Cellobionic Acid Research Articles

Production of oligosaccharides and cellobionic acid by Fibrobacter succinogenes S85 growing on sugars, cellulose and wheat straw

Extracellular culture fluid of Fibrobacter succinogenes S85 grown on glucose, cellobiose, cellulose or wheat straw was analysed by 2D-NMR spectroscopy. Cellodextrins did not accumulate in the culture medium of cells grown on cellulose or straw. Maltodextrins and maltodextrin-1P were identified in the culture medium of glucose, cellobiose and cellulose grown cells. New glucose derivatives were identified in the culture fluid under all the substrate conditions. In particular, a compound identified as cellobionic acid accumulated at high levels in the medium of F. succinogenes S85 cultures. The production of cellobionic acid (and cellobionolactone also identified) was very surprising in an anaerobic bacterium. The results suggest metabolic shifts when cells were growing on solid substrate cellulose or straw compared to soluble sugars.

4-O-β-d-GLUCOPYRANOSYL-d-GLUCONIC ACID (CELLOBIONIC ACID) PRODUCED BY OZONATION OF CELLOBIOSE: ISOLATION BY HPLC AND ASSIGNMENT OF NMR CHEMICAL SHIFTS

Mild ozonation of cellobiose in acetic acid almost exclusively favors oxidation of the reducing end, thus yielding 4-O-β-d-glucopyranosyl-d-gluconic acid (“cellobionic acid”). This compound can be quantitatively separated from the substrate by preparative HPLC using acetonitrile/phosphate buffer on an aminopropyl silica column. The product was identified using one and two dimensional NMR techniques. 13C and 1H NMR chemical shifts are reported. †Current address: Södra Cell R&D, NO-3482 Tofte, Norway. ††Current address: The Norwegian Pulp and Paper Research Institute (PFI), Høgskoleringen 6B, NO-7491 Trondheim, Norway.

Degradation of Cellulose Models During an Ozone Treatment. Ozonation of Glucose and Cellobiose With Oxygen or Nitrogen as Carrier Gas at Different pH

Many parameters can influence the kinetics of cellulose models ozonation. In this work the effect of pH and oxygen as gas carrier on the evolution of the reaction was studied. The involvement of free radical species seemed to be unimportant in high ozone concentration available in water, even at high pH with regard to the reaction rate and by-product formation. Conversely, in basic pH conditions better reaction selectivity with formation in a higher yield of gluconic acid and cellobionic acid was found during the glucose and the cellobiose ozonation. The participation of the solvent and the partial ionization of the hydroxyl group at the Cl position explain this enhanced selectivity. Furthermore, the formation of compounds with a low molecular weight has been proved to be due to a hydrotrioxide fragmentation mechanism involving oxygen.

Direct 1H NMR evidence for conversion of β- d-cellobiose to cellobionolactone by cellobiose dehydrogenase from Phanerochaete chrysosporium

The α- and β-anomers of d-cellobiose were resolved by 1H NMR spectroscopy. Addition of cellobiose dehydrogenase purified from the white-rot P. chrysosporium led to selective conversion of β- d-cellobiose. The product was identical to cellobionolactone as synthesized from Ca-cellobionate. Overnight incubation of the product led to an altered NMR spectrum, which was also obtained by incubation of cellobionolactone. The new spectrum matched that for Ca-cellobionate. The instability of cellobionolactone explains the detection of cellobionic acid as product in earlier studies.

Creation of metal-complexing agents, reduction of manganese dioxide, and promotion of manganese peroxidase-mediated Mn(III) production by cellobiose:quinone oxidoreductase from Trametes versicolor.

Culture supernatants of the white-rot fungus Trametes versicolor were found to contain Mn(III)-complexing agents able to effectively promote manganese-dependent peroxidase (MnP)-mediated oxidation of phenol red. The high molecular weight fractions of these supernatants contained carbohydrate polymers that functioned as effective Mn(III)-complexing agents. Gluconic and glucuronic acids were also found to be effective Mn(III)-complexing ligands capable of supporting MnP-mediated phenol red oxidation, as was the cellobionic acid formed from cellobiose by cellobiose:quinone oxidoreductase (CBQase) (EC 1.1.5.1). The formation of cellulose-derived sugar acid manganese-complexing agents by CBQase increased in the presence of wood pulp and cellulolytic enzymes. CBQase, while oxidizing cellobiose, reduced insoluble Mn(IV)O2 to Mn(II) or Mn(III) by a reaction whose rate is determined by the nature of the manganese-complexing agents present. A model is proposed to explain how oxidation of carbohydrate and reduction of MnO2 and quinones by CBQase may complement oxidation by MnP, thus promoting lignin biodegradation.

Cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile.

Both cellobiose dehydrogenases of Sporotrichum (Chrysosporium) thermophile, ATCC 42464, obtained after fractionation with DEAE-Trisacryl chromatography and named cellobiose dehydrogenase I and II have been purified to homogeneity by different chromatographic techniques. Both enzymes are slightly glycosylated flavocytochrome-b proteins with similar catalytic properties but with distinct molecular masses (91 kDa and 192 kDa for enzymes I and II, respectively) and isoelectric point (4.1 versus 3.45). Examination by SDS/PAGE clearly showed that the larger enzyme II is a homodimer, whose subunit is close to, but different from dehydrogenase I which is homogeneous by this technique. After limited digestion of both enzymes with papain, two main fractions with residual activity are formed, one carrying the heme, the other being the flavin component; each fraction is characterized by its particular chromatographic behaviour. The flavin carrying component shows an atypical (for flavoprotein) three-banded spectrum indicative of the presence of a flavin derivative. Both enzymes react very slowly with oxygen clearly forming some superoxide radicals and possibly hydrogen peroxide. Cellobiose and other cellodextrins are oxidized at their reducing glycosyl moiety to the corresponding aldonic acid. With the use of the autooxidable phenazinemethosulphate, cellulose (either in a hydrated form or crystalline) is also oxidized at free reducing ends so that appreciable amounts of cellobionic acid are released upon enzymatic hydrolysis.

Conversion of cellulose to sugars and cellobionic acid by the extracellular enzyme system of Chaetomium cellulolyticum

Hydrolyses of cellulosics by crude enzyme yielded glucose, cellobiose, and cellobionic acid. A cellobiose oxidizing enzyme was detected in culture broth of Chaetomium cellulolyticum and identified as a cellobiose dehydrogenase.

Characterization of a cellobiose dehydrogenase in the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile.

An extracellular enzyme from culture filtrates of Sporotrichum (Chrysosporium) thermophile (A.T.C.C. 42 464) after growth on cellulose or cellobiose was shown to oxidize cellobiose to cellobionic acid in vitro. Lactose and cellodextrins were also efficiently oxidized, but the enzyme was not active against most mono- and di-saccharides. Several redox substances could act as electron acceptors, but molecular oxygen, tetrazolium salts and NAD(P) were not reduced. Activity was stimulated up to 2-fold in the presence of 0.05 M-Mg2+. The pH optimum of the enzymic reaction was acidic when the activity was tested with dichlorophenol-indophenol or Methylene Blue, but was neutral to alkaline for 3,5-di-t-butyl-1,2-benzoquinone or phenazine methosulphate as electron acceptors. As the enzyme was formed inductively in parallel with the endocellulase, its possible function in relation to cellulolysis is discussed.

Induction and Catabolite Repression of Cellulase Synthesis in the Thermophilic Fungus Sporotrichum thermophile

SUMMARY: A strain of Sporotrichum thermophile (var. 2), grown in submerged culture at 44°C, decayed crystalline cellulose by the secretion of cellulolytic enzymes into the culture medium. Dialysed culture filtrates, after growth on cellobiose, were also effective in degrading crystalline cellulose (Avicel). Cellulase, measured viscometrically with carboxymethylcellulose as substrate and therefore referred to as CM-cellulase, was induced by cellulose, glucomannan, cellobiose and, to a limited extent, by laminaribiose and cellobionic acid. Low CM-cellulase activities were also detectable when the organism was grown on other carbon sources. In culture media supplemented with readily metabolized non-inducing substrates, such as glucose, CM-cellulase activity did not increase after their exhaustion indicating that no derepression of constitutive CM-cellulase occurred. CM-cellulase synthesis induced during growth on cellobiose was strongly inhibited, although not completely suppressed, by glucose and other carbon sources. In cultures grown on glucose and cellobiose, glucose was utilized first and CM-cellulase synthesis was repressed; cellobiose utilization occurred only after glucose exhaustion and triggered CM-cellulase formation. When glucose was added to a culture growing on cellobiose, further utilization of the latter was prevented and CM-cellulase synthesis was inhibited. The use of glucose analogues gave some indications that cellobiose did not compete with glucose for the same transport carrier and that glucose catabolism was a prerequisite for the inhibition of CM-cellulase synthesis.

Strahlenchemie von Kohlenhydraten, IV. γ-Radiolyse von Cellobiose in N2O-gesättigter wäßriger Lösung / γ-Radiolysis of Cellobiose in N2O Saturated Aqueous Solution

After γ-radiolysis of N2O saturated deoxygenated aqueous solutions of cellobiose (10-2Μ) the following products have been determined: Glucose, gluconic acid, 5-keto-glucose, 4-deoxy-glucose, 5-deoxy-gluconic acid, 2-deoxy-gluconic acid, 3-deoxy-4-keto-glucose, 2-deoxy-5-keto-glucose, 4-deoxy-5-keto-glucose, arabinose, 2-deoxy-ribose, ribose, erythrose, threose, 2-deoxy-tetrose, dihydroxy acetone, 3-deoxy-pentulose, 2-butanone-1,4-diol, 5-hydroxymethyl-furfural, carbon monoxid, and C12-acids (probably cellobionic acid and 2-deoxy-cellobionic acid). Analytical techniques were column chromatography, t. l. c. and g. l. c. combined with m. s.