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1.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(6):1217-1220
Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10?h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent KM values of 0.60?mM and 0.65?mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12?h with a half-life of 5.80?h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads. 相似文献
2.
Summary An extracellular -amylase has been isolated from a continuous culture of a thermophilic strain of Bacillus brevis. This enzyme was purified eightfold and obtained in electrophoretically homogenous form. The enzyme had a molecular weight of about 58000, a pH optimum from 5.0 to 9.0 and a temperature optimum at 80°C. The half-life of the purified enzyme in the presence of 5 mM CaCl2 at 90° C and pH 8.0 was 20 min. The K
m
value for soluble starch was calculated to be 0.8 mg/ml. 相似文献
3.
German A. B. Neklyudov A. D. Ivankin A. N. Berdutina A. V. 《Applied Biochemistry and Microbiology》2002,38(6):517-520
Hydrolysis of lipids from beef fat by pancreatic lipase was studied. The maximum release of free fatty acids was shown to occur at 40°C for the first 3 h of the experiment. After this, transetherification was predominant. The main kinetic parameters were the following: maximum hydrolysis rate, V = 1.25 ± 0.1 mg fat/ml min; Michaelis constant, K
M
H = 100 ± 12 mg fat/ml; constant of substrate inhibition, K
S = 10.0 ± 0.8 mg fat/ml; equilibrium constant, K
P = 277 ± 170 mg fat/ml; and activation energy of beef fat hydrolysis by pancreatic lipase, E
a = 19.1 ± 1.1 kJ/mole. The kinetic method used could be applied to development of the method for biotransformation of poorly assimilated fats into more valuable products. 相似文献
4.
An immobilized d-hydantoinase was characterized and employed to produce n-carbamoyl-d-p-hydroxyphenylglycine (CpHPG) in a repeated batch process. The Vmax and Km of the immobilized d-hydantoinase at 50°C were 6.28 mm min−1 g−1 biocatalyst and 71.6 mm, respectively. The product CpHPG did not inhibit the activity of d-hydantoinase. Optimal reaction temperature was 60°C. A decrease in activity of immobilized d-hydantoinase due to thermal inactivation could be described as first-order decay; the deactivation energy was 23.97Kcal mol−1. Under process conditions (50°C, 10% w/v substrate, and pH 8.5), the half-life of the immobilized d-hydantoinase was eight batches. The attrition of immobilized d-hydantoinase particles with a large amount of insoluble substrate particles during stirring resulted in fine biocatalyst particles. In addition to the thermal inactivation, the loss of fine biocatalyst particles during the recovery step contributed to the low operational stability. 相似文献
5.
The racemic resolution of l-valine and l-serine by fungal aminoacylase has been evaluated by comparing the performance of various reactor configurations including an anion exchange nylon tangential flow membrane reactor, a tubular reactor with aminoacylase adsorbed onto DEAE-Sephadex as support and a continuous stirred tank reactor with enzyme recycling using a flat ultrafiltration module (CSTR/UF). Among the substrates tested, the N-chloroacetyl-d,l-amino acids were the preferred substrates, showing the highest catalytic efficiency (Vm/Km).Optimum reactor operational conditions obtained in discontinuous assays were selected to study the behaviour of the reactors in a continuous mode. DEAE-Sephadex loaded six-fold more enzyme than anion exchange nylon (60 and 10 gE/litre, respectively, related to reactor volume), whereas enzyme concentration within the CSTR/UF reactor was limited only by enzyme solubility.The tangential flow membrane reactor configuration with a 10 g/litre enzyme concentration produced higher productivity values (0·35 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t
= 161 days) than the CSTR/UF reactor (0·24 kg l-valine/litre per day, and 80% conversion degree) performing with the same enzyme concentration. The tubular reactor with the enzyme adsorbed onto DEAE-Sephadex (60 g/litre enzyme load) showed higher productivity values (1·9 kg l-valine/litre per day, and 80% conversion degree) and operational stability (t
= 70 days) than the CSTR/UF reactor (1·05 kg l-valine/litre per day, and 80% conversion degree). However, the CSTR/UF reactor was the preferred configuration, as it had the highest enzyme load and productivity (1·95 kg l-valine/litre per day of reactor volume, and 80% conversion degree), a half-life of 55 days at 50°C, and the possibility of easy continuous enzyme addition. 相似文献
6.
A thermodynamic study of the inclusion process between 2-chlorobenzophenone (2ClBP) and cyclomaltoheptaose (β-cyclodextrin, β-CD) was performed using UV–vis spectroscopy, reversed-phase liquid chromatography (RP-HPLC), and molecular modeling (PM6). Spectrophotometric measurements in aqueous solutions were performed at different temperatures. The stoichiometry of the complex is 1:1 and its apparent formation constant (Kc) is 3846 M−1 at 30 °C. Temperature dependence of Kc values revealed that both enthalpy (ΔH° = −10.58 kJ/mol) and entropy changes (ΔS° = 33.76 J/K mol) are favorable for the inclusion process in an aqueous medium. Encapsulation was also investigated using RP-HPLC (C18 column) with different mobile-phase compositions, to which β-CD was added. The apparent formation constants in MeOH–H2O (KF) were dependent of the proportion of the mobile phase employed (50:50, 55:45, 60:40 and 65:35, v/v). The KF values were 419 M−1 (50% MeOH) and 166 M−1 (65% MeOH) at 30 °C. The thermodynamic parameters of the complex in an aqueous MeOH medium indicated that this process is largely driven by enthalpy change (ΔH° = −27.25 kJ/mol and ΔS° = −45.12 J/K mol). The results of the study carried out with the PM6 semiempirical method showed that the energetically most favorable structure for the formation of the complex is the ‘head up’ orientation. 相似文献
7.
A flat-membrane dialyzer was used as enzyme reactor by introducing enzyme solution into one of the membrane-separated chambers. The apparent Michaelis constant Km(app) of urease was always larger (ten times at [urease] = 1 mg/ml) than that of free enzyme because the permeation of substrate through the membrane was rate determining. Km(app) for urease decreased from 125 to 20mM with increasing flow rate of the substrate solution because of the turbulent flow near the membrane. In the case of glucose oxidase or creatine kinase, the reaction rate was limited by the permeation of less permeable substrates such as oxygen or ATP. Therefore, Km(app) of more permeable substrates such as glucose or creatine became smaller than that of free enzyme. The reaction amount calculated from the permeation data agreed well with experimental results. By designing spacers for the reactor to give turbulence to the solution, the effectiveness of the reactor was improved fivefold. 相似文献
8.
A hyper-thermostable, alkaline lipase from Pseudomonas sp. with the property of thermal activation 总被引:1,自引:0,他引:1
A hyper-thermostable, alkaline lipase from a newly-isolated, mesophilic Pseudomonas sp. was optimal at pH 11 and at 90 °C. It had a half-life of more than 13 h at 90 °C. It was activated by 30% when heated at 90 °C for 2 h. The enzyme had a greater affinity for mustard oil (K
m=40 mg ml–1) than for olive oil (K
m=140 mg ml–1). 相似文献
9.
Eremin A. N. Metelitsa D. I. Moroz I. V. Pavlovskaya Zh. I. Mikhailova R. V. 《Applied Biochemistry and Microbiology》2002,38(4):322-327
A comparative kinetic study of extracellular catalases produced by Penicillium piceum F-648 and their variants adapted to H2O2 was performed in culture liquid filtrates. The specific activity of catalase, the maximum rate of catalase-induced H2O2 degradation (V
max), V
max/K
M ratio, and the catalase inactivation rate constant in the enzymatic reaction (k
in, s–1) were estimated in phosphate buffer (pH 7.4) at 30°C. The effective constant representing the rate of catalase thermal inactivation (k
in
*, s–1) was determined at 45°C. In all samples, the specific activity and K
M for catalase were maximum at a protein concentration in culture liquid filtrates of (2.5–3.5) × 10–4 mg/ml. The effective constants describing the rate of H2O2 degradation (k, s–1) were similar to that observed in the initial culture. These values reflected a twofold decrease in catalase activity in culture liquid filtrates. We hypothesized that culture liquid filtrates contain two isoforms of extracellular catalase characterized by different activities and affinities for H2O2. Catalases from variants 5 and 3 with high and low affinities for H2O2, respectively, had a greater operational stability than the enzyme from the initial culture. The method of adaptive selection for H2O2 can be used to obtain fungal variants producing extracellular catalases with improved properties. 相似文献
10.
A maltose-limited chemostat culture was used to investigate the expression and excretion of amylopullulanase by Thermoanaerobacter ethanolicus 39E (formerly Clostridium thermohydrosulfuricum 39E). In maltose-limited continuous culture, amylopullulanase was produced and secreted at tenfold higher levels than in batch culture. The extracellular amylopullulanase was purified to homonogeneity by using an inhibitor-linked affinity column matrix. The purified amylopullulanase had a specific activity of 480 units (U)/mg protein for pullulanase and 175 U/mg protein for -amylase. -Cyclodextrin inhibited both -amylase and pullulanase activities, with a substrate inhibition constant (K
i) of 0.065 mg/ml.Amylopullulanase had a relative molecular mass (Mr) of 140 000 using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and an Mr of 133 000 using gel-filtration chromatography. The N-terminal sequence of the enzyme was Glu-Thr-Asp-Thr-Ala-Pro-Ala. The purified enzyme displayed Michaelis constant (K
m) values of 0.35 mg/ml for pullulan and 1.00 mg/ml for amylose. The enzyme had an isoelectric point (pI) of 4.0, and displayed an optimum pH for stability and activity of 6.2 and 5.5, respectively. The enzyme was stable up to 85° C in the presence of Ca2+, and had a half-life of 40 min at 90° C (pH 6.2). Ca2+ was required for thermal stability, but not for activity. Amylose, glycogen, and amylopectin were degrade to maltose, maltotriose, and maltotetraose, whereas only maltotriose was formed from pullulan.
Correspondence to: J. G. Zeikus 相似文献
11.
Mona A. Esawy Yannick Combet-Blanc 《World journal of microbiology & biotechnology》2006,22(3):197-200
Milk-clotting enzyme from Bacillus licheniformis 5A1 was immobilized on Amberlite IR-120 by ionic binding. Almost all the enzyme activity was retained on the support. The
immobilized milk-clotting enzyme was repeatedly used to produce cheese in a batch reactor. The production of cheese was repeated
5 times with no loss of activity. The specific activity calculated on a bound-protein basis was slightly higher than that
of free enzyme. The free and immobilized enzyme were highly tolerant to repeated freezing and thawing. The optimum temperature
for milk-clotting activity was 70 °C with the free enzyme whereas, it was ranged from 70 to 80 °C with the immobilized milk-clotting
enzyme. The activation energy (E
A) of the immobilized milk-clotting enzyme was lower than the free enzyme (E
A = 1.59 and 1.99 Kcal mol−1 respectively). The immobilized milk-clotting enzyme exhibited great thermal stability. The milk-clotting optimum pH was 7.0
for both free and immobilized enzyme. The Michaelis constant K
m of the immobilized milk-clotting enzyme was slightly lower than the free enzyme. 相似文献
12.
Thanaporn Laothanachareon Verawat Champreda Pornpimol Sritongkham Mithran Somasundrum Werasak Surareungchai 《World journal of microbiology & biotechnology》2008,24(12):3049-3055
Cross-linked enzyme crystals of organophosphate hydrolase (CLEC-OPH) prepared from crude recombinant E. coli cell lysate was used for the development of an electrochemical biosensor for the detection of organophosphate pesticides.
CLEC-OPH showed an increased V
max of 0.721 U mg protein−1 and a slightly lower K
m of 0.083 mM on paraoxon compared to the crude enzyme, resulting in an improved catalytic efficiency (k
cat/K
m = 4.17 × 105 M−1 min−1) with a remarkable increase on thermostability. An amperometric biosensor was constructed based on glutaraldehyde and albumin
cross-linkage of CLEC-OPH with carbon nanotubes. The sensor exhibited greater sensitivity and operational stability with a
lower limit of detection when compared with a sensor using an equivalent loading of crude OPH in a non-crystal form. The application
of crude enzyme-based CLEC would offer a simple and economical approach for the fabrication of efficient electrochemical biosensors. 相似文献
13.
Resistance of Rosa microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin 总被引:1,自引:0,他引:1
The inhibition of the polymerization of tubulin from cultured cells of rose (Rosa. sp. cv. Paul's scarlet) by colchicine and the binding of colchicine to tubulin were examined in vitro and compared with data obtained in parallel experiments with bovine brain tubulin. Turbidimetric measurements of taxol-induced polymerization of rose microtubules were found to be sensitive and semiquantitative at low tubulin concentrations, and to conform to some of the characteristics of a nucleation and condensation-polymerization mechanism for assembly of filamentous helical polymers. Colchicine inhibited the rapid phase of polymerization at 24°C with an apparent inhibition constant (K
i) of 1.4·10-4 M for rose tubulin and an apparent K
i=8.8·10-7 M for brain tubulin. The binding of [3H]colchicine to rose tubulin to form tubulin-colchicine complex was mildly temperature-dependent and slow, taking 2–3 h to reach equilibrium at 24°C, and was not affected by vinblastine sulfate. The binding of [3H]colchicine to rose tubulin was saturable and Scatchard analysis indicated a single class of low-affinity binding sites having an apparent affinity constant (K) of 9.7·102 M-1 and an estimated molar binding stoichiometry (r) of 0.47 at 24°C. The values for brain tubulin were K=2.46·106 M-1 and r=0.45 at 37°C. The binding of [3H]colchicine to rose tubulin was inhibited by excess unlabeled colchicine, but not by podophyllotoxin or tropolone. The data demonstrate divergence of the colchicine-binding sites on plant and animal tubulins and indicate that the relative resistance of plant microtubule polymerization to colchicine results from a low-affinity interaction of colchicine and tubulin.Abbreviations MT
microtubule
- TC
tubulin-colchicine complex 相似文献
14.
Vinod. V. Sardana Jeffrey T. Blue Joan Zugay-Murphy Mohinder K. Sardana Lawrence C. Kuo 《Protein expression and purification》1999,16(3):440
The N-terminal domain of the hepatitis C virus (HCV) polyprotein containing the NS3 protease (residues 1027 to 1206) was expressed in Escherichia coli as a soluble protein under the control of the T7 promoter. The enzyme has been purified to homogeneity with cation exchange (SP-Sepharose HR) and heparin affinity chromatography in the absence of any detergent. The purified enzyme preparation was soluble and remained stable in solution for several weeks at 4°C. The proteolytic activity of the purified enzyme was examined, also in the absence of detergents, using a peptide mimicking the NS4A/4B cleavage site of the HCV polyprotein. Hydrolysis of this substrate at the expected Cys–Ala scissile bond was catalyzed by the recombinant protease with a pseudo second-order rate constant (kcat/KM) of 205 and 196,000 M−1 s−1, respectively, in the absence and presence of a central hydrophobic region (sequence represented by residues 21 to 34) of the NS4A protein. The rate constant in the presence of NS4A peptide cofactor was two orders of magnitude greater than reported previously for the NS3 protease domain. A significantly higher activity of the NS3 protease–NS4A cofactor complex was also observed with a substrate mimicking the NS4B/5A site (kcat/KM of 5180 ± 670 M−1 s−1). Finally, the optimal formation of a complex between the NS3 protease domain and the cofactor NS4A was critical for the high proteolytic activity observed. 相似文献
15.
Summary The effect of three parameters (initial acetate concentration, temperature and pH) on the acetoclastic reaction was studied with the thermophilic methanogenic bacterium Methanosarcina sp. MSTA-1. The optimum temperature for growth ranged around 55° C, and optimum pH was 6.5–7.5, giving a minimum generation time of 12.6–13.9 h (µmax = 0.050–0.055 h–1) and a maximum value of the specific acetate consumption rate (q
infs
supps
) of 14–20 mmol/g cells per hour. Contrary to the methane yield, the growth yield was found to be dependent on culture conditions, especially on incubation temperature. Methanosarcina sp. MSTA-1 showed a low affinity for acetate substrate. Growth at 55° C and at constant pH 7 resulted in a K
m value and a threshold acetate concentration of 10.7 mM and 0.7 mM, respectively.
Offprint requests to: R. Moletta 相似文献
16.
Silgia A. Costa Tzanko Tzanov Filipa Carneiro Georg M. Gübitz Artur Cavaco-Paulo 《Biotechnology letters》2002,24(3):173-176
Catalase was immobilized on alumina carrier and crosslinked with glutaraldehyde. Storing stability, temperature and pH profiles of enzyme activity were studied in a column reactor with recirculation and in a batch stirred-tank reactor. The immobilized enzyme retained 44% of its activity at pH 11, 30 °C and 90% at 80 °C, pH 7. The half-life time of the immobilized catalase was increased to 2 h at pH 12, and 60 °C. Acceptable results were achieved when the residual water from the washing process of H2O2-bleached fabrics was treated with the immobilized enzyme and then reused for dyeing. 相似文献
17.
Rochelle Carpenter Livermore Jerffrey R. Hazel 《Journal of comparative physiology. B, Biochemical, systemic, and environmental physiology》1988,158(3):363-368
Summary Acyl-CoA: lysophosphatidylcholine acyltransferase (LPCAT) (EC 2.3.1.23) activity was assayed in liver microsomes from rainbow trout,Salmo gairdneri, acclimated to 5°C and 20°C to assess its contribution to the temperature-induced restructuring of phospholipid acyl chain composition. The synthesis of phosphatidylcholine (PC) (from lyso-PC) was threefold the synthesis of phosphatidylethanolamine (PE) (from lyso-PE) under similar assay conditions. LPCAT activity (i) displayed an absolute requirement for lysophosphatidylcholine (LPC) and was enhanced by the presence of ATP, MgCl2 and CoA (which reduced the impact of endogenous acyl-CoA hydrolase activity by regenerating the acyl-CoA substrate) in the assay medium; (ii) remained linear with time up to 30 min; and (iii) increased linearly with microsomal protein concentration up to 0.2 mg/ml for the 20°C assay and 0.4 mg/ml for the 5°C assay. There was no difference in Km or Vmax values due to the acclimation history of the fish, but there were obvious differences due to assay temperature. The apparent Km values for LPC were 58.54±7.24 M and 12.26±2.14 M when assayed at 5°C and 20°C respectively; values for oleoyl-CoA were 9.11±0.78 M and 1.23±0.25 M under the same assay conditions. Activity was 1.99±0.31 nmol min–1 mg protein–1 when assayed at 5°C, and 3.8±0.45 nmol min–1 mg protein–1 when assayed at 20°C. These findings indicate that adjustments in the activity of LPCAT play no significant role in the temperature-induced restructuring of PC molecular species composition. However, the marked temperature dependence of the Km values for LPC and oleoyl CoA suggest that patterns of fatty acid incorporation (i.e. substrate preference) may vary with assay temperature, and in this way LPCAT could contribute to the restructuring response.Abbreviations
PC
phosphatidylcholine
-
PE
phosphatidylethanolamine
-
LPCAT
acyl-CoA: lysophosphatidylcholine acyltransferase
-
LPEAT
acyl-CoA: lysophosphatidylethanolamine acyltransferase
-
LPC
1-palmitoyl,2-lysophosphatidylcholine 相似文献
18.
Immobilisation of laccase on Eupergit supports and its application for the removal of endocrine disrupting chemicals in a packed-bed reactor 总被引:1,自引:0,他引:1
Laccase from Myceliophthora thermophila was covalently immobilised on Eupergit C and Eupergit C 250L yielding specific activities of up to 17 and 80 U/g, respectively.
Due to its superior activity, Eupergit C 250L was chosen for further research. The somewhat lower catalytic efficiency (based
on the ratio between the turnover number and the Michaelis constant, kcat/KM) of the immobilised enzyme in comparison with that of the free enzyme was balanced by its increased stability and broader
operational window related to temperature and pH. The feasibility of the immobilised laccase was tested by using a packed
bed reactor (PBR) operating in consecutive cycles for the removal of Acid Green 27 dye as model substrate. High degrees of
elimination were achieved (88, 79, 69 and 57% in 4 consecutive cycles), while the levels of adsorption on the support varied
from 18 to 6%, proving that dye removal took place mainly due to the action of the enzyme. Finally, a continuous PBR with
the solid biocatalyst was applied for the treatment of a solution containing the following endocrine disrupting chemicals:
estrone (E1), 17β-estradiol (E2) and 17α-ethinylestradiol (EE2). At steady-state operation, E1 was degraded by 65% and E2
and EE2 were removed up to 80% and only limited adsorption of these compounds on the support, between 12 and 22%, was detected.
In addition, a 79% decrease in estrogenic activity was detected in the effluent of the enzymatic reactor while only 14% was
attained by inactivated laccase. 相似文献
19.
Marta E. Farías Ana M. Strasser de Saad Aída A. Pesce de Ruiz Holgado Dr. Guillermo Oliver 《Current microbiology》1991,22(4):205-211
l-Serine dehydratase fromLactobacillus fermentum was purified 100-fold. It was stabilized by the presence of 1 mM
l-cysteine in 50 mM phosphate buffer. Mr=150,000 was determined by gel filtration. The enzyme consists of four apparently identical subunits (Mr=40,000) that were observed after treatment with sodium dodecyl sulfate. The apparent Km forl-serine was 65 mM. Fe++ was required for the enzymatic activity, and the apparent Km value for this reaction was 0.55 mM. Maximum enzymatic activity was observed at 45°C and pH 8.0 in 50 mM phosphate buffer. At pH values different from the optimum, a positive cooperativity between substrate molecules was observed. The activation energy of the reaction was 11,400 and 22,800 cal × mol–1 for temperature values more than and less than 35°C respectively. The purified enzyme showed a maximum absorption between 400 and 420 nm, indicating the presence of pyridoxal-5-phosphate (PLP) as a prosthetic group. The PLP concentration was 0.027 µmoles per milligram of protein. The data suggest that there is 1 mol of PLP for each protein subunit. 相似文献