18875-34-8

Basic information

• Product Name: D-FRUCTOSE-UL-14C
• Synonyms: D-FRUCTOSE-UL-14C;D-FRUCTOSE, [14C(U)]- 250-350 MCI(9.25-12.95 GBQ)/MMOL, DELIVERED >= 97% PURE WITH HPLC RADIOCHROMATOGRAM
• CAS NO: 18875-34-8
• Molecular Formula: C6H12O6
• Molecular Weight: 192.26
• Mol File: 18875-34-8.mol
• Article Data: 352

Chemical Properties

• Appearance: /aqueous ethanol solution (1:1)
• Storage Temp.: 2-8°C
• CAS DataBase Reference: D-FRUCTOSE-UL-14C(CAS DataBase Reference)
• NIST Chemistry Reference: D-FRUCTOSE-UL-14C(18875-34-8)
• EPA Substance Registry System: D-FRUCTOSE-UL-14C(18875-34-8)

Safety Data

• Hazard Codes: F,R
• Statements: 11
• Safety Statements: 7-16
• RIDADR: UN 1170 3/PG 2
• WGK Germany: 1
• Hazardous Substances Data: 18875-34-8(Hazardous Substances Data)

Upstream product

• 91-22-5 quinoline 
• 50-99-7 D-glucose 
• 91-63-4 2-methylquinoline 
• 64-18-6 formic acid 
• 141-53-7 sodium formate 
• 69-65-8 mannitol 
• 50-70-4 D-sorbitol 
• 50-99-7 glucose 
• 127-09-3 sodium acetate 
• 64-19-7 acetic acid 
• 57-04-5 dihydroxyacetone phosphate 
• 453-17-8 D-Glyceraldehyde 
• 551-68-8 D-psicose 
• 643-71-0 D-glucosone 

Downstream Products

• 6817-87-4 2-propylamino-2-deoxy-α-D-glucopyranose 
• 1820-84-4 2-ethyl-β-D-fructofuranoside 
• 81024-99-9 ethyl α-D-fructofuranoside 
• 31105-03-0 N-(1-deoxy-D-fructos-1-yl)-L-phenylalanine monohydrate 
• 112072-85-2 N-D-glucose-2-yl-L-phenylalanine 
• 121990-82-7 arabino-[2]hexosulose-bis-(ethyl-phenyl-hydrazone) 
• 119698-21-4 D-arabino-[2]hexosulose-bis-(butyl-phenyl-hydrazone) 
• 119640-22-1 D-arabino-[2]hexosulose-bis-(phenyl-propyl-hydrazone) 
• 100212-78-0 D-arabino-[2]hexosulose-bis-(2-iodo-phenylhydrazone) 
• 108752-53-0 5-hydroxymethyl-furan-2-carbaldehyde-p-tolylimine 
• 115049-21-3 (E)-(5-((phenylimino)methyl)furan-2-yl)methanol 
• 51009-13-3 N-phenyl-β-D-fructopyranosylamine 
• 23275-67-4 lyxo-[2]Hexosulose-bis-phenylhydrazon 
• 100063-31-8 5-hydroxymethyl-furan-2-carbaldehyde-(4-hydroxy-phenylimine) 

Relevant articles and documents

Purification, characterization and N-terminal sequence analysis of betel leaf (Piper betle) invertase

The present study is the first report describing the purification, enzymatic properties and N-terminal amino acid sequence of a native invertase in betel leaf. The invertase was purified as a monomeric glycoprotein of molecular mass (Mr) 68 kDa. The enzyme was capable to attack β-fructofuranoside linkages from the fructose end of sucrose, raffinose and stachyose indicating it as an authentic β-D-fructofurano-sidase with high specificity for sucrose (Km 4.83 mM). The maximum activity was detected at pH 5.2 and 37 °C. Glucose and fructose showed typical inhibitory effect on the enzyme activity where as lectin was found to be effective activators of the enzyme. Significant inhibition by heavy metal ion Hg2+ and sulf-hydryl group modifying agents suggesting that free sulfhydryl group containing amino acid, cysteine is necessary for the catalytic activity of the invertase. A BLAST search of the N-terminal amino acid sequence of betel leaf invertase showed significant homology with the homologous invertases in database.

Integrated chemo-enzymatic production of 5-hydroxymethylfurfural from glucose

Sweets for my sweet: The production and isolation of 5- hydroxymethylfurfural (HMF) in high yield and purity is demonstrated by using a combination of glucose-fructose isomerization with sweetzyme in wet tetraethylammonium bromide (TEAB) and clean fructose dehydration to HMF catalyzed by using HNO3 under moderate conditions, which allow the reuse of any unreacted glucose and TEAB. Copyright

Anti-cariogenic Characteristics of Rubusoside

Streptococcus mutans plays an important role in the development of dental caries in humans by synthesizing adhesive insoluble glucans from sucrose by mutansucrase activity. To explore the anti-cariogenic characteristics of rubusoside (Ru), a natural sweetener component in Rubus suavissimus S. Lee (Rosaceae), we investigated the inhibitory effect of Ru against the activity of mutansucrase and the growth of Streptococcus mutans. Ru (50 mM) showed 97% inhibitory activity against 0.1 U/mL mutansucrase of S. mutans with 500 mM sucrose. It showed competitive inhibition with a Ki value of 1.1 ± 0.2 mM and IC50 of 2.3 mM. Its inhibition activity was due to hydrophobic and hydrogen bonding interactions based on molecular docking analysis. Ru inhibited the growth of S. mutans as a bacteriostatic agent, with MIC and MBC values of 6 mM and 8 mM, respectively. In addition, Ru showed synergistic anti-bacterial activity when it was combined with curcumin. Therefore, Ru is a natural anti-cariogenic agent with anti-mutansucrase activity and antimicrobial activity against S. mutans.

Simple preparation method for Mg–Al hydrotalcites as base catalysts

In this paper, we report a very simple preparation method for Mg–Al hydrotalcites (HTs) as base catalysts via the hydration of their corresponding metal oxides (MgO and Al2O3) without any particular controlled variables. We also propose a reasonable explanation for a series of reaction pathways in which Mg–Al HTs are formed from their metal oxides. The Mg–Al HTs were formed by the reaction of Mg2+ and Al(OH)4?, which resulted from the hydration of both metal oxides. The prepared Mg–Al HTs retained their unique properties, including their layered hydroxide structure and memory effect and showed considerable catalytic activity in the isomerization of glucose to fructose. Consequently, this simple hydration of both metal oxides successfully synthesized Mg–Al HTs, which can be used as base catalysts or as catalyst precursors for calcined HTs in various base-catalyzed reactions.

Synergy of boric acid and added salts in the catalytic dehydration of hexoses to 5-hydroxymethylfurfural in water

In combination with various salts boric acid, B(OH)3, was shown to be an efficient, weak Lewis acid catalyst in the aqueous dehydration of fructose to 5-hydroxymethylfurfural (HMF) due to strong complexation between the boron atom and the hexoses. In the dehydration of a highly concentrated aqueous fructose solution (30 wt%), a HMF yield of 60% was achieved at 92% fructose conversion and a HMF selectivity of 65% with 100 g L-1 B(OH) 3 and 50 g L-1 sodium chloride in the aqueous phase and methyl-isobutylketone (MIBK) as extracting solvent. Furthermore, the dehydration of glucose resulted in a yield of 14% HMF at 41% glucose conversion after 5 h at similar conditions. These results are highly competitive with currently reported aqueous HMF dehydration systems. In combination with the non-corrosive and non-toxic nature of the boric acid, compared to other well known homogeneous catalysts applicable for the dehydration process (e.g., H2SO 4 and HCl), clearly, the boric acid-salt mixture is a very attractive catalyst system.

Efficient and selective oxidation of D -glucose into gluconic acid under low-frequency ultrasonic irradiation

The production of gluconic acid from D-glucose represents an attractive target for industry. In this work, we propose an efficient, eco-friendly, and selective method to oxidize D-glucose with aqueous H2O2 in the presence of FeSO4 as a catalyst. The reaction was improved significantly under ultrasonic (US) activation, which led to excellent yields in only a few minutes. In addition, we determined that the reaction occurred through a sono-Fenton process by studying the production of hydroxyl radicals and optimizing the experimental conditions of the reaction. The energy consumption of the process was also investigated in this study.

New Boronic Acid Fluorescent Reporter Compounds. 2. A Naphthalene-Based On-Off Sensor Functional at Physiological pH

(Matrix presented) A new boronlc acid fluorescent on-off reporter compound (1) was synthesized. This fluorescent sensor shows a 41-fold emission intensity increase upon addition of 50 mM fructose in 0.1 M aqueous phosphate buffer at pH 7.4.

Supramolecular stabilization of acid tolerant l-arabinose isomerase from Lactobacillus sakei

l-Arabinose isomerase stability is a crucial criterion for the industrial application of this biocatalyst. Noria and NoriaPG are capable of increasing the l-arabinose isomerase stability not only at high temperatures but also at low pH. Such results highlight, for the first time, the use of the Noria series of molecules for protein stabilization and activation.

Hydrogenation of Glucose to Sorbitol over Nickel and Ruthenium Catalysts

Hydrogenation of aqueous glucose solution was performed in batch and continuous reactors using supported nickel and ruthenium catalysts. Preparation methods were precipitation, impregnation, solgel and template syntheses, and SiO2, TiO2, Al2O3 and carbon were used as support materials. A procedure for the one-step synthesis of templated metal on support catalysts was established. The influence of support material and preparation methods was studied and the results were compared with those of an industrial nickel catalyst. In a detailed study taking more than 1100 h time on stream we investigated the deactivation of the industrial catalyst and a supported ruthenium catalyst, and evaluated the deactivation mechanisms. As yet unpublished side products of the glucose hydrogenation were identified and a scheme of the reaction network was set up.

Solvent-Activated Hafnium-Containing Zeolites Enable Selective and Continuous Glucose–Fructose Isomerisation

The isomerisation of glucose to fructose is a critical step towards manufacturing petroleum-free chemicals from lignocellulosic biomass. Herein we show that Hf-containing zeolites are unique catalysts for this reaction, enabling true thermodynamic equilibrium to be achieved in a single step during intensified continuous operation, which no chemical or biological catalyst has yet been able to achieve. Unprecedented single-pass yields of 58 % are observed at a fructose selectivity of 94 %, and continuous operation for over 100 hours is demonstrated. The unexpected performance of the catalyst is realised following a period of activation within the reactor, during which time interaction with the solvent generates a state of activity that is absent in the synthesised catalyst. Mechanistic studies by X-ray absorption spectroscopy, chemisorption FTIR, operando UV/Vis and 1H–13C HSQC NMR spectroscopy indicate that activity arises from isolated HfIV atoms with monofunctional acidic properties.

Tandem production of levan and ethanol by microbial fermentation

Tandem production of levan and ethanol by microbial fermentation using sucrose substrate was investigated. The tandem process involves fermentation of Bacillus subtilis (natto) Takahashi in a sucrose medium to produce levan, separation of the levan product from glucose by-product by ultrafiltration and fermentation of the glucose remnant from levan production by Zymomonas mobilis to produce ethanol. After cultivation of B. subtilis (natto) Takahashi for 48 h, 56.0 ± 0.6 g l-1 of levan was produced in a medium containing 250 g l-1 sucrose, which was 45 ± 0.5% yield on available fructose. After removing the cells, the fermentation broth was concentrated by ultrafiltration through a membrane of 5 kDa cutoff. The filtrate which contained 179 ± 3 g l-1 of hexose was diluted, supplemented with yeast extract (5 g l-1), (NH4)2SO4 (1 g l-1), MgSO4·7H2O (0.5 g l-1), KH2PO4 (1 g l-1) and used for alcohol fermentation by Z. mobilis. Incubation of Z. mobilis in the medium containing glucose remnant at 30 °C, pH 5.5, 175 rpm for 120 h gave from 21.1 ± 0.3 to 26.5 ± 0.2 g l-1 of bio-ethanol depending on the dilution. The tandem process developed in this study is an eco-friendly process in that the sucrose substrate was fully utilized without wasting any by-products in the process; in addition, two invaluable environmentally-friendly biomaterials (levan and ethanol) were produced. Furthermore, the amount of alcohol required for levan recovery could be reduced to a quarter of that generally used in the conventional precipitation. The Royal Society of Chemistry 2010.

Lactose- and cellobiose-derived branched trisaccharides and a sucrose-containing trisaccharide produced by acceptor reactions of Weissella confusa dextransucrase

Dextran-producing Weissella have received significant attention. However, except for maltose, the acceptor reactions of Weissella dextransucrases with different sugars have not been investigated. The action of recombinant Weissella confusa VTT E-90392 dextransucrase was tested with several potential acceptors, particularly, analogs lactose and cellobiose. The major acceptor products of both disaccharides were identified as branched trisaccharides, with a glucosyl residue α-(1→2)-linked to the acceptor's reducing end. An additional product, isomelezitose (6Fru-α-Glcp-sucrose), was also produced when using lactose as an acceptor. This is the first report of the synthesis of isomelezitose by a dextransucrase. The NMR spectra of the three trisaccharides were fully assigned, and their structures were confirmed by selective enzymatic hydrolysis. The trisaccharides prepared from 13C6glc sucrose and lactose were analyzed by ESI-MSn, and the fragmentation patterns of these compounds were characterized.

From alcohol dehydrogenase to a "one-way" carbonyl reductase by active-site redesign: A mechanistic study of mannitol 2-dehydrogenase from pseudomonas fluorescens

Directional preference in catalysis is often used to distinguish alcohol dehydrogenases from carbonyl reductases. However, the mechanistic basis underpinning this discrimination is weak. In mannitol 2-dehydrogenase from Pseudomonas fluorescens, stabilization of (partial) negative charge on the substrate oxyanion by the side chains of Asn-191 and Asn-300 is a key feature of catalysis in the direction of alcohol oxidation. We have disrupted this ability through individual and combined substitutions of the two asparagines by aspartic acid. Kinetic data and their thermodynamic analysis show that the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol (Kint) was altered dramatically (104- to 10 5-fold) from being balanced in the wild-type enzyme (Kint ≈ 3) to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. The change in Kint reflects a selective slowing down of the mannitol oxidation rate, resulting because Asn → Asp replacement (i) disfavors partial abstraction of alcohol proton by Lys-295 in a step preceding catalytic hydride transfer, and (ii) causes stabilization of a nonproductive enzyme-NAD+-mannitol complex. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pKvalues of 5.3 ± 0.1 and 6.3 ± 0.2, respectively. The mutant incorporating both Asn → Asp substitutions behaved as a slow "fructose reductase" at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8-10. A mechanism is suggested in which polarization of the substrate carbonyl by a doubly protonated diad of Asp and Lys-295 facilitates NADH-dependent reduction of fructose by N191D and N300D under optimum pH conditions. Creation of an effectively "one-way" reductase by active-site redesign of a parent dehydrogenase has not been previously reported and holds promise in the development of carbonyl reductases for application in organic synthesis.

Cloning, expression, properties, and functional amino acid residues of new trehalose synthase from Thermomonospora curvata DSM 43183

A new trehalose synthase (TreS) gene from Thermomonospora curvata DSM 43183 was cloned and expressed in Escherichia coli XL10-Gold. The purified recombinant enzyme (TreS-T.C) could catalyze the reversible interconversion of maltose and trehalose of sucrose into trehalulose without other disaccharides including isomaltulose at an optimum temperature of 35 °C and a pH of 6.5. The Km of TreS-T.C for maltose (96 mM) was lower than those for trehalose (198 mM) and sucrose (164 mM), suggesting that maltose is the optimum substrate. The maximum trehalose and trehalulose yields were 70% and >80%, respectively. Active TreS-T.C is a trimer comprising three identical 60 kDa subunits. Homology modeling analysis revealed that TreS-T.C had a GH13-typical (β/α)8 barrel catalytic domain. Two sites, one determining substrate specificity (L116) and the other affecting product formation (E330), were found near the active center by homology modeling combined with site-directed mutagenesis. TreS-T.C may be used effectively as a potential biocatalyst for the production of trehalose and trehalulose from maltose and sucrose in a one-step reaction, respectively. This study also provides a feasible and effective method for studying functional amino acid residues around TreS without performing crystal structure analysis and high-throughput screening.

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Crystal structure, solubility, and mutarotation of the rare monosaccharide D-psicose

X-ray crystal analysis for D-psicose (C3-position epimer of D-fructose) crystallized from aqueous solution was successfully performed for the first time. It was confirmed that D-psicose crystallized solely as β-D-pyranose with 1C (1C4 (d)) conformation. The crystal system (orthorhombic), space group (#19, P212121), and number of molecules per unit cell (Z = 4) are the same as those for β-D-fructopyranose, α-L-sorbopyranose, and α-D-tagatopyranose. Solubility of D-psicose at 25 °C was 291 g per 100 g water. Mutarotation was further investigated recording the time course of specific rotation [α] at 589 nm after the dissolution of D-psicose in water. It is thought that [α] for β-D-psicopyranose in water may be ca.-85degdm -1g-1 cm3. The time course of absorbance at 280 nm after the dissolution of D-psicose was also measured to see the development of open-chain carbonyl-form in the solution, and the first-order kinetic behavior with the rate constant k = 4.44 ms-1 was confirmed. Assuming the equilibrium content of carbonyl-form of D-psicose as 0.2%, the molar absorption coefficient, ε, for the carbonyl-form was estimated to be 160cm-1 M-1.

Acid-catalyzed inversion of sucrose in the amorphous state at very low levels of residual water

Purpose. Factors affecting the solid-state acid-catalyzed inversion of amorphous sucrose to glucose and fructose in the presence of colyophilized citric acid, with less than 0.1% w/w residual water, have been studied. Methods. Samples of citric acid and sucrose were lyophilized at a weight ratio of 1:10 citric acid:sucrose from solutions with initial pH values of 1.87, 2.03, and 2.43, as well as at a weight ratio of 1:5, at an initial pH of 1.87. Glass transition temperatures, Tg, were measured by DSC and the presence of any possible residual water was monitored by Karl Fischer Titrimetry. The inversion of sucrose was measured by polarimetric analysis after reconstitution of solid samples stored at 50°C under P2O5. Results. Samples of 1:10 citric acid:sucrose at an initial pH of 1.87, 2.03, and 2.43 exhibited the same Tg. The initial rate of reactivity was affected at a 1:10 ratio by the solution pH before lyophilization in the order: 1.87 > 2.03 > 2.43 and by citric acid concentration at pH 1.87 in the order 1:5 > 1:10. Conclusions. Sucrose, colyophilized with an acid such as citric acid, undergoes significant acid-catalyzed inversion at 50°C despite the very low levels of residual water, i.e., 0.1% w/w. At the same ratio of citric acid to sucrose (1:10), and hence the same Tg, the rate of reaction correlates with the initial solution pH indicating that the degree of ionization of citric acid in solution is most likely retained in the solid state. That protonation of sucrose by citric acid is important is shown by the direct relationship between maximum extent of reaction and citric acid composition. It is concluded that colyophilization of acidic substances with sucrose, even in the absence of residual water, can produce reducing sugars capable of further reaction with other formulation ingredients susceptible to reaction with reducing sugars.

Reaction media dominated product selectivity in the isomerization of glucose by chromium trichloride: From aqueous to non-aqueous systems

While chromium trichloride hydrate (CrCl3·6H 2O) effectively catalyzed the isomerization of glucose into fructose in aqueous solution, a general product selectivity behavior was observed independent of reaction variables such as reaction time, temperature, catalyst loading, halide ion, and initial glucose concentration. By studying the mixed solution of dimethylsulfoxide (DMSO) and water at varied DMSO/H2O ratios, it was found that deviation from the general water-phase fructose yield curve, with concomitant 5-hydroxymethylfurfral (HMF) formation, occurred when the fructose concentration in available water became sufficiently high in DMSO rich solvent mix. Therefore, tuning the solvent system was the most effective approach to change the product distribution from CrCl3·6H 2O catalyzed glucose conversion. The apparent activation energy of glucose conversion in the studied system was estimated to be 58.6 kJ mol -1. Special attention was also given to gain some mechanistic insights by control experiments with simple model compounds and additives, 13C NMR and UV-vis spectroscopic analyses.

Combined Action of Enzyme and Metal Catalyst, applied to the Preparation of D-Mannitol

A novel catalytic approach involving co-operation of a bio- and a chemo-catalyst has been applied in the simultaneous enzymatic interconversion and platinum metal-catalysed hydrogenation of D-fructose-D-glucose mixtures (water, pH 7-8, 60 degC, 20 atm hydrogen); this gives an enhanced yield of D-mannitol.

Identification of Active and Spectator Sn Sites in Sn-β Following Solid-State Stannation, and Consequences for Lewis Acid Catalysis

Lewis acidic zeolites are rapidly emerging liquid-phase Lewis acid catalysts. Nevertheless, their inefficient synthesis procedure currently prohibits greater utilization and exploitation of these promising materials. Herein, we demonstrate that SnIV-containing zeolite beta can readily be prepared both selectively and extremely rapidly by solid-state incorporation (SSI) method. Through a combination of spectroscopic (XRD, UV/Vis, X-ray absorption, magic-angle spinning NMR, and diffuse reflectance infrared Fourier transform spectroscopy) studies, we unambiguously demonstrate that site-isolated, isomorphously substituted SnIV sites dominate the Sn population up to a loading of 5 wt % Sn. These sites are identical to those found in conventionally prepared Sn-beta, and result in our SSI material exhibiting identical levels of intrinsic activity (that is, turnover frequency) despite the threefold increase in Sn loading, and the extremely rapid and benign nature of our preparation methodology. We also identify the presence of spectator sites, in the form of SnIV oligomers, at higher levels of Sn loading. The consequences of this mixed population with regards to catalysis (Meerwein-Pondorf-Verley reaction and glucose isomerization) are also identified.

Synthesis of D-sorbose and D-psicose by recombinant Escherichia coli

In the current work, the in vitro synthetic system for rare sugars was successfully transformed into an engineered Escherichia coli with a plasmid containing both the aldolase RhaD and phosphatase YqaB. By taking advantage of the inherent biosynthetic pathways in E. coli, this approach permits the use of simple and cheap glycerol for the synthesis of DHAP in vivo. Moreover, the introduction of the phosphatase into the E. coli system allows for the removal of the phosphate group on the synthetic intermediate to yield the neutral rare sugars, which can be readily secreted to the medium without accumulation in the cell.

Thermodynamics of reactions catalyzed by l-iditol 2-dehydrogenase: The xylose assimilation pathway

Apparent equilibrium constants and calorimetric enthalpies of reaction have been measured for the following enzyme catalyzed (L-iditol 2-dehydrogenase) biochemical reactions in phosphate buffer at pHs near 7.5 and at the temperature 298.15 K D-sorbitol(aq) + NADox(aq) = D-fructose(aq) + NADred(aq), L-iditol(aq) + NADox(aq) = L-sorbose(aq) + NADred(aq), xylitol(aq) + NADox(aq) = D-xylulose(aq) + NADred(aq). Here, NADox is β-nicotinamide-adenine dinucleotide (oxidized form) and NADred is β-nicotinamide-adenine dinucleotide (reduced form). The results are used to calculate equilibrium constants and standard molar enthalpies, entropies, and Gibbs free energies for reference reactions involving specific species. Standard formation properties and standard transformed formation properties of the biochemical reactants are also calculated. The thermodynamics of the xylose assimilation pathway is summarized.

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Photothermal strategy for the highly efficient conversion of glucose into lactic acid at low temperatures over a hybrid multifunctional multi-walled carbon nanotube/layered double hydroxide catalyst

The conversion of carbohydrates into lactic acid has attracted increasing attention owing to the broad applications of lactic acid. However, the current methods of thermochemical conversion commonly suffer from limited selectivity or the need for harsh conditions. Herein, a light-driven system of highly selective conversion of glucose into lactic acid at low temperatures was developed. By constructing a hybrid multifunctional multi-walled carbon nanotube/layered double hydroxide composite catalyst (CNT/LDHs), the highest lactic acid yield of 88.6% with 90.0% selectivity was achieved. The performance of CNT/LDHs for lactic acid production from glucose is attributed to the following factors: (i) CNTs generate a strong heating center under irradiation, providing heat for converting glucose into lactic acid; (ii) LDHs catalyze glucose isomerization, in which the photoinduced OVs (Lewis acid) in LDHs under irradiation further improve the catalytic activity; and (iii) in a heterogeneous-homogeneous synergistically catalytic system (LDHs-OH-), OH- ions are concentrated in LDHs, forming strong base sites to catalyze subsequent cascade reactions.

Biochemical characterization of a recombinant acid phosphatase from Acinetobacter baumannii

Genomic sequence analysis of Acinetobacter baumannii revealed the presence of a putative Acid Phosphatase (AcpA; EC 3.1.3.2). A plasmid construct was made, and recombinant protein (rAcpA) was expressed in E. coli. PAGE analysis (carried out under denaturing/ reducing conditions) of nickel-affinity purified protein revealed the presence of a nearhomogeneous band of approximately 37 kDa. The identity of the 37 kDa species was verified as rAcpA by proteomic analysis with a molecular mass of 34.6 kDa from the deduced sequence. The dependence of substrate hydrolysis on pH was broad with an optimum observed at 6.0. Kinetic analysis revealed relatively high affinity for PNPP (Km = 90 μM) with Vmax, kcat, and Kcat/Km values of 19.2 pmoles s-1, 4.80 s-1(calculated on the basis of 37 kDa), and 5.30 × 104 M-1s-1, respectively. Sensitivity to a variety of reagents, i.e., detergents, reducing, and chelating agents as well as classic acid phosphatase inhibitors was examined in addition to assessment of hydrolysis of a number of phosphorylated compounds. Removal of phosphate from different phosphorylated compounds is supportive of broad, i.e., 'nonspecific' substrate specificity; although, the enzyme appears to prefer phosphotyrosine and/or peptides containing phosphotyrosine in comparison to serine and threonine. Examination of the primary sequence indicated the absence of signature sequences characteristic of Type A, B, and C nonspecific bacterial acid phosphatases.