Showing papers in "Applied Biochemistry and Biotechnology in 1981"

Production of α-keto acids Part I. Immobilized cells ofTrigonopsis variabilis containing D-amino acid oxidase.

Abstract: Production of alfa-Keto Acids. Part I - Immobilized Cells of Trigonopsis variabilis containing D-Amino Acid Oxidase

Entrapment of living microbial cells in covalent polymeric networks : II. A quantitative study on the kinetics of oxidative phenol degradation by entrappedCandida tropicalis cells.

Abstract: The kinetics of oxidative phenol degradation with microbial cellsCandida tropicalis, immobilized in a polyacrylamide and polymethacrylamide matrix, were mathematically simulated assuming zero-order and Michaelis-Menten rate equations. For zero-order kinetics an expanded equation for catalytic effectiveness as a function of the Thiele modulus, Biot number, and partition coefficients was derived and compared with numerical solutions for Michaelis-Menten kinetics. Errors with regard to the zero-order approximation become negligible ifc o/K M >2. Experimentally determined catalyst activities as a function of particle size and cell concentration were compared to calculated ones. Additional experiments to determine the diffusion and oxygen consumption ratios have been carried out in an effort to resolve the physical parameters to be used in the above mentioned calculations. Furthermore, experiments on cell growth during reincubation with nutrients and oxygen are reported; an increase in activity up to a factor of ten was observed. These experiments demonstrate that the microbial cells are entrapped in the polymer matrix in the living state.

Recent advances in homogeneous and separation-free enzyme immunoassays.

Abstract: The development of the versatile and sensitive analytical technique of quantitative radioimmunoassay (RIA) by Yalow and Berson in 1959 (I) has revolutionized diagnostic methodology in clinical medicine (1-4) and has provided a powerful analytical tool in biomedical research (5-8). Recently, there has been interest in the use of non-isotopic labels for monitoring the distribution of free or antibody-bound ligands in immunological assays. For example, the following labels have been used in immunoassays: bacteriophages (9), free radicals (10), fluorescent groups (11), chemiluminescent groups (12), synthetic particles (13), red blood cells (14), electron dense materials (15), metal (16), enzymes (17-22), and prosthetic group (47). Major reasons for current interests in replacing the radioisotopic labels with nonradioactive ones are (a) the relatively short half-life of gamma-emitting isotopes (e.g., J2~ I), which creates logistic problems in the preparation of Iabeled ligands for immunoassays, (b) the impairment of immunological reactivity and specificity owing to the structural damages caused by the gamma-ray radiation of the isotope, (c) the use of expensive, toxic, flammable liquid scintillants for beta-ray emitting isotopes (effective disposal of scintillants can be

Thermomagnetic surgery for cancer.

Abstract: Thermomagnetic Surgery is a unique technique that takes advantage of the phenomenon of hysteresis heating of a ferromagnetic material to produce intense but controlled temperatures within solid organs or tumors to cause coagulation necrosis. By controlling the power of the electromagnetic coil system, the degree of heating of the tumor can be controlled through temperature monitoring that allows limitation of the area of destruction to the disease process and avoids damage to surrounding structures. If the ferromagnetic material is delivered by the arterial route to the tumor or organ, there is an additional beneficial effect of ischemic necrosis of the tissue and in time more concentration of the ferromagnetic particles. This new technique is applicable to selected cases of human cancer because no ill effect has been shown to exposure of the electromagnetic field or the ferromagnetic material in experimental animals.

Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation.

Abstract: Magnetic biospecific affinity adsorbents for immunoglobulin and enzyme isolation have been prepared They were obtained by a “ post-magnetization” procedure involving a simple treatment of the various affinity gels with magnetic ferrofluid The magnetic biospecific adsorbents tested include magnetic protein A-Sepharose for isolation of IgG antibodies, magnetic human serum albumin (HSA)-Sepharose for anti-HSA isolation, and magnetic 2′,5′-ADP for isolation of glucose-6-phosphate dehydrogenase from baker’s yeast and hemolyzates of human red blood cells For the latter enzyme, a 11,000-fold purification was achieved in one step

Denitrification and removal of heavy metals from waste water by immobilized microorganisms

Abstract: Pseudomonas fluorescens, immobilized on soft polyvinyl chloride granules containing up to 35% softeners as carbon source, was used for simultaneous removal of nitrate and heavy metals. In typical continuous column operation, a 100 mg/L nitrate input solution was reduced to a 20 mg/L output at a feeding rate of 1500 mL/h, with a capacity of 14 kg/day/m3, and with an efficiency of 79%. In the same column, Pb(NO3)2 concentration was reduced from 1.0 to 0.05−0.1 mg/L and ZnSO4 concentration was reduced from 10 to 5 mg/L.Pseudomonas aeruginosa immobilized on an O2 plasma-treated melt blown polypropylene web was used for removing 95% of a 1.7 nCi PuCl4 activity from a nuclear plant waste water in a batch operation.

Determination of intrinsic properties of immobilized enzymes : 1. Kinetic studies on sepharose-staphylococcal nuclease in the absence of diffusional limitations.

Abstract: Using a small substrate (thymidine 5′-(p-nitrophenyl phosphate) 3′-phosphate), the kinetics of staphylococcal nuclease insolubilized on CNBr-activated Sepharoses 4B and 6B are affected by internal diffusional limitations. Since we demonstrate that we are working under conditions in which external mass-transfer resistances do not influence the reaction rate, we propose a simple theoretical model that considers only the case of mixed enzymic reaction-internal diffusion kinetics. In the Eadie-Hofstee plots we find very good agreement between theory and experiment. The model accounts very well for the results obtained by changing support texture, reaction conditions, and/or enzyme concentration in the insoluble derivatives, variables that modify the diffusional restrictions of the system.

Support-bound microbial cells.

Abstract: Index Entries: Immobilization; immobilized microbes; carriers; crosslinking; entrapment: biomass accumulation; encapsulation: continuous fermentation; covalent coupling; covalent binding; immobilized microbe technology; supports; continuous reactor; immobilization technology; adsorption; adsorptive forces; stationary phase: growth phase; sequential multi-enzyme reactions; synchronous growth: generation time; intracellular enzyme: enzyme; porous supports; polyacrylamide; collagen; carrageenan; multisequential enzyme reactions; microbe retention; periodicity.

Recycling of NAD(+) using coimmobilized alcohol dehydrogenase andE. coli.

Abstract: The use of immobilized enzymes has opened the possibility of large scale utilization of NAD+-linked dehydrogenases, but the applications of this technique were limited by the necessity of providing the large amounts of NAD+ required by its stoichiometric consumption in the reaction. After immobilization of alcohol dehydrogenase and intactE. coli by glutaraldehyde in the presence of serum albumin, the respiratory chain was found to be capable of regenerating NAD+ from NADH. This NAD+ can be recycled at least 100 times, and thus the method is far more effective than any other, and, moreover, does not require NADH oxydase purification. The total NADH oxidase activity recovered was 10–30% of the initial activity. Although, NADH is unable to cross the cytoplasmic membrane, it was able to reach the active site of NADH dehydrogenase after immobilization. The best yield of NADH oxidase activity with immobilized bacteria was obtained without prior treatment of the bacteria to render them more permeable. The denaturation by heat of NADH oxidase in cells that are permeabilized was similar before and after immobilization. In contrast, the heat denaturation of soluble Β-galactosidase required either a higher temperature or a longer exposure after immobilization. The sensitivity of immobilized NADH oxidase to denaturation by methanol was decreased compared to permeabilized cells. As a result, it is clear that the system can function in the presence of methanol, which is necessary as a solvent for certain water insoluble substrates.

Determination by the enzyme thermistor of cellobiose formed on degradation of cellulose.

Abstract: A calorimetric assay procedure for the determination of cellobiose has been developed. The cellobiose is hydrolyzed by β-glucosidase and the glucose formed is measured calorimetrically by an enzyme thermistor containing co-immobilized glucose oxidase and catalase. The system was optimized with regard to the arrangement of the enzymes, the pH-dependence of the separate enzymic steps, and of the total system. By placing the β-glucosidase in a precolumn that could be switched in and out of the flow through the enzyme thermistor, both cellobiose and glucose present in the sample could be determined. The performance with standard solutions and with crude samples from cellulose degradation experiments was investigated.

EPR characterization of cellulose triacetate fibers used for enzyme immobilization.

Abstract: EPR studies of a nitroxide spin label and of the nitroxide spin-labeled albumin entrapped in cellulose triacetate fibers were carried out.

Immobilization of proteins as a tool for studying primary structure around their cysteinyl residues.

Abstract: The primary structure around the single cysteinyl residue of chicken pepsin was investigated by binding the protein via this residue to an insoluble carrier. Carriers stable towards reagents used for the fragmentation of proteins and sequence analysis were prepared by coupling a spacer arm to polyN-hydroxymethyl acrylamide using a thioether bond that is potentially cleavable by mercuric ions (1). Phenacyl bromide group, attached to the free end of the spacer, reacted rapidly and specifically with the cysteinyl residue of chicken pepsin. Up to 300 mg of the enzyme were bound to 1 g of carrier. The polymer-bound protein was cleaved by trypsin or by cyanogen bromide or by a sequence of both. Fragments of 40–120 amino acid residues, depending on the method of cleavage, remained attached to the polymer through the cysteinyl residue. The compositions and partial sequences of these fragments revealed that the cysteinyl residue is located within or in the vicinity of a loop in the molecule formed by a disulfide bond.

Delayed luminescence of luminol initiated by a membrane-bound peroxidase.

Abstract: The luminescense of the luminol-H2O2 system was initiated by either free or membrane-bound horseradish peroxideae (HRP). The instantaneous luminescene decayed rapidly and was followed by the delayed luminescence in the presence of excess luminol. The delayed luminescence was characterized by a chain reaction, in which luminescence intensity increased exponentially. Membrane-bound HRP demonstrated that the delayed luminescence took place even in the absence of HRP if the instantaneous luminescence was initiated by HRP. A mechanism for the nonenzymatic luminescence is proposed and discussed.

Solid lactoperoxidase in the iodination ofl-tyrosine and albumin

Abstract: Solid lactoperoxidase (LP-sorbent) was prepared from lactoperoxidase and a carrier copolymer of maleic anhydride and butanediol divinylether. The properties of LP-sorbent in the iodination of tyrosine and albumin were examined. For optimizing the pH of the iodination mixture, buffers in the pH range 4.5–8.0 were used. Albumin was iodinated using Na125I and tyrosine using KI. The effect of substrate concentration and the sequential addition of reagents was examined in the iodination of tyrosine. The optimum pH, for iodination of albumin was 6.5 and that, for the iodination, of tyrosine 6.0–6.5. The iodination reactions were effective over a broad pH range around 6.5 resulting in almost equal iodinations. The optimum concentrations expressed as mmol/L/μmol lactoperoxidase/mg sorbent at pH 6.5 were: H2O2, 70;, KI, 64; and tyrosine, 128. The maximum catalytic activity of the LP-sorbent at pH 6.5 was 4.22 μkat/mg LP-sorbent or 28.8 kat/mol(mol of L-3-ITyr/s/mol lactoperoxidase). After the primary reaction of the LP-sorbent with hydrogen peroxide, both the supernatant and the washed solid phase exhibited iodinative activity. The iodination of tyrosine by LP-sorbent was also found to be possible in water.

Immobilization ofmyo-inositol-1-phosphate synthase containing active, self-regenerating coenzyme (NAD(+)) on the same matrix.

Abstract: myo-Inositol-1-phosphate synthase (EC 5.5.1.4) from rat testes, an NAD+-containing enzyme, which convertsd-glucose 6-phosphate to 1l-myo-inositol 1-phosphate, could be immobilized together with its cofactor and bovine serum albumin by crosslinking with glutaraldehyde at pH 4.5. The enzyme bound to the gel showed a specific activity of 5.6% of that of the native enzyme, but the activity could be increased to 21% by pretreatment with urea.

Potentiometric and spectroscopic studies of the reaction between trypsin and its inhibitors on chemically modified solid surfaces

Abstract: The potential of a titanium metal electrode modified with trypsin changes as a result of the complex formation reaction between trypsin and its inhibitor, aprotinin, dissolved in the solution. A similar potential change in the opposite direction occurs by the reaction between aprotinin-modified electrode and trypsin in the solution. The induced changes in both cases depend on the pH of the solution, showing the maximum change at pH = 9.5. The potentiometric response of the trypsin-modified electrode for the consecutive addition of aprotinin and proflavine proves that trypsin bound on the solid surfaces reacts with aprotinin much more strongly than with proflavine. This result is fully consistent with the spectroscopically observed behavior of a trypsin-modified quartz plate against these inhibitors. The surface coverage of trypsin on the quartz plate is also determined by a near-ultraviolet absorption measurement.

Influence of urea and organic solvents on the activity of immobilizedmyo-inositol-1-phosphate synthase containing active, self-regenerating coenzyme (NAD(+)) on the same matrix.

Abstract: myo-Inositol-1-phosphate synthase (EC 5.5.1.4.) from rat testes, an NAD(+)-containing enzyme that convertsD-glucose 6-phosphate to 1L-myo-inositol-1-phosphate was immobilized together with its cofactor and bovine serum albumin by crosslinking with glutaraldehyde at pH 4.5. The cofactor is reduced and reoxidized during the reaction cycle, thus forming a self-regenerating system with respect to the cofactor. The behavior of this immobilized enzyme/cofactor system in presence of organic solvents and urea and the activating effect of these compounds on the enzymatic activity were studied and discussed in the paper.

Rapid estimation of biochemical oxygen demand

Abstract: The feasibility of using columnar reactors containing immobilized microorganisms for the rapid estimation of BOD was demonstrated in this study. Dilutions of three types of industrial effluents were tested by the BOD5 test and by this experimental system. A high degree of correlation (r = 0.98) was observed between results of the two tests. The mean standard error of estimation of the experimental system was 11%.

The analysis of serum urate utilizing immobilized uricase.

Abstract: The evaluation of a method for the estimation of serum urate using immobilized uricase is described, the resultant hydrogen peroxide produced being measured by the oxidative coupling of 3,5-dichloro-2-hydroxybenzenesulfonate and 4-aminophenazone in the presence of peroxidase.

Hen ovomucoid-agarose: A new conjugate for the isolation by affinity chromatography of UDP-galactose: Glycoprotein galactosyl-transferase.

Abstract: A new conjugate for the affinity chromatography of UDP-galactose:glycoprotein galactosyltransferase has been synthesized by coupling hen ovomucoid, a ligand similar to the acceptor substrate, to agarose. The hen ovomucoid-Sepharose conjugate binds galactosyl transferase more tightly that other acceptor-Sepharose conjugates. The new adsorbent gives comparable yields and purifications with those obtained by ligands similar to the nucleotide moiety of the substrate and to the “specifier” protein, α-lactalbumin. The soluble galactosyltransferase from rat ventral prostate is effectively removed from the high speed supernatant by an ovomucoid-Sepharose column. The enzyme can be eluted with buffer containing EDTA andN-acetylglucosamine in a high yield (75–80%) and in a purified form (4000-fold purification). The stability of ovomucoid to heat and to high concentrations of urea and its inhibition of some proteases makes the conjugate easy to operate with an quite useful even with rather crude preparations.

Analysis of a theoretical model for anisotropic enzyme membranes application to enzyme electrodes.

Abstract: A theoretical model of diffusion and reaction in an anisotropic enzyme membrane is presented with particular emphasis on the application of such membranes in enzyme electrodes. The dynamic response of systems in which the kinetics are linear, which comprises the practical operating regime for enzyme electrodes in analysis, is investigated via an analytic solution of the governing differential equations. The response is presented as a function of a single dimensionless group, Μ, that is the membrane modulus.

Protection of immobilized sulfhydryl groups against autooxidation by alterations in their microenvironment.

Abstract: Immobilized sulfhydryl groups were prepared by partial thiolation of NH2-glass beads. The microenvironment of the immobilized SH groups was varied by different chemical modifications of neighboring NH2 groups. Introduction of a strong charge in the surroundings of immobilized sulfhydryls results in their dramatic stabilization against autooxidation. This effect is due to the salting of O2 from the surface microlayer of the thiolated beads.

Energy-transducing membrane : II. Roles of liquid crystal in the photoresponse of a chlorophyll liquid crystal membrane.

Abstract: The states of chlorophyll a (Chl a) incorporated in a liquid crystal membrane were investigated by spectrophotometry in the visible and IR regions.N-(p-methoxybenzylidene)-p′-butylaniline (MBBA) was used as the liquid crystal. The Chl a-MBBA (1∶3 in molar ratio) showed the dihydrate-Chl a aggregate peak at 743 nm under excess water conditions. IR spectroscopic evidence indicated that the Chl a-MBBA membrane was hydrated in the dihydrate stoichiometry [Chl a-3MBBA-2H2O], via the C-10 ester OC…H(H)O…Mg and the C-9 keto OC…H(H)O…Mg bondings.