A high field NMR study of the products ensuing from konjak glucomannan C(6)-oxidation followed by enzymatic C(5)-epimerization

Konjak glucomannan (KGM) is a water-soluble linear copolymer of (1-->4) linked beta-D-mannopyranosyl and beta-D-glucopyranosyl units. It has been selectively C6-oxidized by a 2,2,6,6-tetramethylpiperidin-1-oxy mediated reaction to obtain the corresponding uronan. Oxidized KGM has been treated with three different C-5 epimerases, AlgE4, AlgE6, and AlgE1, to obtain uronans with a various content of alpha-L-gulopyranuronate residues, namely, KGME4, KGME6, and KGME1. By use of 1D selective and 2D NMR techniques, a full assignment of the high field (600 MHz) NMR spectra of the purified native KGM and of the oxidized and epimerized derivatives has been obtained. Since in the anomeric region of the (1)H NMR spectrum of native KGM, diads sensitivity is present, the glucose-glucose, glucose-mannose, mannose-mannose, and mannose-glucose distribution has been obtained. In the (13)C spectrum of oxidized KGM, due to the presence of triad sensitivity on the C-4 resonance of glucuronic and mannuronic units, a better sequential investigation has been possible. As a result the average length of mannuronic blocks, N(M) is obtained. When AlgE4, AlgE6, and AlgE1 enzymes are used for the epimerization of oxidized KGM, the reaction products differ significantly both in the proportion and in the distribution of the mannuronic and guluronic residues. In epimerized KGM derivatives, a careful deconvolution of (1)H spectra allows the measurement of the degree of epimerization. In the case of KGME1 and KGME6, the average blocks length, N(G), of the guluronic blocks introduced in the polysaccharidic chain with the epimerization has also been calculated. Due to the shortness of mannuronic blocks in the oxidized KGM before the epimerization, N(G) in the epimerized compounds is also very low.     

C(6)-oxidation followed by C(5)-epimerization of guar gum studied by high field NMR

Guar gum, a beta-D-(1-->4)-linked D-mannan with alpha-D-galactopyranosyl units attached as side groups, was treated with alpha-galactosidase, an enzyme that splits off the alpha-D-galactosyl units to obtain a galactomannan with a low galactose content. The galactose-depleted polysaccharide was then selectively oxidized in C(6) position and epimerized using mannuronan C(5)-epimerases, namely AlgE1, AlgE4, AlgE6, and their mixtures, obtaining new pseudo-alginates. In this paper, we report a full high field 1D and 2D NMR study of guar gum as such and of the galactose-depleted, oxidized and epimerized compounds, respectively. From the 1H NMR spectra, the degree of epimerization, the distribution of mannuronic acid (M) and guluronic acid (G) residues and the average G-block length, N(G>1), were obtained. By means of NMR diffusion experiments, it was also shown that no significant degradation of the polysaccharide occurs as a consequence of the epimerization reactions.     

Substrate-dependent chemoselective aldose-aldose and aldose-ketose isomerizations of carbohydrates promoted by a combination of calcium ion and monoamines.

Epimerization of aldoses at C-2 has been extensively investigated by using various metal ions in conjunction with diamines, monoamines, and aminoalcohols. Aldoses are epimerized at C-2 by a combination of alkaline-earth or rare-earth metal ions (Ca(2+), Sr(2+), Pr(3+), or Ce(3+)) and such monoamines as triethylamine. In particular, the Ca(2+)-triethylamine system proved effective in promoting aldose-ketose isomerization as well as C-2 epimerization of aldoses. 13C NMR studies using D-(1-(13)C)glucose and D-(1-(13)C)galactose with the CaCl(2) system in CD(3)OD revealed that the C-2 epimerization proceeds via stereospecific rearrangement of the carbon skeleton, or 1,2-carbon shift, and ketose formation proceeds partially through an intramolecular hydrogen migration or 1,2-hydride shift and, in part, via an enediol intermediate. These simultaneous aldose-aldose and aldose-ketose isomerizations showed interesting substrate-dependent chemoselectivity. Whereas the mannose-type aldoses having 2,3-erythro configuration (D-mannose, D-lyxose, and D-ribose) showed considerable resistance to both the C-2 epimerization and the aldose-ketose isomerization, the glucose-type sugars having 2,3-threo and 3,4-threo configurations, D-glucose and D-xylose, are mainly epimerized at C-2 and those having the 2,3-threo and 3,4-erythro configurations, D-galactose and D-arabinose, were mostly isomerized into 2-ketoses. These features are of potential interest in relevance to biomimic sugar transformations by metal ions.     

Mechanism of dihydroneopterin aldolase. NMR, equilibrium and transient kinetic studies of the Staphylococcus aureus and Escherichia coli enzymes

Dihydroneopterin aldolase (DHNA) catalyzes both the cleavage of 7,8-dihydro-D-neopterin (DHNP) to form 6-hydroxymethyl-7,8-dihydropterin (HP) and glycolaldehyde and the epimerization of DHNP to form 7,8-dihydro-L-monapterin (DHMP). Whether the epimerization reaction uses the same reaction intermediate as the aldol reaction or the deprotonation and reprotonation of C2' of DHNP has been investigated by NMR analysis of the reaction products in a D2O solvent. No deuteration of C2' was observed for the newly formed DHMP. This result strongly suggests that the epimerization reaction uses the same reaction intermediate as the aldol reaction. In contrast with an earlier observation, the DHNA-catalyzed reaction is reversible, which also supports a nonstereospecific retroaldol/aldol mechanism for the epimerization reaction. The binding and catalytic properties of DHNAs from both Staphylococcus aureus (SaDHNA) and Escherichia coli (EcDHNA) were determined by equilibrium binding and transient kinetic studies. A complete set of kinetic constants for both the aldol and epimerization reactions according to a unified kinetic mechanism was determined for both SaDHNA and EcDHNA. The results show that the two enzymes have significantly different binding and catalytic properties, in accordance with the significant sequence differences between them.     

C-2 Epimerization of aldonolactones promoted by magnesium iodide: a new way towards non-enzymatic epimerization

The first example of a non-enzymatic C-2 epimerization of aldonolactones is reported. The reaction of 2,3,4,6-tetra-O-benzyl-d-gluconolactone or 2,3,4,6-tetra-O-benzyl-d-mannonolactone with MgI(2) in EtOH afforded their respective C-2 epimer. Studies conducted in EtOD showing the incorporation of a deuterium atom only at the C-2 position of the epimerized product reveal an epimerization rather than a racemization reaction. A mechanism involving a chelation with a magnesium species is proposed to explain this C-2 inversion reaction.     

Time-resolved 1H and 13C NMR spectroscopy for detailed analyses of the Azotobacter vinelandii mannuronan C-5 epimerase reaction

AlgE2, AlgE4, and AlgE6 are members of a family of mannuronan C-5 epimerases encoded by Azotobacter vinelandii, and are active in the biosynthesis of alginate, where they catalyze the post-polymerization conversion of beta-D-mannuronic acid residues into alpha-L-guluronic acid residues. To study the kinetics and mode of action of these enzymes, homopolymeric mannuronan and other alginate samples with various composition were epimerized by letting the enzymatic reaction take place in an NMR tube. Series of 1H NMR spectra were recorded to obtain a time-resolved picture of the epimerization progress and the formation of specific monomer sequences. Starting from mannuronan, guluronic acid contents of up to 82% were introduced by the enzymes, and the product specificity, substrate selectivity, and reaction rates have been investigated. To obtain direct information of the GulA-block formation, similar experiments were performed using a 13C-1-enriched mannuronan as substrate. The NMR results were found to be in good agreement with data obtained by a radioisotope assay based on 3H-5-labeled substrates.     

Tailor-made alginate bearing galactose moieties on mannuronic residues: selective modification achieved by a chemoenzymatic strategy

1-Amino-1-deoxygalactose (12%, mole) has been chemically introduced on a mannuronan sample via an N-glycosidic bond involving the uronic group of the mannuronic acid (M) residues. The unsubstituted M residues in the modified polymer were converted into guluronic moieties (G) by the use of two C-5 epimerases, resulting in an alginate-like molecule selectively modified on M residues. The molecular details of the newly formed polymer, in terms of both composition and molecular dimensions, were disclosed by use of (1)H NMR, intrinsic viscosity, and high-performance size-exclusion chromatography-multiple-angle laser light scattering (HPSEC-MALLS). Circular dichroism has revealed that the modified alginate-like polymer obtained after epimerization was able to bind calcium due to the introduction of alternating and homopolymeric G sequences. The gel-forming ability of this M-selectively modified material was tested and compared with an alginate sample containing 14% galactose introduced on G residues. Mechanical spectroscopy pointed out that the modified epimerized material was able to form stable gels and that the kinetics of the gel formation was similar to that of the unsubstituted sample. In contrast, the G-modified alginate samples showed a slower gel formation, eventually leading to gel characterized by a reduced storage modulus. The advantage of the selective modification on M residues was confirmed by measuring the Young's modulus of gel cylinders of the different samples. Furthermore, due to the high content in alternating sequences, a marked syneresis was disclosed for the modified-epimerized sample. Finally, calcium beads obtained from selectively M-modified alginate showed a higher stability than those from the G-modified alginate, as evaluated upon treatment with nongelling ions.     

Incorporation of galactomannans in the diet of newly weaned piglets: effect on bacteriological and some morphological characteristics of the small intestine

In search of substances replacing antibiotics as growth promoters for farm animals, non-digestible oligosaccharides (NDO) or non-starch polysaccharides (NSP) have been proposed as possible alternatives. In this context, the influence of galactomannans on bacteriological and morphological aspects of the gastrointestinal tract in weanling pigs was investigated. Four groups of five newly weaned piglets received one of the following diets: control feed (C), C supplemented with guar gum (1%), C supplemented with locust bean gum (1%) and C supplemented with 10% of carob tree seeds meal as source of locust bean gum. The animals were euthanized after 11-12 days and digesta were sampled in stomach, jejunum (proximal and distal) and caecum, while mucosal scrapings and ring shaped tissue samples were taken of proximal and distal jejunum. On these samples bacteriological, biochemical and morphological determinations were carried out. Total count of bacteria in digesta and mucosal scrapings was not influenced by the different diets, with the exception of the proximal jejunum where a small decrease (0.5 log10 CFU) was noted with the guar gum and carob tree seeds diet. The number of E. coli increased by feeding both gums and carob tree seeds. With the latter diet, higher counts of streptococci were observed. In agreement with the lower concentration of lactic acid in jejunal contents, guar gum decreased the number of lactobacilli. Locust bean gum decreased the molar proportion of acetate in caecal contents while butyrate and valerate were augmented. Feeding the carob tree seeds resulted in shorter villi and a lower villus height/crypt depth ratio in the jejunum mucosa, which was an indication for a faster renewal rate of the epithelium. Both locust bean gum feeds significantly lowered the mitotic index in the crypts of the small intestine. Only with the carob tree seeds diet, viscosity of jejunal contents was increased. In conclusion, the effects of the addition of 1% of pure guar gum or locust bean gum were inconsistent and not very outspoken, whereas 10% of carob tree seeds meal in the diet resulted in influences on intestinal characteristics at the bacteriological and morphological level.     

Fourier transform near-infrared vibrational circular dichroism used for on-line monitoring the epimerization of 2,2-dimethyl-1,3-dioxolane-4-methanol: A pseudo racemization reaction

Near-infrared (near-IR) Fourier transform vibrational circular dichroism (FT-VCD) spectroscopy has been used to monitor the epimerization of (S)-(+)-2,2-dimethyl-1,3-dioxolane-4-methanol (S-DDM). The near-IR-VCD spectra display clear isolated VCD bands at the range of 4700-5050 cm(-1) resulting from the OH stretch-bend combination bands of S-DDM, which were found to decrease in intensity with increasing reaction time. The near-IR-VCD spectra of 10 reference samples obtained were subjected to partial least-squares (PLS) regression analysis, and the results were used to build predictive models for enantiomeric excess (EE) determination. Multivariate regression was carried out on three different sets of spectra, corresponding to the epimerization of S-DDM in three different solvents: methylcyclohexane, carbon tetrachloride and tetrahydrofuran. The effects of solvent in DDM epimerization are discussed in terms of the relative stabilization of the reaction intermediate of the DDM epimerization reaction. The results of these near-IR-VCD studies for the determination of EE highlights the potential of VCD for in situ real-time process monitoring of the reaction kinetics of chiral molecules in solution.     

Epimerization of moxalactam by albumin and simulation of in vivo epimerization by a physiologically based pharmacokinetic model

We investigated the mechanism of epimerization (R to S or S to R) of moxalactam in serum of rats, dogs, and humans. The epimerization of moxalactam occurred in the serum of these animals, but not in the serum filtrate. The albumin fraction of human serum purified by gel filtration catalysed the epimerization of moxalactam at an identical rate to serum, but other fractions (i.e., lipoproteins and globulins) showed slower epimerization. alpha 1-acid glycoprotein, which was eluted in the same fraction with albumin by G-200 gel filtration, did not epimerize moxalactam. The presence of 2 mM warfarin decreased the binding of R- and S-moxalactam and decreased the epimerization of moxalactam in human serum. These results demonstrate moxalactam was epimerized on the warfarin binding site on albumin in serum. Additionally, a physiologically based pharmacokinetic model shows that the epimerization of moxalactam after administration in dogs is simulated by the epimerization in serum.     

Incorporation of galactomannans in the diet of newly weaned piglets: Effect on bacteriological and some morphological characteristics of the small intestine

In search of substances replacing antibiotics as growth promoters for farm animals, non-digestible oligosaccharides (NDO) or non-starch polysaccharides (NSP) have been proposed as possible alternatives. In this context, the influence of galactomannans on bacteriological and morphological aspects of the gastrointestinal tract in weanling pigs was investigated. Four groups of five newly weaned piglets received one of the following diets: control feed (C), C supplemented with guar gum (1%), C supplemented with locust bean gum (1%) and C supplemented with 10% of carob tree seeds meal as source of locust bean gum. The animals were euthanized after 11?–?12 days and digesta were sampled in stomach, jejunum (proximal and distal) and caecum, while mucosal scrapings and ring shaped tissue samples were taken of proximal and distal jejunum. On these samples bacteriological, biochemical and morphological determinations were carried out. Total count of bacteria in digesta and mucosal scrapings was not influenced by the different diets, with the exception of the proximal jejunum where a small decrease (0.5 log₁₀ CFU) was noted with the guar gum and carob tree seeds diet. The number of E. coli increased by feeding both gums and carob tree seeds. With the latter diet, higher counts of streptococci were observed. In agreement with the lower concentration of lactic acid in jejunal contents, guar gum decreased the number of lactobacilli. Locust bean gum decreased the molar proportion of acetate in caecal contents while butyrate and valerate were augmented. Feeding the carob tree seeds resulted in shorter villi and a lower villus height/crypt depth ratio in the jejunum mucosa, which was an indication for a faster renewal rate of the epithelium. Both locust bean gum feeds significantly lowered the mitotic index in the crypts of the small intestine. Only with the carob tree seeds diet, viscosity of jejunal contents was increased. In conclusion, the effects of the addition of 1% of pure guar gum or locust bean gum were inconsistent and not very outspoken, whereas 10% of carob tree seeds meal in the diet resulted in influences on intestinal characteristics at the bacteriological and morphological level.     

Endo-[beta]-Mannanase Activity Present in Cell Wall Extracts of Lettuce Endosperm prior to Radicle Emergence

Lettuce (Lactuca sativa L.) endosperm cell walls isolated prior to radicle emergence underwent autohydrolysis, the rate of which was correlated with whether radicle emergence would subsequently occur. Extraction of endosperm cell walls with 6 M LiCl suppressed autohydrolysis, and the desalted extract possessed activity that was capable of hydrolyzing purified locust bean galactomannan but not arabinogalactan, carboxymethylcellulose, glucomannan, polygalacturonic acid, tomato galactomannan, or native lettuce endosperm cell walls. Some hydrolytic activity was detected on endosperm cell walls if they were modified by partial trifluoroacetic acid hydrolysis or pretreatment with guanidinium thiocyanate. In extended incubations the cell wall enzyme extract released only large molecular mass fragments from locust bean galactomannan, indicating primarily endo-activity. Galactomannan-hydrolyzing activity in the cell wall extract increased as a function of imbibition time and was greatest just prior to radicle emergence. Thermoinhibition (imbibition at 32[deg]C) or treatment with abscisic acid at a temperature optimal for germination (25[deg]C) suppressed both germination and endosperm cell wall mannanase activity, whereas alleviation of thermoinhibition with gibberellic acid was accompanied by significant enhancement of mannanase activity. We conclude that a cell wall-bound endo-[beta]-mannanase is expressed in lettuce endosperm prior to radicle emergence and is regulated by the same conditions that govern germination.     

Gelling interactions of phycocolloids extracted from red algae with a galactomannan from locust bean and a glucomannan from konjac tuber

The synergistic interaction between three red algae extracts and the galactomannan from locust bean (Ceratonia siliqua L.) and the glucomannan from the konjac tuber (Amorphophallus konjac C. Koch (syn.A. rivieri Durien var. konjac (C. Kock) Engler)) has been characterized in terms of gel properties. The extract obtained fromEucheuma alvarezii Doty (E. cottonii of commerce) was highly synergistic with bothkonjac flour and locust bean gum.Furcellaria fastigiata (Huds.) Lamour andEucheuma gelatinae (Esper) extracts were only slightly synergistic with locust bean gum, but were found to be highly synergistic with konjac flour.     

31P NMR and computational studies on stereochemistry of conversion of phosphoramidate diesters into the corresponding phosphotriesters

31P NMR spectroscopy was used to investigate a stereochemical course of a nitrite-promoted conversion of phosphoramidate diesters into the corresponding phosphotriesters. It was found that this reaction occurred with almost complete epimerization at the phosphorus center and at the C1 atom in the amine moiety. On the basis of the 31P NMR data, a plausible mechanism for the reaction was proposed. The density functional theory calculation of the key step of the reaction, i.e., breaking of the P-N bond and formation of the P-O bond, suggested a one-step S(N)2(P) process with retention of configuration at the phosphorus center.     

Associative phenomena in galactomannan-deacetylated xanthan systems

The interaction between mesquite seed galactomannan (MSG; D-mannose to D-galactose ratio (M/G) approximately 1.1) and deacetylated xanthan (DX) in 5 mM NaCl leading to synergistic gel formation at 25 degrees C was investigated and compared with the far more studied system made of xanthan and locust bean gum (LBG; M/G approximately 3.5). Rheology and differential scanning calorimetry were used to measure temperatures of gel formation and transition enthalpy as a function of polymer composition, while circular dichroism was used to probe the conformation of DX in the LBG-DX system. MSG and DX associate at 25 degrees C with a well defined stoichiometry of 0.6:1.0 (w/w) at low ionic strength favouring the disordered coil state of DX. When LBG was used in place of MSG in water or 5 mM NaCl, two types of mechanisms of interpolymeric association are envisaged.     

A highly active endo-β-1,4-mannanase produced by Cellulosimicrobium sp. strain HY-13, a hemicellulolytic bacterium in the gut of Eisenia fetida

A xylanolytic gut bacterium isolated from Eisenia fetida, Cellulosimicrobium sp. strain HY-13, produced an extracellular glycoside hydrolase capable of efficiently degrading mannose-based substrates such as locust bean gum, guar gum, mannotetraose, and mannopentaose. The purified mannan-degrading enzyme (ManK, 34,926 Da) from strain HY-13 was found to have an N-terminal amino acid sequence of DEATTDGLHVVDD, which has not yet been identified. Under the optimized reaction conditions of 50°C and pH 7.0, ManK exhibited extraordinary high specific activities of 7109 IU/mg and 5158 IU/mg toward locust bean gum and guar gum, respectively, while the enzyme showed no effect on sugars substituted with p-nitrophenol and various non-mannose carbohydrates. Thin layer chromatography revealed that the enzyme degraded locust bean gum to mannobiose and mannotetraose. No detectable amount of mannose was produced from hydrolytic reactions with the substrates. ManK strongly attached to Avicel, β-cyclodextrin, lignin, and poly(3-hydroxybutyrate) granules, but not bound to chitin, chitosan, curdlan, or insoluble oat spelt xylan. The aforementioned characteristics of ManK suggest that it is a unique endo-β-1,4-mannanase without additional carbohydrolase activities, which differentiates it from other well-known carbohydrolases.     

Neutral Proteinases I and II of Aspergillus sojae

Epimerization of (?)-isodihydrocarvone (I) to (?)-dihydrocarvone (II) by Pseudomonas fragi IFO 3458 was studied. I was easily epimerized to II by the growing cells, the resting cells or the cell-free extracts.

An epimerase catalyzing the conversion of I to II was partially purified from the bacterial extracts about 56-fold with heat-treatment, ammonium sulfate precipitation and DEAE-Sephadex A-50 column chromatography. By the action of this epimerase, the ratio of I to II becomes about 25: 75 (K=3). It appeared that the epimerase is very stable to heat; the activity of epimerization remains 66 and 36% after treatment at 97°C for 60 and 120 min, respectively.     

Stereochemical assignment of fusiccocadiene from NMR shielding constants and vibrational circular dichroism spectroscopy

The absolute configuration (AC) of the common precursor of the fusicoccane family of terpenoids, fusicocca‐2,10(14)‐diene (FCdiene), had only been deduced by a lengthy total synthesis, or indirectly from crystal structures of fusicoccin A. However, in particular the AC determinations based on downstream products of the terpene synthase intrinsically overlook potential epimerization reactions. In this contribution, we confirm the relative stereochemistry of FCdiene by comparison of experimental and predicted ¹³C–NMR chemical shifts, and finally determine the absolute configuration from an analysis of its infrared and vibrational circular dichroism spectra.     

Synthesis and Fungicidal Activity of Diaryl 1,1 -Dichloroethyl-phosphonates

Non-Newtonian behavior and dynamic viscoelasticity of a series of aqueous mixed solutions of xanthan and locust bean gum were measured using a rheogoniometer, and the rheological properties were analysed. A gelation occurred in the mixture at the concentration of 0.2% total gums at room temperature. The flow curves of the mixture solutions showed a yield value and approximated to plastic behavior at 50°C. The maximum dynamic modulus was obtained when the mixing ratio of xanthan to locust bean gum was 1:2, while comparable high moduli were also obtained in the mixing ratio of 1: 3 or 1:4. A mixture of deacetylated xanthan and locust bean gum showed the highest dynamic modulus, about two times that of the mixture of native or Na-form xanthan. The dynamic modulus of the mixtures decreased rapidly with increasing temperature. In contrast, the dynamic viscosity was scarcely changed during increasing temperature in the mixing ratio of 2: 1. The dynamic modulus was decreased by addition of urea (4.0 M), NaCl (0.1%) and MgCl₂. We concluded that the intermolecular interaction between xanthan and locust bean gum might occur between the side chains of the former and backbone of the latter, as in a lock-and-key effect.     

Structure and mechanism of the amphibolic enzyme D-ribulose-5-phosphate 3-epimerase from potato chloroplasts

Ribulose-5-phosphate 3-epimerase (EC 5.1.3.1) catalyzes the interconversion of ribulose-5-phosphate and xylulose-5-phosphate in the Calvin cycle and in the oxidative pentose phosphate pathway. The enzyme from potato chloroplasts was expressed in Escherichia coli, isolated and crystallized. The crystal structure was elucidated by multiple isomorphous replacement and refined at 2.3 A resolution. The enzyme is a homohexamer with D3 symmetry. The subunit chain fold is a (beta alpha)8-barrel. A sequence comparison with homologous epimerases outlined the active center and indicated that all members of this family are likely to share the same catalytic mechanism. The substrate could be modeled by putting its phosphate onto the observed sulfate position and its epimerized C3 atom between two carboxylates that participate in an extensive hydrogen bonding system. A mutation confirmed the crucial role of one of these carboxylates. The geometry together with the conservation pattern suggests that the negative charge of the putative cis-ene-diolate intermediate is stabilized by the transient induced dipoles of a methionine sulfur "cushion", which is proton-free and therefore prevents isomerization instead of epimerization.