these enzymes have been classified into family 2 of the glycosyl hydrolases [11], except for the Pyrococcus furiosus b-mannosidase, which belongs to family 1 [7]. a-Galactosidase hydrolyses terminal a-1,6-linked galactose residues fr...
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