Galactoglucomannan-degrading enzymes from Aspergillus niger

Detta är en avhandling från Department of Biochemistry, Lund University

Sammanfattning: Galactoglucomannan and galactomannan are among the most abundant plant polysaccharides in nature. The central enzymes involved in their degradation, i.e., endo-1,4-beta-mannanase (beta-mannanase, EC, beta-mannosidase (EC, and alpha-galactosidase (EC were purified from the filamentous fungus Aspergillus niger and characterized with respect to physical properties and substrate specificity. The beta-mannanase degraded polymeric ivory nut mannan to mainly mannobiose and mannotriose, and NMR analysis of the hydrolysis of mannopentaose showed that it acts by the retaining mechanism. Unlike some other beta-mannanases it probably lacks a cellulose-binding domain, since it was unable to adsorb on cellulose. The preferred substrates for the beta-mannosidase (MndA) were linear manno-oligosaccharides, but it also hydrolyzed terminal non-reducing mannose residues from polymeric mannan and galactomannan. It was able to cleave up to, but not beyond, a galactose side group. Two alpha-galactosidases (AglB and AglC) with different substrate specificities were purified. AglC had a strong preference for terminal non-reducing galactose residues, whereas AglB hydrolyzed galactose residues linked to terminal as well as internal residues of the substrate main chain. The cooperation of individual enzymes during degradation of polymeric mannan, galactomannan, and galactomanno-oligosaccharides were investigated. The genes encoding AglC and MndA were cloned, characterized, and overexpressed in A. niger. On the basis of sequence comparisons, AglC could be assigned to family 36 of the glycosyl hydrolases and MndA could be assigned to family 2. The expression patterns of three alpha-galactosidase genes (aglA, aglB, and aglC) and a beta-mannosidase gene (mndA) during growth of A. niger on different carbon sources were studied by Northern analysis. The aglB and mndA genes were expressed early and in high levels on galactomannan, indicating that the corresponding enzymes play a key role in the degradation of this polymer.

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