Fluid immobilized cellulase

Summary Fluid immobilized cellulase was prepared using polyethyleneglycols and hexamethylene diisocyanate, and its properties studied. The cellulase activity of the immobilized enzymes varied with monomer composition and molecular weight of polyethyleneglycols. The enzyme activity was affected by the viscosity of the carrier. A solid substrate (cellulose powder) can be hydrolyzed with the fluid immobilized enzyme.     

Enzyme immobilization by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperatures.

Enzyme immobilization by radiation-induced polymerization of hydrophilic glass-forming monomers, such as 2-hydroxyethyl methacrylate, was studied. Enzyme radiation damage could be sufficiently retarded at low temperatures. The immobilized enzyme activity yield was markedly higher at low temperature than at higher temperature polymerization. At low temperatures the polymerized composite had a porous structure owing to ice crystallization which depends on the monomer concentration. It was deduced that the enzyme was partially trapped on the polymer surface, partially isolated in the pore, and partially occluded inside the polymer matrix. A decrease in activity caused by enzyme leakage was observed with repeated use in enzyme reactions where the composites had a large porosity. The activity yield showed a maximum at certain optimum porosities, i.e., at optimum monomer concentrations. Continuous enzyme reaction was preferably carried out using immobilized enzyme columns.     

Polyacrylamide gel immobilization of porcine liver esterase for the enantioselective production of levofloxacin

Porcine liver esterase was immobilized in polyacrylamide gel for the enantioselective production of levofloxacin from ofloxacin butyl ester. The initial activity of immobilized esterase was found to be significantly affected by the polyacrylamide gel composition. The optimum concentrations of monomer and crosslinker were determined to be 20% and 8.3%, respectively. The activity of immobilized esterase was 55.4% compared to a free enzyme. Enantiomeric excess was maintained at 60%, almost the same level as that of free enzyme. In addition, the immobilized esterase could be used repeatedly up to 10 times without experiencing any severe loss of activity and enantioselectivity.     

Latex particles with thermo-flocculation and magnetic properties for immobilization of alpha-chymotrypsin

Core-shell-type latex particles composed of styrene, N-isopropylacrylamide (NIPAAm), and N-acryloxysuccinimide (NAS) were synthesized by surfactant-free emulsion polymerization. The latex particles show thermo-flocculation behavior due to the presence of temperature-sensitive monomer NIPAAm and could be used for immobilization of alpha-chymotrypsin through covalent bonding with the reactive ester groups of NAS. Enzyme recycle could be accomplished in this immobilized enzyme system by sedimentation of the thermo-flocculated latex particles in 20 min at 30 degrees C by raising the salt (NaCl) concentration to 0.5 M. To further enhance the sedimentation rate, ultrafine magnetite particles were prepared and included during polymerization to produce magnetic temperature-sensitive latex particles (MTLP), which could be recovered 6 times faster after thermo-flocculation by applying a low magnetic field. However, a higher salt concentration was necessary to flocculate the MTLP under the same condition as a result of its increased surface hydrophilicity, which originates from different polymerization conditions and the incorporation of magnetite. The immobilized enzyme shows high activity even against macromolecular substrates (hemoglobin and casein) owing to limited diffusion resistance, with full activity retention for nonmagnetic latex but one-half reduction in activity if the magnetic property was introduced. Optimal enzyme immobilization pH and enzyme loading were determined, and properties of the immobilized enzyme were characterized. The immobilized enzyme was used in 10 repeated batch hydrolyses of casein with successive flocculation/dispersion cycles and showed less than 15% activity decrease at the end. Overall, introducing the magnetic property to the latex could effectively enhance the solid-liquid separation rate after thermo-flocculation and maintain enzyme activity after repeated use but adversely influence the activity of the immobilized enzyme.     

Immobilization of phosphorylase B and study of its enzymatic and immunological properties]

The properties of phosphorylase B (PhB) immobilized on an agar derivative were studied. It was shown that the enzyme activity makes up to 15-20% as compared to that of the soluble enzyme, the Km value for glucose-1-phosphate is increased 1.5-fold and the pH optimum remains unchanged, whereas the thermostability of enzyme shows a considerable increase. PhB immobilized on a highly activated sorbent completely losses its enzymatic activity but retains its antigenic properties and binds 1.6-2 mol antibodies (per monomer). Using immunosorbents, purified antibodies homogeneous during electrophoresis in polyacrylamide gel were isolated. The immunosorbent capacity is 500-800 mg of antibodies per 1 g of dry weight. The purified antibodies are characterized by a lower inhibitory power upon interaction with soluble PhB. The type of inhibition of both immobilized and soluble enzyme is similar. It is assumed that immobilization produces conformational changes only at the active site of enzyme, which is spatially separated from the antibody binding site.     

Immobilization of Streptomyces phaerochromogenes by radiation-induced polymerization of glass-forming monomers.

Immobilization of Streptomyces phaerochromogenes was studied by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperatures. Radiation damage of the enzyme could be avoided by choosing irradiation at low temperatures. The enzymatic activity of immobilized cells increased remarkably with a decrease in the irradiation temperature of about -24 degrees C. In constrast to the case of cell-free enzyme immobilization, the most characteristic case was than in these immobilized cells, the enzymatic activity did not decrease with repeated use even in the composite obtained at much lower monomer concentrations. Another characteristic of immobilized cells was the increase in enzymatic activity in the initial stage of repeated use, which could be attributed to the swelling effect of the polymer matrix, thereby increasing the enzymatic activity of whole cells.     

Properties of Trichoderma reesei cells immobilized by the irradiation technique

Aerobic cells of Trichoderma reesei have been immobilized by the radiation polymerization technique using fibrous substances and hydroxyethyl methacrylate. The enzyme [cellulase, 1,4-(1,3;1,4)-β-d-glucan 4-glucanohydrolase, EC 3.2.1.4] productivity and growth of the cells in the immobilized growing cells have been studied. The enzyme (filter paper) activity in the immobilized cells was comparable to that of the intact cells, showing that the cells immobilized with fibrous materials grow and become adhered to the surface of the fibrils. The filter paper activity of the immobilized cells was affected mainly by monomer concentration and the content of the fibrous materials, as well as the irradiation dose. It was demonstrated that in repeated batch culture of the immobilized cells the filter paper activity gave a constant value, and leakage of the cells was not observed.     

A Method for Preparing Bead Shaped Immobilized Enzyme

Monomer solution containing enzyme was frozen to a small bead shape by cool solvent and polymerized by Co–60 gamma ray with a dose of 125~850 Krad.

Thus, bead shaped immobilized enzymes with various diameters (0.02 – 15mm) were obtained.

The bead had spongy structure and large surface area. Consequently, even large sized bead shaped immobilized enzyme showed high enzymic activity.

Retained activity of bead shaped immobilized invertase was 16~73% depending on bead size.

Bead shaped lipase (1 mm diameter) showed 13.5~42.5% of retained activity depending on monomer combinations.     

Evidence that the catalytic and regulatory functions of carbamylphosphate synthetase from Escherichia coli are not dependent on oligomer formation.

Carbamyl-phosphate synthetase from Escherichia coli is an allosteric enzyme which undergoes reversible association reactions in phosphate buffer. The positive allosteric effectors, ornithine, inosine 5'-monophosphate (IMP), and ammonia, facilitate oligomer formation, whereas uridine 5'-monophosphate (UMP), a negative effector, prevents or decreases oligomer formation. When the enzyme is immobilized by reaction with activated Sepharose, under conditions where the enzyme exists only as a monomer, nearly full catalytic activity is retained and the effects of ornithine, IMP, and UMP on the catalytic activity as a function of MgATP concentration are not significantly altered. Gel-filtration chromatography on Sephadex G-200 of catalytic quantities of the enzyme in the presence of all substrates showed that the elution volume was the same as that measured for the enzyme under conditions where it is known to exist in the monomer form. The specific activity of the enzyme does not increase when the concentration of the enzyme is increased 100-fold from a concentration at which the enzyme exists as monomer to a level at which the enzyme exists predominantly as oligomer. These results indicate that the monomer form of the enzyme is the principle active species and that oligomer formation is not directly related to enzyme activity or enzyme regulation.     

Immobilization of glucose oxidase in thin polypyrrole films: influence of polymerization conditions and film thickness on the activity and stability of the immobilized enzyme

Using monomers that polymerize to form electrically conducting polymers, one can control the thickness of the polymer film and the amount of enzyme that can be immobilized in the films. First, an investigation of the major variables that influence the immobilization of glucose oxidase by entrapment in polypyrrole films, prepared by electropolymerization from aqueous solutions containing the enzyme and monomer, was carried out. Then the optimized conditions were used to assess the effects of film thickness on the activity and stability of immobilized enzyme. For the films ranged in thickness from 0.1 mum to 1.6 mum, the resulting apparent activity and stability of the immobilized enzyme were found to be a strong function of the polymer film thickness. Above a thickness of 1.0 mum, the apparent activity of the immobilized enzyme increases linearly with increasing film thickness. The nonlinearity observed for films of thickness less than 1.0 mum can be attributed to the changes observed in the morphology of the resulting polypyrrole films. Furthermore, it was noted that when the glucose oxidase/polypyrrole films are stored in phosphate buffer, at 4 degrees C, the observed rate of loss in apparent activity of the immobilized enzyme is highest for the first few days, also being higher for the thinner films. However, after the loosely entrapped enzyme is leached from the polymer film, the rate of loss in activity is very low indicating that the well-entrapped enzyme, as well as the polypyrrole films, exhibit good stability. Finally, the reproducibility of the immobilization technique is excellent. (c) 1993 John Wiley & Sons, Inc.     

Properties of thermolysin immobilized in polymer matrix by radiation polymerization

Thermolysin (Bacillus thermoproteolyticus neutral proteinase, EC 3.4.24.4) has been immobilized by radiation polymerization of hydrophilic and hydrophobic monomers, and its properties, such as enzyme activity, thermal stability and durability, have been studied. The activity of the immobilized enzymes increased with an increase in the hydrophilicity of the polymer matrix and with a decrease in monomer concentration. Immobilization with hydrophilic monomers increased the thermal stability of the enzymes, but the thermal stability of the enzymes immobilized with hydrophobic monomers was comparable with that of native enzymes. The durability of the immobilized enzymes was examined by continuous hydrolysis of casein; enzymes immobilized with a high concentration (90%) of hydrophilic monomers appeared to be stabilized and could be used for long times.     

Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase

Sepharose-bound tetrameric, dimeric and monomeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase were prepared, as well as immobilized hybrid species containing (by selective oxidation of an active center cysteine residue with H2O2) one inactivated subunit per tetramer or dimer. The catalytic properties of these enzyme forms were compared in the forward reaction (glyceraldehyde-3-phosphate oxidation) and reverse reaction (1,3-bisphosphoglycerate reductive dephosphorylation) under steady-state conditions. In the reaction of glyceraldehyde-3-phosphate oxidation, immobilized monomeric and tetrameric forms exhibited similar specific activities. The hybrid-modified dimer contributed on half of the total activity of a native dimer. The tetramer containing one modified subunit possessed 75% of the activity of an unmodified tetramer. In the reaction of 1,3-bisphosphoglycerate reductive dephosphorylation, the specific activity of the monomeric enzyme species was nearly twice as high as that of the tetramer, suggesting that only one-half of the active centers of the oligomer were acting simultaneously. Subunit cooperativity in catalysis persisted in an isolated dimeric species. The specific activity of a monomer associated with a peroxide-inactivated monomer in a dimer was equal to that of an isolated monomeric species and twice as high as that of a native immobilized dimer. The specific activity of subunits associated with a peroxide-inactivated subunit in a tetramer did not differ from that of a native immobilized tetramer; this indicates that interdimeric interactions are involved in catalytic subunit cooperativity. A complex was formed between the immobilized glyceraldehyde-3-phosphate dehydrogenase and soluble phosphoglycerate kinase. Three monomers of phosphoglycerate kinase were bound per tetramer of the dehydrogenase and one per dimer. Evidence is presented that if the reductive dephosphorylation of 1,3-bisphosphoglycerate proceeds in the phosphoglycerate kinase - glyceraldehyde-3-phosphate dehydrogenase complex, all active sites of the latter enzyme act independently, i.e. subunit cooperativity is abolished.     

Immobilization and characterization of D-amino acid oxidase.

Optimal conditions with respect to pH, concentration of glutaraldehyde and enzyme, and order of addition of enzyme and crosslinking reagent were established for the immobilization of hog kidney D-amino acid oxidase to an attapulgite support. Yields of 40 to 70% were generally attained although when low concentrations of enzyme were used yields were consistently greater than 100%. It is suggested that this is due to a dimer leads to monomer shift at low protein concentrations. The stability of soluble D-amino acid oxidase was dependent on the buffer in which it was stored (pyrophosphate-phosphate greater than borate greater than Tris). Stability of immobilized enzyme was less than soluble in pyrophosphate-phosphate buffer, but storage in the presence of FAD improved stability. In addition, treatment of stored, immobilized enzyme with FAD before assay restored some of its activity. The immobilized D-amino acid oxidase was less stable to heat (50 degrees C) than the soluble enzyme from pH 6 to 8 but was more stable above and below these values. Apparent Km values for D-alanine, D-valine, and D-tryptophan decreased for the immobilized enzyme compared to the soluble.     

Immobilization of polyphenol oxidase in conducting copolymers and determination of phenolic compounds in wines with enzyme electrodes

Polyphenol oxidase (PPO) was immobilized in copolymers of thiophene functionalized menthyl monomer (MM) with pyrrole. Immobilization of enzyme was performed via entrapment in conducting copolymers during electrochemical polymerization of pyrrole. Maximum reaction rates, Michaelis-Menten constants and temperature, pH and operational stabilities of enzyme electrodes were investigated. Total amount of phenolic compounds in red wines of Turkey were analyzed by using these electrodes.     

Immobilization of alpha-chymotrypsin on poly(urethane-graft-acrylic acid)

A study was made of the immobilization of alpha-chymotrypsin (alpha-CT) onto a previously well characterized synthetic polyurethane grafted with acrylic acid P(U-g-AA). The P(U-g-AA) had previously been prepared using 2,2'-azo-bis-isobutyronitrile (AIBN) as a radical initiator and acrylic acid as monomer in the presence of an unsaturated polyurethane in solution at 60 degrees C. Some kinetic parameters of both the native enzyme and the enzyme immobilized on the P(U-g-AA) were evaluated. Using a Lineweaver-Burk plot (double reciprocal), it was found that the Michaelis-Menten constant (Km(for the immobilized enzyme was (4.0 +/- 0.9) x 10(-3) M and that of the free enzyme was (3.0 +/- 0.2) x 10(-3) M. The enzyme alpha-chymotrypsin was immobilized on the grafted polyurethane micelles/aggregates with about 45% retention of activity. Also the immobilized alpha-CT retained this activity for at least 6 weeks. The immobilized enzyme was found to have a maximum stability at 43 degrees C compared with 36 degrees C in the case of free enzyme, and the pH optimum was shifted from pH 6.6 to pH 8.2. The long-term operational stability of the enzyme was investigated and this is of interest since the enzyme is probably trapped physically in a micellar environment. The assay of the enzyme was carried out in 0.01 M phosphate buffer, pH 7.5, using p-nitrophenyl acetate as a substrate. No inhibition of alpha-CT in the presence of the synthetic ungrafted and grafted polyurethane was observed.     

Continuous production ofL-malic acid by immobilizedBrevibacterium ammoniagenes cells

Summary Continuous production ofL-malic acid from fumaric acid using immobilized microbial cells was investigated. Several microorganisms having fumarase activity were immobilized into a polyacrylamide gel lattice. Among the microorganisms tested, immobilizedBrevibacterium ammoniagenes IAM 1645 showed the highest enzyme activity, but produced an unwanted by-product, succinic acid. Conditions for suppression of this side reaction were investigated, and bile extract treatment of immobilized cells was found to be effective.The bile extract treatment of immobilized cells also resulted in a marked increase of reaction rate forL-malic acid formation.No difference was observed between the native enzyme and immobilized cells in optimal pH and temperature of the enzyme reaction.The effect of temperature on the reaction rate and the stability of fumarase activity of an immobilized cell column were investigated under conditions of continuous enzyme reaction. The decay of enzyme activity during continuous enzyme reaction was expressed by an exponential relationship. Half-life of the fumarase activity of the immobilized cell column at 37°C was calculated to be 52.5 days.Presented at the Annual Meeting of the Society of Fermentation Technology, Japan, Osaka, Japan, October 30, 1975.     

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.     

Preparation of a stable biocatalyst of bovine liver catalase using immobilization and postimmobilization techniques

Bovine liver catalase was immobilized on different supports. The tetrameric nature of this enzyme was found to cause its rapid inactivation in diluted conditions due to subunit dissociation, a fact that may rule out its industrial use. Multi-subunit immobilization using highly activated glyoxyl agarose was not enough to involve all enzyme subunits. In fact, washing the derivative produced a strong decrease in the enzyme activity. Further cross-linking of previously immobilized enzyme with tailor-made dextran-aldehyde permitted the multimeric structure to be fully stabilized using either multisubunit preparations immobilized onto highly activated glyoxyl-agarose support or one subunit enzymes immobilized onto poorly activated glyoxyl-agarose. The highest stability of the final biocatalyst was observed using the multisubunit immobilized derivative cross-linked with dextran-aldehyde. The optimal derivative retained around 60% of the immobilized activity, did not release any enzyme subunits after boiling in the presence of SDS, and did not lose activity during washing, and its stability did not depend on the dilution. This derivative was used for 10 cycles in the destruction of 10 mM hydrogen peroxide without any decrease in the enzyme activity.     

Enantioselective production of levofloxacin by immobilized porcine liver esterase

Porcine liver esterase, which cleaves ofloxacin butyl ester enantioselectively to levofloxacin, was successfully immobilized in calcium alginate and polyacrylamide gel. Immobilized esterase in 5% (w/v) calcium alginate exhibited 58% immobilization efficiency and could be reused five times without severe loss of enzyme activity. On the other hand, entrapped esterase in polyacrylamide gel, composed of 20% of total monomer and 8.3% of cross-linking agent, could be reused 10 times, and 51% of enzyme activity remained after the 10th batch without decrease of enantioselectivity. Compared with entrapped methods, significant reduction of enzyme activity was found in the case of physical adsorption on to QAE-Sephadex.     

Structure and properties of enzyme graft copolymers: Temperature effects on HRP immobilization mechanism

The possibility of obtaining immobilized horseradish peroxidase (HRP) materials with K'(m) values close to that of the native enzyme, but with good thermal stability, was investigated. The photochemical reaction was used as the immobilization methodology. Temperature and catalyst concentration were found to be the main parameters able to control the immobilization reaction mechanism more than type of functional monomer, polymer-matrix, and enzyme-polymer ratios. By carrying out the immobilization reaction at 35 degrees C and using either bisacryloylpiperazine (BAP) or hexhydro-1,3,5-triacryloyl-s-triazine (HTsT) as the functional monomer, materials with a good thermal stabilization (the retained activity after 240 min at 60 degrees C was between 65-25%) as well as kinetic constants (0.6-0.8 x 10(-4)M) similar to that of the free enzyme (0.57 x 10(-4)M) were obtained. Since low K'(m) values were obtained also using a high polymer content (pBAP copolymers, 25%; pHTsT copolymers, 30%) and neither limitation to substrate diffusion nor a reduction of the enzyme mobility was found, the enzyme should be linked to the matrix during the last steps of monomer polymerization, and it should have an external disposition with respect to the support.