Comparison of pectolytic enzymes covalently bound to synthetic ion exchangers using different methods of binding

Several methods have been compared for the immobilization of a crude commercial pectolytic enzyme product (Pectofoetidin G3X) on derivatives of technical ion exchangers. The conditions were optimized for maximum retention of the enzyme activity and the efficiency of the hydrolysis of pectin solution was measured for all the immobilized enzyme preparations. The merits and drawbacks of the methods used are discussed.     

Immobilized pectolytic enzymes on acrylic supports

The paper deals with the pectolytic enzymes immobilization on different acrylic supports, and the application of immobilized preparations in the apple juice pectinization process. The correlation between the protein content (C_p) and specific catalytic activities of immobilized enyzme preparations suggest a specific immobilization process only in the case of PONILEX ASH type acrylic supports. The active immobilization degree on PONILEX ASH type supports of Ultrazym 100 G and technical pectinase extract ranged from 99.80 to 296.40% for the Pectinesterase (PE) activity, and from 101.85 to 252.94% for the chain splitting (CS) activity, proving that the ionic immobilization process is a selective one. The simulated operational stability of the immobilized pectolytic enzymes tested by the PE and CS activity values proves the preservation of enzyme catalytic activity.     

Structure-function relationships in immobilized chymotrypsin catalysis

Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six-carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy.     

Structure-function relationships in immobilized chymotrypsin catalysis

Specific activities and the amounts of active immobilized enzyme were determined for several different preparations of alpha-chymotrypsin immobilized on CNBr-activated Sepharose 4B. Electron paramagnetic resonance (EPR) spectroscopy of free and immobilized enzyme with a spin label coupled to the active site was used to probe the effects of different immobilization conditions on the immobilized enzyme active site configuration. Specific activity of active enzyme decreased and rotational correlation time of the spin label increased with increasing immobilized enzyme loading. Enzyme immobilized using an intermediate six-carbon spacer arm exhibited greater specific activity and spin label mobility than directly coupled enzyme. The observed activity changes due to immobilization were completely consistent with corresponding active site structure alterations revealed by EPR spectroscopy.     

Characteristics of the immobilized pectin lyase activity from a commercial pectolytic enzyme preparation

A commercial pectolytic enzyme preparation has been immobilized onto a nylon-polyethyleneimine copolymer, in order ot study the contribution of the pectin lyase activity inthe overall pectindegrading activitg. Optimal conditions for the immobilization process of the pectin lyase activity, such as chemical modification of the support, coupling pH and protein concentration, were determined. Kinetic parameters and temperature behaviour of both the soluble and immobilzied pectin lyase activitgy of the derivative were also determined. OPerational stability of the pectin lyase and overall viscosity reducring activities resulting in a halflife times of 3.8 and 8.5 days, respectively, for both pectolytic activities, when the immobilized derivatives were tested in a cross-flow reactor, using a highly esterified pedtin as substrate.     

Urease immobilization on macroporous silicas

Urease was immobilized on macroporous silicas using gamma-aminopropyl triethoxysilane and glutaraldehyde. The amount of protein on the surface, the structure of pores of the support and the purity of the initial enzyme were varied, the enzymic activity of the immobilized preparations being controlled. After the immobilization of sufficiently large quantities of the enzyme (about 3 mg protein per m2 support) about 35% of the specific activity was retained. The maximum activity per unit weight of the support was observed for silicagels and silochromes with the mean diameter of pores 70-90 nm and the specific surface area about 70 m2/g. The use of purified urease produced highly active preparations of the immobilized enzyme (17,000 U per g dry support). Freeze-drying of the immobilized enzyme in the presence of sorbitol yielded dry preparations retaining their activity.     

金属螯合载体定向固定化木瓜蛋白酶的研究

以磁性金属螯合琼脂糖微球为载体,利用金属螯合配体（IDACu²⁺）与蛋白质表面供电子氨基酸相互作用的原理,定向固定了木瓜蛋白酶。固定化最适条件为Cu²⁺1.5×10^-2mol/g载体、固定化时间4h、固定化pH7.0、给酶量30mg/g载体。固定化酶的最适反应温度70℃、最适反应pH8.0,固定化酶的热稳定性明显高于溶液酶,固定化酶活力回收为68.4％,且有较好的操作稳定性,载体重复使用5次后固定化酶酶活为首次固定化酶79.71%。     

Immobilization of thermostable β-glucosidase from Sulfolobus shibatae by cross-linking with transglutaminase

Thermostable β-glucosidase from Sulfolobus shibatae was immobilized on silica gel modified or not modified with 3-aminopropyl-triethoxysilane using transglutaminase as a cross-linking factor. Obtained preparations had specific activity of 3883 U/g of the support, when measured at 70 °C using o-nitrophenyl β-d-galactopyranoside (GalβoNp) as substrate. The highest immobilization yield of the enzyme was achieved at pH 5.0 in reaction media. The most active preparations of immobilized β-glucosidase were obtained at a transglutaminase concentration of 40 mg/ml at 50 °C. The immobilization was almost completely terminated after 100 min of the reaction and prolonged time of this process did not cause considerable changes of the activity of the preparations. The immobilization did not influence considerably on optimum pH and temperature of GalβoNp hydrolysis catalyzed by the investigated enzyme (98 °C, pH 5.5). The broad substrate specifity and properties of the thermostable β-glucosidase from S. shibatae immobilized on silica-gel indicate its suitability for hydrolysis of lactose during whey processing.     

Immobilization of horseradish peroxidase onto Purolite® A109 and its anthraquinone dye biodegradation and detoxification potential

Horseradish peroxidase (HRP) is a highly specific enzyme with great potential for use in the decolorization of synthetic dyes. A comprehensive study of HRP immobilization using various techniques such as adsorption and covalent immobilization on the novel carrier Purolite® A109 with a special focus on enzymatic decolorization and toxicity of artificially colored wastewater. The immobilized preparations with an activity of 156.21 ± 1.41 U g⁻¹ and 85.71 ± 1.62 U g⁻¹ after the HRP adsorption and covalent immobilization, respectively, were obtained. Stability and reusability of the immobilized preparations were also evaluated. A noteworthy decolorization level (~90%) with immobilized HRP was achieved. Phytotoxicity testing using Mung bean seeds and acute toxicity assay with Artemia salina has confirmed the applicability of the obtained immobilized preparation in industrial wastewater plants for the treatment of colored wastewater.     

Immobilization of cellulolytic and hemicellulolytic enzymes on inorganic supports

Commercial cellulase preparations from Trichoderma viride and Aspergillus niger were immobilized on porous silica glass and ceramics such as alumina and titania with titanium tetrachloride (TiCl(4)) and on their silanized derivatives with glutaraldehyde (GLUT). The amounts of the immobilized enzymes were in the range 10-50 mg/g carrier (dry) depending on the kind of carrier and immobilization method. Their activities toward carboxymethyl cellulose (CMC), xylan, aryl-beta-glucoside, and aryl-beta-xyloside were 3-53% of those of the native enzymes. The optimum pH of the enzymes shifted to the acidic side in most cases, whereas the optimum temperatures were nearly the same as those of native ones. The activity of immobilized enzyme preparations towards CMC did not change significantly during continuous operation over a periods of 60 days. Finally, xylan was hydrolyzed with the immobilized enzymes, and the sugars formed were investigated.     

Immobilization and stabilization of a cyclodextrin glycosyltransferase by covalent attachment on highly activated glyoxyl-agarose supports

Covalent immobilization of cyclodextrin glycosyltransferase on glyoxyl-agarose beads promotes a very high stabilization of the enzyme against any distorting agent (temperature, pH, organic solvents). For example, the optimized immobilized preparation preserves 90% of initial activity when incubated for 22 h in 30% ethanol at pH 7 and 40 degrees C. Other immobilized preparations (obtained via other immobilization protocols) exhibit less than 10% of activity after incubation under similar conditions. Optimized glyoxyl-agarose immobilized preparation expressed a high percentage of catalytic activity (70%). Immobilization using any technique prevents enzyme inactivation by air bubbles during strong stirring of the enzyme. Stabilization of the enzyme immobilized on glyoxyl-agarose is higher when using the highest activation degree (75 micromol of glyoxyl per milliliter of support) as well as when performing long enzyme-support incubation times (4 h) at room temperature. Multipoint covalent immobilization seems to be responsible for this very high stabilization associated to the immobilization process on highly activated glyoxyl-agarose. The stabilization of the enzyme against the inactivation by ethanol seems to be interesting to improve cyclodextrin production: ethanol strongly inhibits the enzymatic degradation of cyclodextrin while hardly affecting the cyclodextrin production rate of the immobilized-stabilized preparation.     

A new method for spectrophotometric assay of activity of cross-linked penicillin acylase aggregates

A new method for monitoring reactions catalyzed by an immobilized enzyme, cross-linked penicillin acylase aggregates (PA CLEA), is suggested. Appropriate chromogenic substrates for spectrophotometric assay of catalytic activity of immobilized enzyme were chosen and their kinetic parameters determined. Active sites in PA CLEA preparations were titrated by the suggested method; it is shown that almost all active sites are retained during immobilization. This method is characterized as highly expressive, simple, and precise and may be used for control of PA immobilization efficiency as well as for study of operational, thermal, and pH stability of immobilized enzyme preparations.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

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.     

Immobilization of lipases on hydrophobic supports involves the open form of the enzyme

The lipases from Thermomyces lanuginosus and Pseudomonas cepacia have been immobilized on octyl and cyanogen bromide (CNBr) agarose beads. The immobilization on octyl-agarose is slowed with increasing ionic strength, while the immobilization on CNBr is not significantly affected by the ionic strength. The inhibition of the immobilized preparations with diethyl p-nitrophenylphosphate (D-pNPP) was analyzed. The inhibition was more rapid using octyl-lipase preparations than using covalent preparations, and the covalent preparations were much more sensitive to the reaction medium. The addition of detergent increased the inhibition rate of the covalent preparation while an increase on the ionic strength produced a slowdown of the inhibition rate by D-pNPP for both lipases. The effect of the medium on the activity versus fully soluble substrate (methyl mandelate) was in the same direction. The octyl preparations presented a slight decrease in activity when comparing the results using different concentrations of sodium phosphate buffer (between 0.025 and 1 M), while the CNBr preparations suffered drastic drops in its activity at high ionic strength.The results confirm that the lipases immobilized on octyl agarose presented their open form stabilized while the covalent preparation maintains a closing/opening equilibrium that may be modulated by altering the medium.     

Immobilization of polygalacturonase from Aspergillus niger onto activated polyethylene and its application in apple juice clarification

The present work is focused on efficient immobilization of polygalacturonase on polyethylene matrix, followed by its application in apple juice clarification. Immobilization of polygalacturonase on activated polyethylene and its use in apple juice clarification was not reported so far. Aspergillus niger Van Tieghem (MTCC 3323) produced polygalacturonase when grown in modified Riviere's medium containing pectin as single carbon source by fed-batch culture. The enzyme was precipitated with ethanol and purified by gel filtration chromatography (Sephacryl S-100) and immobilized onto glutaraldehyde-activated polyethylene. The method is very simple and time saving for enzyme immobilization. Various characteristics of immobilized enzyme such as optimum reaction temperature and pH, temperature and pH stability, binding kinetics, efficiency of binding, reusability and metal ion effect on immobilized enzymes were evaluated in comparison to the free enzyme. Both the free and immobilized enzyme showed maximum activity at a temperature of 45 degrees C and pH 4.8. Maximum binding efficiency was 38%. The immobilized enzyme was reusable for 3 cycles with 50% loss of activity after the third cycle. Twenty-four U of immobilized enzyme at 45 degrees C and 1 h incubation time increased the transmittance of the apple juice by about 55% at 650 nm. The immobilized enzyme can be of industrial advantage in terms of sturdiness, availability, inertness, low price, reusability and temperature stability.     

Evaluation of the performance of immobilized penicillin G acylase using active-site titration

Penicillin G acylase from Escherichia coli was immobilized on Eupergit C with different enzyme loading. The activity of the immobilized preparations was assayed in the hydrolysis of penicillin G and was found to be much lower than would be expected on the basis of the residual enzyme activity in the immobilization supernatant. Active-site titration demonstrated that the immobilized enzyme molecules on average had turnover rates much lower than that of the dissolved enzyme. This was attributed to diffusion limitations of substrate and product inhibition. Indeed, when the immobilized preparations were crushed, the activity increased from 587 U g-1 to up to 974 U g-1. The immobilized preparations exhibited up to 15% lower turnover rates than the dissolved enzyme in cephalexin synthesis from 7-ADCA and D-(-)-phenylglycine amide. The synthesis over hydrolysis ratios of the immobilized preparations were also much lower than that of the dissolved enzyme. This was partly due to diffusion limitations but also to an intrinsic property of the immobilized enzyme because the synthesis over hydrolysis ratio of the crushed preparations was much lower than that of the dissolved enzyme.     

Immobilization of pectawamorine G10x by gel entrapment

Polyacrylamide gel immobilization of pectawamorine G10x was investigated. Its pectinesterase and polygalacturonase activity and stability in storage were measured. The degree of pectawamorine binding during gel immobilization was 80--90%, 55% of initial activity being retained. Thermal stability of the immobilized and native preparations was equal. Pectinesterase activity of the gel immobilized enzyme increased during storage.     

Preparation of immobilized/stabilized biocatalysts of β-glucosidases from different sources: Importance of the support active groups and the immobilization protocol

β-Glucosidases from two different commercial preparations, Pectinex Ultra SP-L and Celluclast® 1.5L, were immobilized on divinylsulfone (DVS) supports at pH 5.0, 7.0, 9.0, and 10. In addition, the biocatalysts were also immobilized in agarose beads activated by glyoxyl, and epoxide as reagent groups. The best immobilization results were observed using higher pH values on DVS-agarose, and for Celluclast® 1.5L, good results were also obtained using the glyoxil-agarose immobilization. The biocatalyst obtained using Pectinex Ultra SP-L showed the highest thermal stability, at 65°C, and an operational stability of 67% of activity after 10 reuses cycles when immobilized on DVS-agarose immobilized at pH 10 and blocked with ethylenediamine. The β-glucosidase from Celluclast® 1.5L produced best results when immobilized on DVS-agarose immobilized at pH 9 and blocked with glycine, reaching 7.76-fold higher thermal stability compared to its free form and maintaining 76% of its activity after 10 successive cycles. The new biocatalysts obtained by these protocols showed reduction of glucose inhibition of enzymes, demonstrating the influence of immobilization protocols, pH, and blocking agent.     

Tyrosine hydroxylase activity of immobilized tyrosinase on enzacryl-AA and CPG-AA supports: Stabilization and properties

Frog epidermis tyrosinase has been immobilized on Enzacryl-AA (a polyacrylamide-based support) and CPG(zirclad)-Arylamine (a controlled pore glass support) in order to stabilize the tyrosine hydroxylase activity of the enzyme; in this way, the immobilized enzyme could be used to synthesize L-dopa from L-tyrosine. The activity immobilization yield Y(IME) (act) (higher than 86%), coupling efficiency (up to 90%), storage stability (no loss in 120 days), and reaction stability (t(1/2) was higher than 20 h in column reactors) were measured for tyrosinase after its immobilization. The results showed a noticeable improvement (in immobilization yield, coupling efficiency, and storage and operational stabilities) over previous reports in which tyrosinase was immobilized for L-dopa production. The activity and stability of immobilized enzyme preparations working in three different reactor types have been compared when used in equivalent conditions with respect to a new proposed parameter of the reactor (R(p)), which allows different reactor configurations to be related to the productivity of the reactor during its useful life time. The characteristic reaction inactivation which soluble tyrosinase shows after a short reaction time has been avoided by immobilization, and the stabilization was enhanced by the presence of ascorbate. However, another inactivation process appeared after a prolonged use of the immobilized enzyme. The effects of reactor type and operating conditions on immobilized enzyme activity and stability are discussed.