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1.
Laccase-catalyzed oxidation of N-substituted phenothiazines and N-substituted phenoxazines was investigated at pH 5.5 and
25°C. The recombinant laccase from Polyporus pinsitus (rPpL) and the laccase from Myceliophthora thermophila (rMtL) were used. The dependence of initial reaction rate on substrate concentration was analyzed by applying the laccase
action scheme in which the laccase native intermediate (NI) reacts with a substrate forming reduced enzyme. The reduced laccase
produces peroxide intermediate (PI) which in turn decays to the NI. The calculated constant (kox) values of the PI formation are (6.1±3.1)×105 M−1s−1 for rPpL and (2.5±0.9)×104 M−1s−1 for rMtL. The bimolecular constants of the reaction of the native intermediate with electron donor (kred) vary in the interval
from 2.2×105 to 2.1×107 M−1s−1 for rPpL and from 1.3×102 to 1.8×105 M-1s−1 for rMtL. The larger reactivity of rPpL in comparison to rMtL is associated with the higher redox potential of type I Cu
of rPpL. The variation of kred values for both laccases correlates with the change of the redox potential of substrates. Following outer sphere (Marcus)
electron transfer mechanism the calculated activationless electron transfer rate and the apparent reorganization energy are
5.0×107 M−1s−1 and 0.29 eV, respectively. 相似文献
2.
Kinetics of 1-hydroxypyrene (1-HP) oxidation catalyzed with recombinant Coprinus cinereus (rCiP) and horseradish (HRP) peroxidases was investigated with a special emphasis for developing a nanomolar hydrogen peroxide
(H2O2) detection system. At pH 8.0 the bimolecular constants of 1-HP oxidation with the ferryl compounds of rCiP and HRP were equal
to (1.0 ± 0.3) × 108 M−1 s−1 and (0.6 ± 0.2) × 108 M−1 s−1, respectively. High bimolecular constants and fluorescence quantum yield of 1-HP (0.66) permitted detection as low as 21
nM of H2O2. To optimize the detection system 1-HP oxidation was modeled at steady-state conditions in the range pH 5.0 to pH 8.0. The
1-HP based detection system was compared with the Amplex Red system. The peroxidase-catalyzed 1-HP oxidation system was used
for determination of ozone in the air. 相似文献
3.
This article reports rate constants for thiol–thioester exchange (k
ex), and for acid-mediated (k
a), base-mediated (k
b), and pH-independent (k
w) hydrolysis of S-methyl thioacetate and S-phenyl 5-dimethylamino-5-oxo-thiopentanoate—model alkyl and aryl thioalkanoates, respectively—in water. Reactions such as
thiol–thioester exchange or aminolysis could have generated molecular complexity on early Earth, but for thioesters to have
played important roles in the origin of life, constructive reactions would have needed to compete effectively with hydrolysis
under prebiotic conditions. Knowledge of the kinetics of competition between exchange and hydrolysis is also useful in the
optimization of systems where exchange is used in applications such as self-assembly or reversible binding. For the alkyl
thioester S-methyl thioacetate, which has been synthesized in simulated prebiotic hydrothermal vents, k
a = 1.5 × 10−5 M−1 s−1, k
b = 1.6 × 10−1 M−1 s−1, and k
w = 3.6 × 10−8 s−1. At pH 7 and 23°C, the half-life for hydrolysis is 155 days. The second-order rate constant for thiol–thioester exchange
between S-methyl thioacetate and 2-sulfonatoethanethiolate is k
ex = 1.7 M−1 s−1. At pH 7 and 23°C, with [R″S(H)] = 1 mM, the half-life of the exchange reaction is 38 h. These results confirm that conditions
(pH, temperature, pK
a of the thiol) exist where prebiotically relevant thioesters can survive hydrolysis in water for long periods of time and
rates of thiol–thioester exchange exceed those of hydrolysis by several orders of magnitude. 相似文献
4.
Theodoros Goulas Athanasios Goulas George Tzortzis Glenn R. Gibson 《Applied microbiology and biotechnology》2009,82(6):1079-1088
Four different β-galactosidases (previously named BbgI, BbgII, BbgIII and BbgIV) from Bifidobacterium bifidum NCIMB41171 were overexpressed in Escherichia coli, purified to homogeneity and their biochemical properties and substrate preferences comparatively analysed. BbgI was forming
a hexameric protein complex of 875 kDa, whereas BbgII, BbgIII and BbgIV were dimers with native molecular masses of 178, 351
and 248 kDa, respectively. BbgII was the only enzyme that preferred acidic conditions for optimal activity (pH 5.4–5.8), whereas
the other three exhibited optima in more neutral pH ranges (pH 6.4–6.8). Na+ and/or K+ ions were prerequisite for BbgI and BbgIV activity in Bis–Tris-buffered solutions, whereas Mg++ was strongly activating them in phosphate-buffered solutions. BbgII and BbgIII were slightly influenced from the presence
or absence of cations, with Mg++, Mn++ and Ca++ ions exerting the most positive effect. Determination of the specificity constants (k
cat/K
m) clearly indicated that BbgI (6.11 × 104 s−1 M−1), BbgIII (2.36 × 104 s−1 M−1) and especially BbgIV (4.01 × 105 s−1 M−1) are highly specialised in the hydrolysis of lactose, whereas BbgII is more specific for β-d-(1→6) galactobiose (5.59 × 104 s−1 M−1) than lactose (1.48 × 103 s−1 M−1). Activity measurements towards other substrates (e.g. β-d-(1→6) galactobiose, β-d-(1→4) galactobiose, β-d-(1→4) galactosyllactose, N-acetyllactosamine, etc.) indicated that the β-galactosidases were complementary to each other by hydrolysing different substrates and thus contributing in a different
way to the bacterial physiology. 相似文献
5.
Kinetics of the steady-state oxidation of n–alkylferrocenes (alkyl = H, Me, Et, Bu and C5H11) by H2O2 to form the corresponding ferricenium cations catalyzed by horseradish peroxidase has been studied in micellar systems of
Triton X-100, CTAB, and SDS, mostly at pH 6.0 and 25 °C. The rate of oxidation of ferrocenes with longer alkyl radicals is
too slow to be measured. The reaction obeying the [RFc]:[H2O2] = 2 : 1 stoichiometry is strictly first-order in both HRP and RFc in a wide concentration range. The corresponding observed
second-order rate constants k, which refer to the interaction of the peroxidase compound II (HRP-II) with RFc, decrease with the elongation of the alkyl
substituent R, and this in turn is accompanied by an increase in the formal redox potentials E°′ in the same medium. Increasing the surfactant concentration lowers the rate constants k, the effect being due to the nonproductive binding of RFc to micelles rather than to enzyme inactivation. The micellar effects
are accounted for in terms of the Berezin pseudo-phase model of micellar catalysis applied to the interaction of enzyme with
organometallic substrates. The oxidation was found to occur primarily in the aqueous pseudo-phase and the calculated intrinsic
second-order rate constants k
w are (1.9 ± 0.5)×105, (2.7 ± 0.1)×104, and (5.9 ± 0.6)×103 M–1 s–1 for HFc, EtFc, and n–BuFc, respectively. The data obtained were used for estimating the self-exchange rate constants for the HRP-II/HRP couple
in terms of the Marcus formalism.
Received: 15 July 1996 / Accepted: 15 November 1996 相似文献
6.
Tiecheng Qiao Robert Witkowski Robin Henderson G. McLendon 《Journal of biological inorganic chemistry》1996,1(5):432-438
The kinetics of methemoglobin reduction by cytochrome b
5 has been studied by stopped-flow and saturation transfer NMR. A forward rate constant k
f = 2.44×104 M–1 s–1 and a reverse rate constant k
b = 540 M–1s–1 have been observed at 10 mm, pH 6.20, 25 °C. The ratio k
f/k
b = k
eq = 43.6 is in good agreement with the equilibrium constant calculated from the electrochemical potential between cyt b
5 and methemoglobin. A bimolecular collisional mechanism is proposed for the electron transfer from cyt b
5 to methemoglobin based on the kinetic data analysis. The dependence of the rate constants on ionic strengths supports such
collisional mechanism. It is also found that the reaction rate strongly depends on the conformations of methemoglobin.
Received: 20 February 1996 / Accepted: 4 June 1996 相似文献
7.
Cloning and characterization of a new laccase from Bacillus licheniformis catalyzing dimerization of phenolic acids 总被引:1,自引:0,他引:1
Koschorreck K Richter SM Ene AB Roduner E Schmid RD Urlacher VB 《Applied microbiology and biotechnology》2008,79(2):217-224
A new laccase gene (cotA) was cloned from Bacillus licheniformis and expressed in Escherichia coli. The recombinant protein CotA was purified and showed spectroscopic properties, typical for blue multi-copper oxidases. The
enzyme has a molecular weight of ~65 kDa and demonstrates activity towards canonical laccase substrates 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic
acid) (ABTS), syringaldazine (SGZ) and 2,6-dimethoxyphenol (2,6-DMP). Kinetic constants K
M and k
cat for ABTS were of 6.5 ± 0.2 μM and 83 s−1, for SGZ of 4.3 ± 0.2 μM and 100 s−1, and for 2,6-DMP of 56.7 ± 1.0 μM and 28 s−1. Highest oxidizing activity towards ABTS was obtained at 85°C. However, after 1 h incubation of CotA at 70°C and 80°C, a
residual activity of 43% and 8%, respectively, was measured. Furthermore, oxidation of several phenolic acids and one non-phenolic
acid by CotA was investigated. CotA failed to oxidize coumaric acid, cinnamic acid, and vanillic acid, while syringic acid
was oxidized to 2,6-dimethoxy-1,4-benzoquinone. Additionally, dimerization of sinapic acid, caffeic acid, and ferulic acid
by CotA was observed, and highest activity of CotA was found towards sinapic acid. 相似文献
8.
Jose Neptuno Rodriguez-Lopez Andrew T. Smith R. N. F. Thorneley 《Journal of biological inorganic chemistry》1996,1(2):136-142
Horseradish peroxidase isoenzyme C (HRPC) mutants were constructed in order to understand the role of two key distal haem
cavity residues, histidine 42 and arginine 38, in the formation of compound I and in substrate binding. The role of these
residues as general acid-base catalysts, originally proposed for cytochrome c peroxidase by Poulos and Kraut in 1980 was assessed for HRPC. Replacement of histidine 42 by leucine [(H42L)HRPC*] decreased
the apparent bimolecular rate constant for the reaction with hydrogen peroxide by five orders of magnitude (k
1 = 1.4×102 M–1s–1) compared with both native-glycosylated and recombinant forms of HRPC (k
1 = 1.7×107 M–1s–1). The first-order rate constant for the heterolytic cleavage of the oxygen-oxygen bond to form compound I was estimated to
be four orders of magnitude slower for this variant. Replacement of arginine 38 by leucine [(R38L)HRPC*] decreased the observed
pseudo-first-order rate constant for the reaction with hydrogen peroxide by three orders of magnitude (k
1 = 1.1×104 M–1s–1), while the observed rate constant of oxygen bond scission was decreased sixfold (k
2 = 142 s–1). These rate constants are consistent with arginine 38 having two roles in catalysing compound I formation: firstly, promotion
of proton transfer to the imidazole group of histidine 42 to facilitate peroxide anion binding to the haem, and secondly,
stabilisation of the transition state for the heterolytic cleavage of the oxygen-oxygen bond. These roles for arginine 38
explain, in part, why dioxygen-binding globins, which do not have an arginine in the distal cavity, are poor peroxidases.
Binding studies of benzhydroxamic acid to (H42L)HRPC* and (R38L)HRPC* indicate that both histidine 42 and arginine 38 are
involved in the modulation of substrate affinity.
Received: 21 July 1995 / Accepted: 27 November 1995 相似文献
9.
J. K. Sharma M. Yadav N. P. Singh K. D. S. Yadav 《Applied Biochemistry and Microbiology》2011,47(5):532-537
Extracellular secretion of lignin peroxidase from Pycnoporus sanguineus MTCC-137 in the liquid culture growth medium amended with lignin containing natural sources has been shown. The maximum secretion
of lignin peroxidase has been found in the presence of saw dust. The enzyme has been purified to homogeneity from the culture
filtrate of the fungus using ultrafiltration and anion exchange chromatography on DEAE-cellulose. The purified lignin peroxidase
gave a single protein band in sodium dodecylsulphate polyacrylamide gel electrophoresis corresponding to the molecular mass
40 kDa. The K
m, k
cat and k
cat/K
m values of the enzyme using veratryl alcohol and H2O2 as the substrate were 61 M, 2.13 s−1, 3.5 × 104 M−1s−1 and 71 M, 2.13 s−1, 3.0 × 104 M−1 s−1 respectively at the optimum pH of 2.5. The temperature optimum of the enzyme was 25°C. 相似文献
10.
Both native Trametes hirsuta laccase and the same laccase modified with palmytic chains to turn it more hydrophobic were prepared and studied with cyclic
voltammetry and Raman spectroscopy. Native laccase immobilized in the monoolein cubic phase was characterized with resonance
Raman spectroscopy, which demonstrated that the structure at the “blue” copper site of the protein remained intact. The diamond-type
monoolein cubic phase prevents denaturation of enzymes on the electrode surface and provides contact of the enzyme with the
electrode either directly or through the mediation by electroactive probes. Direct electron transfer for both laccases incorporated
into a lyotropic liquid crystal was obtained under anaerobic conditions, whereas bioelectrocatalytic activity was shown only
for the native enzyme. The differences in electrochemical behavior of native and hydrophobic laccase as well as possible mechanisms
of direct and mediated electron transfers are discussed. The Michaelis constant for 2,2′-azinobis(3-ethylbenzothiazoline-6-sulfonate)
diammonium salt (ABTS2−), K
Mapp, and the maximal current, I
max, for the native enzyme immobilized onto the electrode were estimated to be 0.24 mM, and 5.3 μA, respectively. The maximal
current density and the efficiency of the catalysis, I
max/K
Mapp, were found to be 73 μA cm−2 and 208.2 μA cm−2 mM−1, respectively, and indicated a high efficiency of oxygen electroreduction by the enzyme in the presence of ABTS2− in the cubic-phase environment. Rate constants were calculated to be 7.5 × 104 and 3.6 × 104 M−1 s−1 for native and hydrophobic laccase, respectively. 相似文献
11.
The antioxidant properties of hydroxycinnamic acid derivatives (HCA) were studied by laser flash photolysis. The transient species with maximum absorption at 360 nm were assigned to the phenoxyl radical of HCA. The SO4•− induced oxidation of HCA was also investigated. It was shown that the interaction of SO4•− with HCA resulted in the formation of HCA phenoxyl radicals with rate constants of 2.0–3.9×109 M−1 s−1. The reactions of HCA with triplet state of benzophenone were analyzed and quenching rate constants of 4.3–7.8×109 M−1 s−1 were determined. 相似文献
12.
Quaratino D Federici F Petruccioli M Fenice M D'Annibale A 《Antonie van Leeuwenhoek》2007,91(1):57-69
Extracellular laccase from Panus tigrinus CBS 577.79 was produced in a bubble-column reactor using glucose-containing medium supplemented with 2,5-xylidine under conditions of nitrogen sufficiency. The main laccase isoenzyme was purified to apparent homogeneity by ultra-filtration, anion-exchange chromatography and gel filtration that led to a purified enzyme with a specific activity of 317 IU (mg protein)−1 and a final yield of 66%. Laccase was found to be a monomeric protein with a molecular mass of 69.1 kDa, pI of 3.15 and 6.9% N-glycosylation of the high mannose type. Temperature and pH optima were 55°C and 3.75 (2,6-dimethoxyphenol as substrate). At 50 and 60°C, the enzyme half-lives were 281 and 25 min, respectively. The P. tigrinus laccase oxidized a wide range of both naturally occurring and synthetic aromatic compounds: the highest catalytic efficiencies were for 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonic) acid and 2,6-dimethoxyphenol (5.99 × 106 and 3.07 × 106 M−1 s−1, respectively). Catalytic rate constants for typical N–OH redox mediators, such as 1-hydroxybenzotriazole (2.6 s−1), violuric acid (8.4 s−1) and 2,2,6,6-tetramethylpiperidin-N-oxide radical (7.8 s−1), were found to be higher than those reported for other high redox potential fungal laccases. 相似文献
13.
S. C. J. Meskers C. Dennison Gerard W. Canters Harry P. J. M. Dekkers 《Journal of biological inorganic chemistry》1998,3(6):663-670
The dynamic quenching of the luminescence of racemic Eu(III)(pyridine-2,6-dicarboxylate=dpa)3
3– by the title proteins is investigated and the enantioselectivity of the proteins in the quenching of the Δ and Λ enantiomers
of Eu(dpa)3
3– is determined. The two diastereomeric quenching rate constants pertaining to azurin (k
q
Δ=3.3×106, k
q
Λ=2.7×106 M–1 s–1, pH 7.2, ionic strength I=22 mM) are lower than for its Met→44Lys mutant (k
q
Δ=1.9×107, k
q
Λ=1.4×107 M–1 s–1, same pH and I), indicating that energy transfer occurs from Eu(dpa)3
3– to the Cu(II) centre when the luminophore is bound to the hydrophobic patch of the protein near residue 44. The enantioselectivity
remains unaltered by the mutation: k
q
Δ/k
q
Λ=1.27±0.04, so Lys44 is probably not in direct contact with the Eu chelate. The I and pH dependence of k
q indicate that the lysine residue interacts electrostatically with Eu(dpa)3
3–. For plastocyanin the quenching rates are of the order of 106 M–1 s–1; for amicyanin they are two orders of magnitude larger (k
q
Δ=12×107, k
q
Λ=11×107 M–1 s–1, pH 7.2, I=22 mM). The variation of k
q is attributed to differences in the charge distribution on the proteins, which influences the binding of the luminophore
to the protein surface. For amicyanin the anion binding site near Lys59 and Lys60 may be involved in the energy transfer.
Received: 16 June 1998 / Accepted: 18 September 1998 相似文献
14.
John Ejnik Amalia Muñoz Tong Gan C. Frank Shaw III D. H. Petering 《Journal of biological inorganic chemistry》1999,4(6):784-790
− 1 s − 1 at 25 °C and pH 7.4 in Tris.HCl buffer and 0.1 M KCl. At 25 °C, Zn7-metallothionein also exchanged metal ions with Cd-carbonic anhydrase with a rate constant of 0.33 ± 0.02 M − 1 s − 1 to reconstitute enzymatically active protein. Cd-carbonic anhydrase reacted within the time of mixing with the peptide sequence
49–61 of rabbit metallothionein 2 which contains four cysteinyl residues, leading to the exchange of most of the Cd2+ into the peptide. At pH 7.4 and 25 °C, Cd2+ has higher affinity for apometallothionein than for the apo-peptide.
Received: 25 February 1999 / Accepted: 17 September 1999 相似文献
15.
trans -[PtCl4(NH3)(thiazole)] (1), trans-[PtCl4(cha)(NH3)] (2), cis-[PtCl4(cha)(NH3)] (3) (cha =cyclohexylamine), and cis-[PtCl4(NH3)2] (4) has been investigatedat 25 °C in a 1.0 M aqueous medium at pH 2.0–5.0 (1) and 4.5–6.8 (2–4) using stopped-flow spectrophotometry. The redox reactions follow the second-order rate law , where k is a pH-dependent rate constant and [GSH]tot the total concentration of glutathione. The reduction takes place via parallel reactions between the platinum(IV) complexes
and the various protolytic species of glutathione. The pH dependence of the redox kinetics is ascribed to displacement of
these protolytic equilibria. The thiolate species GS− is the major reductant under the reaction conditions used. The second-order rate constants for reduction of compounds 1–4 by GS− are (1.43±0.01)×107, (3.86±0.03)×106, (1.83±0.01)×106, and (1.18±0.01)×106 M−1 s−1, respectively. Rate constants for reduction of 1 by the protonated species GSH are more than five orders of magnitude smaller. The mechanism for the reductive elimination
reactions of the Pt(IV) compounds is proposed to involve an attack by glutathione on one of the mutually trans coordinated chloride ligands, leading to two-electron transfer via a chloride-bridged activated complex. The kinetics results
together with literature data indicate that platinum(IV) complexes with a trans Cl-Pt-Cl axis are reduced rapidly by glutathione as well as by ascorbate. In agreement with this observation, cytotoxicity
profiles for such complexes are very similar to those for the corresponding platinum(II) product complexes. The rapid reduction
within 1 s of the platinum(IV) compounds with a trans Cl-Pt-Cl axis to their platinum(II) analogs does not seem to support the strategy of using kinetic inertness as a parameter
to increase anticancer activity, at least for this class of compounds.
Received: 8 December 1999 / Accepted: 15 February 2000 相似文献
16.
Water conductance of the cuticular membrane (CM) of mature sweet cherry fruit (Prunus avium L. cv. Sam) was investigated by monitoring water loss from segments of the outer pericarp excised from the cheek of the fruit.
Segments consisted of epidermis, hypodermis and several cell layers of the mesocarp. Segments were mounted in stainless-steel
diffusion cells with the mesocarp surface in contact with water, while the outer cuticular surface was exposed to dry silica
(22 ± 1 °C). Conductance was calculated by dividing the amount of water transpired per unit area and time by the difference
in water vapour concentration across the segment. Conductance values had a log normal distribution with a median of 1.15 × 10−4 m s−1 (n=357). Transpiration increased linearly with time. Conductance remained constant and was not affected by metabolic inhibitors
(1 mM NaN3 or 0.1 mM carbonylcyanide m-chlorophenylhydrazone) or thickness of segments (range 0.8–2.8 mm). Storing fruit (up to 42 d, 1 °C) used as a source of
segments had no consistent effect on conductance. Conductance of the CM increased from cheek (1.16 ± 0.10 × 10−4 m s−1) to ventral suture (1.32 ± 0.07 × 10−4 m s−1) and to stylar end (2.53 ± 0.17 × 10−4 m s−1). There was a positive relationship (r2=0.066**; n=108) between conductance and stomatal density. From this relationship the cuticular conductance of a hypothetical astomatous
CM was estimated to be 0.97 ± 0.09 × 10−4 m s−1. Removal of epicuticular wax by stripping with cellulose acetate or extracting epicuticular plus cuticular wax by dipping
in CHCl3/methanol increased conductance 3.6- and 48.6-fold, respectively. Water fluxes increased with increasing temperature (range
10–39 °C) and energies of activation, calculated for the temperature range from 10 to 30 °C, were 64.8 ± 5.8 and 22.2 ± 5.0 kJ
mol−1 for flux and vapour-concentration-based conductance, respectively.
Received: 23 March 2000 / Accepted: 28 July 2000 相似文献
17.
J. B. K. Leonard J. F. Norieka B. Kynard S. D. McCormick 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1999,169(4-5):287-295
To assess the energetics of migration in an anadromous fish, adult American shad (Alosa sapidissima) were swum in a large respirometer at a range of speeds (1.0–2.3 body lengths (BL) s−1, 13–24 °C). Metabolic rate (MO2) was logarithmically related to swimming speed (Bl s−1; r
2 = 0.41, slope = 0.23 ± 0.037) and tailbeat frequency (beats × min−1; r
2 = 0.52, slope = 0.003 ± 0.0003). Temperature had a significant effect on metabolic rate (r
2 = 0.41) with a Q10 of 2.2. Standard metabolic rate (SMR), determined directly after immobilization with the neuroblocker gallamine triethiodide,
ranged from 2.2–6.2 mmolO2 kg−1 h−1 and scaled with mass (W) such that SMR = 4.0 (±0.03)W0.695(±0.15). Comparison of directly determined and extrapolated SMR suggests that swimming respirometry provides a good estimate of SMR
in this species, given the differences in basal activity monitored by the two methods. Overall, American shad metabolic rates
(MO2 and SMR) were intermediate between salmonids and fast-swimming perciforms, including tunas, and may be a result of evolutionary
adaptation to their active pelagic, schooling life history. This study demonstrates variability in metabolic strategy among
anadromous fishes that may be important to understanding the relative success of different migratory species under varying
environmental conditions.
Accepted: 3 March 1999 相似文献
18.
G. J. Grobben W. H. M. van Casteren H. A. Schols A. Oosterveld G. Sala M. R. Smith J. Sikkema J. A. M. de Bont 《Applied microbiology and biotechnology》1997,48(4):516-521
The exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus NCFB 2772 grown in defined medium were investigated. At equal cell densities, the strain produced 95 mg l−1 exopolysaccharides with glucose and 30 mg l−1 with fructose as the carbohydrate source. High-performance size-exclusion chromatography of the exopolysaccharides produced
on glucose showed the presence of two fractions with relative molecular masses (M
r) of 1.7 × 106 and 4 × 104 in almost equal amounts. The exopolysaccharides produced on fructose contained mainly a fraction of low M
r of 4 × 104. The high-M
r fraction of the purified exopolysaccharides produced on glucose appeared to have a sugar composition of galactose, glucose
and rhamnose in the molar ratio of 5:1:1, whereas the low-M
r weight fraction contained galactose, glucose and rhamnose in the molar ratio of approximately 11:1:0.4. The purified exopolysaccharide
fractions produced on fructose showed comparable ratios. The high-molecular-mass fractions contained terminally linked galactose,
1,2,3-linked galactose, 1,3,4-linked galactose, 1,3-linked glucose and terminally linked rhamnose. The low-molecular-mass
fractions contained mainly 1,3-linked galactose and 1,6-linked galactose and lower amounts of other sugar linkages. The production
of the high-M
r fractions appeared to be dependent on the carbohydrate source, whereas the low-M
r fractions were produced more continuously.
Received: 30 April 1997 / Received revision: 11 June 1997 / Accepted: 14 June 1997 相似文献
19.
Nicola D’Amelio Luca Fontanive Fulvio Uggeri Furio Suggi-Liverani Luciano Navarini 《Food biophysics》2009,4(4):321-330
Caffeine complexation by chlorogenic acid (3-caffeoylquinic acid, CAS Number [327-97-9]) in aqueous solution as well as caffeine–chlorogenate
complex in freshly prepared coffee brews have been investigated by high-resolution 1H-NMR. Caffeine and chlorogenic acid self-associations have also been studied and self-association constants have been determined
resorting to both classical isodesmic model and a recently introduced method of data analysis able to provide also the critical
aggregation concentration (cac). Furthermore, caffeine–chlorogenate association constant was measured. For the caffeine, the
average value of the self-association constant determined by isodesmic model (K
i = 7.6 ± 0.5 M−1) is in good agreement with the average value (K
a = 10 ± 1.8 M−1) determined with the method which permits the determination of the cac (8.43 ± 0.05 mM). Chlorogenic acid shows a slight
decreased tendency to aggregation with a lower average value of association constants (K
i = 2.8 ± 0.6 M−1; K
a = 3.4 ± 0.6 M−1) and a critical concentration equal to 24 ± 1 mM. The value of the association constant of the caffeine–chlorogenate complex
(30 ± 4 M−1) is compatible with previous studies and within the typical range of reported association constants for other caffeine–polyphenol
complexes. Structural features of the complex have also been investigated, and the complex conformation has been rediscussed.
Caffeine chemical shifts comparison (monomeric, complexed, coffee brews) clearly indicates a significant amount of caffeine
is complexed in beverage real system, being chlorogenate ions the main complexing agents. 相似文献
20.
This is the first report describing the gene structure and the enzymatic properties of a β-fructosidase of a hyperthermophilic
organism. The bfrA gene of the ancestral bacterium Thermotoga maritima MSB8 codes for a 432-residue, polypeptide of about 50 kDa, with significant sequence similarity to other β-fructosidases.
On the basis of its primary structure, BfrA can be assigned to glycosyl hydrolase family 32. The bfrA gene was expressed in Escherichia coli and the recombinant enzyme was purified and characterised. BfrA was specific for the fructose moiety and the β-anomeric configuration
of the glycosidic linkages of its substrates. The enzyme released fructose from sucrose and raffinose, and the fructose polymer
inulin was hydrolysed quantitatively in an exo-type fashion. BfrA displayed similar catalytic efficiencies for the hydrolysis
of sucrose and inulin with k
cat/K
m values (at 75 °C, pH 5.5) of about 4.1 × 104 M−1s−1 and 3.1 × 104 M−1s−1 respectively. BfrA had an optimum temperature of 90–95 °C (10-min assay) and was extremely insensitive to thermo-inactivation.
During 5 h at temperatures up to 80 °C at pH 7, the enzyme retained at least 85% of its initial activity. Thus, BfrA is the
most thermostable β-fructosidase and also the most thermostable inulinase described to date. In conclusion, the T. maritima enzyme can be classified as an exo-β-d-fructofuranosidase (EC 3.2.1.26) with invertase and inulinase activity. Its catalytic properties along with the extreme thermostability
recommend it for use in biotechnology.
Received: 28 August 1997 / Received revision: 19 January 1998 / Accepted: 24 January 1998 相似文献