Isolation, characterization and enzymatic hydrolysis of water-soluble wood polysaccharides

University dissertation from Henrik Stålbrand, dep. Biochemistry Lund University

Abstract: The need for new renewable products has enhanced the interest in cellulose and hemicellulose as raw material. The wood plant cell wall is a complex mixture of cellulose, hemicellulose, lignin and minor amounts of pectin, proteins and extractives. Several interactions between the compounds make them difficult to separate without modification. O-acetyl-galactoglucomannan (O-acetyl-GGM), the major hemicellulose in spruce, has been the main focus of this thesis. Heat-fractionation with microwave irradiation and size-fractionation with size exclusion chromatography have been used for the isolation of O-acetyl-GGM. HPLC, NMR and MALDI-MS were used as characterization methods. The structure of O-acetyl-GGM is sensitive to temperature, residence time and pH during the heat-fractionation. Considering MW, yield of GGM and sugar composition the best conditions for extracting O-acetyl-GGM using microwave irradiation were found to be a pH between 4-5, a temperature of 190°C and a residence time of 5 min. Cellulose- and hemicellulose-degrading enzymes are valuable tools for the investigation of O-acetyl-GGM. The possibility to characterize O-acetyl-GGM is improved by the purification and characterization of a beta-mannosidase (Man2A) from Aspergillus niger. Man2A has the capacity to catalyze the cleavage of mannosyl residues from the non-reducing end of manno-oligosaccharides, soluble and insoluble mannans and glucomannans. Man2A was shown to remove acetylated mannosyl units, but not galactosyl substituted mannosyl units from the substrate. The endoglucanase Cel7B and beta-mannanase Man5A from Trichoderma reesei have the ability to catalyze the cleavage of O-acetyl-GGM. Characterization methods were studied to determine the substitution pattern of ethyl(hydroxyethyl)cellulose (EHEC) by means of enzymatic degradation and chromatographic methods. The action of Cel7B and Cel12A from T. reesei on EHEC was found to be limited due to the high degree of substitution.

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