Enzyme immobilization by radiation-induced polymerization of hydrophobic glass-forming monomers at low temperatures.

Enzyme immobilization was studied by means of radiation-induced polymerization of hydrophobic glass-forming monomers at low temperatures. The polymerized hydrophobic composite was generally obtained in microspheric form. Enzymatic activity showed little decrease with repeated use in these systems. The particle size of the microsphere increased with increasing monomer concentration, and activity yield had a maximum at an optimum monomer concentration. Immobilization by copolymerization of hydrophilic and hydrophobic comonomers was also investigated and a maximum activity yield was found at a certain monomer concentration. A model scheme for immobilization at low temperatures was proposed and discussed.     

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.     

Immobilization of enzymes masks their active site

We studied the effect of immobilizing cellulase to carboxycellulose sodium by radiation polymerization on the masking of the active site of the enzyme. Masking of the enzyme during the preparation of immobilized enzyme was assayed at tow temperature. The activity of immobilized enzyme was retained during repeated batch reactions, indicating that the enzyme was firmly trapped in the polymer matrix. Various compounds (designated monomers) were used to dissolve the carboxymethylcellulose; enzyme activity was affected by the nature of the monomer, by the monomer concentration, and by the solubility of the substrate in monomer.     

Immobilization of lipase onto a photo-crosslinked polymer network: Characterization and polymerization applications

Abstract

The recovery of activity of lipases immobilized onto a photo-crosslinked polymer network was 76.0% and 41.0% for entrapment and adsorption methods, respectively. Both entrapped and adsorbed immobilized enzymes were very stable, retaining more than 60% of their activity over the range of temperatures studied. Immobilization by either method protected their relative activities nearly 96% at 70°C. The optimum pH was 8.0 for immobilized enzymes and 6.0 for the free enzyme at 40°C, while the relative activities after storage at 0–4°C for 30 days were 98% and 75% using entrapment and adsorption methods, respectively. These results indicated that lipase immobilized by entrapment and adsorption not only had good activity recovery, but also remarkable stability, better reusability and application adaptability than free lipase. Also, it can be safely stated that, photo-crosslinked polymer network can be used as alternative supports for immobilization of lipase for enzymatic polymerization reactions. In the ring-opening polymerization of ?-caprolactone, polymerization rates were clearly affected as monomer conversions were 58% and 49% and the highest molecular weights (M_n) obtained were 7890 and 5600 gmol^{? 1} for entrapment and adsorption methods, respectively.     

Immobilization of Streptomyces phaeochromogenes cells at a high concentration by radiation-induced polymerization of glass-forming monomers

Summary Characteristics of Streptomyces phaeochromogenes cells immobilized by radiation-induced polymerization of 2-hydroxyethyl methacrylate at low temperatures were studied. It was found that very high concentrations of cells could be trapped effectively on the surface of the polymer matrix. Glucose isomerase activity of immobilized cells increased with increasing cell concentration. No cell leakage from the matrix was observed with repeated use, even at very high cell concentration and low monomer concentrations. The Km value of immobilized cells decreased with increasing cell concentration and with decreasing monomer concentration; it was close to that of intact cells.     

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.     

Modeling of lipase catalyzed ring-opening polymerization of epsilon-caprolactone

Enzymatic ring-opening polymerization of epsilon-caprolactone by various lipases was investigated in toluene at various temperatures. The determination of molecular weight and structural identification was carried out with gel permeation chromatography and proton NMR, respectively. Among the various lipases employed, an immobilized lipase from Candida antartica B (Novozym 435) showed the highest catalytic activity. The polymerization of epsilon-caprolactone by Novozym 435 showed an optimal temperature of 65 degrees C and an optimum toluene content of 50/50 v/v of toluene and epsilon-caprolactone. As lipases can degrade polyesters, a maximum in the molecular weight with time was obtained due to the competition of ring opening polymerization and degradation by specific chain end scission. The optimum temperature, toluene content, and the variation of molecular weight with time are consistent with earlier observations. A comprehensive model based on continuous distribution kinetics was developed to model these phenomena. The model accounts for simultaneous polymerization, degradation and enzyme deactivation and provides a technique to determine the rate coefficients for these processes. The dependence of these rate coefficients with temperature and monomer concentration is also discussed.     

固定化青霉素V酰化酶的制备及性质

尖镰孢(Fusarium oxysporum)FP941青霉素V酰化酶经γ氧化铝吸附洗脱、硫酸铵沉淀和脱盐处理后,固定在环氧丙烯聚合物载体上,湿固定化酶表现活力为217 IU/g,固定化产率为53%。固定化酶作用最适温度为55℃,最适pH为80;在pH50～110及50℃以下稳定;37℃使用25次后,酶活力保留90%。     

Immobilization of Glucose Isomerase-Containing Streptomyces phaeochromogenes Cells in Fine-Particle Form

A new preparation method for immobilizing Streptomyces phaeochromogenes cells in fine-particle form was investigated using radiation-induced polymerization at low temperatures with previously salted out hydrophilic monomers. Using this method, it was found that the glucose isomerase activity of the immobilized cell particles was markedly higher than that of immobilized cells in block form obtained without salting out of the monomer. The diameter of the particles was varied by changing the irradiation temperature or the concentrations of monomer and salt. The magnitude of the enzymatic activity increased with decreasing particle diameter. K_m values of the immobilized cell particles were close to that of the intact cell. These facts suggested that the cells were trapped on the surface of the particle.     

Characterization of alpha-galactosidase from Lactobacillus fermentum

α-Galactosidase activity was studied in Lactobacillus fermentum strains. The optimum temperature was found to be 45°C. The enzyme was inactivated at temperatures higher than 55°C, but remained active during storage at low temperatures (0, -30 and -70°C) for 5 months. Enzyme activity was observed within a 5.0–6.5 pH range, while optimum pH was dependent on the particular strain assayed. The addition of Zn²⁺ to the reaction buffer exerted a slight negative effect upon the activity, while Hg²⁺ and p -chloromercuribenzoate produced a strong inhibition. These results would indicate the presence of -SH groups in the catalytic site of the enzyme.     

Immobilization of Aspergillus oryzae tannase and properties of the immobilized enzyme

Tannase enzyme from Aspergillus oryzae was immobilized on various carriers by different methods. The immobilized enzyme on chitosan with a bifunctional agent (glutaraldehyde) had the highest activity. The catalytic properties and stability of the immobilized tannase were compared with the corresponding free enzyme. The bound enzyme retained 20·3% of the original specific activity exhibited by the free enzyme. The optimum pH of the immobilized enzyme was shifted to a more acidic range compared with the free enzyme. The optimum temperature of the reaction was determined to be 40 °C for the free enzyme and 55 °C for the immobilized form. The stability at low pH, as well as thermal stability, were significantly improved by the immobilization process. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilized enzyme retained about 85% of the initial catalytic activity, even after being used 17 times.     

Immobilization of Amaranthus leaf oxalate oxidase on alkylamine glass

A membrane bound oxalate oxidase from leaves of Amaranthus spionsus has been partially purified and immobilized on alkylamine glass with a yield of 9.2 mg protein/g support. The enzyme retained 99.4% of initial activity of free enzyme after immobilization. There was no change in the optimum pH (3.5) and Vmax but the temperature for maximum activity was slightly decreased (35 degrees C) and energy of activation (Ea) and Km for oxalate were increased after immobilization. The immobilized enzyme preparation was stable for 6 months, when stored in distilled water at 4 degrees C. Presence of Cl- did not affect the activity of immobilized enzyme.     

A selective molecularly imprinted polymer for immobilization of acetylcholinesterase (AChE): an active enzyme targeted and efficient method

In the present study, we immobilized acetylcholinesterase (AChE) enzyme onto acetylcholine removed imprinted polymer and acetylcholine containing polymer. First, the polymers were produced with acetylcholine, substrate of AChE, by dispersion polymerization. Then, the enzyme was immobilized onto the polymers by using two different methods: In the first method (method A), acetylcholine was removed from the polymer, and then AChE was immobilized onto this polymer (acetylcholine removed imprinted polymer). In the second method (method B), AChE was immobilized onto acetylcholine containing polymer by affinity. In method A, enzyme‐specific species (binding sites) occurred by removing acetylcholine from the polymer. The immobilized AChE reached 240% relative specific activity comparison with free AChE because the active enzyme molecules bounded onto the polymer. Transmission electron microscopy results were taken before and after immobilization of AChE for the assessment of morphological structure of polymer. Also, the experiments, which include optimum temperature (25–65°C), optimum pH (3–10), thermal stability (4–70°C), kinetic parameters, operational stability and reusability, were performed to determine the characteristic of the immobilized AChE. Copyright © 2015 John Wiley & Sons, Ltd.     

Enzyme immobilization on colloidal liquid aphrons (CLAs): the influence of system parameters on activity

The novel technique of immobilization of beta-galactosidase on colloidal liquid aphrons (CLAs) was investigated. CLAs are oil-in-water macroemulsions stabilised by a mixture of ionic and nonionic surfactants. Enzyme retention was found to be unaffected by changes in bulk phase pH and ionic strength, indicating that beta-galactosidase immobilization was due primarily to hydrophobic interactions. However, by varying the polarity of the internal solvent core, and the charge of the surfactants used in the formation of the CLAs, it was found that immobilization could be improved to almost 100% under certain conditions indicating that electrostatic interactions also affected immobilization to a lesser degree. Upon immobilization, it was found that there was a shift in the pH optimum of the enzyme, with the immobilized enzyme showing a broader range, and a maximal activity at higher pH. The immobilized beta-galactosidase displayed normal Michaelis-Menten dependence on substrate concentration, whilst also exhibiting superactivity for increased substrate concentrations. Activation energy was determined for the CLA immobilized enzyme, and it was found to decrease indicating that a conformational change had occurred that may account for the observed increase in activity. Finally, although the temperature profile of the immobilized enzyme was similar to the free enzyme, it was very stable, with a potential half-life of 3.6 years at 30 degrees C.     

l-alanine decarboxylase in Camellia sinensis

l-Alanine decarboxylase, extracted from the cotyledon and root of tea seedling, had a pH optimum of 6.25. Pyridoxal-5-phosphate did not activate the enzyme. The enzyme was partially stabilized by l-alanine. Enzyme activity in green seedlings was higher than in etiolated seedlings.     

Immobilization of Escherichia coli cells containing aspartase activity with kappa-carrageenan. Enzymic properties and application for L-aspartic acid production.

Whole cells of Escherichia coli having high aspartase (L-asparate ammonialyase, EC 4.3.1.1) activity were immobilized by entrapping into a kappa-carrageenan gel. The obtained immobilized cells were treated with glutaraldehyde or with glutaraldehyde and hexamethylenediamine. The enzymic properties of three immobilized cell preparations were investigated, and compared with those of the soluble aspartate. The optimum pH of the aspartase reaction was 9.0 for the three immobilized cell preparations and 9.5 for the soluble enzyme. The optimum temperature for three immobilized cell preparations was 5--10 degrees C higher than that for the soluble enzyme. The apparent Km values of immobilized cell preparations were about five times higher than that of the soluble enzyme. The heat stability of intact cells was increased by immobilization. The operational stability of the immobilized cell columns was higher at pH 8.5 than at optimum pH of the aspartase reaction. From the column effluents, L-aspartic acid was obtained in a good yield.     

Catalytic properties of the immobilized Aspergillus tamarii xylanase

Xylanase from Aspergillus tamarii was covalently immobilized on Duolite A147 pretreated with the bifunctional agent glutaraldehyde. The bound enzyme retained 54.2% of the original specific activity exhibited by the free enzyme (120 U/mg protein). Compared to the free enzyme, the immobilized enzyme exhibited lower optimum pH, higher optimum reaction temperature, lower energy of activation, higher K_m (Michaelis constant), lower V_max (maximal reaction rate). The half-life for the free enzyme was 186.0, 93.0, and 50.0 min for 40, 50, and 60°C, respectively, whereas the immobilized form at the same temperatures had half-life of 320, 136, and 65 min. The deactivation rate constant at 60°C for the immobilized enzyme is about 6.0 × 10⁻³, which is lower than that of the free enzyme (7.77 × 10⁻³ min). The energy of thermal deactivation was 15.22 and 20.72 kcal/mol, respectively for the free and immobilized enzyme, confirming stabilization by immobilization. An external mass transfer resistance was identified with the immobilization carrier (Duolite A147). The effect of some metal ions on the activity of the free and immobilized xylanase has been investigated. The immobilized enzyme retained about 73.0% of the initial catalytic activity even after being used 8 cycles.     

Stability improvement of immobilized lactoperoxidase using polyaniline polymer

Enzyme engineering via immobilization techniques is perfectly compatible against the other chemical or biological approximate to improve enzyme functions and stability. In this study lactoperoxidase was immobilized onto polyaniline polymer activated with glutaraldehyde as a bifunctional agent, to improve enzyme properties. Polyaniline polymer was used due its unique physical and chemical properties to immobilize lactoperoxidase (LPO). The optimum activity of immobilized LPO was observed at pH 6 and 55?°C, which has been increased about 10?°C for the immobilized enzyme. The immobilized enzyme maintained absolutely active for 60?days whereas the native enzyme lost 80?% of its initial activity within this period of time. Moreover, the immobilized enzyme can be reused for several times without loss of activity. The kinetic parameter studies showed slight differences between free and immobilized enzymes. The K_m and K_m.app were calculated to be 0.6 and 0.4; also V_max and V_max.app were 1.3 and 0.9 respectively.     

Factors affecting the production of prednisolone by immobilization of Bacillus pumilus E601 cells in poly(vinyl alcohol) cryogels produced by radiation polymerization

To increase the yield percent of prednisolone from hydrocortisone (cortisol), Bacillus pumilus E601 (a radioresistant microorganism) was incorporated into poly(vinyl alcohol) (PVA) cryogel grafted with hydroxyethyl-methacrylate (HEMA) as a crosslinking agent. The polymer was prepared by a radiation polymerization technique at 20 kGy from Co-60 source. The optimum temperature for the biotransformation of hydrocortisone by free cells, poly(PVA)/HEMA, and poly(PVA)/HEMA /N-isopropylacrylamide (N-IPAAm) was 30 °C. The highest yield % of prednisolone was obtained by immobilization of the cells on poly(PVA/HEMA), the addition of N-IPAAm to poly(PVA/HEMA) protected the immobilized cells from temperatures above 35 °C during the fermentation process. The optimal pH (buffered pH) of the biotransformation of hydrocortisone by immobilized and free cells was 7.0, but the maximum yield of prednisolone (60%) was obtained by immobilized cells in comparison with free cells (42%) also at pH 7.0. The prednisolone yield reached 60–65% with 1,2-propanediol cosolvent containing media and 60–62% in the case of ethanediol cosolvent containing media at 1% (v/v) of both cosolvents. 10 mg/50 ml Tween 80 the medium increased the prednisolone yield by only 1.1-fold compared with the control. The maximum bioconversion efficiency was obtained at a substrate concentration of 20 mg/50 ml medium. Stability studies showed that the immobilized cells can be used for seven times without any significant decrease in activity.     

Immobilization of oxalate decarboxylase to Eupergit and properties of the immobilized enzyme

Oxalate decarboxylase, an oxalate degradation enzyme used for medical diagnosis and decreasing the oxalate level in the food or paper industry, was covalently immobilized to Eupergit C. Different immobilization parameters, including ratio of enzyme to support, ammonia sulfate concentration, pH, and incubation time, were optimized. Under the condition of enzyme/support ratio at 1:20, pH 9, with 1.5?mol/L (NH(4))(2)SO(4), room temperature, and shaking at 30?rpm for 24?hr, activity recovery of immobilized Oxdc reached 90% with an apparent specific activity of 0.44?U/mg support. The enzymatic properties of immobilized Oxdc were investigated and compared with those of the soluble enzyme. Both shared a similar profile of optimum conditions; the optimum pH and temperature for soluble and immobilized Oxdc were 3.5 and 50°C, respectively. The immobilized enzyme was more stable at lower pH and higher temperatures. The kinetic parameters for soluble and immobilized enzyme were also determined.