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1.
The present study explores the efficiency of Talaromyces thermophilus β-xylosidase, in the production of xylose and xylooligosaccharides. The β-xylosidase was immobilized by different methods namely ionic binding, entrapment and covalent coupling and using various carriers. Chitosan, pre-treated with glutaraldehyde, was selected as the best support material for β-xylosidase immobilization; it gave the highest immobilization and activity yields (94%, 87%, respectively) of initial activity, and also provided the highest stability, retaining 94% of its initial activity even after being recycled 25 times. Shifts in the optimal temperature and pH were observed for the immobilized β-xylosidase when compared to the free enzyme. The maximal activity obtained for the immobilized enzyme was achieved at pH 8.0 and 53 °C, whereas that for the free enzyme was obtained at pH 7.0 and 50 °C. The immobilized enzyme was more thermostable than the free β-xylosidase. We observed an increase of the Km values of the free enzyme from 2.37 to 3.42 mM at the immobilized state. Native and immobilized β-xylosidase were found to be stimulated by Ca2+, Mn2+ and Co2+ and to be inhibited by Zn2+, Cu2+, Hg2+, Fe2+, EDTA and SDS. Immobilized enzyme was found to catalyze the reverse hydrolysis reaction, forming xylooligosaccharides in the presence of a high concentration of xylose. In order to examine the synergistic action of xylanase and β-xylosidase of T. thermophilus, these two enzymes were co-immobilized on chitosan. A continuous hydrolysis of 3% Oat spelt xylan at 50 °C was performed and better hydrolysis yields and higher amount of xylose was obtained.  相似文献   

2.
The extracellular β-agarase LSL-1 produced by an agar-liquefying, soil bacterium Acinetobacter sp., AG LSL-1 was purified to homogeneity by combination of ion-exchange and size exclusion chromatography with final yield of 44%. The enzyme has a specific activity of 397 U mg−1 protein and with a molecular mass of 100 kDa. The agarase was active in the pH range of 5.0–9.0, optimally at pH 6.0 and temperature between 25 °C and 55 °C and optimal at 40 °C. The enzyme retained 63% of native activity at 50 °C suggesting it is a thermostable. The activity of the agarase was completely inhibited by metal ions, Hg2+, Ag+ and Cu2+, whereas 25–40% of native activity was retained in the presence of Zn2+, Sn2+ and SDS. Neoagarobiose was the final product of hydrolysis of both agarose and neoagarohexaose by the purified agarase LSL-1. Based on the molecular mass and final products of agarose hydrolysis, the β-agarase LSL-1 may be further grouped under group III β-agarases and may be a member of GH-50 family. This is the first report on the purification and biochemical characterization of β-agarase from an agar-liquefying Acinetobacter species.  相似文献   

3.
The relation that exist between the Pi-PPi exchange reaction and pyrophosphate hydrolysis by the membrane-bound pyrophosphatase of chromatophores ofRhodospirillum rubrum was studied. The two reactions have a markedly different requirement for pH. The optimal pH for hydrolysis was 6.5 while the Pi-PPi exchange reaction was at 7.5; the pH affects mainly theK m of Mg2+ or Pi for the enzyme; Mn2+ and Co2+ support the Pi-PPi exchange reaction partially (50%), but the reaction is slower than with Mg2+; other divalent cations like Zn2+ or Ca2+ do not support the exchange reaction. In the hydrolytic reaction, Zn2+, at low concentration, substitutes for Mg2+ as substrate, and Co2+ also substitutes in limited amount (50%). Other cations (Ca2+, Cu2+, Fe2+, etc.) do not act as substrates in complex with PPi. The Zn2+ at high concentrations inhibited the hydrolytic reaction, probably due to uncomplexed free Zn2+. In the presence of high concentration of substrate for the hydrolysis (Mg-PPi) the divalent cations are inhibitory in the following order: Zn2+>Mn2+>Ca2+Co2+>Fe2+>Cu2+>Mg2+. The data in this work suggest that H+ and divalent cations in their free form induced changes in the kinetic properties of the enzyme.  相似文献   

4.
In vitro selection of Zn2+-dependent RNA-cleaving DNAzymes with activity at 90°C has yielded a diverse spool of selected sequences. The RNA cleavage efficiency was found in all cases to be specific for Zn2+ over Pb2+, Ca2+, Cd2+, Co2+, Hg2+, and Mg2+. The Zn2+-dependent activity assay of the most active sequence showed that the DNAzyme possesses an apparent Zn2+-binding dissociation constant of 234 μM and that its activity increases with increasing temperatures from 50–90°C. A fit of the Arrhenius plot data gave Ea = 15.3 kcal mol−1. Surprisingly, the selected Zn2+-dependent DNAzymes showed only a modest (∼3-fold) activity enhancement over the background rate of cleavage of random sequences containing a single embedded ribonucleotide within an otherwise DNA oligonucleotide. The result is attributable to the ability of DNA to sustain cleavage activity at high temperature with minimal secondary structure when Zn2+ is present. Since this effect is highly specific for Zn2+, this metal ion may play a special role in molecular evolution of nucleic acids at high temperature.  相似文献   

5.
The antagonistic effect of calcium (Ca2+), zinc (Zn2+) and selenium (Se4+) at different concentrations (10−2–10−6 M) against cadmium (Cd2+) induced genotoxic effects in root cells of Hordeum vulgare were studied. The results showed that 10−3–10−5 M could induce chromosomal aberrations and micronuclei formation. But in the treatment with 10−2–10−6 M of Ca2+, Zn2+ and Se4+ together with Cd2+ (10−3–10−5 M), respectively, the frequencies of chromosomal aberrations and micronuclei effectively decreased after 48 h of treatment. The treatment with 10−4–10−6 M of Ca2+ together with 10−4–10−5 M Cd2+, 10−6 M of Zn2+ together with 10−5 M Cd2+ and 10−6 M of Se4+ together with 10−5 M Cd2+ suggested rather obvious antagonistic effects. The order of the antagonisms of Ca2+, Se4+ and Zn2+ against Cd2+ toxicity was Ca2+>Se4+>Zn2+. The degree of antagonisms of Ca2+, Se4+ and Zn2+ against Cd2+ related to their concentration ratio.  相似文献   

6.
Rate constants have been determined for hydrolysis of the acetate, glutarate, and phthalate monoesters of 2-hydroxy-1,10-phenanthroline in water at 30°C and μ = 0.1 M with KCl. The hydrolysis reactions of the esters are hydroxide ion catalyzed at pH > 9. The phthalate and glutarate monoesters have in addition pH-independent reactions from pH 5.5 to 9 that involve intramolecular participation by the neighboring carboxylate anion. The pH-independent reaction of the glutarate monoester is 5-fold faster than that of the phthalate monoester. The plots of log kobsd vs pH for hydrolysis of the carboxyl substituted esters are bell shaped at pH < 5, which indicates a rapid reaction of the zwitterionic species (carboxyl anion and protonated phenanthroline nitrogen). The divalent metal ions, Cu2+, Ni2+, Zn2+, and Co2+, complex strongly with the esters; saturation occurs at metal ion concentrations less than 0.01 M. The 1:1 metal ion complexes have greatly enhanced rates of hydrolysis; the second-order rate constants for the OH reactions are increased by factors of 105 to 108 by the metal ion. The pH-rate constant profiles for the phthalate and glutarate ester metal ion complexes have a sigmoidal region below pH 6 that can be attributed to a metal ion-promoted carboxylate anion nucleophilic reaction. The carboxyl group reactions are enhanced 102 - to 103 -fold by the metal ions, which allows the neighboring group reaction to be competitive with the favorable metal ion-promoted OH reaction at pH < 6, but not at pH > 6. The half-lives of the pH-independent neighboring carboxyl group reactions of the Cu(II) complexes at 30°C are l2 s. The other metal ion complexes are only slightly less reactive (half-lives vary from 2.5 to 40 s). These are the most rapid neighboring carboxyl group reactions that have been observed in ester hydrolysis.  相似文献   

7.
To clarify the kinetic characteristics and ionic requirements of the tonoplast H+-translocating inorganic pyrophosphatase (H+-PPiase), PPi hydrolysis and PPi-dependent H+ transport were studied in tonoplast vesicles isolated from leaf mesophyll tissue of Kalanchoë daigremontiana Hamet et Perrier de la Bâthie. The tonoplast H+-PPiase showed an absolute requirement for a monovalent cation and exhibited hyperbolic kinetics with respect to cation concentration. H+-PPiase activity was maximal in the presence of K+ (K50 approximately 3 millimolar), with PPi-dependent H+ transport being more selective for K+ than PPi hydrolysis. When assayed in the presence of 50 millimolar KCl at fixed PPi concentrations, H+-PPiase activity showed sigmoidal kinetics with respect to total Mg concentration, reflecting a requirement for a Mg/PPi complex as substrate and free Mg2+ for activation. At saturating concentrations of free Mg2+, H+-PPiase activity exhibited Michaelis-Menten kinetics towards MgPPi2− but not Mg2PPi, demonstrating that MgPPi2− was the true substrate of the enzyme. The apparent Km (MgPPi2−) for PPi hydrolysis (17 micromolar) was significantly higher than that for PPi-dependent H+ transport (7 micromolar). Free Mg2+ was shown to be an allosteric activator of the H+-PPiase, with Hill coefficients of 2.5 for PPi hydrolysis and 2.7 for PPi-dependent H+ transport. Half-maximal H+-PPiase activity occurred at a free Mg2+ concentration of 1.1 millimolar, which lies within the range of accepted values for cytosolic Mg2+. In contrast, cytosolic concentrations of K+ and MgPPi2− appear to be saturating for H+-PPiase activity. We propose that one function of the H+-PPiase may be to act as an ancillary enzyme that maintains the proton-motive force across the vacuolar membrane when the activity of the tonoplast H+-ATPase is restricted by substrate availability. As ATP levels decline in the cytosol, free Mg2+ would be released from the MgATP2− complex, thereby activating the tonoplast H+-PPiase.  相似文献   

8.
A fourth molecular from of α-galactosidase, designated LIV, an alkaline α-galactosidase, was isolated from leaves of Cucurbita pepo and purified 165-fold. It was active over a narrow pH range with optimal hydrolysis of p-nitrophenyl-α-d-galactoside and stachyose at pH 7.5. The rate of stachyose hydrolysis was 10 times that of raffinose. Km determinations in McIlvaine buffer (200 millimolar Na2-phosphate, 100 millimolar citric acid, pH 7.5) for p-nitrophenyl-α-d-galactoside, stachyose, and raffinose were 1.40, 4.5, and 36.4 millimolar, respectively. LIV was partially inhibited by Ca2+, Mg2+, and Mn2+, more so by Ni2+, Zn2+, and Co2+, and highly so by Cu2+, Ag2+, Hg2+ and by p-chloromercuribenzoate. It was not inhibited by high concentrations of the substrate p-nitrophenyl-α-d-galactoside or by myo-inositol, but α-d-galactose was a strong inhibitor. As observed for most other forms of α-galactosidase, LIV only catalyzed the hydrolysis of glycosides possessing the α-d-galactose configuration at C1, C2, and C4, and did not hydrolyze p-nitrophenyl-α-d-fucoside (α-d-galactose substituted at C6). The enzyme was highly sensitive to buffers and chelating agents. Maximum hydrolytic activity for p-nitrophenyl-α-d-galactoside was obtained in McIlvaine buffer (pH 7.5). In 10 millimolar triethanolaminehydrochloride-NaOH (pH 7.5) or 10 millimolar Hepes-NaOH (pH 7.5), hydrolytic activity was virtually eliminated, but the addition of low concentrations of either ethylenediaminetetraacetate or citrate to these buffers restored activity almost completely. Partial restoration of activity was also observed, but at higher concentrations, with pyruvate and malate. Similar effects were found for stachyose hydrolysis, but in addition some inhibition of LIV in McIlvaine buffer, possibly due to the high phosphate concentration, was observed with this substrate. It is questionable whether the organic acid anions possess any regulatory control of LIVin vivo. It was possible that the results reflected the ability of these anions, and ethylene-diaminetetraacetate, to restore LIV activity through coordination with some toxic cation introduced as a buffer contaminant.  相似文献   

9.
An aminopeptidase was isolated from the mid-gut gland of Patinopecten yessoensis. The enzyme was purified from an acetone-dried preparation by extracting, ammonium sulfate precipitation, Hi-Load Q column chromatography, isoelectric focusing, and POROS HP2 and HQ column chromatography. The molecular weight of the enzyme was estimated to be 61 kDa by SDS-polyacrylamide gel electrophoresis and 59 kDa by gel permeation chromatography. The isoelectric point of the enzyme was 5.2 and the optimum pH was 7.0 toward leucine p-nitroanilide (Leu-pNA). The enzyme was inhibited by o-phenanthroline. The activity of the enzyme treated with o-phenanthroline was completely recovered by adding excess Zn2+. Relative hydrolysis rates of amino acid-pNAs and amino acid-4-methylcoumaryl-7-amides (amino acid-MCAs) indicated that the enzyme preferred substrates having Ala or Met as an amino acid residue. The enzyme had a Km of 32.2 μM and kcat of 29.5 s−1 with Ala-pNA and a Km of 11.1 μM and kcat of 9.49 s−1 with Ala-MCA. The enzyme sequentially liberated amino acids from the amino-termini of Ala–Phe–Tyr–Glu.  相似文献   

10.
Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg2+, SDS, p-chloromercuribenzoate, Cu2+ and Mn2+ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca2+, Co2+ and Mg2+ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg2+ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.  相似文献   

11.
The rate constant for the hydrolysis of prostacyclin (PGI2) to 6-keto-PGF was measured by monitoring the UV spectral change, over a pH range 6 to 10 at 25°C and the total ionic strength of 0.5 M. The first-order rate constant (kobs) extrapolated to zero buffer concentration follows an expression, kobs = kH+ (H+), where kH+ is a second-order rate constant for the specific acid catalyzed hydrolysis. The value of kH+ obtained (3.71 × 104 sec−1 M−1) is estimated approximately 700-fold greater than a kH+ value expected from the hydrolysis of other vinyl ethers. Such an unusually high reactivity of PGI2 even for a vinyl ether is attributed to a possible ring strain release that would occur upon the rate controlling protonation of C5. A Brønsted slope (α) of 0.71 was obtained for the acid (including H3O+) catalytic constants, from which a pH independent first-order rate constant for the spontaneous hydrolysis (catalyzed by H2O as a general acid) was estimated to be 1.3 × 10−6 sec−1. An apparent activation energy (Ea) of 11.85 Kcal/mole was obtained for the hydrolysis at pH 7.48, from which a half-life of PGI2 at 4°C was estimated to be approximately 14.5 min. when the total phosphate concentration is 0.165 M (cf. 3.5 min. at 25°C).  相似文献   

12.
Tu SI  Nungesser E  Brauer D 《Plant physiology》1989,90(4):1636-1643
The substrate requirement of the H+-ATPase in purified corn root tonoplast vesicles was investigated. The coupled activities, ATP hydrolysis and proton pumping, were simultaneously supported only by Mg2+ or Mn2+. The presence of Ca2+ or Ba2+ did not significantly affect the coupled activities. The addition of Cd2+, Co2+, Cu2+, and Zn2+ inhibited both the hydrolysis of Mg-ATP and the proton transport. However, the inhibition of proton pumping was more pronounced. Based on equilibrium analysis, both ATP-complexed and free forms of these cations were inhibitory. Inhibition of the hydrolysis of Mg-ATP could be correlated to the concentrations of the ATP-complex of Zn. On the other hand, the free Cu2+ and Co2+ were effective in inhibiting hydrolysis. For proton pumping, the ATP complexes of Co2+, Cu2+, and Zn2+ were effective inhibitors. However, this inhibition could be further modulated by free Co2+, Cu2+, and Zn2+. While the equilibrium concentrations of Cd-ATP and free Cd2+ were not estimated, the total concentration of this cation needed to inhibit the coupled activities of the H+-ATPase was found to be in the range of 10 to 100 micromolars. The presence of free divalent cations also affected the structure of the lipid phase in tonoplast membrane as demonstrated by the changes of emission intensity and polarization of incorporated 1,6-diphenyl-1,3,5-hexatriene. The differential inhibition caused by these cations could be interpreted by interactions with the protogenic domain of the membrane as previously proposed in “indirect-link” mechanism.  相似文献   

13.
The acute effects of aqueous solutions of As, Cd, Cu, Pb, F, and Zn ions at concentrations from 0.01 to 100 micrograms per milliliter and solutions adjusted to pH 2 to 6 with nitric or sulfuric acid were studied with respect to acetylene reduction, net photosynthesis, respiration rate, and chlorophyll content in Vernal alfalfa (Medicago sativa L. cv. Vernal). The effects of the various treatments on acetylene reduction varied from no demonstrable effect by any concentration of F and 42% inhibition by 100 micrograms Pb2+ per milliliter, to 100% inhibition by 10 micrograms Cd2+ per milliliter and 100 micrograms per milliliter As, Cu2+, and Zn2+ ions. Zn2+ showed statistically significant inhibition of activity at 0.1 micrograms per milliliter. Acid treatments were not inhibitory above pH 2, at which pH nitric acid inhibited acetylene reduction activity more than did sulfuric acid. The inhibition of acetylene reduction by these ions was Zn2+ > Cd2+ > Cu2+ > AsO3 > Pb2+ > F. The sensitivity of acetylene reduction to the ions was roughly equal to the sensitivity of photosynthesis, respiration, and chlorophyll content when Pb2+ was applied, but was 1,000 times more sensitive to Zn2+. The relationship of the data to field conditions and industrial pollution is discussed.  相似文献   

14.
Purification and properties of α-d-mannosidase from jack-bean meal   总被引:1,自引:1,他引:0  
1. α-Mannosidase from jack-bean meal was purified 150-fold. β-N-Acetyl-glucosaminidase and β-galactosidase were removed from the preparation by treatment with pyridine. Zn2+ was added during the purification to stabilize the α-mannosidase. 2. At pH values below neutrality, α-mannosidase undergoes reversible spontaneous inactivation at a rate dependent on the temperature, the degree of dilution and the extent of purification. The enzyme is also subject to irreversible inactivation, which is prevented by the addition of albumin. 3. Reversible inactivation of α-mannosidase is accelerated by EDTA and reversed or prevented by Zn2+. Other cations, such as Co2+, Cd2+ and Cu2+, accelerate inactivation; an excess of Zn2+ again exerts a protective action, and so does EDTA in suitable concentration. 4. Neither Zn2+ nor EDTA has any marked effect in the assay of untreated enzyme. In an EDTA-treated preparation, however, Zn2+ reactivates the enzyme during assay. 5. It is postulated that α-mannosidase is a dissociable Zn2+–protein complex in which Zn2+ is essential for enzyme activity.  相似文献   

15.
Tripositive-pyrophosphate [M(III)-PPi] complexes were used to investigate the role of free divalent cations on the membrane-bound pyrophosphatase. Divalent cations remain free and the M(III)-PPi complexes were employed as substrates. Formation of a La-PPi complex was studied by fluorescence, and the fact that Zn2+ and Mg2+ remain free in the solution was validated. Hydrolysis of La-PPi is stimulated by the presence of fixed concentrations of free Mg2+ or Zn2+ and this stimulation depends on the concentration of the cations when the La-PPi complex is fixed. The divalent cation stimulation order is Zn2+ > Co2+ > Mg2+ > Mn2+ > Ca2+ (at 0.5 mm of free cation). With different M(III)-PPi complexes, Zn2+ produces the same K m, for all the complexes and Mg2+ stimulates with a different K m. The results suggest that both Mg2+ and Zn2+ activate the membrane-bound pyrophosphatase but through different mechanisms.  相似文献   

16.
Summary Efflux of36Cl from frog sartorius muscles equilibrated in depolarizing solutions was measured. Cl efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases. In depolarized muscles fromRana pipiens, the pH-dependent Cl efflux has an apparent pK a near 6.4.The reduction of Cl efflux by external Zn2+ was determined at different external pHs and chloride activities. The effect of external chloride activity on the pH-dependent Cl efflux was also examined.At pH 6.5 and a membrane potential of –22 mV, increasing external Cl activity from 0.108 to 0.28m decreased inhibition of the pH-dependent Cl efflux at all activities of Zn2+. The Zn2+ activity needed to reduce Cl efflux by half increased from 0.39×10–3 to 2.09×10–3 m. By contrast, external Cl activity had no measurable effect on the apparent pK a of the pH-dependent efflux.At constant Cl activity less than 0.21m, increasing external pH from 6.5 to 7.5 decreased inhibition by low Zn2+ activities with either a slight increase or no change in the Zn2+ activity producing half-inhibition. In other words, for relatively low Cl activities, protection against inhibition of Cl efflux by low Zn2+ activities was obtained by raising, not lowering, external pH; this is not what is expected if H+ and Zn2+ ions compete at the same site to produce inhibition of Cl efflux. We conclude that Zn2+ and low pH inhibit Cl efflux by separate and distinct mechanisms.By contrast, the protection against Zn2+ inhibition produced by high external Cl activity (0.28m) was partially reversed by raising external pH from 6.5 to 7.5 at all Zn2+ activities. The half-inhibition Zn2+ activity decreased from 2.09×10–3 to 0.68×10–3 m.The results can be simulated quantitatively by a model in which single Cl channel elements are in equilibrium with sextets of associated single-channel elements, each sextet having a conductance six times that of a single-channel element. The association into sextets is promoted by OH or Cl binding to a control site on the single-channel elements. Both the single Cl channel element and the sextet of Cl channel elements are closed when this same control site instead binds ZnOH+. The sextet has a much higher affinity for ZnOH+ than does the single Cl channel element.  相似文献   

17.
A novel raw starch degrading α-cyclodextrin glycosyltransferase (CGTase; E.C. 2.4.1.19), produced by Klebsiella pneumoniae AS-22, was purified to homogeneity by ultrafiltration, affinity and gel filtration chromatography. The specific cyclization activity of the pure enzyme preparation was 523 U/mg of protein. No hydrolysis activity was detected when soluble starch was used as the substrate. The molecular weight of the pure protein was estimated to be 75 kDa with SDS-PAGE and gel filtration. The isoelectric point of the pure enzyme was 7.3. The enzyme was most active in the pH range 5.5–9.0 whereas it was most stable in the pH range 6–9. The CGTase was most active in the temperature range 35–50°C. This CGTase is inherently temperature labile and rapidly loses activity above 30°C. However, presence of soluble starch and calcium chloride improved the temperature stability of the enzyme up to 40°C. In presence of 30% (v/v) glycerol, this enzyme was almost 100% stable at 30°C for a month. The Km and kcat values for the pure enzyme were 1.35 mg ml−1 and 249 μM mg−1 min−1, respectively, with soluble starch as the substrate. The enzyme predominantly produced α-cyclodextrin without addition of any complexing agents. The conditions employed for maximum α-cyclodextrin production were 100 g l−1 gelatinized soluble starch or 125 g l−1 raw wheat starch at an enzyme concentration of 10 U g−1 of starch. The α:β:γ-cyclodextrins were produced in the ratios of 81:12:7 and 89:9:2 from gelatinized soluble starch and raw wheat starch, respectively.  相似文献   

18.
A hydrogen peroxide permselective membrane with asymmetric structure was prepared and -glucose oxidase (EC 1.1.3.4) was immobilized onto the porous layer. The activity of the immobilized -glucose oxidase membrane was 0.34 units cm−2 and the activity yield was 6.8% of that of the native enzyme. Optimum pH, optimum temperature, pH stability and temperature stability were found to be pH 5.0, 30–40°C, pH 4.0–7.0 and below 55°C, respectively. The apparent Michaelis constant of the immobilized -glucose oxidase membrane was 1.6 × 10−3 mol l−1 and that of free enzyme was 4.8 × 10−2 mol l−1. An enzyme electrode was constructed by combination of a hydrogen peroxide electrode with the immobilized -glucose oxidase membrane. The enzyme electrode responded linearly to -glucose over the concentration 0–1000 mg dl−1 within 10 s. When the enzyme electrode was applied to the determination of -glucose in human serum, within day precision (CV) was 1.29% for -glucose concentration with a mean value of 106.8 mg dl−1. The correlation coefficient between the enzyme electrode method and the conventional colorimetric method using a free enzyme was 0.984. The immobilized -glucose oxidase membrane was sufficiently stable to perform 1000 assays (2 to 4 weeks operation) for the determination of -glucose in human whole blood. The dried membrane retained 77% of its initial activity after storage at 4°C for 16 months.  相似文献   

19.
The effects of phosphorus, Zn2+, CO2, and light intensity on growth, biochemical composition, and the activity of extracellular carbonic anhydrase (CA) in Isochrysis galbana were investigated. A significant change was observed when the concentration of phosphorus in the medium was increased from 5 μmol/L to 1000 μmol/L affecting I. galbana’s cell density, biochemical composition, and the activity of extracellular CA. Phosphorous concentration of 50 μmol/L to 500 μmol/L was optimal for this microalgae. The Zn2+ concentration at 10 μmol/L was essential to maintain optimal growth of the cells, but a higher concentration of Zn2+ (≥ 1000 μmol/L) inhibited the growth of I. galbana. High CO2 concentrations (43.75 mL/L) significantly increased the cell densities compared to low CO2 concentrations (0.35 mL/L). However, the activity of extracellular CA decreased significantly with an increasing concentration of CO2. The activity of extracellular CA at a CO2 concentration of 43.75 mL/L was approximately 1/6 of the activity when the CO2 concentration was at 0.35 mL/L CO2. Light intensity from 4.0 mW/cm2 to 5.6 mW/cm2 was beneficial for the growth, biochemical composition and the activity of extracellular CA. The lower and higher light intensity was restrictive for growth and changed its biochemical composition and the activity of extracellular CA. These results indicate that phosphorus, Zn2+, CO2, and light intensity are important factors that impact growth, biochemical composition and the activity of extracellular CA in I. galbana.  相似文献   

20.
A fluorescent ATP analog, β-naphthyl triphosphate, was hydrolyzed to β-naphthyl diphosphate and orthophosphate by heavy meromyosin ATPase. In the process of hydrolysis the fluorescence intensity of β-naphthyl triphosphate changed remarkably. Thus, the rate of β-naphthyl triphosphate hydrolysis is evaluated directly and continuously by measuring the time course of fluorescence intensity.In the presence of Ca2+, the Michaelis constant (Km) of β-naphthyl triphosphate hydrolysis by heavy meromyosin was similar to that of ATP hydrolysis. While, in the presence of Mg2+ the Km of β-napthyl triphosphate hydrolysis was 9.0·10−6 M, much larger than the value of ATP hydrolysis, indicating that the apparent affinity of the enzyme for β-naphthyl triphosphate is less than that for ATP.The pH dependence of β-naphthyl triphosphatase activity resembled that of ATPase activity, suggesting a similarity in the mechanism of hydrolysis of the two substrates.  相似文献   

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