Immobilization of fumarase and malate dehydrogenase on agarose efficiency of activity as a function of the enzyme ratio

Two enzymes, L-malate dehydrogenase, L-malate: NAD⁺ oxidoreductase (EC 1.1.1.37) and fumarase, L-malate hydrolyase (EC 4.2.1.2) were immobilized on a (Sepharose 4B) resin by the cyanogen bromide method. Studies showed that the matrix-immobilized fumarase retains the same characteristics as the free enzyme, while the matrix-immobilized malate dehydrogenase has reduced activity. The activity of the coupled enzymes is more enzymeconcentration dependent than the free enzymes, and at a ratio of 0.3 (fumarase: malate dehydrogenase) the simultaneously coupled immobilized enzymes become a better catalytic system. Individually immobilized enzymes, mixed to form a coupled system, yielded the poorest catalytic action.     

Synthesis of the bifuncttonal dinucleotide AMP-ATP and its application in general ligand affinity chromatography

A new AMP derivative substituted with spacer arms both at position N⁶ and C8 of the adenine moiety was synthesized and immobilized to Sepharose. To the immobilized ligand was subsequently coupled C8-substituted ATP in a solid-phase synthesis fashion yielding the bifunctional general ligand AMP-ATP. This affinity material was used in the separation of two major groups of enzymes, dehydrogenases and kinases. It was found that on passage of crude homogenates obtained from mouse kidney through the affinity column, several dehydrogenases and kinases were bound, which could be eluted separately using pulses of NADH and ATP, respectively. In the fractions obtained on NADH elution, lactate dehydrogenase, malate dehydrogenase, and α-glycerol phosphate dehydrogenase were found, whereas ATP eluted 3-phosphoglyceric acid kinase, pyruvate kinase, and aldolase.     

A coupled enzyme nylon tube reactor pyruvate kinase-lactate dehydrogenase system

A coupled enzyme nylon tube reactor has been made by simultaneously immobilizing rabbit muscle pyruvate kinase (EC 2.7.1.40) and lactate dehydrogenase (EC 1.1.1.27) onto the inside surface of a nylon tube initially derivatized with poly (scl)-lysine to serve as a spacer molecule. The enzymes were covalently linked to the amino groups of the poly lysine spacer molecule by cross-linking with glutaraldehyde. The coupled enzyme system may be used in routine analysis to determine the concentrations of PEP, ADP, pyruvate, and NADH in a given solution and also to modify radioactively labeled nucleotides. The kinetic properties of the enzymes appear to be partially diffusion controlled as shown by an inverse relationship ofK _m(app) values and activities to the flow rate of substrates through the tube reactor. This coupled enzyme system may be used as an indicator system when used in conjunction with other enzymes to complete a sequence of catalytic steps. An example of this is demonstrated in this paper by linking a nylon tube supported acetate kinase (EC 2.7.2.1) to this coupled enzyme system so that the three enzymes function in a series that facilitates the estimation of acetate.     

Recycling of NADP+ using immobilizedE. coli and glucose-6-phosphate dehydrogenase

Recycling of NADP⁺ using immobilized wholeEscherichia coli cells as source of respiratory chain, glucose-6-phosphate, and soluble yeast glucose-6-phosphate dehydrogenase (1.1.1.49) is described. NADP⁺ was recycled more than 10-fold. We demonstrated NADPH respiration at pH 5.8 inE. coli membrane vesicles. The respiratory chain was involved most probably in NADPH oxidation.

1. The respiratory activity is localized at the level of the inner bacterial membrane. The active site for NADPH facing the cytoplasm.
2. NADPH respiration is inhibited by 10 mM cyanide, similar to the conditions of inhibition of NADH respiration.
3. NADPH dehydrogenase activity seems to be the limiting step of the respiratory chain:K _M for NADPH respiration and NADPH dehydrogenase activity are similar. The pH optima for these two activities are also comparable (around pH 5.8). Furthermore, the following properties are rather in favor of a common NADH dehydrogenase and NADPH dehydrogenase activity (1.6.99.2).

o| li](1)|At saturating concentrations of NADH and NADPH, neither respiration nor dehydrogenase activities were additive. li](2)|Similar heat inactivation kinetics were observed for NADH and NADPH dehydrogenase-activity. Protection against heat inactivation was obtained for the two activities with NAD⁺, NADP⁺, NADH, and NADPH. All these results suggested the possibility of recycling of NADP⁺ under similar conditions to those previously described for NAD⁺ (Burstein et al., 1981). It becomes thus possible to use various NAD⁺ and NADP⁺-dependent dehydrogenases in enzyme technology.     

Immobilized citrate synthase

Kinetic properties of pig heart citrate synthase immobilized on Sepharose were determined. Compared to the free enzyme the K_m values for both acetyl-CoA and oxalacetate were increased. The kinetic pattern of the Lineweaver-Burk plots of both substrates for the immobilized enzyme was that of lines intersecting on thex axis. This is the same as that obtained for the free enzyme and indicates that there is no change in the kinetic mechanism of the reaction. The pH response and the Arrhenius plot of both the immobilized and free enzyme were the same. The enzymes show a break in their Arrhenius plots. The immobilized enzyme exhibits greater heat stability than does the free enzyme.     

Kinetics of immobilized pig heart fumarase

The kinetics of immobilized pig heart fumarase are described and compared with the properties of the enzyme free in solution.

1. An analogous pH dependence of initial activity is found for free and immobilized enzyme.
2. Immobilized and free fumarase deviate from classical Michaelis-Menten kinetics in the same way. The apparent K_m values are three to eight times higher for the immobilized (2 mg/g gel) enzyme.
3. The specific activity of immobilized fumarase is dependent on the final enzyme concentration on the gel; normal specific activities are observed when 50 ?g fumarase is immobilized per gram of gel, whereas the specific activity decreases with increasing enzyme concentration.
4. The activation energies for free and immobilized fumarase (50 ?g/g gel) were found to be identical between 22 and 32?C and with L-malate as substrate (E_a = 12,290 cal/mol at pH 7.9). Upon increasing the concentration of fumarase on the gel, the activation energy decreases.

Our results indicate that the true catalytic properties of fumarase are not affected by immobilization of this enzyme. The slight differences observed when fumarase is immobilized at concentrations higher than 50 ?g/g gel must be attributed to diffusional limitation at the surface of the Sepharose matrix.     

Characterization of an immobilized antibody-enzyme complex

Antiserum specific for propanediol dehydrogenase, an enzyme found inNeisseria gonorrhoeae cells, has been immobilized to glass. When mixed withN gonorrhoeae cell lysates, the immobilized antibody (IMA) binds the enzyme. Over 70% of the calculated adsorbed activity can be recovered from the immobilized antibody-enzyme (IMA-E) complex. When mixed with bacterial lysates prepared from different organisms having propanediol dehydrogenase-like activity, the IMA specifically adsorbed the enzyme from theN gonorrhoeae lysate. IMA-E complexes have been prepared and their kinetic, temperature and chemical stability, and antigenic properties investigated. These studies demonstrated the feasibility of using an immobilized antibody in the detection of the propanediol dehydrogenase enzyme.     

Grease immobilized on polyethyleneimine cotton cloth

Jack bean urease was immobilized on polyethylenimine-coated cotton cloth by adsorption following by crosslinking with dimethyl suberimidate. Of the various methods used, crosslinking with dimethyl suberimidate was found to stabilize the enzyme with minimal inactivation. Cloth-bound urease showed a shift in pH optimum towards the acidic side without appreciable change in temperature optimum and thermostability. Cloth-bound urease could be used repeatedly for urea hydrolysis without appreciable loss in activity. Alternatively, urease cloth could be stitched in the form of a bag containing ammonia adsorbent and used for urea hydrolysis with simultaneous removal of ammonia.     

Micellar solubilization of biopolymers in hydrocarbon solvents. I. a structural model for protein-containing reverse micelles

Three proteins (horse liver alcohol dehydrogenase, ribonuclease, lysozyme) were solubilized in hydrocarbon with the help of reverse micelles formed by aqueous di(2-ethyl-hexyl) sodium sulfosuccinate (AOT). Sedimentation and diffusion coefficients of the micellar aggregates were measured with an analytical ultracentrifuge. Partial specific volumes were also evaluated from density measurements. The molecular weight of the protein-containing reverse micelles (M _t ) could thus be determined for each protein system at various w₀ values (w₀ - [H₂O]/[AOT]). For horse liver alcohol dehydrogenase at w₀ = 46.4, for example,M _t is ca. 2,670,000 Daltons; for lysozyme at wo = 22.5,M _t is ca. 323,000 Daltons and increases by increasing w₀. On the basis of these experimentally determined molecular weights, a structural model for the protein-containing reverse micelle is proposed. The model is based upon the assumption that the protein is confined in the water pool of a spherical micelle, and that the inner core volume is the sum of the protein volume and the volume of all water molecules present in a micelle. It is possible then to calculate the micellar structure at each w₀ value. For example, in the case of ribonuclease at w₀ = 20, the inner core radius is ca. 37.5 A, and a layer of water of ca. 22 A separates the protein surface from the surfactant layer. The possible implications of this model for the reactivity of enzymes solubilized in hydrocarbons by reverse micelles are discussed.     

Analysis of the reaction kinetics of single esterase sepharose beads by microfluorometry of the fluorogenic substrate turnover

Heterogeneous catalysis of esterase (E.C. 3.1.1.1) immobilized on CNBr-Sepharose 4B was analyzed by microfluorometry. The hydrolysis of fluorescein diacetate was measured within single esterase-Sepharose beads during steady-state turnover. Fluorescence intensity profiles, total intensities, and reaction rates of single beads were measured by means of a microfluorometer. Their suitability for determining the kinetic constants of the system was evaluated by comparison with the theoretical values of the kinetic model. The computation was based on the kinetic equations describing the interaction of internal diffusion and enzymatic reaction with noncompetitive product inhibition within spherical particles. A method is described which can be used to derive the true K_m of the immobilized enzyme from a correlation of intensity- and turnover-dependent apparent K_m—s. Effectiveness factors and Damkohler numbers for individual esterase-Sepharose beads were determined from relative total fluorescence intensities and fluorescence intensity profiles, respectively.     

Biological conversion of coal gas to methane

Biological conversion of low-Btu coal synthesis gas to higher Btu methane was demonstrated using both pure co-cultures and/or adapted-mixed anaerobic bacteria.Peptostreptococcus productus metabolized coal gas to mainly acetate and CO₂. The co-cultures containing methanogens converted these products to methane. In mixed culture studies, CH₄ and small amounts of acetate were produced. Reactor studies using stirred-tank and immobilized cell reactors exhibited excellent potential to convert CO, CO₂ and H₂ to methane at higher gas flow rates. Gas retention times ranging from 0.7 to 2 hours and high agitation were required for 90 percent CO conversion in these systems. This paper also illustrates the potential of biological methanation and demonstrates the need for good mass transfer in converting gas phase substrates.     

Characterization of a Novel Agarose–Nickel Composite Matrix for Protein Nanoparticles Affinity Chromatography in Expanded Bed

The novel agarose–nickel (Ag–Ni) expanded bed matrix was investigated with regard to suitability for practical recovery of nano-bioproducts (NBPs) such as protein nanoparticles as drug delivery carriers. The matrix was immobilized by Reactive Green 19 (RG19) dye–ligand and was subjected to biochemical evaluation through batch adsorption studies (isotherm and kinetic studies) and column chromatography of bovine serum albumin nanoparticles (BSA NPs) with average size of 85–95 nm as a model system. Based on adsorption isotherm investigations, the adsorption phenomenon appeared to follow the Langmuir isotherm model with maximum binding capacity of 24.9 mg/ml adsorbent. Subsequently adsorption data were modeled using the pseudo-first-order and pseudo-second-order kinetics equation. The results demonstrated that the adsorption process kinetics followed the pseudo-first-order kinetic model. The dynamic binding capacity (DBC) for BSA NP adsorption was calculated at various flow velocities which showed favorable column efficiency at relatively high flow rates. BSA NPs recovery was studied in the expanded bed column which resulted in 74 % recovery. The results indicated that the novel resin is a promising chromatographic medium for protein nanoparticle separation with high adsorption capacity and column efficiency at reasonably high flow rates. The generic application of such dye–ligand immobilized composite matrix for the adsorption and purification of BSA NPs as a nanoparticulate bioproduct was discussed.     

Efficiency of two enzymes immobilized to the same surface and acting in sequence

Phosphoglucomutase and glucose-6-phosphate dehydrogenase were immobilized to s-triazine trichloride activated cellulose. The optimal conditions for binding the immobilized enzymes were determined and the kinetic and physical properties were investigated.The final ratio of the two enzymes immobilized to the surface was determined by the physical properties of the enzymes as well as by the ratio of the enzymes present in the attachment solution. The immobilized enzymes were found to retain at least 60% of the original activity for at least 40 days when stored at 4°C and in the presence of substrates and cofactors. Immobilized phosphoglucomutase and glucose-6-phosphate dehydrogenase also were much more stable at 58°C, retaining 28 and 13% of the original activity, respectively, after 75 min. The apparentK _m’s were 5.4 to 1.5 times higher for immobilized phosphoglucomutase and glucose-6-phosphate dehydrogenase, respectively than for the soluble enzymes.     

Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

Immobilization of photosystem I or II complexes on electrodes for preparation of photoenergy-conversion devices

Photosystems, PSI and PSII isolated from Thermosynechococcus elongatus were successfully immobilized on a TiO₂ nanostructured film for use in dye-sensitized biosolar cells (DSBCs). The photosystem complexes were also immobilized on an ITO electrode modified with 3-aminopropyltriethoxysilane by utilizing the interactions between the electrode and the surface of the PSI or PSII polypeptide. Illumination of the PSI and PSII complexes immobilized on the ITO electrode resulted in action spectra in the presence of methyl viologen, which corresponded to the absorption spectra of the complexes. Compared with the ITO electrode, PSI or PSII complexes assembled on the TiO₂ electrode had much higher energy-conversion efficiency in the presence of an iodide/triiodide redox system of an ionic-liquid-based electrolyte. This could have interesting applications in the development of DSBCs.     

Ion beam irradiation of ZnS dispersed in PMMA and its photocatalytic application

ZnS nanoparticles implanted with 45 keV O⁵⁺ ion beam exhibited 83.6 % degradation of methyl blue in 2 h. This idea was utilized to fabricate nanocomposite system of ZnS and PMMA where ZnS nanoparticles were immobilized in PMMA film and irradiated with 45 keV O⁵⁺ ion beam at particle fluence of 2.5 × 10¹⁵, 1 × 10¹⁶ and 4 × 10¹⁶ particles/cm². These irradiated batches of ZnS nanoparticle immobilized in PMMA batches revealed formation of porous structure characterized by scanning electron microscopy and these batches exhibited 54 % photocatalytic degradation of methyl blue in 80 min which was higher as compared to the pristine ZnS nanoparticles.     

Pigeonpea (Cajanus cajan L.) urease immobilized on glutaraldehyde-activated chitosan beads and its analytical applications

Urease from pigeonpea (Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was observed at 4°C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05 M Trisacetate buffer, pH 7.3, at 4°C had a t _1/2 of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized urease showed a significantly higher Michaelis constant (8.3 mM) compared to that of the soluble urease (3.0 mM). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77°C, compared with 47°C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations.     

Purification and Characterization of Paraoxonase 1 (PON1) from Swiss Black,Holstein, and Montofon Bovines

Paraoxonase 1 (PON1: EC 3.1.8.1) is a calcium-dependent enzyme associated with high-density lipoproteins (HDLs) and has a protective effect against oxidation of low-density lipoproteins (LDLs) in mammals. PON1 is the best-studied member of a family of enzymes called serum paraoxonases, or PONs, identified in mammals and other vertebrates as well as in invertebrates. PONs exhibit a range of important activities, including drug metabolism and detoxification of organophosphates such as nerve agents. This study reports, for the first time, purification and biochemical characterization of serum PON1 from different bovine breeds namely Swiss Black, Holstein, and Montofon. Bovine serum PON1s were purified using ammonium sulfate precipitation followed by Sepharose-4B-l-tyrosine-1-naphthylamine hydrophobic interaction chromatography. SDS–polyacrylamide gel electrophoresis of the purified enzymes indicates a single band with an apparent MW of 43 kDa. The purified enzymes had a specific activity of 10.78, 27.00, and 22.38 U/mg for Swiss Black, Holstein, and Montofon bovines, respectively. The overall purification rates of our method were 262.47-, 2,476.90-, and 538.06-fold for Swiss Black, Holstein, and Montofon bovines, respectively. Furthermore, using phenyl acetate as a substrate, we determined the K _M and V _max values of the purified enzymes, as 0.80 mM, 1428.5 U/ml for Swiss Black; 0.40 mM, 714.3 U/ml for Holstein; and 0.50 mM, 1,111.1 U/ml for Montofon bovine. The present study has revealed that there is no substantial difference in PON1 activities among the studied bovine breeds.     

Preparation,characterization, and luminescence of (SBA-15) immobilized pepsin

SBA-15 mesoporous silica was synthesized by hydrothermal method and its surface was methylated by treatment with methyltrimethoxysilane. Pepsin was immobilized on the obtained materials giving host-guest composite materials (SBA-15)-pepsin and (methylated SBA-15)-pepsin. The optimum conditions for preparation of these materials were established. Methylated SBA-15 (M-SBA-15) has improved immobilization efficiency of enzyme compared to initial SBA-15 silica. It was shown that with the gradual increase of NaCl solution ionic strength the immobilized amount of enzyme was reduced. Powder X-ray diffraction and Fourier transform infrared spectroscopy showed that the host frameworks in the prepared host-guest composite materials are intact and the ordered structure was retained. Scanning electron microscopic studies revealed fibrous morphologic characteristics of the SBA-15 and the immobilized pepsin composite materials. The average particle diameter of (SBA-15)-pepsin composite was 338 ± 10 and 343 ± 10 nm for (M-SBA-15)-pepsin. The low temperature N₂ adsorption-desorption study at 77 K showed that the pore sizes and specific surface areas of the host-guest composite materials were smaller than those before the introduction of the enzyme, suggesting that the immobilized enzyme occupied a definite position in the host material pore channels. The UV-vis solid diffuse reflectance and luminescence studies showed that the enzyme was successfully immobilized on to the host material and that after the immobilization of enzyme on SBA-15 the conformation of pepsin macromolecule has not been changed.     

Experimental studies of immobilized-yeast,packed-bed reactors with reduced CO2 entrapment

A horizontal packed-bed reactor with baffles (HPBR) and a continuously stirred tank reactor with intermittent paddle agitation have been shown to considerably reduce the CO₂ entrapment when glucose is fermented with immobilized baker’s yeast in calcium alginate beads. Using high cell contents in the gel resulted in internal mass transfer hindrance. The highest productivity was obtained with the HPBR giving 29 g EtOH/Lh at an ethanol yield of 90%. The substrate used was 100 g/L glucose. Fermentation of lactose and deproteinized whey by coimmobilized baker’s yeast and |3-galactosidase resulted in much lower productivity-about 5 g EtOH/L.h because of the slow fermentation of galactose.