Henrik Stålbrand

Characterization of acetylated 4-O-methylglucuronoxylan isolated from aspen employing 1H and 13C NMR spectroscopy

Water-soluble hemicelluloses were extracted from milled aspen wood (Populus tremula) employing microwave oven treatment at 180 degrees C for 10 min. The final pH of this extract was 3.5. From this extract oligo- and polysaccharides were isolated and subsequently fractionated by size-exclusion chromatography. The structures of the saccharides in three of the fractions obtained were determined by 1H and 13C NMR spectroscopy, using homonuclear and heteronuclear two-dimensional techniques. The polysaccharides present in the two fractions eluted first were O-acetyl-(4-O-methylglucurono)xylans. The average degree of acetylation of the xylose residues in these compounds was 0.6. The structural element -->4)[4-O-Me-alpha-D-GlcpA-(1-->2)][3-O-Ac]-beta-D-Xylp-(1 --> could also be identified. On the average, these two xylans were composed of the following (1-->4)-linked beta-D-xylopyranosyl structural elements: unsubstituted (50 mol%), 2-O-acetylated (13 mol%), 3-O-acetylated (21 mol%), 2,3-di-O-acetylated (6 mol%) and [MeGlcA alpha-(1-->2)][3-O-acetylated] (10 mol%). Most of the 4-O-methylglucuronyl and acetyl substituents in the isolated polysaccharides survived the microwave oven treatment. The third fraction, eluted last, contained acetylated xylo-oligosaccharides, with minor contamination by an acetylated mannan. In the case of these xylo-oligosaccharides, the average degree of acetylation was 0.3.

Purification and characterization of two β-mannanases from Trichoderma reesei

Five enzymes with mannanase activity were separated from Trichoderma reesei culture filtrate using analytical isoelectric focusing and subsequently detected with the zymogram technique. The crude enzymes had isoelectric points in the range of 3.6–6.5. Two of the mannanases with pI values of 4.6 and 5.4 were purified using ion-exchange chromatography, affinity chromatography and chromatofocusing. The molecular weights determined with SDS-PAGE were 51 000 (mannanase pI 4.6) and 53 000 (mannanase pI 5.4). The two enzymes had similar properties with respect to pH optimae and pH stabilities. Both mannanases hydrolyzed ivory nut mannan mainly to mannotriose and mannobiose. The specific activities (against locust bean gum) of the purified enzymes were 1860 and 1430 nkat mg−1 for the pI 4.6 and pI 5.4 mannanases, respectively.

Isolation and characterization of galactoglucomannan from spruce (Picea abies)

Water-soluble hemicelluloses were extracted from spruce chips by microwave heat fractionation. The chips were impregnated with water at different pH values. Screening of heat-fractionation conditions, i.e. impregnation medium, temperature and residence time was performed with the aim to extract O-acetyl-galactoglucomannan. The impregnation and heat fractionation conditions were evaluated on the basis of the yield of dissolved mannan (oligo- and polysaccharides), molecular weight of the carbohydrates and amount of dissolved lignin. Increasing temperature and residence time increases the yield of mannan and decreases the molecular weight of dissolved carbohydrates. For a structural study of the extracted carbohydrates the chips were impregnated with water and treated at 200oC for 2min. Oligo- and polysaccharides were fractionated with preparative size-exclusion chromatography from the filtered extract.The structure of the obtained saccharides in two fractions 8 and 9 was determined by 1H NMR spectroscopy. The polysaccharides in the fractions were O-acetyl-galactoglucomannan with a degree of polymerization ~20 and ~11 for fractions 8 and 9, respectively. The molar ratio for galactose:glucose:mannose was approximately 0.1:1:4. About one-third of the d-mannosyl units are substituted by O-acetyl groups almost equally distributed between C-2 and C-3. (Less)

How the walls come crumbling down : recent structural biochemistry of plant polysaccharide degradation

The recent years have witnessed considerable developments in the interpretation of the three-dimensional structures of plant polysaccharide-degrading enzymes in the context of their functional specificity. A plethora of new structures of catalytic, carbohydrate-binding and protein-scaffolding modules involved in (hemi)cellulose catabolism has emerged in harness with sophisticated biochemical analysis. Despite significant advances, a full understanding of the intricacies of substrate recognition and catalysis by these diverse and specialised enzymes remains an important goal, especially if the application potential of these biocatalysts is to be fully realised.

Softwood hemicellulose-degrading enzymes from Aspergillus niger: Purification and properties of a β-mannanase

The enzymes needed for galactomannan hydrolysis, i.e., beta-mannanase, alpha-galactosidase and beta-mannosidase, were produced by the filamentous fungus Aspergillus niger. The beta-mannanase was purified to electrophoretic homogeneity in three steps using ammonium sulfate precipitation, anion-exchange chromatography and gel filtration. The purified enzyme had an isoelectric point of 3.7 and a molecular mass of 40 kDa. Ivory nut mannan was degraded mainly to mannobiose and mannotriose when incubated with the beta-mannanase. Analysis by 1H NMR spectroscopy during hydrolysis of mannopentaose showed that the enzyme acts by the retaining mechanism. The N-terminus of the purified A. niger beta-mannanase was sequenced by Edman degradation, and comparison with Aspergillus aculeatus beta-mannanase indicated high identity. The enzyme most probably lacks a cellulose binding domain since it was unable to adsorb on cellulose.

Characterization of galactoglucomannan extracted from spruce (Picea abies) by heat-fractionation at different conditions

Water-soluble hemicelluloses were extracted from spruce chips by microwave heat-fractionation. The galactoglucomannan (GGM) extraction was evaluated on the basis of weight-average molecular weight (MW), yield and carbohydrate composition of the GGM. The MW was determined by size-exclusion chromatography with column calibration using off-line MALDI-MS analysis, and determination of mannan content in the fractions collected. Water impregnated spruce chips were heat-fractionated at three different temperatures (180, 190, and 200oC). The spruce chips were also impregnated in NaOH solutions of different concentrations, and then heat-fractionated at 190oC for 5min. The highest mannan yield (78% based on the amount in the raw material) was obtained from water impregnated spruce chips heat-fractionated at 190oC for 5min (MW of 3800). The highest MW (14,000) was obtained from impregnation with 2% NaOH (190oC, 5min), but the yield of mannan was very low (3%). Impregnation with 0.025% NaOH and heat-fractionation at 190oC for 5min resulted in extraction of GGM with MW of 9500 and a mannan yield of 31%. When the spruce chips were impregnated with =<0.05% NaOH an O-acetyl-galactoglucomannan was extracted, whereas when higher NaOH charges were used in the impregnation, the extracted GGM lacked acetyl groups. (Less)

A cellulose-binding module of the Trichoderma reesei β-mannanase Man5A increases the mannan-hydrolysis of complex substrates

Endo-beta-1,4-D-mannanases (beta-mannanase; EC 3.2.1.78) are endohydrolases that participate in the degradation of hemicellulose, which is closely associated with cellulose in plant cell walls. The beta-mannanase from Trichoderma reesei (Man5A) is composed of an N-terminal catalytic module and a C-terminal carbohydrate-binding module (CBM). In order to study the properties of the CBM, a construct encoding a mutant of Man5A lacking the part encoding the CBM (Man5ADeltaCBM), was expressed in T. reesei under the regulation of the Aspergillus nidulans gpdA promoter. The wild-type enzyme was expressed in the same way and both proteins were purified to electrophoretic homogeneity using ion-exchange chromatography. Both enzymes hydrolysed mannopentaose, soluble locust bean gum galactomannan and insoluble ivory nut mannan with similar rates. With a mannan/cellulose complex, however, the deletion mutant lacking the CBM showed a significant decrease in hydrolysis. Binding experiments using activity detection of Man5A and Man5ADeltaCBM suggests that the CBM binds to cellulose but not to mannan. Moreover, the binding of Man5A to cellulose was compared with that of an endoglucanase (Cel7B) from T. reesei.

Characterization of water-soluble hemicelluloses from spruce and aspen employing SEC/MALDI mass spectroscopy

Partly depolymerized hemicelluloses isolated from wood chips of spruce and aspen employing microwave treatment were resolved using size-exclusion chromatography (SEC) into oligo- and polysaccharide fractions containing components with a narrow range of sizes, as determined by MALDI mass spectroscopy. The degree of substitution with acetyl moieties (DS) was also calculated on the basis of the MALDI-MS spectra obtained prior to and following deacetylation. For spruce hemicelluloses, the low molecular mass fraction contained small arabino-4-O-methylglucuronoxylan oligosaccharides, with DP values ranging from 4 to approximately 20, separated primarily on the basis of their charge density. The fraction eluted last consisted of an O-acetyl-(galacto)glucomannan polysaccharide of peak-average DP value (DP(p)) 14. The degree of substitution with acetyl groups (DS) decreased with decreasing DP, a value DS of 0.39 being obtained for the fraction with DP(p) 12. For the aspen hemicelluloses, the SEC fractions eluted first contained an acidic O-acetyl-4-O-methylglucuronoxylan polysaccharide with DP ranging from 10 to approximately 28 and an average DS of approximately 0.75. The fractions eluted last consisted of oligosaccharide mixtures composed primarily of small neutral O-acetyl-xylooligosaccharides (DP(p) 6, DS 0.41), together with minor quantities of an O-acetyl-glucomannan.

Multiple alpha-galactosidases from Aspergillus niger: purification, characterization, and substrate specificities

Enzymes with α-galactosidase activity are produced by many organisms, often in multiple forms. Here we compare the biochemical and hydrolytic properties of four major α-galactosidase forms (α-gal I-IV) that were purified from the culture filtrate of Aspergillus niger. α-Gal II, III and IV appear to be isoforms of the same enzyme, and N-terminal amino acid sequence data suggest that they are closely related or identical to A. niger AglB in family 27 of the glycosyl hydrolases. α-Gal I is a completely different enzyme that belongs to family 36. α-Gal I had an isoelectric point of 4.15 and appears to be a tetramer composed of four 94-kDa subunits. α-Gal II, III and IV were dimers with monomeric molecular masses of 64 kDa and isoelectric points of 4.5, 4.7 and 4.8, respectively. α-Gal II-IV were stable when incubated for 17 h at 50°C and pH 2–5, whereas α-gal I was most stable at pH 5–6. All enzymes had maximal catalytic activity at pH 4.5 and 60°C, and hydrolyzed melibiose, raffinose and stachyose. α-Gal II-IV also degraded galactomanno-oligosaccharides and released 66% of the galactose side groups from polymeric locust bean gum galactomannan. α-Gal I released galactose from locust bean gum only in combination with A. niger β-mannosidase. Kinetic experiments showed that α-gal I hydrolyzed p-nitrophenyl-α-Image-galactopyranoside and melibiose more efficiently than α-gal II-IV. The distinct hydrolytic and biochemical properties of α-gal I and α-gal II-IV further signifies the difference between α-galactosidases of family 27 and 36. (Less)

endo-β-1,4-Mannanases from blue mussel, Mytilus edulis: purification, characterization, and mode of action

Two variants of an endo-β-1,4-mannanase from the digestive tract of blue mussel, Mytilus edulis, were purified by a combination of immobilized metal ion affinity chromatography, size exclusion chromatography in the absence and presence of guanidine hydrochloride and ion exchange chromatography. The purified enzymes were characterized with regard to enzymatic properties, molecular weight, isoelectric point, amino acid composition and N-terminal sequence. They are monomeric proteins with molecular masses of 39&#;216 and 39&#;265 Da, respectively, as measured by MALDI-TOF mass spectrometry. The isoelectric points of both enzymes were estimated to be around 7.8, however slightly different, by isoelectric focusing in polyacrylamide gel. The enzymes are stable from pH 4.0 to 9.0 and have their maximum activities at a pH about 5.2. The optimum temperature of both enzymes is around 50-55oC. Their stability decreases rapidly when going from 40 to 50oC. The N-terminal sequences (12 residues) were identical for the two variants. They can be completely renatured after denaturation in 6 M guanidine hydrochloride. The enzymes readily degrade the galactomannans from locust bean gum and ivory nut mannan but show no cross-specificity for xylan and carboxymethyl cellulose. There is no binding ability observed towards cellulose and mannan (Less)