Purification and characterization of an extracellular α-l-arabinofuranosidase from Fusarium oxysporum

An α-l-arabinofuranosidase from Fusarium oxysporum F3 was purified to homogeneity by a two-step ion exchange intercalated by a gel filtration chromatography. The enzyme had a molecular mass of 66 kDa and was optimally active at pH 6.0 and 60°C. It hydrolyzed aryl α-l-arabinofuranosides and cleaved arabinosyl side chains from arabinoxylan and arabinan. There was a marked synergistic effect between the α-l-arabinofuranosidase and an endo-(1 →4)-β-d-xylanase produced by F. oxysporum in the extensive hydrolysis of arabinoxylan.     

Preparation of optical active aliphatic <Emphasis Type="Italic">α</Emphasis>-amino acid from fatty acid: synthesis of <Emphasis Type="SmallCaps">l</Emphasis>-norvaline and <Emphasis Type="SmallCaps">d</Emphasis>-norvaline

A new synthesis of l-norvaline is described. Using valeric acid as the raw material, α-brominevaleryl chloride was achieved by acyl chlorination and α-position bromination in one-pot with the yield of 84.7%. The yields of the following ammoniation of α-brominevaleryl chloride and resolution of dl-norvaline were 88.7% and 26.7%, respectively. d-Norvaline was also obtained in a similar method from the waste resolution solution. Other optical active amino acids, valine, α-aminobutyric acid and alanine were also synthesized in similar ways.     

Determination of the Enantiomeric Purity of l-Carnitine and Acetyl-l-Carnitine by Chiral Liquid Chromatography

A chiral liquid chromatographic method for determination of the enantiomeric purity of both l-carnitine and acetyl-l-carnitine is described. Separation of the enantiomers of dl-carnitine and acetyl-dl-carnitine was achieved on a commercial chiral column (Chiralcel OD-R) after derivatization with (alpha-bromo)methyl phenyl ketone. Introduction of this lipophilic UV chromophoric group to the carnitine and acetylcarnitine molecules improved their retention, resolution, and UV detection. The mobile phase was 74:26 (v/v) 0.5 mol L^-1 sodium perchlorate–acetonitrile, pH 3.8, and the flow rate was 0.4 mL min^-1. Detection was performed at 235 nm. The method is selective and reliable for determination of the enantiomeric purity of bulk drug substances l-carnitine and acetyl-l-carnitine.     

Glycosylation of betulin acetates with glycals

Betulin 2-deoxy-α-d-, 2-deoxy-α-l-, and 2,6-dideoxy-α-l-arabino-hexopyranosides were synthesized by acid-catalyzed glycosylation (cationite in the H⁺ form, LiBr) of betulin 3- and 28-monoacetates with glycal acetates. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 531–534, March, 1998.     

Stereoselective synthesis of triterpene 2-deoxy-α-d-lyxo-hexopyranosides

2-Deoxy-α-d-lyxo-hexopyranosides of 18β-glycyrrhetic acid, its 11-deoxo derivative and allobetulin were synthesized by glycosylation of oleonane-type triterpene alcohols withd-galactal acetate in the presence ofN-iodosuccinimide followed by deiodination and deprotection. Translated fromIzvestiya Akademii Nauk. Seriya Khimicheskaya, No. 3, pp. 596–600. March, 1997.     

Influence of reaction medium composition on enzymatic synthesis of galactooligosaccharides and lactulose from lactose concentrates prepared from whey permeate

The paper presents the results of investigations into the technological possibilities of controlling the transgalactosylation process of lactose in permeate after whey ultrafiltration in order to improve the efficiency of galactooligosaccharides or lactulose synthesis. The synthesis efficiency was influenced by the selection of a β-galactosidase preparation, substrate concentration and, in the synthesis of lactulose, also by the ratio of lactose and fructose added to the reaction mixture. The obtained synthesis efficiency of GOS and, most of all, of lactulose (65 g L⁻¹), may be found satisfactory. The study also resulted in a proposed GOS or lactulose concentrates (concentrated or dried) production technology using permeate after ultrafiltration of milk or whey as lactose sources. Presented at the 35th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 26–30 May 2008.     

Quantitative determination of protein of bacterial origin on the basis ofd-aspartic acid andd-glutamic acid content

Summary In recent decades several methods have been developed for determination of the proportion of nitrogen-containing substances passed from the rumen into the abomasum, or small intestine, which are of microbial origin. Recently, when examining thed-amino acid content of foodstuffs, particularly milk and milk products, it was observed that, in addition tod-alanine (d-Ala,d-glutamic acid (d-Glu) andd-aspartic acid (d-Asp) can also be detected in similar quantities, primarily in products which have links with bacterial activity. This gave rise to the idea of examining the diaminopimelic acid (DAPA),d-Glu, andd-Asp content of bacteria extracted from the rumen of cattle, and that of chyme from the same cattle, to establish whetherd-Asp andd-Glu can be used to estimate protein of bacterial origin. The investigations performed have provided evidence that bothd-Asp andd-Glu might be appropriate for determination of protein of bacterial origin. The results obtained using these two bacterial markers (d-Asp andd-Glu) proved to the approximately 10% lower than those obtained using DAPA; this was not because of to error attributable to the new markers but rather to the unreliability of determination using DAPA Analyses performed on samples of known bacterial protein content indicate thatd-Asp andd-Glu gave almost identical results for bacterial protein content which were very close to the theoretical (calculated) values. Presented at Balaton Symposium '01 on High-Performance Separation Methods, Siófok, Hungary, September 2–4, 2001.     

Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (K _M and V _max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higher K _M and lower V _max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.     

Characterization and complementation of a Pichia stipitis mutant unable to grow on d-xylose or l-arabinose

Pichia stipitis CBS 6054 will grow on d-xylose, d-arabinose, and l-arabinose. d-Xylose and l-arabinose are abundant in seed hulls of maize, and their utilization is important in processing grain residues. To elucidate the degradation pathway for l-arabinose, we obtained a mutant, FPL-MY30, that was unable to grow on d-xylose and l-arabinose but that could grow on d-arabinitol. Activity assays of oxidoreductase and pentulokinase enzymes involved in d-xylose, d-arabinose, and l-arabinose pathways indicated that FPL-MY30 is deficient in d-xylitol dehydrogenase (D-XDH), d- and l-arabinitol dehydrogenases, and d-ribitol dehydrogenase. Transforming FPL-MY30 with a gene for xylitol dehydrogenase (PsXYL2), which was cloned from CBS 6054 (Gen Bank AF127801), restored the D-XDH activity and the capacity for FPL-MY30 to grow on l-arabinose. This suggested that FPL-MY30 is critically deficient in XYL2 and that the d-xylose and l-arabinose metabolic pathways have xylitolas a common intermediate. The capacity for FPL-MY30 to grow on d-arabinitol could proceed through d-ribulose.     

The electrochemical polymerization of <Emphasis Type="Italic">o</Emphasis>-phenylenediamine on <Emphasis Type="SmallCaps">l</Emphasis>-tyrosine functionalized glassy carbon electrode and its application

The polymerization of o-phenylenediamine (OPD) on l-tyrosine (Tyr) functionalized glassy carbon electrode (GCE) and its electro-catalytic oxidation towards ascorbic acid (AA) had been studied in this report. l-Tyrosine was first covalently grafted on GCE surface via electrochemical oxidation, which was followed by the electrochemical polymerization of OPD on the l-tyrosine functionalized GCE. Then, the poly(o-phenylenediamine)/l-tyrosine composite film modified GCE (POPD-Tyr/GCE) was obtained. X-ray photo-electron spectroscopy (XPS), field emission scanning electron microscope (SEM), and electrochemical techniques have been used to characterize the grafting of l-tyrosine and the polymerization and morphology of OPD film on GCE surface. Due to the doping of the carboxylic functionalities in l-tyrosine molecules, the POPD film showed good redox activity in neutral medium, and thus, the POPD-Tyr/GCE exhibited excellent electrocatalytic response to AA in 0.1 mol l⁻¹ phosphate buffer solution (PBS, pH 6.8). The anode peak potential of AA shifted from 0.58 V at GCE to 0.35 V at POPD-Tyr/GCE with a greatly enhanced current response. A linear calibration graph was obtained over the AA concentration range of 2.5 × 10⁻⁴–1.5 × 10^–3 mol l⁻¹ with a correlation coefficient of 0.9998. The detection limit (3δ) for AA was 9.2 × 10⁻⁵ mol l⁻¹. The modified electrode showed good stability and reproducibility and had been used for the determination of AA content in vitamin C tablet with satisfactory results.     

l-Glutamate biosensor for estimation of the taste of tomato specimens

An amperometric biosensor has been developed for measurement of Umami, or the taste based on the amount of L-glutamate, in tomato foods. The biosensor is based on an enzyme-mediator system in which L-glutamate oxidase is used for biochemical oxidation of L-glutamate and a tetrafulvalene-tetracyanoquinodimethane (TTF-TCNQ) paste, prepared from the mixture of TTF-TCNQ salt, graphite powder, and silicone oil, serves as the mediator. The limit of detection, calculated by use of a four-parameter logistic model, was 0.05 mmol L(-1), and the limit of quantification was 0.15 mmol L(-1). The correlation coefficient (R2) was 0.990 and the relative standard deviation was no more than 1% (n=5). The response time (tau (95)) was 20-50 s, depending on concentration. The repeatability of the sensor was better than 5% (n=10). The sensor developed was stable for more than ten days.     

Substrate selectivity of Gluconobacter oxydans for production of 2,5-diketo-D-gluconic acid and synthesis of 2-keto-L-gulonic acid in a multienzyme system

Substrate selectivity of Gluconobacter oxydans (ATCC 9937) for 2,5-diketo-d-gluconic acid (2,5-DKG) production was investigated with glucose, gluconic acid, and gluconolactone in different concentrations using a resting-cell system. The results show that gluconic acid was utilized favorably by G. oxydans as substrate to produce 2,5-DKG. The strain was coupled with glucose dehydrogenase (GDH) and 2,5-DKG reductase for synthesis of 2-keto-l-gulonic acid (2-KLG), a direct precursor of l-ascorbic acid, from glucose. NADP and NADPH were regenerated between GDH and 2,5-DKG reductase. The mole yield of 2-KLG of this multienzyme system was 16.8%. There are three advantages for using the resting cells of G. oxydans to connect GDH with 2,5-DKG reductase for production of 2-KLG: gluconate produced by GDH may immediately be transformed into 2,5-DKG so that a series of problems generally caused by the accumulation of gluconate would be avoided; 2,5-DKG is supplied directly and continuously for 2,5-DKG reductase, so it is unnecessary to take special measures to deal with this unstable substrate as it was in Sonoyama’s tandem fermentation process; and NADP(H) was regenerated within the system without any other components or systems.     

Determination of serum <Emphasis Type="SmallCaps">d</Emphasis>-lactic and <Emphasis Type="SmallCaps">l</Emphasis>-lactic acids in normal subjects and diabetic patients by column-switching HPLC with pre-column fluorescence derivatization

d-Lactic and l-lactic acids were simultaneously determined by means of a column-switching high-performance liquid chromatography (HPLC) with fluorescence detection. As a fluorescence reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ) was employed for the fluorescence derivatization of lactic acid. The proposed HPLC system adopted both octylsilica (Cadenza CD-C8) and amylose-based chiral columns (CHIRALPAK AD-RH), which proved to give a sufficient enantiomeric separation of the lactic acid derivatives with a separation factor () of 1.32 and a resolution (R_s) of 1.98. Moreover, the features of the first elution of d-lactic acid peak in the proposed HPLC were convenient for the determination of trace amount of serum d-lactic acid, which is known to increase under diabetes. Intra-day and inter-day accuracies were in the range of 90.5–101.2 and 89.0–100.7%, and the intra-day and inter-day precisions were 0.3–1.2 and 0.4–4.8%, respectively. The proposed method was applied to determine d-lactic and l-lactic acids in human serum of normal subjects and diabetic patients, showing that both d-lactic and l-lactic acid concentrations were significantly increased in the serum of diabetic patients (n=31) as compared with normal subjects (n=21). This fact was found for the first time owing to the development of the proposed HPLC method which is able to determine d-lactic and l-lactic acid simultaneously. Finally, serum d-lactic acid concentrations determined by the proposed HPLC method were compared with those from a reported enzymatic assay, and the smaller p value between normal subjects and diabetic patients was shown by the proposed HPLC method.     

Macroligandd-alanyl-d-alanine-dextran for vancomycin purification

Water soluble macroligands for vancomycin purification in affinity ultrafiltration have been prepared by coupling the ligandd-alanyl-d-alanine to dextran activated by tosyl chloride, carbonyldiimidazole, and chloroformate, respectively. Centrifugal ultrafiltration has been used to study the equilibrium binding of vancomycin for the macroligands. The affinity binding can be described as Langmuir type adsorption and is strongly affected by temperature. The binding between vancomycin and macroligand is an unusual endothermic process that binding capacity of macroligand increases with temperature. Vancomycin has also been successfully purified from fermentation liquor using the macroligand in a centrifugal ultrafiltration device.     

Volumetric and conductometric studies on the interactions of dipeptides with sodium acetate and sodium butyrate in aqueous solutions at T = 298.15 K

Densities and conductivity data for the sodium carboxylate (sodium acetate and sodium butyrate)–dipeptides {(glycyl-l-glutamine and l-alanyl-l-glutamine) + water} systems were determined at T = 298.15 K. The apparent molar volumes of the peptides and the molar conductivity (Λ) of sodium acetate and sodium butyrate have been calculated. These data have been utilized to deduce the standard partial molar volumes (), standard partial molar volumes of transfer for dipeptide from water to aqueous sodium carboxylate solutions (Δ_tV^°), volumetric interaction coefficient, the limiting molar conductivity (Λ_°), and Walden product (Λ_°η). Both and Δ_tV^° for the dipeptides increase with increasing concentration of sodium carboxylate. The interpretation is that this result arises from the dominant interactions of the sodium carboxylate with the charged group and polar groups of peptides. The decrease in Λ_° of sodium carboxylate with increasing dipeptide concentration and nonconstant Walden product are attributed to the interactions of sodium carboxylate with peptide and friction resistance of the solvent medium.     

Presence and origin of large amounts of d-proline in the urine of mutant mice lacking d-amino acid oxidase activity

Using a column-switching HPLC system combining a micro-ODS column and a chiral column, the amounts of d-proline (d-Pro) have been determined in 18 tissues, plasma and urine of mice. To avoid the enzymatic degradation of d-amino acids in vivo, a mutant mouse strain lacking d-amino acid oxidase activity (ddY/DAO⁻ mouse) was used. In the brain, relatively large amounts of d-Pro were observed in the anterior pituitary, posterior pituitary and pineal glands. In the peripheral tissues, the amounts of d-Pro were high in the pancreas and kidney. Above all, it is surprising that the ddY/DAO⁻ mice excreted large amounts of d-Pro in their urine (433 nmol/mL, 20 times that of l-Pro). The origin of d-Pro has also been investigated. By comparing germ-free mice and gnotobiotic mice, intestinal bacteria were shown to have no effect on the urinary d-Pro amount. Concerning the dietary origin, a notable amount of d-Pro was still excreted in the urine after starvation for 4 days, suggesting that some of the d-Pro is produced in the mice. Age-dependent changes in the urinary d-Pro amount have also been investigated from the postnatal 1st month up to 12 months, and ddY/DAO⁻ mice were found to excrete large amounts of d-Pro in the urine constantly throughout their lives. Kenji Hamase is Associate Professor in the Department of Bioanalytical Chemistry, Graduate School of Pharmaceutical Sciences at Kyushu University. His current research interests focus on the development of analytical methods for d-amino acids and the study of their physiological functions and diagnostic values. He received the Japanese Society for Analytical Chemistry Award for Young Scientists in 2003, and the PSJ Award for Young Scientists in 2006.     

Structural characterization of trimethylsilyl methyl glycosides derivatives by high-resolution mass spectrometry,linked scans and MIKE experiments

Gas chromatography–mass spectrometry investigations have been carried out for the structural analysis of trimethylsilyl methyl derivatives of keto-deoxy sugars and sialic acids studied under electron ionization. Fragmentation patterns were determined. The three derivatives undergo some common fragmentation pathways. Formation of fragment ions possessing cyclic resonance-stabilized structures was demonstrated. As the sialic acid derivative contained a N-acetyl substituent, some additional fragmentations occurred and were also elucidated. Thus, the mechanism of fragmentation was revealed for these derivatives and new findings concerning some series of fragment ions are presented. Presented at the Annual French National Symposium on Mass Spectrometry, Electrophoresis and Proteomics, 20–23 September 2007 in Pau, France.     

Kinetics and mechanism of catalytic oxidation of alcohols to carbonyl compounds with dioxygen in the Pd-containing aqua system

The oxidation of lower aliphatic alcohols C₁–C₄ with dioxygen to form the corresponding carbonyl compounds in the presence of the PdII tetraaqua complexes and Fe^II-Fe^III aqua ions in an aqueous medium was studied at 40–80 °C. The introduction of an aromatic compound (acetophenone, benzonitrile, phenylacetonitrile, o-cyanotoluene, nitrobenzene) and Fe^II aqua ion instead of the Fe^III aqua ion into the reaction system increases substantially the catalytic activity and the yield of the carbonyl compound. The key role of the Pd species in the intermediate oxidation state stabilized by the aromatic additive in the catalytic cycle of alcohol oxidation with dioxygen to the carbonyl compound was shown. An increase in the kinetic isotope effect with an increase in the temperature of methanol oxidation indicates a change in the rate-determining step of alcohol oxidation with dioxygen in the presence of Pd^II-Fe^II-Fe^III and the aromatic compound. At temperatures below 60 °C, the catalytically active palladium species are mainly formed upon the reduction of the Pd^II tetraaqua complex with the Fe^II aqua ion, whereas at higher temperatures the reaction between the alcohol and Pd^II predominates. The mechanism and kinetic equation of the process were proposed. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 5, pp. 842–848, May, 2007.     

Structural modeling of glucanase–substrate complexes suggests a conserved tyrosine is involved in carbohydrate recognition in plant 1,3-1,4-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucanases

Glycosyl hydrolase family 16 (GHF16) truncated Fibrobacter succinogenes (TFs) and GHF17 barley 1,3-1,4-β-d-glucanases (β-glucanases) possess different structural folds, β-jellyroll and (β/α)₈, although they both catalyze the specific hydrolysis of β-1,4 glycosidic bonds adjacent to β-1,3 linkages in mixed β-1,3 and β-1,4 β-d-glucans or lichenan. Differences in the active site region residues of TFs β-glucanase and barley β-glucanase create binding site topographies that require different substrate conformations. In contrast to barley β-glucanase, TFs β-glucanase possesses a unique and compact active site. The structural analysis results suggest that the tyrosine residue, which is conserved in all known 1,3-1,4-β-d-glucanases, is involved in the recognition of mixed β-1,3 and β-1,4 linked polysaccharide.     

N,S,O-Containing organic ligands based on salicylaldehyde and 2-thiosubstituted ethylamines and their complexes with CoII, NiII, and CuII

The reaction of 2,2′-di(2-hydroxybenzaliminoethyl) disulfide (H₂L¹) and 2-[(2-thioethyl)iminomethyl]phenol (H₂L²) with MCl₂·xH₂O (M = Co, Ni, Cu) afforded the [M₂(L¹)Cl₂] and [M(L²)]₂ complexes, respectively. Their structures were determined by the data of electronic and IR spectroscopy and PM3 quantum chemical calculations. The H₂L¹ ligand and the complexes were studied by electrochemistry (CV and using a rotating disk electrode). The primary electronic changes are localized on the ligand fragment upon the electrochemical oxidation and reduction of the complexes. Published in Russian in Izvestiya Akademii Nauk. Seriya Khimicheskaya, No. 7, pp. 1325–1330, July, 2007.