Cross validation of liquid chromatography-mass spectrometry and lectin array for monitoring glycosylation in fed-batch glycoprotein production.

Glycosylation analysis of recombinant glycoproteins is of importance for the biopharmaceutical industry and the production of glycoprotein pharmaceuticals. A commercially available lectin array technology was evaluated for its ability to present a reproducible fingerprint of a recombinant CTLY4-IgG fusion glycoprotein expressed in large scale CHO-cell fermentation. The glycosylation prediction from the array was compared to traditional negative mode capillary LC-MS of released oligosaccharides. It was shown that both methods provide data that allow samples to be distinguished by their glycosylation pattern. This included information about sialylation, the presence of reducing terminal galactose ß1-, terminal N-acetylglucosamine ß1-, and antennary distribution. With both methods it was found that a general trend of increased sialylation was associated with an increase of the antenna and reduced amount of terminal galactose ß1-, while N-acetylglucosamine ß1- was less affected. LC-MS, but not the lectin array, provided valuable information about the sialic acid isoforms present, including N-acetylneuraminic acid, N-glycolylneuraminic acid and their O-acetylated versions. Detected small amounts of high-mannose structures by LC-MS correlated with the detection of the same epitope by the lectin array.

Molecular origin of two polysaccharides of Campylobacter jejuni 81116.

The nature of the polysaccharide molecules of the human enteric pathogen Campylobacter jejuni has been the subject of debate. Previously, C. jejuni 81116 was shown to contain two different polysaccharides, one acidic (polysaccharide A) and the other neutral (polysaccharide B), occurring in a 3 : 1 ratio, respectively. The aim of this study was to determine the molecular origin of these polysaccharides. Using a combination of centrifugation, gel permeation chromatography, chemical assays, and (1)H-NMR analysis, polysaccharide B was shown to be derived from lipopolysaccharide and polysaccharide A from capsular polysaccharide. Thus, C. jejuni 81116 produces both lipopolysaccharide-like molecules and capsular polysaccharide.

The structure of the O-polysaccharide of the Pseudoalteromonas rubra ATCC 29570T lipopolysaccharide containing a keto sugar.

The structure of the phenol-soluble polysaccharide from Pseudoalteromonas rubra type strain ATCC 29570T has been elucidated using 1H and 13C NMR spectroscopy, including 2D COSY, TOCSY, gNOESY, ROESY, 1H,13C gHMQC and gHMBC experiments. It is concluded that the trisaccharide repeating unit of the polysaccharide has the following structure: [carbohydrate structure: see text] where Sug is 2-acetamido-2,6-dideoxy-D-xylo-hexos-4-ulose, Am is acetimidoyl and Acyl is a malic acid residue, which is O-acetylated in approximately 70% of the units.

Structure of the O-specific polysaccharide from Shewanella japonica type strain KMM 3299T containing the rare amino sugar Fuc4NAc.

An acidic O-specific polysaccharide (PS) of the agar-digesting bacterium Shewanella japonica with the type strain KMM 3299(T) was obtained by mild acid hydrolysis of the lipopolysaccharide. The polysaccharide was studied by component analysis, methylation analysis, (1)H and (13)C NMR spectroscopy, including 2D NMR experiments. The PS was determined to have the following structure involving three unusual amino sugars:

Comparison of wild-type and UV-mutant beta-glucanase-producing strains of Talaromyces emersonii with potential in brewing applications.

A screen of 46 UV-mutant strains of the moderately thermophilic fungus Talaromyces emersonii yielded two mutants (TC2, TC5) that displayed gross morphological differences to the parent strain and enhanced activity against mixed linkage cereal beta-glucans. Activity against beta-(1, 3)(1, 4)-D: -glucan from barley (BBGase) was measured during growth of the mutant and wild-type strains on a variety of carbon sources, ranging from solka floc to crude cereal fractions. In liquid culture, TC2 and TC5 secreted 1.2- to 8.6-fold more BBGase than the parent strain and markedly less beta-glucosidase (exo-activity); enzyme levels were dependent on the carbon source. Cellulose induced high BBGase. However, beet pulp, wheat bran, carob and tea-leaves were cheap and effective inducers. T. emersonii wild-type, TC2 and TC5 crude enzyme preparations achieved similar end-points during the hydrolysis of commercial barley beta-glucan (13.0-16.9%), but were more active against crude beta-glucan from barley (16.0-24.2% hydrolysis). The products of hydrolysis were quantified by high-performance anion-exchange chromatography. Mash trials indicated that enzyme preparations from all three organisms effected a significant reduction in wort viscosity and residual mash beta-glucan. Finally, TC2 and TC5 produce more efficient beta-glucan-depolymerizing enzymes; and wheat bran and solka floc can be used to provide inexpensive and potent enzyme cocktails with potential in brewing applications.

Chemical structure of aeromonas gum--extracellular polysaccharide from Aeromonas nichidenii 5797.

Aeromonas (A) gum, an extracellular heteropolysaccharide produced by the bacterium Aeromonas nichidenii strain 5797, was studied by 1H and 13C NMR spectroscopy including 2D COSY, TOCSY, 1H, 13C HMQC, HMBC and ROESY experiments after O-deacetylation and Smith degradation. These investigations revealed the presence of an O-acetylated pentasaccharide repeating unit composed of mannose, glucose, xylose and glucuronic acid, and it has the following structure: [Image: see text]

Structure of an acidic O-specific polysaccharide from marine bacterium Shewanella fidelis KMM 3582T containing Nepsilon-[(S)-1-carboxyethyl]-Nalpha-(D-galacturonoyl)-L-lysine.

The O-specific polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of the marine bacterium Shewanella fidelis type strain KMM 3582T and studied by sugar analysis along with 1H and 13C NMR spectroscopy including one-dimensional NOE in difference mode and two-dimensional experiments. The polysaccharide was found to consist of linear tetrasaccharide repeating units containing Nepsilon-[(S)-1-carboxyethyl]-Nalpha-(D-galacturonoyl)-L-lysine and having the following structure: [See text.] The amide of D-galacturonic acid with Nepsilon-[(S)-1-carboxyethyl]-L-lysine ('alaninolysine', 2S,8S-AlaLys) was found for the first time in nature as a component of the O-specific polysaccharide of Providencia rustigianii O14 (Carbohydr. Res. 2003, 338, 1009-1016).

Structure of the O-polysaccharide of Idiomarina zobellii KMM 231T containing two unusual amino sugars with the free amino group, 4-amino-4,6-dideoxy-D-glucose and 2-amino-2-deoxy-L-guluronic acid.

Mild acid degradation of the lipopolysaccharide of the bacterium Idiomarina zobellii, type strain KMM 231T, with aq 2% HOAc at 100 degrees C, yielded an oligosaccharide, which represents one repeating unit of the O-polysaccharide. A polysaccharide was obtained by mild base degradation of the lipopolysaccharide. The following structure of the O-polysaccharide was elucidated by 1H and 13C NMR spectroscopy of the oligosaccharide and base-degraded lipopolysaccharide, including COSY, TOCSY, ROESY, 1H, 13C HSQC, HSQC-TOCSY and HMBC experiments: [-->3)-alpha-D-Quip4N-(1-->4)-alpha-D-GlcpA-(1-->6)-alpha-D-GlcpNAc-(1-->4)-alpha-L-GulpNA-(1-->3)-beta-D-FucpNAc-(1-->] The O-polysaccharide is distinguished by the presence of two unusual amino sugars, 4-amino-4,6-dideoxy-D-glucose (D-Qui4N) and 2-amino-2-deoxy-L-guluronic acid (L-GulNA), both having the free amino group. The unexpectedly high acid lability of the glycosidic linkage of 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc) could be associated with the presence of a free amino group adjacent to the site of attachment of FucNAc to Qui4N.

Chemical components and molecular mass of six polysaccharides isolated from the sclerotium of Poria cocos.

Six polysaccharides were extracted sequentially from the fresh sclerotium of Poria cocos cultivated in China using 0.9% NaCl (PCS1), hot water (PCS2), 0.5M NaOH (PCS3-I and PCS3-II), and 88% formic acid (PCS4-I and PCS4-II). Their chemical and physical characteristics were determined using infrared spectroscopy (IR), gas chromatography (GC), GC-MS methylation analysis, 13C NMR spectroscopy, elementary analysis (EA), protein analysis, size exclusion chromatography combined with laser light scattering (SEC-LLS), light scattering (LS), and viscometry. The results indicated that the polysaccharides PCS1, PCS2, and PCS3-I were heteropolysaccharides containing D-glucose, D-galactose, D-mannose, D-fucose, and D-xylose; the predominant monosaccharide was D-glucose except for PCS1 where it was D-galactose. PCS3-II, the main component of the sclerotium of P. cocos, was a linear (1-->3)-beta-D-glucan of high purity. PCS4-I consisted of (1-->3)-beta-D-glucan with some beta-(1-->6) linked branches. PCS4-II was mainly composed of (1-->3)-beta-D-glucan containing some glucose branches. The M(w) values of the six polysaccharides PCS1, PCS2, PCS3-I, PCS4-I in 0.2M NaCl aqueous solution, PCS3-II, and PCS4-II in dimethyl sulfoxide (Me(2)SO) were determined to be 11.6 x 10(4), 20.8 x 10(4), 17.1 x 10(4), 9.1 x 10(4), 12.3 x 10(4), and 21.1 x 10(4), respectively. The six polysaccharides in aqueous solution or Me(2)SO exist as flexible chains.

Structures of polysaccharides and oligosaccharides of some Gram-negative marine Proteobacteria.

The chemical structures of polysaccharides and LPS core oligosaccharides, isolated from various Gram-negative marine bacteria from the genera Pseudoalteromonas and Shewanella belonging to the Alteromonadaceae family and gamma-subclass of Proteobacteria, are reviewed. The polysaccharides are distinguished by the acidic character (e.g., due to the presence of hexuronic and aldulosonic acids and their derivatives) and the occurrence of unusual sugars, including N-acyl derivatives of 6-deoxyamino sugars, such as N-acetyl-D-quinovosamine, N-acetyl-L-fucosamine and N-acetyl-6-deoxy-L-talosamine, and higher sugars like 2,6-dideoxy-2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-D-galactopyranose (shewanellose). Many constituent sugars have various uncommon non-sugar substituents, such as alanine, formic, lactic and hydroxybutyric acids, sulfate, phosphate, and 2-aminopropane-1,3-diol.

Catalytic properties and mode of action of three endo-beta-glucanases from Talaromyces emersonii on soluble beta-1,4- and beta-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).

Structure of an acidic polysaccharide from the marine bacterium Pseudoalteromonas flavipulchra NCIMB 2033(T).

An acidic polysaccharide was isolated from Pseudoalteromonas flavipulchra type strain NCIMB 2033(T) and found to consist of 6-deoxy-L-talose (L-6dTal), D-galactose and 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo). The identities of the monosaccharides were ascertained by sugar analysis and 1D 1H and 13C NMR spectroscopy in conjunction with 2D COSY, TOCSY, ROESY and 1H, 13C HMQC experiments, which enabled determination of the following structure of the trisaccharide repeating unit of the polysaccharide:-->3)-alpha-L-6dTalp4Ac-(1-->3)-beta-D-Galp-(1-->7)-alpha-Kdop-(2-->.

Structural investigation of the O-specific polysaccharides of Morganella morganii consisting of two higher sugars.

The lipopolysaccharide of the bacterium Morganella morganii (strain KF 1676, RK 4222) yielded two polysaccharides, PS1 and PS2, when subjected to mild acid degradation followed by GPC. The polysaccharides were studied by 1H and 13C NMR spectroscopy, including two-dimensional COSY, TOCSY, NOESY, 1H,(13)C HMQC, and HMBC experiments. Each polysaccharide was found to contain a disaccharide repeating unit consisting of two higher sugars, 5-acetamidino-7-acetamido-3,5,7,9-tetradeoxy-L-glycero-D-galacto-non-2-ulosonic acid (a derivative of 8-epilegionaminic acid, 8eLeg5Am7Ac) and 2-acetamido-4-C-(3'-carboxamide-2',2'-dihydroxypropyl)-2,6-dideoxy-D-galactose (shewanellose, She). The two polysaccharides differ only in the ring size of shewanellose and have the following structures:Shewanellose has been previously identified in a phenol-soluble polysaccharide from Shewanella putrefaciens A6, which shows a close structural similarity to PS2.

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]

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.