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1.
Rabbit muscle lactate dehydrogenase (EC 1.1.1.27) was attached covalently to the inner surface of nylon tubing; a modified technique, involving benzidine and glutaraldehyde, was used, and the resulting immobilized enzyme showed no loss of activity over a period of several months. An experimental study was made of the flow kinetics for the reaction between pyruvate and reduced nicotinamide adenine dinucleotide in two limiting cases, one substrate in excess and the concentration of the other one varied. A range of flow rates and temperatures was covered. The results were analyzed in various ways on the basis of the Kobayashi--Laidler treatment of flow systems. It was concluded that the kinetics are largely diffusion-controlled, especially at the lower substrate concentrations and flow rates. The values of the apparent Michaelis constants vary with flow rate vf, being linear in vf-1/3, and the values extrapolated to infinite flow rate (vf-1/3 = 0) approach the values for the enzyme in free solution. Analysis of the rates led to activation energies for the diffusion of the two substrates. 相似文献
2.
Telefoncu A 《Biotechnology and bioengineering》1983,25(3):713-724
Styrene-methacrylic acid copolymers of varying combinations crosslinked with p-DVB (1-2%) and porous structure were synthesized to be used as carriers in trypsin immobilization. The styrene-methacrylic acid copolymers containing free carboxy groups were activated by conversion into the mixed carbonic anhydride with N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline (EEDQ) at pH 4.0. The degree of activation of copolymers were determined from the amount of p-aminobenzoic acid each could bind. The activated copolymers were incubated with trypsin in phosphate buffer (pH 8.0) at 4 degrees C for 24 h. The optimum conditions for enzymatic activity measurements determined and the activity tests were carried out in 1.5 x 10(-2)M CaCl(2) solution (pH 8.0) at 0.05 ionic strength with a pH-stat instrument. The dependence of the activity of styrene-methacrylic acid (SMA)/trypsin derivatives to pH was investigated and it was observed that the optimum pH of the immobilized trypsin derivatives moved to the basic region compared to the native trypsin. It was found that as the ionic strength increased, the shift in the optimum pH decreased and the activity increased. The Michaelis constants for the SMA-trypsin derivatives were determined with aid of Lineweaver-Burk diagrams. The thermal, storage, and operational stabilities of SMA-trypsin derivatives were assessed. It was found that the above stabilities for all the immobilized trypsin derivatives were better than that for the native trypsin. 相似文献
3.
Urease was immobilized on O-alkylated nylon tubes coated with polyaminated derivatives of starch or dextran. The specific activity of the enzyme coil and the relative stability of the immobilized enzyme, compared with immobilized urease derived from other nylon tube modifications, were enhanced. Also, the nonspecific binding of urease to O-alkylated nylon tubes was virtually eliminated by the coating process. 相似文献
4.
The preparation and properties of ficin chemically attached to carboxymethylcellulose 总被引:9,自引:8,他引:1
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1. Purified ficin has been coupled to four CM-celluloses by reaction with their acid azide derivatives. Insoluble products containing 1.8-4.7mg. of ficin/100mg. of product and retaining 8.0-12.0% of the free enzyme's esterase activity have been obtained. 2. The amount of bound ficin in these preparations is dependent on the degree of carboxymethyl substitution of the CM-cellulose to which the ficin is attached. 3. A shift of the alkaline limb of the pH-activity curve of ficin when chemically attached to CM-cellulose has been shown. 4. Only a small loss has been observed in the enzymic activity of these products when stored at 2 degrees for 4 months. They are more resistant than free enzyme to heat denaturation. 5. Columns of CM-cellulose-ficin have been packed. The degree of hydrolysis of perfused substrate has been measured for different flow rates through the column. 6. The properties of these derivatives have been discussed. 相似文献
5.
B Svensson 《Biochimica et biophysica acta》1976,429(3):954-963
Analogous soluble and insoluble derivatives of subtilisin Novo (EC 3.4.21.14) were prepared by coupling the enzyme to CNBr-activated DEAE-dextran and DEAE-Sephadex, respectively. The DEAE-dextran-subtilisin displayed pH optima and Km values for ester hydrolysis similar to subtilisin, whereas the pH versus activity profiles obtained with DEAE-Sephadex-subtilisin were shifter towards the alkaline pH region and the Km values were increased. Compared with subtilisin, DEAE-dextran-subtilisin showed a 40-65% reduction of kcat for hydrolysis of N-acetyl-L-tyrosine ethyl ester, p-tosyl-L-arginine methyl ester and benzyloxycarbonyl-glycyl-L-tyrosinamide and its maximum velocities for digestion of casein and clupein also amounted to 40-60% of the subtilisin values. With Deae-sephadex-subtilisin, in contrast, the maximum velocity of hydrolysis decreased to a greater extent for polypeptide substrates compared to ester substrates. The present results indicate that the chemical nature of a support can effect intrinsic properties of a matrix-bound enzyme in addition to the steric and diffusional effects usually observed with polymer-attached enzymes. 相似文献
6.
7.
Preparation and properties of solid-supported urease 总被引:1,自引:0,他引:1
8.
Acetylcholinesterase has been attached covalently to the inner surface of nylon tubing. An experimental study has been carried out on the flow kinetics; solutions of acetylthiocholine at various concentrations were passed through tubing at various flow rates, and measurements made of the rates of formation of product. The results were analyzed in the light of the theoretical treatment of Kobayashi and Laidler, four different methods of analysis being employed. It is found that at lower substrate concentrations and flow rates the reactions are largely diffusion controlled. The Km(app) values are substantially higher than the Km value for diffusion-free conditions, but approach it as the flow rate is increased, when the diffusion layer becomes less important. The results are entirely consistent with the Kobayaski-Laidler theory, and provide guidelines for the design of open tubular heterogeneous enzyme reactors, both for industrial and analytic purposes. 相似文献
9.
Flow kinetics of L-asparaginase attached to nylon tubing 总被引:1,自引:0,他引:1
L -Asparaginase has been attached by chemical means to the inner surface of nylon tubing. An experimental study has been carried out of the flow kinetics for such a system, asparagine solutions at various concentrations being passed through two lengths of tubing at various flow rates. Measurements were made of the concentration of the product ammonia at the tube exit, and of the rate of formation of ammonia, under the various conditions. Apparent Michaelis constants, Km(app), were some three orders of magnitude higher than the Km for the enzyme in free solution (~13 × 10?6JM). The results were analyzed with respect to the theoretical treatment described in the preceding paper (Kobayashi and Laidler), three different methods being employed. It is concluded that at lower substrate concentrations and flow rates the reactions are largely diffusion-controlled, the enhanced Km(app) values being largely if not entirely due to the diffusion control; ionic strength studies showed electrostatic repulsion effects to be unimportant. At high concentrations and high flow rates (when the diffusion layer is of negligible thickness) the diffusional effects are minimized, and Km(app) approaches the true Km value for the immobilized enzyme. 相似文献
10.
11.
Affinity chromatography and immunosorption with acetylcholine receptor attached to nylon tubes. 总被引:1,自引:0,他引:1
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Nylon-linked proteins were used for affinity trapping and chromatography. As representative examples purified acetylcholine receptor, alpha-cobratoxin and bovine serum albumin were coupled to the activated matrix to serve as biospecific ligands. In particular, acetylcholine receptor was coupled without significant loss of biochemical properties. The resulting affinity tubes bind receptor-specific ligands including immunoglobulins and thus can be used for affinity-chromatographic purposes and immunoassays. 相似文献
12.
L Shemer R Granot A Freeman M Sokolovsky L Goldstein 《Biotechnology and bioengineering》1979,21(9):1607-1627
Urease was bound to commercially available nonwoven nylon fabric filters. Multilayer immobilized-enzyme filter reactors were constructed by packing varying numbers of urease-nylon filters in a column. Owing to the relatively open structure and high mechanical strength of the filter fabric, compaction and pressure drop effects were minimal. The reactors could be operated in a wide range of substrate concentrations and flow rates under conditions where mass-transfer limitations could be neglected. The kinetic behavior of the immobilized-enzyme filter reactors could be described by a linear form of the integrated Michaelis-Menten equation using a model based on the sequential action of the enzyme filters. 相似文献
13.
14.
Poly (acrylonitrile-methylmethacrylate-sodium vinylsulfonate) membranes were subjected to seven different chemical modifications. The amounts of new groups incorporated in the membranes with the modifications were determined. Urease was covalently immobilized on the modified membranes. Both the amount of bound protein and relative activity of immobilized urease were measured. The highest activity was found for urease bound to membranes modified with hydroxylammonium sulfate (68%) and hydrazinium sulfate (67%). Optimum pH of free urease was determined to be 5.8. For positively charged membranes, pH optimum was shifted to higher values, while for negatively charged membranes-to lower pH. The charge of the matrix affected also the rate of the enzyme reaction. The highest rate was measured with urease immobilized on membranes modified with hydroxylammonium sulfate and hydrazinium sulfate. The major part of the immobilized enzyme on different modified membranes remained stable-only ca. 20% of enzyme activity was lost for 4 h at 70 degrees C while the free enzyme was totally inactivated. 相似文献
15.
Studies on immobilized enzymes. IX. Preparation and properties of aminoacylase covalently attached to halogenoacetylcelluloses 总被引:2,自引:0,他引:2
Immobilization of mold aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) was investigated by covalently binding the enzyme to halogenoacetylcelluloses. As a result, the iodoacetylcellulose was found to be the best carrier among the halogenoacetylcelluloses. The yield of activity of the insoluble aminoacylase relative to that of the native aminoacylase used was 40–50%, and the specific activities of both enzyme preparations were the same within the limits of error of the estimation. 相似文献
16.
17.
Some properties and purifications of urease 总被引:4,自引:0,他引:4
K R Lynn 《Biochimica et biophysica acta》1967,146(1):205-218
18.
Urease was encapsulated within kappa-carrageenan beads. Various parameters, such as amount of kappa-carrageenan and enzyme activity, were optimized for the immobilization of urease. Immobilized urease was thoroughly characterized for pH, temperature, and storage stabilities and these properties were compared with the free enzyme. The free urease activity quickly decreased and the half time of the activity decay was about 3 days at 4 degrees C. The immobilized urease remained very active over a long period of time and this enzyme lost about 70.43% of its orginal activity over the period of 26 days for storage at 4 degrees C. The Michaelis constant (Km) and maximum reaction velocity (Vmax) were calculated from Lineweaver-Burk plots for both free and immobilized enzyme systems. Vmax = 227.3 U/mg protein, Km = 65.6 mM for free urease and Vmax = 153.9 U/mg protein, Km = 96.42 mM for immobilized urease showed a moderate decrease of enzyme specific activity and change of substrate affinity. 相似文献
19.
Handa T Hirose A Akino T Watanabe K Tsuchiya H 《Biotechnology and bioengineering》1983,25(12):2957-2967
In this report, alpha-Amylase originating from Bacillus subtilis (liquefying type) was immobilized on partially imidoesterized polyacrylonitrile (PAN) by covalent bonding. For the preparation of immobilized alpha-amylase, which has a high activity and high stability to repeated use, the optimum conditions for the preparation reaction were investigated. The optimum conditions for the preparation reaction were quantified on the basis of the enzymatic activity, the preservation of the activity during repeated use in batch process and the protein content on the support. Further-more, enzymatic properties of immobilized alpha-amylase prepared at optimum conditions were compared with the native enzyme. The optimum temperature and reaction time for the imidoes-terification reaction were 30 degrees c and 6 h, respectively, whereas those of the amidinatin reaction were 30-40 degrees C and more than 3 h, respectively; the optimum pH range was 9-10. Immobilized alpha-amylase prepared at the optimum conditions was very stable against the repeated use and had more than 90% of relative to activity of the first use after the tenth procedure. The initial reaction rate of immobilized alpha-amylase was lower than native alpha-amylase, but same amount of reducing sugars were produced after the reaction passed for more than 90 min. The immobilized alpha-amylase was less stabel at the high temperature and the more basic media. However, after long incubation time, immobilized alpha-amylase was more stable than the native enzyme in exposure to heat and a storng base. 相似文献
20.
Thomas Jahns 《Antonie van Leeuwenhoek》1995,68(3):209-214
Urease (EC 3.5.1.5) catalyses the hydrolysis of urea to ammonia and carbon dioxide. The enzyme fromSporobolomyces roseus was enriched 780-fold and purified to apparent homogeneity using heat treatment, ion exchange chromatography on Q-Sepharose fast flow, hydrophobic interaction chromatography on Phenyl-Sepharose, size exclusion chromatography on Sephacryl S 300 HR, and ion exchange chromatography on MonoQ. Analysis of the purified enzyme by SDS-PAGE demonstrated the presence of subunits with a molecular weight of 90 (± 4) kDa. The M
r
of the native enzyme was estimated by size exclusion chromatography to be 340 (± 30) kDa, suggesting a tetrameric structure different from other ureases isolated so far from both prokaryotes and eukaryotes. The enzyme was heat-stable, showing no loss of activity after incubation at 70 °C for 15 min. The highest urease activities were observed after growth on media containing urea as the sole source of nitrogen. 相似文献