Role of Ser216 in the mechanism of action of membrane-bound lytic transglycosylase B: further evidence for substrate-assisted catalysis

Lytic transglycosylases cleave the beta-(1-->4)-glycosidic bond in the bacterial cell wall heteropolymer peptidoglycan between the N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) residues with the concomitant formation of a 1,6-anhydromuramoyl residue. Based on sequence alignments, Ser216 in Pseudomonas aeruginosa membrane-bound lytic transglycosylase B (MltB) was targeted for replacement with alanine to delineate its role in the enzyme's mechanism of action. The specific activity of the Ser216-->Ala MltB derivative was less than 12% of that for the wild-type enzyme, while its substrate binding affinity remained virtually unaltered. These data are in agreement with a role of Ser216 in orienting the N-acetyl group on MurNAc at the -1 subsite of MltB for its participation in a substrate-assisted mechanism of action.     

Characterisation of a novel amylosucrase from Deinococcus radiodurans

The BLAST search for amylosucrases has yielded several gene sequences of putative amylosucrases, however, with various questionable annotations. The putative encoded proteins share 32-48% identity with Neisseria polysaccharea amylosucrase (AS) and contain several amino acid residues proposed to be involved in AS specificity. First, the B-domains of the putative proteins and AS are highly similar. In addition, they also reveal additional residues between putative beta-strand 7 and alpha-helix 7 which could correspond to the AS B'-domain, which turns the active site into a deep pocket. Finally, conserved Asp and Arg residues could form a salt bridge similar to that found in AS, which is responsible for the glucosyl unit transfer specificity. Among these found genes, locus NP_294657.1 (dras) identified in the Deinococcus radiodurans genome was initially annotated as an alpha-amylase encoding gene. The putative encoded protein (DRAS) shares 42% identity with N. polysaccharea AS. To investigate the activity of this protein, gene NP_294657.1 was cloned and expressed in Escherichia coli. When acting on sucrose, the pure recombinant enzyme was shown to catalyse insoluble amylose polymer synthesis accompanied by side-reactions (sucrose hydrolysis, sucrose isomer and soluble maltooligosaccharide formation). Kinetic analyses further showed that DRAS follows a non-Michaelian behaviour toward sucrose substrate and is activated by glycogen, as is AS. This demonstrates that gene NP_294657.1 encodes an amylosucrase.     

Impact and efficiency of GH10 and GH11 thermostable endoxylanases on wheat bran and alkali-extractable arabinoxylans

The results of a comparative study of two thermostable (1-->4)-beta-xylan endoxylanases using a multi-technical approach indicate that a GH11 xylanase is more useful than a GH10 xylanase for the upgrading of wheat bran into soluble oligosaccharides. Both enzymes liberated complex mixtures of xylooligosaccharides. 13C NMR analysis provided evidence that xylanases cause the co-solubilisation of beta-glucan, which is a result of cell-wall disassembly. The simultaneous use of both xylanases did not result in a synergistic action on wheat bran arabinoxylans, but instead led to the production of a product mixture whose profile resembled that produced by the action of the GH10 xylanase alone. Upon treatment with either xylanase, the diferulic acid levels in residual bran were unaltered, whereas content in ferulic and p-coumaric acids were unequally decreased. With regard to the major differences between the enzymes, the products resulting from the action of the GH10 xylanase were smaller in size than those produced by the GH11 xylanase, indicating a higher proportion of cleavage sites for the GH10 xylanase. The comparison of the kinetic parameters of each xylanase using various alkali-extractable arabinoxylans indicated that the GH10 xylanase was most active on soluble arabinoxylans. In contrast, probably because GH11 xylanase can better penetrate the cell-wall network, this enzyme was more efficient than the GH10 xylanase in the hydrolysis of wheat bran. Indeed the former enzyme displayed a nearly 2-fold higher affinity and a 6.8-fold higher turnover rate in the presence of this important by-product of the milling industry.     

Inhibition of β-Glucosidase (Amygdalae dulces) by (+)-Catechin Oxidation Products and Procyanidin Dimers

The sensitivity and specificity of the inhibition of β-glucosidase (Amygdalae dulces) by (+ )-catechin, an oxidized (+)-catechin solution, three dimeric procyanidins, and five (+)-catechin dimers obtained by enzymatic oxidation were evaluated by using a chromatographic method. All the polyphenols tested presented a significant inhibitory effect. Non-competitive inhibition was observed for the oxidized (+)-catechin solution. Some oxidation products were at least as powerful inhibitors as procyanidins which are known for their tanning effect. Yellow oxidation products were among the strongest inhibitors. No marked role of the number of hydroxyl and o-diphenol groups nor of the nature or position of the interflavanic linkage in the inhibitory effect was apparent.     

The glycosylation pattern of a humanized IgGl antibody (D1.3) expressed in CHO cells

A humanized IgG antibody (D1.3) which retains murine complementarity determining regions specific for the antigen lysozyme has been expressed in CHO-DUKX cells. Heavy and light chain containing plasmids were co-transfected into CHO-DUKX cells and stable clones were grown in DMEM/F12 medium supplemented with 5% foetal calf serum. D1.3 antibody was purified from culture supernatants by Protein G chromatography. With the recombinant D1.3 antibody as a model, this cell culture system was shown to glycosylate the IgG Fc region in a similar manner to IgG isolated from serum. The neutral, core fucosylated biantennary oligosaccharides found are present in serum IgG and no novel carbohydrate sequences were detected. The degree of terminal agalactosylation was also similar to normal serum, in contrast to the increased levels found in rheumatoid serum. Furthermore, those oligosaccharides which lack only one terminal Gal are exclusively galactosylated on the GlcNAc(β1,2) Man(α1,6) Man(β1,4) antenna. Unambiguous identification of the exact glycosylation pattern of the antibody was carried out by a combination of specific exoglycosidase digestions, gel permeation chromatography of 2-aminobenzamide derivatives, high pH anion exchange chromatography and methylation analysis followed by gas–liquid chromatography-mass spectrometry. Abbreviations: CDR, complementarity determining region; CHO, chinese hamster ovary; GPC, gel permeation chromatography; 2-AB, 2-aminobenzamide; HPAEC-PAD, high pH anion exchange chromatography with pulsed amperometric detection; GC-MS, gas chromatography with mass spectrometry analysis; PNGase F, peptide-N-glycosidase F; PGC, porous graphitized carbon column; RAAM, reagent array analysis method; NeuAc: N-acetylneuraminic acid; NeuGc: N-glycolylneuraminic acid This revised version was published online in November 2006 with corrections to the Cover Date.     

Characterization of extracellular polymeric substances from denitrifying organism Comamonas denitrificans

Extracellular polymeric substances (EPS) play an important role in the formation and activity of biofilms in wastewater treatment (WWT). The EPS of the denitrifying biomarker Comamonas denitrificans strain 110, produced in different culture media and growth modes, were characterized. The EPS mainly contained protein (3–37%), nucleic acids (9–50%), and carbohydrates (3–21%). The extracellular DNA was found to be important for initial biofilm formation since biofilm, but not planktonic growth, was inhibited in the presence of DNase. The polysaccharide fraction appeared to consist of at least two distinct polymers, one branched fraction (A) made up of glucose and mannose with a molecular weight around 100 kDa. The other fraction (B) was larger and consisted of ribose, mannose, glucose, rhamnose, arabinose, galactose, and N-acetylglucosamine. Fraction B polysaccharides were mainly found in capsular EPS which was the dominant type in biofilms and agar-grown colonies. Fraction A was abundant in the released EPS, the dominant type in planktonic cultures. Biofilm and agar-grown EPS displayed similar overall properties while planktonic EPS showed clear compositional disparity. This study presents results on the physiology of a key WWT organism, which may be useful in the future development of improved biofilm techniques for WWT purposes.     

β-Galactosidase from Lactobacillus plantarum WCFS1: biochemical characterization and formation of prebiotic galacto-oligosaccharides

Recombinant β-galactosidase from Lactobacillus plantarum WCFS1, homologously over-expressed in L. plantarum, was purified to apparent homogeneity using p-aminobenzyl 1-thio-β-d-galactopyranoside affinity chromatography and subsequently characterized. The enzyme is a heterodimer of the LacLM-family type, consisting of a small subunit of 35 kDa and a large subunit of 72 kDa. The optimum pH for hydrolysis of its preferred substrates o-nitrophenyl-β-d-galactopyranoside (oNPG) and lactose is 7.5 and 7.0, and optimum temperature for these reactions is 55 and 60 °C, respectively. The enzyme is most stable in the pH range of 6.5-8.0. The K_m, k_cat and k_cat/K_m values for oNPG and lactose are 0.9 mM, 92 s⁻¹, 130 mM⁻¹ s⁻¹ and 29 mM, 98 s⁻¹, 3.3 mM⁻¹ s⁻¹, respectively. The L. plantarum β-galactosidase possesses a high transgalactosylation activity and was used for the synthesis of prebiotic galacto-oligosaccharides (GOS). The resulting GOS mixture was analyzed in detail, and major components were identified by using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) as well as capillary electrophoresis. The maximal GOS yield was 41% (w/w) of total sugars at 85% lactose conversion (600 mM initial lactose concentration). The enzyme showed a strong preference for the formation of β-(1→6) linkages in its transgalactosylation mode, while β-(1→3)-linked products were formed to a lesser extent, comprising ∼80% and 9%, respectively, of the newly formed glycosidic linkages in the oligosaccharide mixture at maximum GOS formation. The main individual products formed were β-d-Galp-(1→6)-d-Lac, accounting for 34% of total GOS, and β-d-Galp-(1→6)-d-Glc, making up 29% of total GOS.     

The structure of the carbohydrate backbone of the LPS from Shewanella putrefaciens CN32

The lipopolysaccharide (LPS) from a natural rough strain of Shewanella putrefaciens CN32 was analyzed using NMR and mass spectroscopy and chemical methods, and the following structure of its carbohydrate backbone is proposed: beta-Galf-(1-->3)-beta-Gal-(1-->4)-beta-Glc-(1-->4)-alpha-DDHep2PEtN-(1-->5)-alpha-Kdo4P-(1-->6)-beta-GlcN4P-(1-->6)-alpha-GlcN1P     

LC/CE-MS tools for the analysis of complex arabino-oligosaccharides

Recently, various branched arabino-oligosaccharides as present in a sugar beet arabinan digest were characterized using NMR. Although HPAEC often has been the method of choice to monitor the enzymatic degradation reactions of polysaccharides, it was shown that HPAEC was incapable to separate all known linear and branched arabino-oligosaccharides present. As this lack of resolution might result in an incorrect interpretation of the results, other separation techniques were explored for the separation of linear and branched arabino-oligosaccharides. The use of porous-graphitized carbon liquid chromatography with evaporative light scattering and mass detection as well as capillary electrophoresis with laser-induced fluorescence and mass detection demonstrated the superiority of both the techniques toward HPAEC by enabling the separation and unambiguous identification of almost all the linear and branched arabino-oligosaccharides available. The elution behavior of all arabino-oligosaccharides for the three tested separation techniques was correlated with their chemical structures and conclusions were drawn for the retention mechanisms of the arabino-oligosaccharides on the different chromatographic and electrophoretic systems. The combination of the elution/migration behavior on LC/CE and the MS fragmentation patterns of the arabino-oligosaccharides led to the prediction of structures for new DP6 arabino-oligosaccharides in complex enzyme digests.     

Oligosaccharides in feces of breast- and formula-fed babies

So far, little is known on the fate of oligosaccharides in the colon of breast- and formula-fed babies. Using capillary electrophoresis with laser induced fluorescence detector coupled to a mass spectrometer (CE–LIF–MSⁿ), we studied the fecal oligosaccharide profiles of 27 two-month-old breast-, formula- and mixed-fed preterm babies. The interpretation of the complex oligosaccharide profiles was facilitated by beforehand clustering the CE–LIF data points by agglomerative hierarchical clustering (AHC). In the feces of breast-fed babies, characteristic human milk oligosaccharide (HMO) profiles, showing genetic fingerprints known for human milk of secretors and non-secretors, were recognized. Alternatively, advanced degradation and bioconversion of HMOs, resulting in an accumulation of acidic HMOs or HMO bioconversion products was observed. Independent of the prebiotic supplementation of the formula with galactooligosaccharides (GOS) at the level used, similar oligosaccharide profiles of low peak abundance were obtained for formula-fed babies. Feeding influences the presence of diet-related oligosaccharides in baby feces and gastrointestinal adaptation plays an important role herein. Four fecal oligosaccharides, characterized as HexNAc-Hex-Hex, Hex-[Fuc]-HexNAc-Hex, HexNAc-[Fuc]-Hex-Hex and HexNAc-[Fuc]-Hex-HexNAc-Hex-Hex, highlighted an active gastrointestinal metabolization of the feeding-related oligosaccharides. Their presence was linked to the gastrointestinal mucus layer and the blood-group determinant oligosaccharides therein, which are characteristic for the host’s genotype.