title: Hydrolysis of konjac glucomannan by Trichoderma reesei mannanase and endoglucanases Cel7B and Cel5A for the production of glucomannooligosaccharides.
abstract: In this paper we describe the enzymatic hydrolysis of konjac glucomannan for the production of glucomannooligosaccharides using purified Trichoderma reesei mannanase, endoglucanases EGI (Tr Cel7b) and EGII (Tr Cel5a). Hydrolysis with each of the three enzymes produced a different pattern of oligosaccharides. Mannanase was the most selective of the three enzymes in the hydrolysis of konjac mannan and over 99% of the formed oligosaccharides had mannose as their reducing end pyranosyl unit. Tr Cel5A hydrolysate shared similarities with mannanase and Tr Cel7B hydrolysates and the enzyme had the lowest substrate specificity of the studied enzymes. The hydrolysate of Tr Cel7B contained a series of oligosaccharides with non-reducing end mannose (M) and reducing end glucose (G) (MG, MMG, MMMG, and MMMMG). These oligosaccharides were isolated from the hydrolysate by size exclusion chromatography in relatively high purity (86-95%) and total yield (23% of substrate). The isolated oligosaccharides were characterized using acid hydrolysis and HPAEC-PAD (carbohydrate composition), HPLC-RI and HPAEC-MS (to determine the DP of purified oligosaccharides), enzymatic hydrolysis (determination of non-reducing end carbohydrate) and NMR (both 1D and 2D, to verify structure and purity of purified compounds). Hydrolysis of konjac mannan with a specific enzyme, such as T. reesei Cel7B or mannanase, followed by fractionation with SEC offers the possibility to produce glucomannooligosaccharides with defined structure. The isolated oligosaccharides can be utilised as analytical standards, for determination of bioactivity of oligosaccharides with defined structure or as substrates for defining substrate specificity of novel carbohydrate hydrolyzing enzymes.
authors.affiliation: VTT Technical Research Centre of Finland, PO Box 1000, 02044 VTT, Finland.
authors.forename: Atte
authors.initials: A
authors.lastname: Mikkelson
authors.forename: Hannu
authors.initials: H
authors.lastname: Maaheimo
authors.forename: Terhi K
authors.initials: TK
authors.lastname: Hakala
mesh_terms: D002480:Cellulase / Q000378:metabolism*; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006868:Hydrolysis; D009682:Magnetic Resonance Spectroscopy; D008351:Mannans / Q000737:chemistry / Q000378:metabolism*; D008358:Mannose / Q000737:chemistry; D008361:Mannosidases / Q000378:metabolism*; D009844:Oligosaccharides / Q000138:chemical synthesis* / Q000302:isolation & purification; D014242:Trichoderma / Q000201:enzymology*
chemical_list: D008351:Mannans; D009844:Oligosaccharides; C022901:(1-6)-alpha-glucomannan; D008361:Mannosidases; C407024:endoglucanase Cel5A; D002480:Cellulase; D008358:Mannose