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1.
Diffusional and electrostatic effects on the apparent maximum reaction rate Vmapp and the apparent Michaelis constant Kmapp were investigated theoretically for a system in which an enzyme immobilized on the external surface of a solid support catalyzes a reaction according to Michaelis-Menten kinetics. In such a system, the dependence of Vmapp and Kmapp on the substrate concentration can be expressed analytically. When the support and substrate carry charges of the same sign, resulting in a repulsive force between them, both Vmapp and Kmapp decrease with increasing substrate concentration, but they never decrease below the respective intrinsic values. On the other hand, when the support and substrate carry charges of opposite sign and therefore an attractive force occurs, Vmapp decreases towards its intrinsic value, while Kmapp decreases to values below its intrinsic value in the region of high substrate concentration.  相似文献   

2.
Epoxide hydrolase (EC 3.3.2.3) purified from rat liver microsomes has been immobilized by covalent linking to dextran activated by imidazolyl carbamate groups, under mild conditions. Kappm values of free and dextran bound epoxide hydrolase toward benzo(a)pyrene-4,5-oxide were 0.5 and 0.35 μM respectively, while Vappmax was lowered from 300 to 120 nmol min?1mg?1protein. The activity lost upon coupling could not be restored by digestion of the support by dextranase (1,6-α-d-glucan 6-glucanohydrolase, EC 3.2.1.11) treatment. This fact, along with the similarity of the activation energy values for both native and bound epoxide hydrolase, indicated that steric hindrance effects due to the polymer support played only a minor role in this loss of activity. Evidences of changes in the conformation of epoxide hydrolase were obtained by a comparative study of u.v. circular dichroism and tryptophan fluorescence emission spectra of the native and dextran bound enzymes. On the other hand, the enzyme conjugate showed greater resistance than the free enzyme to thermal inactivation.  相似文献   

3.
A yeast growing at 48°C was isolated from soil and the strain was identified as Cryptococcus lactativorus. The aldose reductase which the strain produced was purified 114-fold with an overall recovery of 36%. The stability of the enzyme was higher than that of other aldose reductases. The half life of the enzyme was 800 h and 14 h at 30°C and 50°C, respectively. The enzyme showed the best activity with d-xylose. l-Sorbose and d-fructose were also reduced by the enzyme. The enzyme was active with both NADPH and NADH as a conenzyme, and the activity with NADH was 1.25 times higher than that with NADPH. The Kmapp value for d-xylose was 8.6 mM and the Vmaxapp was 20.8 units/mg NADH was used as a coenzyme. The Kmapp values for NADPH and NADH were 6μM and 170 μM, respectively, when d-glucose was used as a substrate.  相似文献   

4.
Enzyme engineering via immobilization techniques is perfectly compatible against the other chemical or biological approximate to improve enzyme functions and stability. In this study lactoperoxidase was immobilized onto polyaniline polymer activated with glutaraldehyde as a bifunctional agent, to improve enzyme properties. Polyaniline polymer was used due its unique physical and chemical properties to immobilize lactoperoxidase (LPO). The optimum activity of immobilized LPO was observed at pH 6 and 55?°C, which has been increased about 10?°C for the immobilized enzyme. The immobilized enzyme maintained absolutely active for 60?days whereas the native enzyme lost 80?% of its initial activity within this period of time. Moreover, the immobilized enzyme can be reused for several times without loss of activity. The kinetic parameter studies showed slight differences between free and immobilized enzymes. The Km and Km.app were calculated to be 0.6 and 0.4; also Vmax and Vmax.app were 1.3 and 0.9 respectively.  相似文献   

5.
Pectinesterase isolated from Malatya apricot pulp was covalently immobilized onto glutaraldehyde-containing amino group functionalized porous glass beads surface by chemical immobilization at pH 8.0. The amount of covalently bound apricot PE was found 1.721 mg/g glass support. The properties of immobilized enzyme were investigated and compared to those of free enzyme. The effect of various parameters such as pH, temperature, activation energy, heat and storage stability on immobilized enzyme were investigated. Optimum pH and temperature were determined to be 8.0 and 50 °C, respectively. The immobilized PE exhibited better thermostability than the free one. Kinetic parameters of the immobilized enzyme (Km and Vmax values) were also evaluated. The Km was 0.71 mM and the Vmax was 0.64 μmol min?1 mg?1. No drastic change was observed in the Km and Vmax values. The patterns of heat stability indicated that the immobilization process tends to stabilize the enzyme. Thermal and storage stability experiments were also carried out. It was observed that the immobilized enzyme had longer storage stability and retained 50% of its initial activity during 30 days.  相似文献   

6.
Acid phosphatase (EC 3.1.3.2) from rye germs is a glycoprotein of M, 90000 with subunit structure. The pH optimum for pNPP hydrolysis is 5.4. The best substrates for the enzyme are pNPP, PPi and ATP. In the presence of plant lectins an increase in AcPase activity was found. ConA causes a 20% decrease of Kmapp and a 50% increase of Vmaxapp with pNPP as substrate.  相似文献   

7.
Forskolin action was studied using uterine smooth muscle adenylate cyclase, an enzyme form that is slowly and irreversibly activated by treatment with nonhydrolyzable GTP analogs. Activation of the particulate smooth muscle enzyme by prolonged treatment with Gpp[NH]p (guanyl-5′-yl imidodiphosphate) at 24 °C followed simple Michaelis-Menten kinetics with respect to the guanine nucleotide. Under these treatment conditions, forskolin increased both the Vmax and the Km for Gpp[NH]p, suggesting diterpene action affected the guanine nucleotide-binding coupling factor. Sensitivity of a detergent-solubilized form of the enzyme to stimulation by both Gpp[NH]p and forskolin was much more labile at 4 °C than was the Mn+2 sensitivity of the catalytic subunit. In the particulate form, the catalytic subunit was more resistant to the denaturing effects of N-ethylmaleimide than was its sensitivity to stimulation by Gpp[NH]p or forskolin. Forskolin stimulation of the particulate form of the enzyme followed simple Michaelis-Menten kinetics with respect to the concentration of the diterpene. Denaturation of the enzyme by treatment with N-ethylmaleimide lowered the Vmax and increased the Km for forskolin, further suggesting that forskolin had an indirect effect on the activity of the catalytic subunit. These results could be accounted for if the diterpene, like Gpp[NH]p, was bound by the coupling factor.  相似文献   

8.
Summary A quantitative histochemical technique was developed for determining the kinetics of the calcium-activated myosin ATPase (Ca2+-myosin ATPase) reaction in rat skeletal muscle fibres. Using this technique, the maximum velocity (Vmax) and the apparent Michaelis-Menten rate constant for ATP (Kapp) of the Ca2+-myosin ATPase reaction were measured in type-identified fibres of the rat medial gastrocnemius (MG) muscle. The Vmax and the Kapp of the Ca2+-myosin ATPase reaction were lowest in type I fibres and highest (i.e., approx. two times greater) in type IIb fibres. The Kapp in type IIa fibres was similar to that in type I. However, the Vmax was 1.5 times greater in type IIa fibres, compared to type I fibres. Evidence is presented to suggest that the type IIb fibre population in the MG does not represent a single myosin isozyme. In addition, the broad range of Vmax and Kapp values indicates that there is marked heterogeneity in the myosin heavy chain and myosin light chain composition of myosin isozymes among individual fibres.  相似文献   

9.
High throughput covalent urease immobilization was performed through the amide bond formation between the urease and the amino-functional MNPs. The enzyme’s performances, including shelf-life, reusability, enzymatic kinetics, and the enzyme relative activity in organic media was improved. At optimal conditions, the immobilization efficiency was calculated about 95.0% with keeping 94.7% of the urease initial specific activity. The optimal pH for maximum activity of the free and immobilized urease was calculated as 7.0 at 37.0 °C and 8.0 at 60.0 °C, respectively. The kinetics studies showed the Km of 26.0 mM and 8.0 mM and the Vmax of 5.31 μmol mg−1 min−1 and 3.93 μmol mg−1 min−1 for the free and immobilized urease, respectively. The ratio Kcat/Km as a measure of catalytic efficiency and enzyme specificity was calculated as 0.09 mg mL−1 min−1 and 0.22 mg mL−1 min−1 for the free and immobilized urease, respectively, indicating an improvement in the enzymatic kinetics. The shelf-life and operational studies of immobilized urease indicated that approximately 97.7% and 88.5% of its initial activity was retained after 40 days and 17 operational cycles, respectively. The immobilized urease was utilized to urea removal from water samples with an efficiency between 91.5–95.0%.  相似文献   

10.
The utilization of natural mica as a biocatalyst support in kinetic investigations is first described in this study. The formation of lactose caprate from lactose sugar and capric acid, using free lipase (free-CRL) and lipase immobilized on nanoporous mica (NER-CRL) as a biocatalyst, was evaluated through a kinetic study. The apparent kinetic parameters, K m and V max, were determined by means of the Michaelis-Menten kinetic model. The Ping-Pong Bi-Bi mechanism with single substrate inhibition was adopted as it best explains the experimental findings. The kinetic results show lower K m values with NER-CRL than with free-CRL, indicating the higher affinity of NER-CRL towards both substrates at the maximum reaction velocity (V max,app>V max). The kinetic parameters deduced from this model were used to simulate reaction rate data which were in close agreement with the experimental values.  相似文献   

11.
(S)-1-Phenylethanol derivatives, which are the precursors of many pharmacological products, have also been used as anti-Alzheimer drugs. Bioreduction experiments were performed in a batch and packed-bed bioreactor. Then, the kinetics constants were determined by examining the reaction kinetics in the batch system with free and immobilized carrot cells. Also, the effective diffusion coefficient (De) of acetophenone in calcium alginate-immobilized carrot cells was investigated. Kinetics constants for free cells, which are intrinsic values, are reaction rate Vmax?=?0.052?mmol?L?1?min?1, and constants of the Michaelis–Menten KM?=?2.31?mmol?L?1. Kinetics constants for immobilized cells, which are considered apparent values, are Vmax, app?=?0.0407?mmol?L?1 min?1, KM, app?=?3.0472?mmol?L?1 for 2?mm bead diameter, and Vmax, app?=?0.0453?mmol?L?1 min?1, KM, app?=?4.9383?mmol?L?1 for 3?mm bead diameter. Average value of effective diffusion coefficient of acetophenone in immobilized beads was determined as 1.97?×?10?6?cm2?s?1. Using immobilized carrot cells in an up-flow packed-bed reactor, continuous production of (S)-1-phenylethanol through asymmetric bioreduction of acetophenone was performed. The effects of the residence time and concentrations of substrate were investigated at pH 7.6 and 33°C. Enantiomerically pure (S)-1-phenylethanol (ee?>?99%) was produced with 75% conversion at 4-hr residence time.  相似文献   

12.
Cellulase extracted from seeds of Cowpea (Vigna sinensis L var VITA-4) was partially purified and immobilized on brick dust as solid support via glutaraldehyde. The percentage retention of the enzyme activity on brick dust was nearly 85%. After immobilization specific activity of the enzyme increased from 0.275 to 0.557 U mg?1 protein with about 2 fold enrichment. The optimum pH and temperature of soluble enzyme were determined as pH 4.6 and WC, respectively whereas immobilized enzyme showed at pH 5.0 and 37°C, respectively. The Vmax values for soluble and immobilized enzyme were determined as 6.67 and 1.25 mg min?1, respectively whereas Km values were 4.35 and 4.76 mg ml?1, respectively. The immobilized enzyme displayed higher thermal stability than soluble enzyme and retained about 50% of its initial activity after 12 reuses. Immobilized enzyme was packed in an indigenously designed double walled glass bed reactor for continuous production of reducing sugars.  相似文献   

13.
Bacillus subtilis SHS0133 cephalosporin-C deacetylase (CAH) overexpressed in Escherichia coli was immobilized on an anion-exchange resin, KA-890, using glutaraldehyde. The activity yield of immobilized enzyme was approximately 55% of the free enzyme. The pH range for stability of the immobilized enzyme (pH 5–10) was broader than that for free enzyme. The Kmapp value of immobilized enzyme for 7-aminocephalosporanic acid (7-ACA) was similar to that of the free enzyme. This immobilized enzyme obeyed Michaelis–Menten kinetics similar to those of the free enzyme. A batch-type reactor with a water jacket was employed for deacetylation of 7-ACA using CAH immobilized on KA-890. Ten kilograms of 7-ACA were completely converted to deacetyl 7-ACA at pH 8.0 within 90 min. The reaction kinetics agreed well with a computer simulation model. Moreover, the immobilized enzyme exhibited only a slight loss of the initial activity even after repeated use (52 times ) over a period of 70 days. This reaction will thus be useful for the production of cephalosporin-type antibiotics.  相似文献   

14.
The stability and, consequently, the lifetime of immobilized enzymes (IME) are important factors in practical applications of IME, especially so far as design and operation of the enzyme reactors are concerned. In this paper a model is presented which describes the effect of intraparticle diffusion on time stability behaviour of IME, and which has been verified experimentally by the two-substrate enzymic reaction. As a model reaction the ethanol oxidation catalysed by immobilized yeast alcohol dehydrogenase was chosen. The reaction was performed in the batch-recycle reactor at 303 K and pH-value 8.9, under the conditions of high ethanol concentration and low coenzyme (NAD+) concentration, so that NAD+ was the limiting substrate. The values of the apparent and intrinsic deactivation constant as well as the apparent relative lifetime of the enzyme were calculated.The results show that the diffusional resistance influences the time stability of the IME catalyst and that IME appears to be more stabilized under the larger diffusion resistance.List of Symbols C A, CB, CE mol · m–3 concentration of coenzyme NAD+, ethanol and enzyme, respectively - C p mol · m3 concentration of reaction product NADH - d p mm particle diameter - D eff m2 · s–1 effective volume diffusivity of NAD+ within porous matrix - k d s–1 intrinsic deactivation constant - K A, KA, KB mol · m–3 kinetic constant defined by Eq. (1) - K A x mol · m–3 kinetic constant defined by Eq. (5) - r A mol · m–3 · s–1 intrinsic reaction rate - R m particle radius - R v mol · m–3 · s–1 observed reaction rate per unit volume of immobilized enzyme - t E s enzyme deactivation time - t r s reaction time - V mol · m–3 · s–1 maximum reaction rate in Eq. (1) - V x mol · m–3 · s–1 parameter defined by Eq. (4) - V f m3 total volume of fluid in reactor - w s kg mass of immobilized enzyme bed - factor defined by Eqs. (19) and (20) - kg · m–3 density of immobilized enzyme bed - unstableness factor - effectiveness factor - Thiele modulus - relative half-lifetime of immobilized enzyme Index o values obtained with fresh immobilized enzyme  相似文献   

15.
Ca-polygalacturonate is a demethoxylated component of pectins which are constitutive of plant root mucigel. In order to define the role of root mucigel in myrosinase immobilization and activity at root level, a myrosinase enzyme which had been isolated from Sinapis alba seeds was immobilized into Ca-polygalacturonate. The activity profile for the immobilized and free enzyme was evaluated using the pH-Stat method as a function of time, temperature, and pH. The Michaelis-Menten kinetic parameters change between the immobilized (V max ?=?127?±?13 U mg?1 protein; K M ?=?6.28?±?0.09?mM) and free (V max ?=?17?±?1 U mg?1 protein; K M ?=?0.96?±?0.01?mM) forms of myrosinase, probably due to conformational changes involving the active site as a consequence of enzyme immobilization. Immobilized enzyme activity evaluated as a function of different substrates gave the highest value with nasturtin, the glucosinolate that is typical of several brassicaceae plant roots containing the glucosinolate-myrosinase defensive system. No feedback regulation mechanism was found in the presence of an excess of enzymatic reaction products (i.e. allyl isothiocyanate or sulphate). The high enzyme immobilization yield into Ca-polygalacturonate and its activity preservation under different conditions suggest that the enzyme released by plants at root level could be entrapped in root mucigel in order to preserve its activity.  相似文献   

16.
Pyridoxine kinase purified from sheep liver was found to consist of a single polypeptide chain with a molecular weight of 60,000 as determined by gel filtration, sedimentation equilibrium ultracentrifugation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The isoelectric pH of the enzyme was 5.1, and the pH optimum was between 5.5 and 6.0. The enzyme required divalent cations for activity. At cation concentrations of 80 μm, the enzyme activity with each cation was in the order of Zn2+ > Mn2+ > Mg2+. At cation concentrations of 400 μm, the enzyme activity with each cation was in the order of Mn2+ > Zn2+ > Mg2+. Excess free divalent cation inhibited the enzyme. Pyridoxine kinase also required monovalent cations. The enzyme activation was greatest with K+, then Rb+ and NH4+, whereas the enzyme had very little activity with Na+, Li+, or Cs+. Na+ did not interfere with the activation by K+. The activation of the kinase by K+, NH4+, and Rb+ followed Michaelis-Menten kinetics, and the apparent Km values for the cations were 8.9, 3.7, and 5.3 mm, respectively. Increasing the potassium concentration lowered the apparent Km value of the enzyme for pyridoxine and had little or no effect on the Km for ZnATP2? or the V of the kinase-catalyzed reaction.  相似文献   

17.
The allosteric properties of platelet actomyosin and myosin have been further studied. At pH 7.2, both exhibit sigmoid kinetics with at least two interacting ATP binding sites. At pH 8.9, the velocity versus substrate curve is shifted to the right and becomes more sigmoidal. In contrast, at pH 5.5, the enzyme appears to follow hyperbolic kinetics and the Km is reduced. In the presence of 1.4 m urea, the sigmoidicity is lost and the enzyme obeys Michaelis-Menten kinetics. The effect of ADP on the ATPase activity was also investigated. ADP shows characteristics of a competitive inhibitor; it increases Km (shifts sigmoid curve to the right) without affecting V. When the enzyme is desensitized by low pH (5.5) or urea (1.4 m), the allosteric interaction is abolished without impairing the catalytic activity and ADP is no longer inhibitory. These findings suggest that platelet myosin possesses two interacting sites and that ADP binds to the allosteric site which appears to be different from the catalytic site.  相似文献   

18.
A procedure is described for purification of NAD malic enzyme (EC 1.1.1.39) to near homogeneity from potato tuber mitochondria. The purified enzyme is active with either NAD or NADP, and functions with either Mg2+ or Mn2+. Vapp is greatest when the enzyme is assayed with Mg2+ and NAD. When Mn2+ replaces Mg2+ the Vapp of the NAD-linked reaction decreases but the Km values for all substrates drop substantially. When NADP is used in place of NAD, the Vapp of the Mg2+-linked reaction decreases and the Km values for most substrates increase. The pH optimum of the enzyme depends on the metal ion and cofactor used and varies between 6.4 and 6.8. At pH 6.8, with saturating levels of Mg2+ and NAD, the turnover number of the enzyme is 37,000 min?1. The shape of the pH profile indicates the involvement of two to three protons in the activation of the enzyme, whereas only one proton is involved in the inactivation process. The molecular weight of the enzyme in the presence of 5 mm dithiothreitol and 2 mm MgCl2 is 490,000 as determined by gel filtration. A lower molecular weight form of the enzyme predominates in gel filtration at lower levels of dithiothreitol and in native gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis of the enzyme reveals two main bands with molecular weights of 61,000 and 58,000, suggesting that the subunit stoichiometry of the high-molecular-weight form may be α4β4. However, given the possibility that the smaller subunit may be a proteolytic artifact, the enzyme may prove to be an octamer of identical subunits.  相似文献   

19.
Michaelis-Menten kinetic parameters for H2 consumption by three methanogenic habitats were determined from progress curve and initial velocity experiments. The influences of mass transfer resistance, endogenous H2 production, and growth on apparent parameter estimates were also investigated. Kinetic parameters could not be determined for undiluted rumen fluid and some digestor sludge from gas-phase measurements of H2, since mass transfer of H2 across the gas-liquid interface was rate limiting. However, accurate values were obtained once the samples were diluted. H2 consumption by digestor sludge with a long retention time and by hypereutrophic lake sediment was not phase transfer limited. The Km values for H2 uptake by these habitats were similar, with means of 5.8, 6.0, and 7.1 μM for rumen fluid, digestor sludge, and sediment, respectively. Vmax estimates suggested a ratio of activity of approximately 100 (rumen fluid):10 (sludge):1 (sediment); their ranges were as follows: rumen fluid, 14 to 28 mM h−1; Holt sludge, 0.7 to 4.3 mM h−1; and Wintergreen sediment, 0.13 to 0.49 mM h−1. The principles of phase transfer limitation, studied here for H2, are the same for all gaseous substrates and products. The limitations and errors associated with gas phase determination of kinetic parameters were evaluated with a mathematical model that combined mass transport and Michaelis-Menten kinetics. Three criteria are described which can be used to evaluate the possibility that a phase transfer limitation exists. If it does not exist, (i) substrate consumption curves are Michaelis-Menten and not first order, (ii) the Km is independent of initial substrate concentration, and (iii) the Km is independent of biomass (Vmax) and remains constant with dilution of sample. Errors in the Michaelis-Menten kinetic parameters are caused by endogenously produced H2, but they were <15% for rumen fluid and 10% for lake sediment and digestor sludge. Increases in Vmax during the course of progress curve experiments were not great enough to produce systematic deviations from Michaelis-Menten kinetics.  相似文献   

20.
Bacillus subtilis α-amylase (EC 3.2.1.1) has been immobilized on zirconia-coated alkylamine glass by using the process of glutaraldehyde coupling. The immobilized enzyme preparation exhibited 52% of the initial enzyme activity and a conjugation yield of 28 mg/g support. The Km value of the immobilized α-amylase was decreased by immobilization while Vmax was unaltered. Ea of the enzyme was decreased upon conjugation. The soluble enzyme was optimally active at pH 5.6 while the immobilized enzyme exhibited optimal activity in the pH range 5.4–6.2. The alkylamine-immobilized enzyme has also been characterized through its isoelectric point. The industrial importance of this work is discussed.  相似文献   

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