Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

Purification and immobilization of rabbit liver aldehyde oxidase

Aldehyde oxidase (E.C. 1.2.3.1) was isolated from rabbit liver and two potential bioaffinity ligands, i.e., 3-aminocarbonyl-1-benzyl-6-methylpyridinium bromide and 3-aminocarbonyl-1-benzyl-4,6-dimethylpyridinium chloride, were tested for their applicability in a purification procedure for this enzyme. Various supports and different coupling methods were investigated for the immobilization of aldehyde oxidase. Adsorption to n-hexyl- and n-octylamine-substituted Sepharose 4B and DEAE Sepharose 6B gave the best retention of aldehyde oxidase activity. The storage stability of free enzyme and enzyme immobilized to n-octylamine-substituted Sepharose 4B was studied in several buffers at pH 7.8 and 9.0. This showed that the stability of immobilized enzyme was much less than that of free enzyme. The apparent operational stability of the immobilized enzyme preparation, however, improved substantially compared to soluble enzyme, although the corresponding product yield is still very poor. Coimmobilization of catalase and/or superoxide dismutase provided no significant increase of the apparent operational stability and product yield. A positive effect on both parameters was found for aldehyde oxidase-n-alkylamine Sepharose 4B preparations by increasing the amount of enzyme adsorbed per unit weight of support, whereas the productivity of these preparations remained about constant.     

Some properties of a papain–agarose reactor provided with a pH monitor

A small reactor of immobilized papain was used to gain some knowledge about the effect of immobilization upon the reactivity of the enzyme towards one substrate and various types of inhibitors. A buffer solution containing benzoyl–arginine ethyl ester as substrate was run through a small column of papain immobilized by attachment to agarose beads. The pH of the effluent was measured continuously and provided the data used to calculate the substrate conversion during passage through the reactor. The operation of the system was checked by determining the substrate conversion as a function of flow rate. It proved to operate as theory demanded. The rate and extent of inhibition were measured after addition of various inhibitors to the buffer–substrate solution. The following quantities of immobilized papain were found to be equal within ±20% to those of the free enzyme in solution: the overall activity, the K_m of benzoyl–arginine ethyl ester, the K_i of the competitive inhibitor benzoylamino-acetonitrile, the rate of inactivation by chloroacetic acid and by chloroacetamide, the rate of activation by cysteine of the mixed disulfide of papain and cysteine, and the rate of spontaneous reactivation of the KCNO–papain adduct. The inactivation by KCNO proved to be strongly pH dependent. This may explain why the rate of the latter reaction is only 66% of the rate with free enzyme. It is concluded that the rates and equilibrium constants measured in the present reactor system are within ±20% of the values of the dissolved enzyme, provided that the reactions are not strongly pH dependent. Calculation showed there was no diffusion limitation.     

Coimmobilization of D-amino acid oxidase and catalase by entrapment ofTrigonopsis variabilis in radiation polymerised Polyacrylamide beads

Trigonopsis variabilis induced for D-amino acid oxidase and catalase was immobilized by entrapment in Polyacrylamide beads obtained by radiation polymerisation. Permeabilization of the cells was found to be essential for optimal activity of the enzymes in free cells. However, the process of entrapment itself was found to eliminate the permeability barrier of cells immobilized in Polyacrylamide. The two enzymes exhibited a differential response on Polyacrylamide entrapment. Thus, D-amino acid oxidase activity was stabilized to heat inactivation whereas catalase in the same cells showed a destabilization on entrapment in Polyacrylamide. The coimmobilized enzyme preparation showed an operational half life of 7–9 days after which the D-amino acid oxidase activity remained stable at a value 35–40% of that of the initial activity for a study period of 3 weeks. Coimmobilization of MnO₂ was not effective in enhancing the operational life of the enzyme preparation.     

Non-thermal Effects of a Ceramics Heater Radiation on Xanthine Oxidase Activity

The non-thermal effects of ceramics heater radiation on xanthine oxidase activity have been investigated using the enzyme, substrate, and competitive inhibitors which were irradiated on cooling. The K_m and V_max in the irradiated enzyme system were reduced to 51% and 85%, of the non-irradiated control, respectively. The K_i for a competitive inhibitor, folic acid, in the irradiated enzyme system decreased to 22% of the non-irradiated control. A steady-state molecular kinetic analysis for the reaction estimates that the irradiated enzyme may be kept in a folding state, and the formation of a Michaelis complex has been accelerated, and the activated Michaelis complex has been stabilized, and that a solvation or an electrostriction of xanthine, folate, and an active center of the enzyme with water may be promoted by irradiating the components in an aqueous solution, by which modification of the enzyme activity has been regulated.     

Xanthine Oxidase Catalyzes the Synthesis of Retinoic Acid

Milk xanthine oxidase (xanthine: oxygen oxidore-ductase; XO; EC 1.1.3.22) was found to catalyze the conversion of retinaldehyde to retinoic acid. The ability of XO to synthesize all trans-retinoic acid efficiently was assessed by its turnover number of 31.56 min^?1, determined at pH 7.0 with 1nM XO and all trans-retinaldehyde varying between 0.05 to 2μM. The determination of both retinoid and purine content in milk was also considered in order to correlate their concentrations with kinetic parameters of retinaldehyde oxidase activity. The velocity of the reaction was dependent on the isomeric form of the substrate, the all trans- and 9-cis-forms being the preferred substrates rather than 13-cis-retinaldehyde. The enzyme was able to oxidize retinaldehyde in the presence of oxygen with NAD or without NAD addition. In this latter condition the catalytic efficiency of the enzyme was higher. The synthesis of retinoic acid was inhibited 87% and 54% by 4μM and 2μM allopurinol respectively and inhibited 48% by 10 μM xanthine in enzyme assays performed at 2μM all trans-retinaldehyde. The K_i value determined for xanthine as an inhibitor of retinaldehyde oxidase activity was 4 μM.     

The adsorption of Fusarium flocciferum spores on celite particles and their use in the degradation of phenol

Summary Spores of Fusarium flocciferum were inserted in porous celite beads. The effects of bead size, adsorption time course, washing cycle and spore concentration on spore loading were investigated. Cell loadings up to 50% (dry weight/beads) were obtained. The degradation of phenol using adsorbed cells was studied in batch experiments. The immobilized cell system was shown to efficiently degrade high concentrations of the substrate (up to 2.0 g/l) and to remain active for more than 2 motths. The oxygen uptake rate of free and immobilized cells was determined at various concentrations of phenol. The kinetic constants K _s=85 mg/l, K _i=345 mg/l and SMI=170 mg/l were estimated from the experimental data by linearization of the Haldane function for the free cells. The uptake rates exhibited by the confined cells were lower (30%) than those obtained for free cells and no significant differences were found for phenol concentrations between 150 and 1200 mg/l.     

Preparation and properties of proteases immobilized on anion exchange resin with glutaraldehyde

High activity alkaline protease was obtained when the enzyme was immobilized on Dowex MWA-1 (mesh 20–50) with 10% glutaraldehyde in chilled phosphate buffer (M/15, pH 6.5). Activity yields of the protease and rennet were 27 and 29, respectively. The highest activities appeared at 60°C, pH 10 for alkaline protease and 50°C, pH 4.0 for rennet. The properties of both proteases were not essentially changed by the immobilization except that the K_m values of both enzymes were increased about tenfold as a result of immobilization. Both proteases in the immobilized state were more stable than those in the free state at 60°C. Other peptide hydrolases, β-galactosidase, invertase, and glucoamylase, were successfully immobilized with high activities, but lipase, hexokinase, glucose-6-phosphate dehydrogenase, and xanthine oxidase became inactive.     

Performance of Aspergillus niger B 03 β-xylosidase immobilized on polyamide membrane support

The dynamics of β-xylosidase biosynthesis from Aspergillus niger B 03 was investigated in laboratory bioreactor. Maximum xylosidase activity 5.5 U/ml was achieved after 80 h fermentation at medium pH 4.0. The isolated β-xylosidase was immobilized on polyamide membrane support and the basic characteristics of the immobilized enzyme were determined. Maximum immobilization and activity yield obtained was 30.0 and 6.8%, respectively. A shift in temperature optimum and pH optimum was observed for immobilized β-xylosidase compared to the free enzyme. Immobilized enzyme exhibited maximum activity at 45 °C and pH 4.5 while its free counterpart at 70 °C and pH 3.5, respectively. Thermal stability at 40 and 50 °C and storage stability of immobilized β-xylosidase were investigated at pH 5.0. Kinetic parameters K_m, V_max and K_i were determined for both enzyme forms. Free and immobilized β-xylosidase were tested for xylose production from birchwood xylan. The substrate was preliminarily depolymerized with xylanase to xylooligosaccharides and the amount of xylose obtained after their hydrolysis with free and immobilized β-xylosidase was determined by HPLC analysis. Continuous enzyme hydrolysis of birchwood xylan was performed with xylanase and free or immobilized β-xylosidase. The maximum extent of hydrolysis was 25 and 30% with free and immobilized enzyme, respectively. Immobilized preparation was also examined for reusability in 20 consecutive cycles at 40 °C.     

Bioaffinity Based Oriented Immobilization of Stem Bromelain

Bromelain is a basic, 23.8 kDa thiol proteinase obtained from stem of the pineapple plant (Ananas comosus) and is unique in containing a single oligosaccharide chain attached to the polypeptide. This property allowed its affinity binding and favorable orientation on a Sepharose support pre-coupled with the lectin, concanavalin A (Con A). For comparison, bromelain was also immobilized by covalently coupling to the CNBr-activated Sepharose. The preparation obtained was more resistant to thermal inactivation as evident from the retention of over 50% activity after incubation at 60 for 100 min (as compared to 20% retained by the native enzyme and 30% retained by the covalently immobilized enzyme), exhibited a broader pH-activity profile with the enzyme retaining over 60% activity at pH 11 (as compared to over 25% retained by native and the enzyme immobilized covalently). The native, covalently-coupled and affinity-bound bromelains had apparent K _m values of 1.1, 2 and 0.54 mg/ml, respectively using casein as the substrate. The V _max values remained unaffected on immobilization.     

α-d-Galactosidase immobilized on a soluble polymer

α-d-Galactosidase (α-d-galactoside galactohydrolase, EC 3.2.1.22) from green coffee beans has been immobilized by attachment to cyanogen bromide-activated Dextran T-70. Since this represents the first reported example of the preparation of a water-soluble derivative of an enzyme showing substrate inhibition, the kinetic properties, thermal stability and pH optima were investigated and compared with those of the free enzyme. The K_m, K_s, K_i, V_max, optimum substrate concentration and optimum pH were all lower than those of free enzyme. The enzyme conjugate showed greater resistance than the free enzyme to thermal inactivation. These data, although obtained with the synthetic substrate 4-nitrophenyl-α-d-galactoside, suggest some advantages in using the enzyme conjugate for the removal of terminal α-d-galactopyranosyl groups from the erythrocyte cell surface.     

Active protein and substrate flow effects in a tubular immobilized invertase reactor

Investigations of invertase (EC 3.2.1.26) immobilized inside modified nylon tubes showed that between 4% and 20% (w/w) of the protein exposed to binding sites on the tube was immobilized. An enhanced activity consistent with enzyme purification during immobilization was also evident, suggesting that, in scaled-up commercial applications, nylon tube invertase would be a more economical converter of sucrose than the free enzyme. The quantity and specific activity of the immobilized protein were not stochiometrical with the amount used in the coupling solution and, in the system studied, a concentration of 2 mg ml^?1 was optimal. K_m and V_max values confirmed higher rates of immobilized invertase catalysis when the rates of substrate flow through the reactor were higher. Higher rates of substrate flow imply a shortened residence time in the reactor and would lower the fractional conversion per pass of the substrate, reducing the efficiency of the reactor in flow-through situations. Thus, these higher catalysis rates, attributable at the higher flow rates to a reduction of the diffusion barrier between enzyme and substrate, would not translate into improved economy in the commercial flow-through processes at which the reactor is aimed.     

Adsorption of peroxidase on Celite 545 directly from ammonium sulfate fractionated white radish (Raphanus sativus) proteins

This paper demonstrates the direct immobilization of peroxidase from ammonium sulfate fractionated white radish proteins on an inorganic support, Celite 545. The adsorbed peroxidase was crosslinked by using glutaraldehyde. The activity yield for white radish peroxidase was adsorbed on Celite 545 was 70% and this activity was decreased and remained 60% of the initial activity after crosslinking by glutaraldehyde. The pH and temperature-optima for both soluble and immobilized peroxidase was at pH 5.5 and 40°C. Immobilized peroxidase retained higher stability against heat and water-miscible organic solvents. In the presence of 5.0 mM mercuric chloride, immobilized white radish peroxidase retained 41% of its initial activity while the free enzyme lost 93% activity. Soluble enzyme lost 61% of its initial activity while immobilized peroxidase retained 86% of the original activity when exposed to 0.02 mM sodium azide for 1 h. The K_m values were 0.056 and 0.07 mM for free and immobilized enzyme, respectively. Immobilized white radish peroxidase exhibited lower V_max as compared to the soluble enzyme. Immobilized peroxidase preparation showed better storage stability as compared to its soluble counterpart.     

Kinetic and thermodynamic properties of purified alkaline protease from Bacillus pumilus Y7 and non‐covalent immobilization to poly(vinylimidazole)/clay hydrogel

The characterization of the hydrogel was performed using Fourier‐transform infrared spectroscopy, X‐ray diffraction, and scanning electron microscopy. Purified Bacillus pumilus Y7‐derived alkaline protease was immobilized in Poly (vinylimidazole)/clay (PVI/SEP) hydrogel with 95% yield of immobilization. Immobilization decreased the pH optimum from 9 to 6 for free and immobilized enzyme, respectively. Temperature optimum 3°C decreased for immobilized enzyme. The K_m, V_m, and k_cat of immobilized enzyme were 4.4, 1.7, and 7.5‐fold increased over its free counterpart. Immobilized protease retained about 65% residual activity for 16^th reuse. The immobilized protease endured its 35% residual activity in the material after six cycle's batch applications. The results of thermodynamic analysis for casein hydrolysis showed that the ΔG^≠ (activation free energy) and ΔG^≠_E‐T (activation free energy of transition state formation) obtained for the immobilized enzyme decreased in comparison to those obtained for the free enzyme. On the other hand, the value of ΔG^≠_ES (free energy of substrate binding) was observed to have increased. These results indicate an increase in the spontaneity of the biochemical reaction post immobilization. Enthalpy value of immobilized enzyme that was 2.2‐fold increased over the free enzyme indicated lower energy for the formation of the transition state, and increased ΔS^≠ value implied that the immobilized form of the enzyme was more ordered than its free form.     

Inhibition of choline oxidase by quinoid dyes

Choline oxidase catalyzes the oxidation of choline to glycine-betaine, with betaine-aldehyde as intermediate and molecular oxygen as primary electron acceptor. This study reports on the inhibitory effects of triarylmethanes (cationic malachite green; neutral leukomalachite green), phenoxazines (cationic, meldola blue and nile blue; neutral nile red) and a structurally-related phenothiazine (methylene blue) on choline oxidase, assayed at 25°C in 50 mM MOPS buffer, pH 7, using choline as substrate. Methylene B acted as a competitive inhibitor with K_i = 74 ± 7.2 μM, pointing to the choline–binding site of the enzyme as a target site. Nile B caused noncompetitive inhibition of enzyme activity with K_i = 20 ± 4.5 μM. In contrast to methylene B and nile B, malachite G and meldola B caused complex, nonlinear inhibition of choline oxidase, with estimated K_i values in the micromolar range. The difference in kinetic pattern was ascribed to the differential ability of the dyes to interact (and interfere) with the flavin cofactor, generating different perturbations in the steady-state balance of the catalytic process.     

The properties of exo-1.4-alpha-D-glucosidase (E.C.3.2.1.3) covalently bound to mycelium

Summary The values of K_m, E_a, and K_i (for product inhibition) of glucoamylase covalently bound to the waste mycelium were determined as 7.9mM, 71.4 kJ/mol, and 3.2 M resp. using maltose as the substrate. The bound enzyme is not subject to diffusion limitation in the range of the concentration used.     

Effects of some drugs on the activity of glucose 6-phosphate dehydrogenase from rainbow trout (Oncorhynchus mykiss) erythrocytes in vitro

Inhibitory effects of some drugs on glucose 6-phosphate dehydrogenase from the erythrocytes of rainbow trout (Oncorhynchus mykiss Walbaum, 1792) were investigated. The enzyme was purified 2488-fold in a yield of 76.8% using ammonium sulfate precipitation and 2′,5′-ADP Sepharose 4B affinity gel at 4°C. The drugs pental sodium, MgSO₄, vancomycin, metamizol, marcaine, and prilocaine all exhibited inhibitory effects on the enzyme. While MgSO₄ (K_i = 12.119 mM), vancomycin (K_i = 1.466 mM) and metamizol (K_i = 0.392 mM) showed competitive inhibition, pental sodium (K_i = 0.748 mM) and marcaine (K_i = 0.0446 mM) displayed noncompetitive inhibition.     

Diamine oxidase ofLathyrus sativus seedlings. Purification and properties

Diamine oxidase (EC 1.4.3.6) was purified from 5-day-old etiolated seedlings ofLathyrus sativus by MnCl₂ treatment, (NH₄)₂SO₄ and acetone fractionations, DEAE-Sephadex chromatography followed by gel filtration on Sephadex G-200. A single step purification of the enzyme was achieved by using an immunoaffinity column, wherein rabbit antibodies to the homogeneous diamine oxidase were coupled to CNBr-activated Sepharose. The enzyme thus obtained was homogeneous by electrophoretic, immunological and ultracentrifugal criteria. It had anM _r of 148,000 (6.46S) and was a dimer with similar sub-units (M _r 75,000). Amino acid analysis showed the absence of cysteine residues although it contained five disulphide bonds. The enzyme had copper (2.7 g atom/mol enzyme) but was not a glycoprotein. No absorption maximum in the visible region was detectable. Ethylenediamine 1,3-diaminopropane and histamine were potent competitive inhibitors for the substrate putrescine. The addition of monospecific antibodies to the enzyme increased the K_m for benzyl amine without any change in the V_max Diamine oxidase from pea seedling, partially purified, exhibited complete crossreactivity with the antibodies to theL. sativus enzyme.     

Novel polymeric microspheres: Synthesis,enzyme immobilization,antimutagenic activity,and antimicrobial evaluation against pathogenic microorganisms

New polymeric microspheres containing azomethine ( 1a ‐ 1c and 2a ‐ 2c ) were synthesized by condensation to compare the enzymatic properties of the enzyme glucose oxidase (GOx) and to investigate antimutagenic and antimicrobial activities. The polymeric microspheres were characterized by elemental analysis, infrared spectra (FT‐IR), proton nuclear magnetic resonance spectra, thermal gravimetric analysis, and scanning electron microscopy analysis. The catalytic activity of the glucose oxidase enzyme follows Michaelis‐Menten kinetics. Influence of temperature, reusability, and storage capacity of the free and immobilized glucose oxidase enzyme were investigated. It is determined that immobilized enzymes exhibit good storage stability and reusability. After immobilization of GOx in polymeric supports, the thermal stability of the enzyme increased and the maximum reaction rate (V_max) decreased. The activity of the immobilized enzymes was preserved even after 5 months. The antibacterial and antifungal activity of the polymeric microspheres were evaluated by well‐diffusion method against some selected pathogenic microorganisms. The antimutagenic properties of all compounds were also examined against sodium azide in human lymphocyte cells by micronuclei and sister chromatid exchange tests.     

Studies on the stability of immobilized xanthine oxidase and urate oxidase.

The stability of immobilized preparations of xanthine oxidase and urate oxidase was studied, and optimized, because of the potential joint use of both enzymes in clinical analysis. Xanthine oxidase was immobilized on cellulose, Sepharose, hornblende, Enzacryl-TIO, and porous glass. Thehalf-lives of these preparations at 30 degree C ranged from 40 min to 5.0 hr. In this respect immobilized enzyme resembled soluble enzyme in dilute solution (0.11 mg/ml), when the half-live was about 3.5 hr. More concentrated enzyme solution (1 mg/ml) had a half-life of 64 hr, and was, therefore, considerably more stable than the untreated immobilized xanthine oxidase preparations. Inclusion of albumen in storage and assay buffer increased the half-life of bound xanthine oxidase. So also did treatment with glutaraldehyde: in the case of xanthine oxidase bound to Enzarcyl-TIO such treatment increased the half-life at 30 degree C from 3 hr to about 100 hr. Immobilized xanthine dehydrogenase was more stable than immobilized xanthine oxidase: the dehydrogenase lost no activity during continuous assay for 5 hr at 30 degree C. The stability of immobilized urate oxidase depended on the quantity of enzyme used and on the time of stirring during immobilization: thus a preparation was made (by stirring urate oxidase (48 mg/g support) with Enzacryl-TIO for 24 hr) which lost no activity during 350 hr at 30 degree C.