Angela V. Savage

Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii.

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.

Isolation and characterization of a thermostable endo-β-glucanase active on 1,3-1,4-β-D-glucans from the aerobic fungus Talaromyces emersonii CBS 814.70

Abstract A novel endoglucanase active on 1,3-1,4-β- d -glucans was purified to apparent homogeneity from submerged cultures of the moderately thermophilic aerobic fungus Talaromyces emersonii CBS 814.70. The enzyme is a single subunit glycoprotein with Mr and pI values of 40.7 ± 0.3 kDa and 4.4, respectively, and an estimated carbohydrate content of 77% (w/w). The purified β-glucanase displayed activity over broad ranges of pH and temperature, yielding respective optima values of pH 4.8 and 80°C. This enzyme was markedly thermostable with 15% of the original activity remaining after incubation for 15 min at 100°C. Substrate specificity studies revealed the identity of the enzyme to be a 1,3-1,4-β- d -glucanase. Identical Km values (13.38 mg.ml−1) were obtained with lichenan and BBG, while the Vmax value with lichenan (142.9 IU.mg−1) was approximately twice the value obtained with BBG (79.3 IU.mg−1). Time-course hydrolysis of barley-β-glucan did not proceed linearly with respect to time indicating an ‘endo’ or more processive action for the enzyme. HPAEC fractionation of the products of hydrolysis yielded a range of oligosaccharides, with cellobiose, cellotriose and cellotetraose being the predominant oligosaccharide products.

Chemical structure of a polysaccharide from Campylobacter jejuni 176.83 (serotype O:41) containing only furanose sugars ☆

A neutral polysaccharide was obtained by hot phenol-water extraction of biomass from Campylobacter jejuni 176.83 and subsequently separated from acid-liberated core oligosaccharide of lipopolysaccharide by sequential GPC on Bio-Gel P6 and TSK-40 columns. All sugar components of the trisaccharide repeating unit of the polysaccharide were found to be of the furanose ring form. The major trisaccharide contained beta-L-arabinose, 6-deoxy-beta-D-altro-heptose (beta-D-6d-altHep) and 6-deoxy-beta-L-altrose (beta-L-6d-Alt), whereas in the minor trisaccharide the beta-L-6d-Alt is replaced by its C-5 epimer alpha-D-Fuc. On the basis of 1H and 13C NMR spectroscopic studies, including 2D ROESY, HMQC and HMQC-TOCSY experiments, the following structures of the repeating units were established: [formula: see text]

Structure of an acidic O-specific polysaccharide of Pseudoalteromonas haloplanktis type strain ATCC 14393 containing 2-acetamido-2-deoxy-d- and -l-galacturonic acids and 3-(N-acetyl-d-alanyl)amino-3,6-dideoxy-d-glucose

Abstract An acidic O-specific polysaccharide was obtained from the lipopolysaccharide of Pseudoalteromonashaloplanktis ATCC 14393 and found to contain d -galactose, 3-(N-acetyl- d -alanyl)amino-3,6-dideoxy- d -glucose ( d -Qui3N d AlaAc), 2,4-diacetamido-2,4,6-trideoxy- d -glucose ( d -QuiNAc4NAc), 2-acetamido-2-deoxy- d - and - l -galacturonic acids ( d - and l -GalNAcA), and O-acetyl groups. On the basis of Smith degradation and 1H and 13C NMR spectroscopic studies, including 2D COSY, TOCSY, NOESY, 1H, 13C HMQC, and HMBC experiments, the following structure of the pentasaccharide repeating unit of the polysaccharide was established: →4 )- α - l - Gal p NAcA -(1 →3 )- β - d - Qui p NAc4NAc -(1 →2 )- β - d - Qui p3 N d AlaAc -(1 →4 )- α - d - Gal p NAcA -(1 →4 )- α - d - Gal p2,6 Ac 2 -(1 → where O-acetylation of the galactose residue at each position is partial (50–70%).

Structure of a highly acidic O-specific polysaccharide of lipopolysaccharide of Pseudoalteromonas haloplanktis KMM 223 (44-1) containing l-iduronic acid and d-QuiNHb4NHb

An acidic O-specific polysaccharide was obtained by mild acid degradation of the lipopolysaccharide isolated by phenol-water extraction of Pseudoalteromonas haloplanktis strain KMM 223 (44-1). L-Iduronic acid (IdoA) was found to be a component of the polysaccharide and identified by NMR spectroscopy and after carboxyl-reduction followed by acid hydrolysis and acetylation, by GLC-MS as 2,3,4-tri-O-acetyl-1,6-anhydroidose. On the basis of 1H and 13C NMR spectroscopic studies, including 1D NOE, 2D NOESY, HSQC and HMBC experiments, the following structure of the branched pentasaccharide repeating unit of the polysaccharide was established: -->4)-beta-D-GlcpAI-(1-->4)-beta-D-GlcpAII-(1-->3)-beta-D-++ +QuipNHb4NHbII- (1-->2)-alpha-L-IdopA-(-->4 increases 1 alpha-D-QuipNAc4NAcI where QuiNAc4NAc and QuiNHb4NHb are 2,4-diacetamido-2,4,6-trideoxyglucose and 2,4,6-tri-deoxy-2,4- di[(S)-3-hydroxybutyramido]glucose, respectively. This is the first report of L-iduronic acid in a lipopolysaccharide and of D-QuiNHb4NHb in nature.

High performance anion exchange chromatography of reduced oligosaccharides from sialomucins

High performance anion exchange chromatography on pellicular ion exchange resins under high pH conditions with detection of sugars using a pulsed amperometric detector has been developed as a method for the separation and analysis of reduced oligosaccharides liberated from mucins by alkaline borohydride treatment. Ovine, bovine and porcine submaxillary mucins were used as models to develop the method. Although neutral reduced di-to tetraoligosaccharides were poorly retained on the column, a variety of sialylated reduced oligosaccharides could be separated efficiently. Treatment of the samples with sialidase and rechromatography identified the sialylated compounds in the elution profile. A striking finding was the greatly delayed elution times given byN-glycolylneuraminic acid containing compounds in comparison with the correspondingN-acetylneuraminic acid containing analogues. The elution profiles for the product from the mucins closely corresponded to those expected for the major oligosaccharides from these mucins. The procedures described will be useful for analysing sialomucins on a microscale without resorting to radiolabelling procedures.

Catalytic properties and mode of action of three endo-β-glucanases from Talaromyces emersonii on soluble β-1,4- and β-1,3;1,4-linked glucans

In this paper, we present the first detailed analysis of the modes of action of three purified, thermostable endo-beta-D-glucanases (EG V-VII) against a range of soluble beta-linked glucans. Studies indicated that EG V-VII, purified to homogeneity from a new source, the thermophilic fungus Talaromyces emersonii, are strict beta-glucanases that exhibit maximum activity against mixed-link 1,3;1,4-beta-D-glucans. Time-course hydrolysis studies of 1,4-beta-D-glucan (carboxymethylcellulose; CMC), 1,3;1,4-beta-D-glucan from barley (BBG) and lichenan confirmed the endo-acting nature of EG V-VII and verified preference for 1,3;1,4-beta-D-glucan substrates. The results suggest that EG VI and EG VII belong to EC 3.2.1.6, as both enzymes also exhibit activity against 1,3-beta-glucan (laminaran), in contrast to EG V. Although cellobiose, cellotriose and glucose were the main glucooligosaccharide products released, the range and relative amount of each product was dependent on the particular enzyme, substrate and reaction time. Kinetic constants (Km, Vmax, kcat and kcat/Km) determined for EG V-VII with BBG as substrate yielded similar Km and Vmax values for EG V and EG VI. EG VII exhibited highest affinity for BBG (Km value of 9.1 mg ml(-1)) and the highest catalytic efficiency (kcat/Km of 12.63 s(-1) mg(-1) ml).

Structural investigation of the capsular polysaccharide of Klebsiella serotype k23

Abstract Klebsiella K23 capsular polysaccharide has been investigated by the techniques of hydrolysis, methylation, Smith degradation-periodate oxidation, and base-catalysed degradation, either on the original or the carboxyl-reduced polysaccharide. The structure was found to consist of a tetrasaccharide repeating-unit, as shown below. The anomeric configurations of the sugar residues were determined by 1 H-and 13 C-n.m.r. spectroscopy on the original and degraded polysaccharides.

Structure of an acidic polysaccharide from a marine bacterium Pseudoalteromonas distincta KMM 638 containing 5-acetamido-3,5,7,9-tetradeoxy-7- formamido-L-glycero-L-manno-nonulosonic acid

An acidic polysaccharide was obtained from the lipopolysaccharide of Pseudoalteromonas distincta strain KMM 638, isolated from a marine sponge, and found to contain D-GlcA, D-GalNAc, 2-acetamido-2,6-dideoxy-D-glucose (D-QuiNAc) and two unusual acidic amino sugars: 2-acetamido-2-deoxy-D-galacturonic acid (D-GalNAcA) and 5-acetamido-3,5,7,9-tetradeoxy-7-formamido-L-glycero-L-manno-nonulosonic acid (Pse5Ac7Fo, a derivative of pseudaminic acid). Oligosaccharides were derived from the polysaccharide by partial acid hydrolysis and mild alkaline degradation and characterised by electrospray ionisation (ESI) MS and 1H and 13C NMR spectroscopy. Based on these data and NMR spectroscopic studies of the initial and O-deacetylated polysaccharides, including quaternary carbon detection, 2D COSY, TOCSY, ROESY, H-detected 1H,13C HMQC and HMBC experiments, the following structure of the branched pentasaccharide repeating unit was established: [structure: see text].

Structures of two polysaccharides of Campylobacter jejuni 81116.

Campylobacter jejuni 81116 has been extensively investigated in studies on genes associated with the synthesis of Campylobacter lipopoly/lipooligosaccharides (LPS/LOS). Despite these investigations, data on the chemical structure of polysaccharides from C. jejuni 81116 have been absent. The present study was undertaken to fill that void. Biomass was grown in large quantities on agar medium, harvested and extracted by hot phenol-water extraction. Subsequently, extracts were treated by DNase, RNase and proteinase K to remove contaminants. After mild acid treatment, followed by preparative gel-permeation and anion-exchange chromatography, fractions were isolated and studied by 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, 1H,(13)C HMQC and HMBC experiments. These advanced investigations revealed the occurrence of two different polysaccharides in the approximate ratio of 3:1, each having a tetrasaccharide repeating unit. Polysaccharide A contained glucose, glucuronic acid and mannose, and is O-acetylated. Polysaccharide B contained glucose, galactose and N-acetylglucosamine. Importantly, polysaccharide A is acidic, whereas polysaccharide B is neutral. [carbohydrate structure: see text]