In vitro and in silico inhibition of angiotensin-converting enzyme by carbohydrates and cyclitols

Fifteen carbohydrates (d-mannose, d-glucose, d-galactose, methyl-α-d-glucose, l-rhamnose, d-xylose, d-fructose, d-arabinose, dulcitol, mannitol, β-maltose, α-lactose, melibiose, sucrose, and raffinose) and four cyclitols [l-(+)-bornesitol, myo-inositol, per-O-acetyl-1-l-(+)-bornesitol, and quinic acid] were assayed for in vitro ACE inhibition. Of these molecules, per-O-Acetyl-1-l-(+)-bornesitol, quinic acid, methyl-α-d-glucose, d-rhamnose, raffinose, and the disaccharides were determined to be either inactive or weak ACE inhibitors, whereas l-(+)-bornesitol, d-galactose, d-glucose, and myo-inositol exhibited significant ACE inhibition. Molecular docking studies were performed to investigate interactions between active compounds and human ACE (Protein Data Bank, PDB 1O83). The results of various calculations showed that all active sugars bind to the same enzyme region, which is a tunnel directed towards the active site. With the exception of myo-inositol (K _i = 13.95 μM, IC₅₀ = 449.2 μM), the active compounds presented similar K _i and IC₅₀ values. d-Galactose (K _i = 19.6 μM, IC₅₀ = 35.7 μM) and l-(+)-bornesitol (K _i = 25.3 μM, IC₅₀ = 41.4 μM) were the most active compounds, followed by d-glucose (K _i = 32.9 μM, IC₅₀ = 85.7 μM). Our docking calculations are in agreement with the experimental data and show a new binding region for sugar-like molecules, which may be explored for the development of new ACE inhibitors.     

Simultaneous Determination of Ten Sugars in Tinospora cordifolia by Ultrasonic Assisted Extraction and LC-ELSD

Sugars present in medicinal plants are known for protecting and stimulating the immune system against various biological disorders. Tinospora cordifolia is a reputed Indian herb used for immunity enhancing which is mainly attributed to saccharides. In the present study, a simple, sensitive, and reliable liquid chromatography method based on ultrasonic assisted extraction and evaporative light scattering detection was developed for simultaneous determination of ten sugars comprising of monosaccharides (l-(+)-rhamnose, d-(+)-xylose, d-(?)-arabinose, β-d-(+)-glucose), disaccharides (sucrose, d-(+)-cellobiose, α-lactose), alditols (xylitol, d-(+)-mannitol) and a polyalcohol (myo-inositol) in T. cordifolia. The separation was achieved on Zorbax-NH₂ column (250 mm × 4.6 mm, 5 µm) in gradient elution of acetonitrile: water as mobile phase with flow rate of 0.5 mL min^?1. The drift tube temperature and nitrogen flow-rate were optimized at 70 °C and 2.0 standard litres per minute, respectively. The method was validated for linearity, accuracy, precision, limits of detection and quantification. The calibration equation revealed a good linear relationship (r ²  = 0.959–0.999). The sufficient recovery was observed in the range of 94.1–99.9%. The method showed good reproducibility with intra- and inter-day precision of <0.99 and 0.97% (RSD), respectively. The detection and quantification limits for the compounds were in the range of 8.32–44.29 and 25.23–134.20 μg mL^?1, respectively.     

d-glucose Enhanced 5-Aminolevulinic Acid Production in Recombinant Escherichia coli Culture

In this study, we introduced a new strategy, feeding d-glucose, to overproduce extracellular 5-aminolevulinic acid (ALA) in the recombinant Escherichia coli. We investigated that the d-glucose concentration is dependent on extracellular ALA production. The results indicated that increasing d-glucose concentration in bacteria culture enhanced final cell density and ALA yield and simultaneously decreased the activities of ALA synthase (ALAS) and ALA dehydratase (ALAD); then, the inhibitory effect of d-glucose on ALAS activity was relieved with the metabolism of d-glucose. when 4.0 g/L d-glucose was added at late exponential phase; 1.46 g/L ALA was achieved in shaking culture, which is 47% or 109% higher than the ALA yields with 30 mM levulinic acid of ALAD inhibitor or no inhibitor. In jar fermenter, final extracellular ALA concentration reached 3.1 g/L by feeding with d-glucose.     

Xylitol Production by Genetically Engineered Trichoderma reesei Strains Using Barley Straw as Feedstock

Xylitol, a naturally occurring five-carbon sugar alcohol derived from d-xylose, is currently in high demand by industries. Trichoderma reesei, a prolific industrial cellulase and hemicellulase producing fungus, is able to selectively use d-xylose from hemicelluloses for xylitol production. The xylitol production by T. reesei can be enhanced by genetic engineering of blocking further xylitol metabolism in the d-xylose pathway. We have used two different T. reesei strains which are impaired in the further metabolism of xylitol including a single mutant in which the xylitol dehydrogenase gene was deleted (?xdh1) and a double mutant where additionally l-arabinitol-4-dehydrogenase, an enzyme which can partially compensate for xylitol dehydrogenase function, was deleted (?lad1?xdh1). Barely straw was first pretreated using NaOH and Organosolv pretreatment methods. The highest xylitol production of 6.1 and 13.22 g/L was obtained using medium supplemented with 2 % Organosolv-pretreated barley straw and 2 % d-xylose by the ?xdh1 and ?lad1?xdh1 strains, respectively.     

Neue enzymatische Analysenmethoden zur Bestimmung von Maltose,Stärke und Lactat in der Lebensmittelchemie

1. Determination of Maltose. Maltose is hydrolyzed by the enzyme α-glucosidase into glucose, which is determined by the enzymes hexokinase and glucose-6-phosphate-dehydrogenase. α-Glucosidase is specific for oligosaccharides with α-1,4 and α-1,2 bonds.
2. Determination of Starch and Glycogen. Starch and glycogen are splitted to glucose by the enzyme amylo-glucosidase. Starch has to be dissolved before enzymatic cleavage. A comparison of different methods for preparing starch solutions is given.
3. Determination of d- and l-Lactate. It is possible to determine d-lactate and l-lactate with the specific enzymes d-lactate-dehydrogenase and l-lactate-dehydrogenase. By different samples it is shown that no equal quantities of d- and l-lactate were found in the analyzed foods.

     

Stabilization ofd-amino acid oxidase from yeastTrigonopsis variabilis used for production of glutaryl-7-aminocephalosporanic acid from cephalosporin C

The studies to improve the production of glutaryl-7-ACA from cephalosporin C are described in this paper. During the conversion of cephalosporin C to keto-adipyl-7-aminocephalosporonic acid by d-amino acid oxidase (d-AAO), with the simultaneous production of equimolar amount of hydrogen peroxide, an incomplete nonenzymatic conversion of the keto form into the glutaryl form occurs, where cephalosporin C as well asd-AAO are partly destroyed in the presence of hydrogen peroxide. d-AAO was immobilized to different carriers in order to achieve better enzyme stability. The activity of immobilizedd-AAO on manganese oxide remained above 100% during the first 9 h of a semicontinuous conversion of cephalosporin C. The presence of catalase coimmobilized with D-AAO and coupled to CNBr-activated Sepharose 4B improved the operation stability ofd-AAO. An additional approach for the continuous transformation of cephalosporin C used whole cells ofTrigonopsis variabilis, containingd-AAO, immobilized to magnetic iron oxide particles.     

Mutant d-amino acid oxidase with higher catalytic efficiency toward d-amino acids with bulky side chains

d-Amino acid oxidase from the yeast Trigonopsis variabilis (TvDAAO) is widely used in fine organic synthesis, including the preparation of unnatural l-amino acids and α-keto acids. The analysis of the three-dimensional structure of TvDAAO was carried out with the aim of producing the enzyme specific to d-amino acids with bulky side chains. The analysis revealed the residue Phe54 at the entrance to the active site, which controls the substrate access to this site. The residue Phe54 was replaced by residues Ala, Ser, and Tyr. The cultivation of recombinant E. coli strains expressing TvDAAO mutants showed that the mutein with the Phe54Ala substitution had very low stability. Thus, the inactivation of the enzyme occured within 10 min after the cell disruption. The Phe54Ser TvDAAO and Phe54Tyr TvDAAO mutants were obtained as homogeneous preparations, and their thermal stability and catalytic properties were investigated. The introduction of Phe54Ser and Phe54Tyr substitutions resulted in additional stabilization of the protein macromolecule compared to the wild-type TvDAAO. Thus, the half-inactivation time for the mutant enzymes at 54 °C increased by a factor of 1.5 and 2, respectively. As in the case of wild-type TvDAAO, the thermal inactivation of the muteins proceeds via a two-step dissociative mechanism. The introduction of mutations led to a strong change in the substrate specificity profile. The mutants have no activity toward a series of d-amino acids (Phe54Ser TvDAAO toward d-Ala, d-Ser, d-Val, and d-Thr; Phe54Tyr TvDAAO toward d-Ser, d-Tyr, d-Thr, and d-Lys). The catalytic efficiency (the k _cat/K _M ratio) of the Phe54Ser TvDAAO mutant toward d-amino acids with bulky side chains (d-Lys, d-Asn, d-Phe, d-Tyr, d-Trp, and d-Leu) increased from 2.4 to 7.3 times.     

Synthesis of β-d-glucopyranosyl- and β-d-galactopyranosylamines from 4-bromo-3-methylaniline and 2-amino-5-bromopyridine

The possibility of coupling of d-glucose and d-galactose with 4-bromo-3-methylaniline, 2,4,6-tribromoaniline, and 2-amino-5-bromopyridine was studied. The substituent in the aromatic ring was found to influence the conditions and possibility of the reaction. The yields of β-d-glucopyranosyl- and β-d-galactopyranosylamines from 4-bromo-3-methylaniline and 2-amino-5-bromopyridine were 50–65%; 2,4,6-tribromoaniline did not react at all.     

A method for the determination of d-kynurenine in biological tissues

d-Kynurenine (d-KYN), a metabolite of d-tryptophan, can serve as the bioprecursor of kynurenic acid (KYNA) and 3-hydroxykynurenine, two neuroactive compounds that are believed to play a role in the pathophysiology of several neurological and psychiatric diseases. In order to investigate the possible presence of d-KYN in biological tissues, we developed a novel assay based on the conversion of d-KYN to KYNA by purified d-amino acid oxidase (d-AAO). Samples were incubated with d-AAO under optimal conditions for measuring d-AAO activity (100 mM borate buffer, pH 9.0), and newly produced KYNA was detected by high-performance liquid chromatography (HPLC) with fluorimetric detection. The detection limit for d-KYN was 300 fmol, and linearity of the assay was ascertained up to 300 pmol. No assay interference was noted when other d-amino acids, including d-serine and d-aspartate, were present in the incubation mixture at 50-fold higher concentrations than d-KYN. Using this new method, d-KYN was readily detected in the brain, liver, and plasma of mice treated systemically with d-KYN (300 mg/kg). In these experiments, enantioselectivity was confirmed by determining total kynurenine levels in the same samples using a conventional HPLC assay. Availability of a sensitive, specific, and simple method for d-KYN measurement will be instrumental for evaluating whether d-KYN should be considered for a role in physiology and pathology.     

Comparative investigation on the enthalpy relaxation of four amorphous pentose isomers

The enthalpy relaxation of the pentose isomers (d-xylose, d-ribose, l-arabinose, and d-arabinose) was investigated in terms of the Tool–Narayanaswamy–Moynihan and Adam–Gibbs–Vogel models using differential scanning calorimetry. Single set of parameters for each model was obtained from the multicurve-fitting procedure. The differences between the glass transition and relaxation parameters were discussed in terms of isomeric effect. The cooperativity determined from curve-fitting results was further compared to those obtained via Donth’s formula.     

Synthesis of N-glycyl-β-glycopyranosyl amines,derivatives of natural oligosaccharides — glucose analogs of Lex antigen

Treatment of the natural tri-, tetra-, and pentasaccharides, β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, α-l-Fucp-(1→2)-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, and α-l-Fucp-(1→2)-[α-d-GalNAcp-(1→3)]-β-d-Galp-(1→4)-[α-l-Fucp-(1→3)]-d-Glcp, which are glucose analogs of Le^x, with ammonium carbamate in aqueous methanol gave the corresponding β-glycopyranosyl amines. After their N-acylation with N-Z-glycine N-hydroxysuccinimidyl ester (Z is benzyloxycarbonyl) with subsequent hydrogenolytic removal of Z-group, corresponding N-glycyl-β-glycopyranosyl amines were obtained in yields up to 70%.     

Two-dimensional high-performance liquid chromatographic determination of day–night variation of d-alanine in mammals and factors controlling the circadian changes

d-Alanine (d-Ala) is one of the naturally occurring d-amino acids in mammals, and its amount is known to have characteristic circadian changes. It is a candidate for a novel physiologically active substance and/or a biomarker, and the regulation mechanisms of the intrinsic amounts of d-Ala are expected to be clarified. In the present study, the effects of the possible factors controlling the d-Ala amounts, e.g., diet, d-amino acid oxidase (DAO) and intestinal bacteria, on the day–night changes in the intrinsic d-Ala amounts have been investigated using a highly sensitive and selective two-dimensional high-performance liquid chromatographic system combining a reversed-phase column and an enantioselective column. The circadian rhythm was not changed under fasting conditions. In the mice lacking d-amino acid oxidase activity (ddY/DAO^- mice), clear day–night changes were still observed, suggesting that the factors controlling the d-Ala rhythm were not their food and DAO activity. On the other hand, in the germ-free mice, quite low amounts of d-Ala were detected compared with those in the control mice, indicating that the main origin of d-Ala in the mice is intestinal bacteria. Because the d-Ala amounts in the digesta containing intestinal bacteria did not show the day–night changes, the controlling factor of the circadian changes of the d-Ala amount was suggested to be the intestinal absorption.     

Untersuchungen über die Biosynthese der Cyclite, 20. Mitt.: Über die Bildung vonl-Bornesit inVinca-Arten

Incorporation experiments with radioactively labelledmyo-inositol andd-glucose inVinca minor andVinca rosea make it very probable that in these plantsl-bornesitol is formed through direct methylation ofmyo-inositol.     

Cis-Cycloprolylprolin-Anion-ein konfigurationsstabiles Carbanion

The racemisation ofcyclo-(l-Pro?l-Pro) (2) with metal amides in liq. ammonia was examined. The K-kation causes more extensive racemisation than Na-kation, which in turn is more effective than Li⁺. This, the racemisation of2 int-butyl alcohol with K⁺C₆H₅O^? and the data gained from corresponding deuterated medium show that the racemisation of2 proceeds in two steps: in the first, the less stabletrans-cyclo-(l-Pro?d-Pro) (3) is formed, followed by the rapid conversion of3 to a mixture ofcyclo-(l-Pro?l-Pro) andcyclo-(d-Pro?d-Pro) in the second step.     

Untersuchungen über die Biosynthese der Cyclite, 25. Mitt.: Vorkommen und Bildung von Condurit inMarsdenia-Arten

A systematic study of tropical Asclepiadaceae shows thatMarsdenia abyssinica, M. zambesica, M. erecta, M. angolensis, andDragea Faulknerae contain conduritol as their main cyclitol component. Taking earlier reports into consideration, these findings mean that conduritol can be found only in the subfamily Cynanchoideae of the Asclepiadaceae. This very restricted distribution is in contrast to that of its isomer,l-leucanthemitol, which—at least in traces—can be detected in almost all green plants. Infusion experiments with¹⁴C-labelled possible precursors of conduritol inMarsdenia abyssinica showed incorporation ofd-glucose andd-galactose into the cyclitol. The hexose molecules seem to be built in into conduritol as such, i.e. without being fragmented previously.l-Leucanthemitol is incorporated into conduritol with a much better radiochemical yield than the two hexoses. This result suggests that the last step in the biosynthesis of conduritol is an epimerization ofl-leucanthemitol to conduritol.     

Improvement of Solvent Production from Xylose Mother Liquor by Engineering the Xylose Metabolic Pathway in Clostridium acetobutylicum EA 2018

Xylose mother liquor (XML) is a by-product of xylose production through acid hydrolysis from corncobs, which can be used potentially for alternative fermentation feedstock. Sixteen Clostridia including 13 wild-type, 1 industrial strain, and 2 genetically engineered strains were screened in XML, among which the industrial strain Clostridium acetobutylicum EA 2018 showed the highest titer of solvents (12.7 g/L) among non-genetic populations, whereas only 40 % of the xylose was consumed. An engineered strain (2018glcG-TBA) obtained by combination of glcG disruption and expression of the d-xylose proton-symporter, d-xylose isomerase, and xylulokinase was able to completely utilize glucose and l-arabinose, and 88 % xylose in XML. The 2018glcG-TBA produced total solvents up to 21 g/L with a 50 % enhancement of total solvent yield (0.33 g/g sugar) compared to that of EA 2018 (0.21 g/g sugar) in XML. This XML-based acetone–butanol–ethanol fermentation using recombinant 2018glcG-TBA was estimated to be economically promising for future production of solvents.     

Production of ethanol from sugar cane bagasse hemicellulose hydrolyzate byPichia stipitis

The ability ofPichia stipitis to fermentd-xylose andd-glucose in the acid-hydrolyzed hemicellulose component of sugar cane bagasse depends on the alkali used to neutralize the hydrolyzate to pH 6.5. With NH₄OH and NaOH no fermentation occurred, whereas neutralization with Ca(OH)₂ gave the best results (Q_pmax=0.25 g/L-h; Y_p/s =0.38 g/g sugar). However, the volumetric productivity was still considerably less than observed in a semisynthetic medium with a sugar composition similar to the hydrolyzate. L-arabinose was not fermented but assimilated. Sequential neutralization methods failed to improve the fermentation. Acetic acid and lignin derivatives present in the hydrolyzate were major components that inhibited the fermentation.     

Synthesis ofS-(Carboxymethyl)-d-cysteine by 3-chloro-d-alanine chloride-lyase ofPseudomonas putida CR 1-1

S-(Carboxymethyl)-d-cysteine, which is an important component of semisynthetic cephalosporin, MT-141, was enzymatically synthesized.S-(Ethoxy-carbonyl-methyl)-d-cystein was synthesized from 3-chloro-d-alanine and ethyl thioglycolate by the β-replacement reaction of 3-chloro-d-alanine chloride-lyase fromPseudomonas putida CR 1-1 and subsequently hydrolyzed by alkali. The synthesizedS-(carboxymethyl)-d-cysteine was isolated from a large scale reaction mixture and identified physicochemically. The reaction conditions for the synthesis ofS-(ethoxycarbonylmethyl)-d-cysteine were optimized using resting cells ofP. putida CR 1-1.     

Synthesis and characterization of new N-phenylmaleimide thioglycosides

Thioglycosides derivatives of N-phenylmaleimide have been prepared by the reaction of derivatives of 1-thio-d-glucopyranose, d-galactopyranose, d-lactose, and d-maltose with 3,4-dichloro-N-phenylmaleimide. The reaction of 3,4-dichloro-N-phenyl maleimide with sugar thiols (protected or unprotected) took place by displacement of both chlorine atoms by sulfide nucleophile giving the corresponding bis-thioglycoside products.     

Direct electrochemical non-enzymatic assay of glucose using functionalized graphene

A non-enzymatic amperometric sensor is developed based on the graphite electrode modified with functionalized graphene for the determination of β, d (+)-glucose. Cyclic voltammetry and electrochemical impedance spectroscopy techniques are used to study the behavior. Atomic force microscopy was used to study the surface topography of the working electrode before and after its modification. The sensor enabled the direct electrochemical oxidation of β, d (+)-glucose in alkaline medium and responded linearly to the analyte over the range from 0.5?×?10^?3 to 7.5?×?10^?3?M with a limit of detection of 10?μM. The sensor is found to exhibit a better sensitivity of 28.4?μA?mM^?1?cm^?2, good stability, and shelf life. The sensitivity of the sensor to β, d (+)-glucose was not affected by the commonly co-existing interfering substances such as l-ascorbic acid, dopamine, uric acid, and acetaminophen.