Immobilization of enzyme onto poly(ethylene-vinyl alcohol) membrane

Invertase was ionically bound to the poly(ethylene-vinyl alcohol) membrane surface modified with two aminoacetals with different molecular length, 2-dimethyl-aminoacetoaldehyde dimethylacetal (AAA) and 3-(N, N-dimethylamino-n-propanediamine) propionaldehyde dimethylacetal (APA). Immobilization conditions were determined with respect to enzyme concentration in solution, pH value, ionic strength in immobilization solution, and immobilization time. Various properties of immobilized invertase were evaluated, and thermal stability was found especially to be improved by immobilization. The apparent Michaelis constant, K(m), was smaller for invertase bound by APA with longer molecular lengths than for invertase bound by AAA. We attempted to bind glucoamylase of Rhizopus delemar origin in the same way. The amount and activity of immobilized glucoamylase were much less than of invertase.     

Immobilization of Enzyme into Poly(vinyl alcohol) Membrane

Glucoamylase, invertase, and cellulase were entrapped within poly(vinyl alcohol) (PVA) membrane cross-linked by means of irradiation of ultraviolet light. The conditions for immobilization of glucoamylase were examined with respect to enzyme concentration in PVA, sensitizer (sodium benzoate) concentration in PVA, irradiation time, and membrane thickness. Various characteristics of immobilized glucoamylase were evaluated. Among them, the pH activity curve for the immobilized enzyme was superior to that for the native one, and thermal stability was improved by immobilization with bovine albumin. The apparent K(m) was larger for immobilized glucoamylase than for the native one, while V(max) was smaller for the immobilized enzyme. Also, the apparent K(m) appeared to be affected by the molecular size of the substrate. Further, immobilized invertase and cellulase showed good stabilities in repeating usage.     

Immobilization of invertase onto poly(3-methylthienyl methacrylate)/poly(3-thiopheneacetic acid) matrix

A novel immobilization matrix, poly(3-methylthienyl methacrylate)–poly(3-thiopheneacetic acid) (PMTM–PTAA), was synthesized and used for the covalent immobilization of Saccharomyces cerevisiae invertase to produce invert sugar. The immobilization resulted in 87% immobilization efficiency. Optimum conditions for activity were not affected by immobilization and the optimum pH and temperature for both free and immobilized enzyme were found to be 4.5 and 55 °C, respectively. However, immobilized invertase was more stable at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined using the Lineweaver–Burk plot. The K_m values were 35 and 38 mM for free and immobilized enzyme, respectively. The V_max values were 29 and 24 mg glucose/mg enzyme min for free and immobilized enzyme, respectively. Immobilized enzyme could be used for the production of glucose and fructose from sucrose since it retained almost all the initial activity for a month in storage and retained the whole activity in repeated 50 batch reactions.     

The properties of invertase,covalently immobilized at activated carbon

The covalent immobilization of yeast invertase with glutaraldehyde at activated carbon, modified preliminarily by urea and dimethyl formamide treatment, has been established. Some physicochemical properties of the immobilized and native enzyme in water and water-organic solutions have been studied. Hydrolytic, as well as transferase enzyme characteristics have changed after immobilization. The optimal conditions for hydrolytic and transferase activity of immobilized invertase are pH 6.0 and 7.0, respectively. The optimum temperature for the immobilized enzyme is 30°C. The conversion degree of isoamyl alcohol depends on the substrate and enzyme concentrations in medium, holdup time and organic phase quantity in the reaction medium.     

Properties of a glucose oxidase covalently immobilized on amorphous AlPO4 support

Glucose oxidase (GOD) was covalently immobilized on amorphous AlPO₄ as well as on an AlPO₄/clay mineral Sepiolite system. Immobilization of the enzyme was carried out through the -amino group of lysine residues through an aromatic Schiff's-base. Activation of the support was obtained after reaction of appropriate molecules with support surface –OH groups. The enzymatic activities of native, and different immobilized GOD systems and filtrates, were followed by the amount of liberated -gluconic acid obtained in the enzymatic β- -glucose oxidation with the aid of an automatic titrator. The kinetic properties of native and immobilized GOD were obtained for glucose concentrations in the range of physiological conditions and at different working conditions such as reaction temperature, reaction pH, and enzyme concentration.

The binding percentage of enzymes was in the 50–80% range, with residual and specific activities in the 65–80% and 90–150% ranges, respectively. No change in the pH optimum and only slight changes in the V_max and K_M kinetic parameters with respect to native GOD were observed, so that not only was little deactivation of enzyme obtained throughout the immobilization process but also that the stability of the covalently bound enzyme in the two supports appeared to have increased with respect to the soluble enzyme. GOD immobilization also increased its efficiency and operational stability in repeated uses on increasing the amount of immobilized enzyme.     

Cellulase immobilized by sol–gel entrapment for efficient hydrolysis of cellulose

Cellulase from Trichoderma reesei (Celluclast 1.5 L, Novozyme) was immobilized by sol–gel encapsulation, using binary or ternary mixtures of tetramethoxysilane (TMOS) with alkyl- or aryl-substituted trimethoxysilanes as precursors. Optimization of immobilization conditions resulted in 92 % recovery of total enzymatic activity in the best immobilized preparate. The immobilized cellulase exhibiting the highest activity, obtained from tetramethoxysilane and methyltrimethoxysilane precursors at 3:1 molar ratio, was investigated in the hydrolysis reaction of microcrystalline cellulose (Avicel PH101). Although the optimal values did not change significantly, both temperature and pH stabilities of the sol–gel entrapped cellulase improved compared to the native enzyme. Immobilization also conferred superior resistance against the inactivation effect of glucose. Reuse of the sol–gel entrapped cellulase showed 40 % retention of the initial activity after five batch hydrolysis cycles, demonstrating the potential of this biocatalyst for large-scale application.     

Immobilization and properties of carminomycin 4-O-methyltranferase, the enzyme which catalyzes the final step in the biosynthesis of daunorubicin in Streptomyces peucetius

The carminomycin 4-O-methyltransferase enzyme from Streptomyces peucetius was covalently immobilized on 3M Emphaze ABI-activated beads. Optimal conditions of time, temperature, pH, ionic strength, enzyme, substrate (carminomycin), and cosubstrate (S-adenosyl-L-methionine) concentrations were defined for the immobilization reaction. Protein immobilization yield ranged from 52% to 60%. Including carminomycin during immobilization had a positive effect on the activity of the immobilized enzyme but a strongly negative effect on the coupling efficiency. The immobilized enzyme retained at least 57% of its maximum activity after storage at 4 degrees C for more than 4 months. The properties of the free and immobilized enzyme were compared to determine whether immobilization could alter enzyme activity. Both soluble and bound enzyme exhibited the same pH profile with an optimum near 8.0. Immobilization caused an approximately 50% decrease in the apparent K(m) (K'(m)) for carminomycin while the K'(m) for S-adenosyl-L-methionine was approximately doubled. A 57% decrease in the V(max) value occurred upon immobilization. These changes are discussed in terms of active site modifications as a consequence of the enzyme immobilization. This system has a potential use in bioreactors for improving the conversion of carminomycin to daunorubicin. (c) 1995 John Wiley & Sons, Inc.     

Optimization of invertase immobilization by adsorption in ionic exchange resin for sucrose hydrolysis

This work presents as a main objective to study the immobilization process of yeast invertase by adsorption in the ion exchanging resin Duolite A-568 for invert sugar production. Initially, a kinetic study of the soluble form of the enzyme was carried out. At the sequence was studied the immobilization process of yeast invertase in the weakly exchanging anionic resin Duolite A-568. The influences of the pH, enzyme concentration and temperature in the enzyme immobilization were analyzed through a central composite design (CCD). The results indicated that the retention of the catalytic activity in immobilization was strongly dependent of these variables, being maximum in a pH value of 5.0, with an enzyme concentration of 12.5 g/L (1.875 g of protein per liter) and temperature of 30 °C. The simultaneous influence of pH and temperature on the free and immobilized invertase activity was also studied through a CCD.     

Isolation and properties of prostaglandin H synthetase immobilized in insoluble polyelectrolyte complexes

Immobilization of prostaglandin-H-synthetase (EC 1.14.99.1) of the microsomal fraction of ram vesicular gland in the microparticles of insoluble polyelectrolyte complexes by non-covalent incorporation was studied. It was shown that immobilization occurs with a high efficiency and high activity yield (40-70%). Non-specific reversible inhibition of the enzyme by the polycationic component of the complex was demonstrated. The dependencies of activity of the native and immobilized enzymes on pH, temperature and substrate (adrenaline and arachidonic acid) were studied. The immobilized enzyme had an increased thermal stability as compared to the native one; the thermoinactivation rate constant was decreased 3-7 times. The biexponential type of the curve of the rependence of activity vs. time for the native enzyme and the transformation of the curve into a simple exponential form for the immobilized enzyme were observed.     

Production of levan using recombinant levansucrase immobilized on hydroxyapatite

Levansucrase of Zymomonas mobilis was immobilized onto the surface of hydroxyapatite by ionic binding. Optimum conditions for the immobilization were: pH 6.0, 4 h of immobilization reaction time, and 20 U of enzyme/g of matrix. The enzymatic and biochemical properties of the immobilized enzyme were similar to those of the native enzyme, especially towards the effect of salts and detergents. The immobilized enzyme showed sucrose hydrolysis activity higher as that of the native enzyme, but levan formation activity was 70% of the native enzyme. HPLC analysis of levan produced by immobilized enzyme showed the presence of two different types of levan: high-molecular-weight levan and low-molecular-weight levan. The proportion of low-molecular-weight levan to total levan produced by the immobilized enzyme was much higher than that with the native enzyme, indicating that immobilized levansucrase could be applied to produce low-molecular-weight levan. Immobilized levansucrase retained 65% of the original activity after 6 times of repeated uses and 67% of the initial activity after 40 d when stored at 4 °C.     

Invertase reversibly immobilized onto polyethylenimine-grafted poly(GMA–MMA) beads for sucrose hydrolysis

The epoxy group containing poly(glycidyl methacrylate-co-methylmethacrylate) poly(GMA–MMA) beads were prepared by suspension polymerisation and the beads surface were grafted with polyethylenimine (PEI). The PEI-grafted beads were then used for invertase immobilization via adsorption. The immobilization of enzyme onto the poly(GMA–MMA)–PEI beads from aqueous solutions containing different amounts of invertase at different pH was investigated in a batch system. The maximum invertase immobilization capacity of the poly(GMA–MMA)–PEI beads was about 52 mg/g. It was shown that the relative activity of immobilized invertase was higher then that of the free enzyme over broader pH and temperature ranges. The Michaelis constant (K_m) and the maximum rate of reaction (V_max) were calculated from the Lineweaver–Burk plot. The K_m and V_max values of the immobilized invertase were larger than those of the free enzyme. The immobilized enzyme had a long-storage stability (only 6% activity decrease in 2 months) when the immobilized enzyme preparation was dried and stored at 4 °C while under wet condition 43% activity decrease was observed in the same period. After inactivation of enzyme, the poly(GMA–MMA)–PEI beads can be easily regenerated and reloaded with the enzyme for repeated use.     

Immobilization of chitosan-invertase neoglycoconjugate on carboxymethylcellulose-modified chitin

Saccharomyces cerevisiae invertase, chemically modified with chitosan, was immobilized on a carboxymethylcellulose-coated chitin support via polyelectrolyte complex formation. The yield of immobilized protein was determined to be 72% and the enzyme retained 68% of the initial invertase activity. The optimum temperature for invertase was increased by 5 degrees C and its thermostability was enhanced by about 9 degrees C after immobilization. The immobilized enzyme was stable against incubation in high ionic strength solutions and was 12.6-fold more resistant to thermal treatment at 65 degrees C than the native counterpart. The prepared biocatalyst retained 98% and 100% of the original catalytic activity after 10 cycles of reuse and 70 h of continuous operational regime in a packed bed reactor, respectively. The immobilized enzyme retained 95% of its activity after 50 days of storage at 37 degrees C.     

酶法提取玉米胚中总黄酮的研究

尝试使用α-淀粉酶进行糖苷转移提取玉米中总黄酮,同时对α-淀粉酶的固定化进行研究。对α-淀粉酶固定化的研究表明:以壳聚糖为载体,用体积分数4%戊二醛进行交联,加酶量为3 g.L-1条件下可以获得最佳的固定化效果;与游离酶相比,其最适作用温度范围、pH值范围均比游离酶范围宽;固定化酶的热稳定性优于游离酶,且具有良好的操作稳定性。本试验使用游离酶法、固定化酶法提取玉米中总黄酮,得率分别为2.82%、2.46%。     

Properties of enzymes immobilized by the diazotized m-diaminobenzene method.

Some properties of a number of enzymes immobilized by the diazotized m-diaminobenzene (dDAB) method are described. The pH-activity profiles of beta-D-glucosidase, glucoamylase, peroxidase, uricase, and D-glucose oxidase were virtually unchanged on immobilization while those of catalase and dextranase were significantly altered. beta-D-Glucosidase, glucoamylase, and glucose oxidase were found to be more susceptible to denaturation on lyophilization when immobilized than in the native state; however, sorbitol had a marked protective effect in every case examined. Sorbitol was also found to exert a stabilizing effect when lyophilized immobilized preparations were stored. Immobilization marginally improved the stabilities of a number of enzymes to heating at 60 degrees at pH 8.0. The usefulness for continuous reaction of a column of glucoamylase attached to celite was established. The reuse of the solid supports was demonstrated.     

Characteristics of yeast invertase immobilized on porous cellulose beads.

Invertase from Candida utilis was immobilized on porous cellulose beads by an ionic-quanidino bond. The immobilized invertase showed optimum activity between pH 4.0 and 5.4, while the free enzyme had a sharp optimum at pH 4.1. Both temperature profiles were fairly similar up to 55 degrees C. However, above this temperature the immobilized enzyme was more stable than the free enzyme. From the temperature data, the activation energies were found to be 7,322 and 4,052 cal/g mol for the free and the immobilized enzyme, respectively. Candida invertase shows characteristics of substrate inhibition. Both the Km and Ki for the free and the immobilized enzymes were determined. The apparent Ki for the immobilized invertase was much higher than the Ki of the free enzyme, suggesting a diffusion effect. Immobilized invertase molecules deep in the pores only see sucrose concentrations much less than the bulk concentrations. Immobilization, thus, offers certain processing advantages in this regard.     

基于膨润土的层柱黏土固定β-葡萄糖醛酸苷酶的研究

以膨润土制备的层柱黏土为载体,考察给酶量、固定化pH、温度和时间对固定化β-葡萄糖醛酸苷酶活性的影响,并对其操作稳定性进行研究。结果表明：给酶量为2700U／g,最适pH为3．6,固定化温度40℃,固定化60min条件下固定化酶催化活性较高;酶经固定化后其热稳定性及储存稳定性显著提高。     

Cholesterol oxidation using hollow fiber dialyzer immobilized with cholesterol oxidase: preparation and properties

The surface of polyacrylonitrile hollow fibers were hydrolyzed and covalently bonded with cholesterol oxidase (COD) via glutaraldehyde. The immobilized amount of the COD increased with the concentration of glutaraldehyde. However, COD immobilized with 10% glutaraldehyde had higher activity than with other concentrations. The stabilities of immobilized COD to pH and temperature were higher than those of native enzyme. The immobilized enzyme retained 80% of initial activity after 15 days when stored at 4 degrees C, which was longer than native COD. After being reused six times, the COD-immobilized hollow fiber retained more than 80% of the activity.     

Evaluation of intrinsic immobilized kinetics in hollow fiber reactor systems.

Immobilized cell and enzyme hollow fiber reactors have been developed for a variety of biochemical and biomedical applications. Reported mathematical models for predicting substrate conversion in these reactors have been limited in accuracy because of the use of free-solution kinetic parameters. This paper describes a method for determining the intrinsic kinetics of enzymes immobilized in hollow fiber reactor systems using a mathematical model for diffusion and reaction in porous media and an optimization procedure to fit intrinsic kinetic parameters to experimental data. Two enzymes, a thermophilic beta-galactosidase that exhibits product inhibition and L-lysine alpha-oxidase, were used in the analysis. The intrinsic kinetic parameters show that immobilization enhanced the activity of the beta-galactosidase while decreasing the activity of L-lysine alpha-oxidase. Both immobilized enzymes had higher Km values than did the soluble enzyme, indicating less affinity for the substrate. These results are used to illustrate the significant improvement in the ability to predict substrate conversion in hollow fiber reactors.     

Immobilization of the exo-maltohexaohydrolase by the irradiation method

Exomaltohexaohydrolase (E.C.3.2.1.98) was immobilized by radiocopolymerization of some synthetic monomers which were mixed in various combinations. Irradiation was carried out while the mixture of monomers and enzymes was frozen in petroleum ether-dry-ice bath. Recovery of the immobilized enzyme was 44-75%.The optimum pH of the enzyme slightly shifted to the acidic side. The pH stability was improved remarkably by immobilization. The enzyme was stable retaining more than 90% of its original activity in the range pH 4-11. The optimum reaction temperature of the enzyme increased about 2 degrees C. Heat stability was also improved by immobilization, and that the enzyme retained about 40% of its original activity after treatment at 75 degrees C for 15 min. The immobilized enzyme was stable to the repeated use of 20 cycles. The K(m) value of the enzyme for short-chain amylose was almost the same as that of native enzyme. When soluble starch was used as the substrate, the K(m), value of the enzyme was three times as large as that of native enzyme. Effects of various metal ions and inhibitors on the immobilized enzyme were also studied compared to the native enzyme.     

Characterization of dextransucrase immobilized on calcium alginate beads from Leuconostoc mesenteroides PCSIR-4

Immobilization of dextransucrase from Leuconostoc mesenteroides PCSIR-4 on alginate is optimized for application in the production of dextran from sucrose. Dextransucrase was partially purified by ethanol upto 2.5 fold. Properties of dextransucrase were less affected by immobilization on alginate beads from soluble enzyme. Highest activities of both soluble and immobilized dextransucrase found to be at 35 degrees C and optimum pH for activity remain 5.00. Substrate maxima for immobilized enzyme changed from 125 mg/ml to 200 mg/ml. Incubation time for enzyme-substrate reaction for maximum enzyme activity was increased from 15 minutes to 60 minutes in case of immobilized enzyme. Maximum stability of immobilized dextransucrase was achieved at 25 degrees C with respect to time.