Galactosyl hydrolases from Bifidobacterium adolescentis and Bifidobacterium longum

The human intestine contains many bacteria, among which bifidobacteria. These can have a positive effect on human health. By consuming products containing dietary fibres (prebiotics), the amount of these intestinal bacteria can be stimulated, because they contain enzymes, which are able to degrade the fibres. Knowing which enzymes are present in the bacteria, will help to determine which kind of dietary fibres are suitable for use as a prebiotic. In this research, enzymes present in bifidobacteria, which were able to degrade the fibres galactan and galacto-oligosaccharides, were investigated. Three different enzymes were examined: a beta-galactosidase, an endo-galactanase, and an alpha-galactosidase. The results of this thesis gave more insight in how galactans and galacto-oligosaccharides can be degraded by bifidobacteria

Type I arabinogalactan contains ß-D-Galp-(1ß3)- ß-D-Galp structural elements

Arabinogalactan type I from potato was partially degraded by endo-galactanase from Aspergillus niger. High-performance anion-exchange chromatography revealed that several of the oligomeric degradation products eluted as double peaks. To investigate the nature of these products, the digest was fractionated by Bio-Gel P2 chromatography. The pool that contained tetramers was treated with a ß-d-Galp-(1¿4)-specific galactosidase from Bifidobacterium adolescentis to obtain a dimer with deviating linkage type, which was further purified by BioGel P2 chromatography. By obtaining all 1H and 13C chemical shifts and the presence of intra residual scalar coupling (HMBC) it could be concluded that the dimer contained a ß-(1¿3)-linkage instead of the expected ß-(1¿4)-linkage. Using the same NMR techniques as for the dimer, it was found that the pool of tetramers consisted of the following two galactose tetramers: ß-Galp-(1¿4)-ß-Galp-(1¿4)-ß-Galp-(1¿4)-¿/ß-Galp-OH and ß-Galp-(1¿4)-ß-Galp-(1¿4)-ß-Galp-(1¿3)-¿/ß-Galp-OH. The fact that the deviating ß-(1¿3)-linked galactose was found at the reducing end of the dimer showed that this deviating linkage is present within the backbone. The ß-(1¿3)-galactosyl interruption appeared to be a common structural feature of type I arabinogalactans with a frequency ranging from approximately 1 in 160 (potato, soy, citrus) to 1 in 250 (onion)

Chrysosporium lucknowense C1 arabinofuranosidases are selective in releasing arabinose from either single or double substituted xylose residues in arabinoxylans

Two novel arabinofuranosidases, Abn7 and Abf3 from Chrysosporium lucknowense (C1), belonging to the glycoside hydrolase family 43 and 51 were purified and characterized. Abn7 is exclusively able to hydrolyze arabinofuranosyl residues at position O-3 of double substituted xylosyl residues in arabinoxylan-derived oligosaccharides, an activity rarely found thus far. Abf3 is able to release arabinose from position O-2 or O-3 of single substituted xyloses. Both enzymes performed optimal at pH 5.0 and 40 °C. Combining Abn7 and Abf3 resulted in a synergistic increase in arabinose release from arabinoxylans. This synergistic effect is due to the action of Abf3 on the remaining arabinose residues at position O-2 on single substituted xylosyl residues resulting from the action of Abn7 on double substituted xylosyl residues. Arabinose release was further increased when an endo-1,4-ß-xylanase was present during digestion. The efficiency of these arabinohydrolases from C1 on insoluble arabinoxylan substrates is discussed

ß-Glactosidase from Bifidobacterium adolescentis DSM20083 prefers ß(1,4)-galactosides over lactose

A -galactosidase gene (-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (–) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. -Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that -Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl--d-galactopyranoside as a substrate. The Km and Vmax for Gal(1–4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant -Gal II was highly active towards Gal(1–4)Gal and Gal(1–4)Gal-containing oligosaccharides; only low activity was observed towards Gal(1–3)Gal, lactose, and Gal(1–3)GalOMe. No activity was found towards Gal(1–6)Gal, Gal(1–4)Man, Gal(1–4)Gal, Gal(1–3)Gal(1–4)Gal, cellobiose, maltose and sucrose. -Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(1–4)]2Gal peak area ratio was 9:1

Characterization and mode of action of two acetyl xylan esterases from Chrysosporium lucknowense C1 active towards acetylated xylans

Two novel acetyl xylan esterases, Axe2 and Axe3, from Chrysosporium lucknowense (C1), belonging to the carbohydrate esterase families 5 and 1, respectively, were purified and biochemically characterized. Axe2 and Axe3 are able to hydrolyze acetyl groups both from simple acetylated xylo-oligosaccharides and complex non-soluble acetylglucuronoxylan. Both enzymes performed optimally at pH 7.0 and 40 °C. Axe2 has a clear preference for acetylated xylo-oligosaccharides (AcXOS) with a high degree of substitution and Axe3 does not show such preference. Axe3 has a preference for large AcXOS (DP 9–12) when compared to smaller AcXOS (especially DP 4–7) while for Axe2 the size of the oligomer is irrelevant. Even though there is difference in substrate affinity towards acetylated xylooligosaccharides from Eucalyptus wood, the final hydrolysis products are the same for Axe2 and Axe3: xylo-oligosaccharides containing one acetyl group located at the non-reducing xylose residue remain as examined using MALDI-TOF MS, CE-LIF and the application of an endo-xylanase (GH 10)

Bifidobacterium carbohydrases-their role in breakdown and synthesis of (potential) prebiotics

Abstract There is an increasing interest to positively influence the human intestinal microbiota through the diet by the use of prebiotics and/or probiotics. It is anticipated that this will balance the microbial composition in the gastrointestinal tract in favor of health promoting genera such as Bifidobacterium and Lactobacillus. Carbohydrates like non-digestible oligosaccharides are potential prebiotics. To understand how these bacteria can grow on these carbon sources, knowledge of the carbohydrate-modifying enzymes is needed. Little is known about the carbohydrate-modifying enzymes of bifidobacteria. The genome sequence of Bifidobacterium adolescentis and Bifidobacterium longum biotype longum has been completed and it was observed that for B. longum biotype longum more than 8% of the annotated genes were involved in carbohydrate metabolism. In addition more sequence data of individual carbohydrases from other Bifidobacterium spp. became available. Besides the degradation of (potential) prebiotics by bifidobacterial glycoside hydrolases, we will focus in this review on the possibilities to produce new classes of non-digestible oligosaccharides by showing the presence and (transglycosylation) activity of the most important carbohydrate modifying enzymes in bifidobacteria. Approaches to use and improve carbohydrate-modifying enzymes in prebiotic design will be discussed

The ferulic acid esterases of Chrysosporium lucknowense C1: Purification, characterization and their potential application in biorefinery

Three ferulic acid esterases from the filamentous fungus Chrysosporium lucknowense C1 were purified and characterized. The enzymes were most active at neutral pH and temperatures up to 45 °C. All enzymes released ferulic acid and p-coumaric acid from a soluble corn fibre fraction. Ferulic acid esterases FaeA1 and FaeA2 could also release complex dehydrodiferulic acids and dehydrotriferulic acids from corn fibre oligomers, but released only 20% of all ferulic acid present in sugar beet pectin oligomers. Ferulic acid esterase FaeB2 released almost no complex ferulic acid oligomers from corn fibre oligomers, but 60% of all ferulic acid from sugar beet pectin oligomers. The ferulic acid esterases were classified based on both, sequence similarity and their activities toward synthetic substrates. The type A ferulic acid esterases FaeA1 and FaeA2 are the first members of the phylogenetic subfamily 5 to be biochemically characterized. Type B ferulic acid esterase FaeB2 is a member of subfamily 6

Mode of action of Chrysosporium lucknowense C1 a-l-arabinohydrolases

The mode of action of four Chrysosporium lucknowense C1 a-l-arabinohydrolases was determined to enable controlled and effective degradation of arabinan. The active site of endoarabinanase Abn1 has at least six subsites, of which the subsites -1 to +2 have to be occupied for hydrolysis. Abn1 was able to hydrolyze a branched arabinohexaose with a double substituted arabinose at subsite -2. The exo acting enzymes Abn2, Abn4 and Abf3 release arabinobiose (Abn2) and arabinose (Abn4 and Abf3) from the non-reducing end of reduced arabinose oligomers. Abn2 binds the two arabinose units only at the subsites -1 and -2. Abf3 prefers small oligomers over large oligomers. It is able to hydrolyze all linkages present in beet arabinan, including the linkages of double substituted residues. Abn4 is more active towards polymeric substrate and releases arabinose monomers from single substituted arabinose residues. Depending on the combination of the enzymes, the C1 arabinohydrolases can be used to effectively release branched arabinose oligomers and/or arabinose monomers

ß-Glactosidase from Bifidobacterium adolescentis DSM20083 prefers ß(1,4)-galactosides over lactose

A -galactosidase gene (-Gal II) from Bifidobacterium adolescentis DSM 20083 was cloned into a pbluescript SK (–) vector and expressed in Escherichia coli. The recombinant enzyme was purified from the cell extract by anion-exchange and size-exclusion chromatography. -Gal II had a native molecular mass of 235 kDa and the subunits had a molecular mass of 81 kDa, indicating that -Gal II occurs as a trimer. The enzyme was classified as belonging to glycosyl hydrolase family 42. The optimal pH was 6.0 and the optimal temperature was 50°C, using p-nitrophenyl--d-galactopyranoside as a substrate. The Km and Vmax for Gal(1–4)Gal were 60 mM and 1,129 U/mg, respectively. The recombinant -Gal II was highly active towards Gal(1–4)Gal and Gal(1–4)Gal-containing oligosaccharides; only low activity was observed towards Gal(1–3)Gal, lactose, and Gal(1–3)GalOMe. No activity was found towards Gal(1–6)Gal, Gal(1–4)Man, Gal(1–4)Gal, Gal(1–3)Gal(1–4)Gal, cellobiose, maltose and sucrose. -Gal II was inhibited at high substrate concentrations (100 mg/ml) and no transglycosylation activity was found. At lower substrate concentrations (10 mg/ml) only low transglycosylation activity was found; the Gal/[Gal(1–4)]2Gal peak area ratio was 9:1