Enzyme Immobilization Efficiency Research Articles

Construction of a “Battlements” Structure by Zirconium Glyphosate on Silica Nanospheres and the Catalytic Stability Promotion of Candida lipolytica Lipase on the Hybrid Materials

Different diameter SiO2 coated with zirconium glyphosate (ZrGP) was prepared, and Candida lipolytica lipase was loaded on the surface of ZrGP@SiO2 in this study. ZrGP was in situ synthesized on the surface of SiO2 and existed like “battlements”. Under protection of the battlements structure and organic parts in ZrGP, the immobilization efficiency and activity recovery of Candida lipolytica lipase were improved greatly: 69.0% of the enzyme immobilization efficiency and 71.2% of the activity recovery were found after the lipase was loaded under certain immobilization conditions and the optimum catalytic environment. Besides, the immobilized enzyme was found to have better thermostability (up to 43 °C), wider pH value adaptation (from 6.9 to 8.4), and high reserved activity (over 80% remained after four cycles of catalysis).

Enhanced biocatalytic sustainability of laccase by immobilization on functionalized carbon nanotubes/polysulfone membranes

Chemically functionalized multi-walled carbon nanotubes (CNTs) are used as carriers for laccase immobilization. In this work, CNTs were modified using different approaches with a combination of methods involving hydrothermal oxidation with nitric acid, treatment with 3-aminopropyltriethoxysilane, glutaraldehyde, N-ethyl-N-(3-(dimethylamino)-propyl) carbodiimide hydrochloride and N-hydroxysuccinimide. The enzyme immobilization efficiency and recovered activity were evaluated towards 2,2′-azino-bis(3-ethylbenzathiazoline-6-sulfonic acid) biocatalytic oxidation. The best compromise between immobilization efficiency and recovered activity was obtained using the CNTs functionalized with 0.3 M HNO3, treated with N-ethyl-N-(3-(dimethylamino)propyl) carbodiimide hydrochloride and N-hydroxysuccinimide. This catalyst also showed the best thermal stability (at 50 and 60 °C). The bioconjugate based on this material was characterized by vibrational spectroscopies (FTIR and Raman) and by N2 adsorption. The results from reutilization tests showed that laccase activity was kept above 65% of its initial value after five consecutive cycles of reuse. The biocatalytic performance of the immobilized enzyme was evaluated for the degradation of a mixture of phenolic compounds in water containing phenol, resorcinol, 4-methoxyphenol and 4-chlorophenol. As means of cost efficient to enzyme reutilization, laccase was immobilized over polysulfone membranes blended with the functionalized CNTs and studied in the degradation of 4-methoxyphenol.

Immobilization of α-amylase on chitosan-montmorillonite nanocomposite beads

Enzyme immobilization is a way to increase efficiency of the enzyme and facilitate its recovery. The aim of this study was to immobilize α-amylase on chitosan-montmorillonite nanocomposite beads. Nanocomposite beads were prepared as the carrier for the enzyme stabilization and their surface was modified by Glutaraldehyde. Alpha-amylase was immobilized on nanocomposite beads by covalent bonding. The results of scanning electron microscopy (SEM) showed that particle size range of montmorillonite was 10–30 nm. This study indicated that the enzyme immobilization efficiency was 87%. The activity of free and immobilized enzyme during 40 days of storage at 4 °C decreased 95% and 36%, respectively. The results showed that the immobilized enzyme activity after reusing five times decreased about 47%. This study indicated that the immobilized enzyme activity was higher than the free enzyme at different temperatures. Also the immobilized enzyme was more stable than the free enzyme at lower pH. The results of kinetic parameters showed that Km values of the immobilized enzyme (9.12 μmol/ml) were higher than free enzyme (6.80 μmol/ml). The Vmax values for the free and immobilized enzyme were 1.30 and 0.629 μmol/mg·min, respectively.

Immobilization of alcohol dehydrogenase on ceramic silicon carbide membranes for enzymatic CH3OH production

AbstractBACKGROUNDAlcohol dehydrogenase (ADH; EC 1.1.1.1) catalyzes oxidation of CH3OH to CHOH during NAD+ reduction to NADH. ADH can also accelerate the reverse reaction, which is studied as part of cascadic enzymatic conversion of CO2 to CH3OH. In the present study, immobilization of ADH onto macroporous membranes of silicon carbide (SiC) was investigated for CHOH to CH3OH conversion.RESULTSImmobilization techniques included physical adsorption directly to the membrane and functionalization of the membrane with polyethylenimine (PEI) or (3‐aminopropyl)triethoxysilane (APTES) followed by glutaraldehyde (GA) cross‐linking. Enzyme loadings, flux, NADH conversion, and overall ADH reusability were assessed. Enzyme loadings were similar, but substrate conversion was approximately 2 and 2.5 times higher for APTES‐GA and PEI‐GA, respectively, and the relative activity retention was better than for physical adsorption. Membrane surface treatment with NaOH prior to APTES‐GA immobilization resulted in significant improvement in enzyme loading and a doubling of ADH activity as well as higher activity during recycling as the ADH destabilization rate was unaffected.CONCLUSIONSThe results provided proof‐of‐concept for the use of NaOH‐treated SiC membranes for covalent enzyme immobilization and biocatalytic efficiency improvement of ADH during multiple reaction cycles. These data have implications for the development of robust extended enzymatic reactions. © 2018 Society of Chemical Industry

Stabilization of Lipase in Polymerized High Internal Phase Emulsions.

Candida antarctica lipase B is stabilized in a porous, high internal phase emulsion (HIPE) of polydicyclopentadiene to enable biocatalytic waste stream upcycling. The immobilized lipase is subjected to thorough washing conditions and tested for stability in extreme environments and reusability. A porous internal microstructure is revealed through scanning electron microscopy. After preparation, lipase activity increased to 139 ± 9.7% of its original activity. After 10 cycles of reuse, immobilized lipase retains over 50% activity. Immobilized lipase retains activity after 24 h of exposure to temperatures ranging from 20 to 60 °C and pH values of 3, 7, and 10. In the most extreme environments tested, lipase retained 42.8 ± 21% relative activity after exposure to 60 °C and 49.4 ± 16% relative activity after exposure to pH 3. Polymerized HIPEs stabilize lipase and, thus, extend its working range. Further synthesis optimization has the potential to increase enzyme stability, immobilization efficiency, and uniformity. The reported hierarchical stabilization technique shows promise for use of immobilized lipase in non-ideal, industrially relevant conditions.

Enhancement in Biological Activity of L-Asparginase by its Conjugation on Silica Nanoparticles.

L-asparaginase is a drug of choice in the treatment of Hodgkin's lymphoma and acute lymphoblastic leukemia. Production of its bioconjugates can increase its half-life, stability and decrease its immunogenicity. The aim of the present study was to immobilize this drug on silica nanoparticles by two different cross-linking agents. The drug was conjugated to nanoparticles by two cross-linking agents; 1-ethyl-3-(3- dimethylaminopropyl) carboiimide HCl (EDC) or glutaraldehyde. The effect of the drug to the nanoparticles ratio, the amount of cross-linking agents and the time of conjugation were optimized according to the zeta potential, size particle and the enzyme immobilization efficiency. Conjugation of L-asparaginase to nanoparticles was confirmed by FT-IR and TEM. The activity, kinetic profiles, stability against pH changes, thermal and storage stability of the native and immobilized drug were compared. The results showed significant increase in pH range of the stability and decrease in the km value of the drug after immobilization; indicating an increase in the enzyme tendency for the substrate. The Time of stability of the drug increased after immobilization in plasma and phosphate buffer saline which can increase its half-life of circulation. The activity and stability of immobilized drug by EDC were better than glutaraldehyde.

Covalent immobilization of phytase on the multi-walled carbon nanotubes via diimide-activated amidation: structural and stability study

Phytase is the most widely used feed enzyme in the world. This enzyme inactivates under pelleting conditions such as temperatures above 80 °C. The present study reported covalent attachment of phytase onto functionalized multi-walled carbon nanotubes (F-MWNT). The optimum enzyme loading and immobilization efficiency were 110 µg/mg of functionalized multi-walled carbon nanotubes and 62%, respectively. Characterization, structural changes and immobilization of phytase were studied by scanning electron microscopy (SEM), circular dichroism (CD), zeta potential measurement and Fourier transform infrared (FTIR) spectroscopy. Immobilized phytase exhibited improved stability towards temperature than the free phytase. The free phytase retain 3% and 27% of relative activity at 90 and 80 °C, respectively after 2 min of incubations. While immobilized phytase retained about 33% and 51% at the same condition. Also the results showed that the presence of small quantities of NaCl (<1 M) in the reaction media increased enzyme activity of immobilized phytase up to 78% but free phytase activity was not significantly changed until 1 M NaCl which increased by 30%. Higher concentrations of 1 M NaCl drastically reduced both free and immobilized phytase activities.

Immobilization of Candida rugosa lipase onto graphene oxide Fe3O4 nanocomposite: Characterization and application for biodiesel production

The purpose of this work is to develop a magnetically recyclable immobilized lipase for biodiesel production to meet the need of green and clean production. To achieve this, magnetic Fe3O4 nanoparticles were encapsulated in graphene oxides (GO), and then employed as a magnetic carrier for the immobilization of lipase from Candida rugosa via the interfacial activation of enzyme on hydrophobic surfaces. By using this immobilization strategy, the activity recovery of 64.9% and enzyme immobilization efficiency of 85.5% could be achieved. The as-prepared graphene oxide Fe3O4 nanocomposite (MGO) and immobilized lipase microsphere were fully characterized by means of enzyme activity assays, transmission electron microscopy (TEM), X-ray powder diffraction (XRD), X-ray photoelectron spectroscopy (XPS), Fourier transform infrared (FT-IR) spectroscopy, vibrating-sample magnetometer (VSM) and nitrogen adsorption–desorption techniques. It was shown that the Fe3O4 nanoparticles were successfully encapsulated into the GO, and moreover the lipase was tethered on the magnetic support. The immobilized lipase could efficiently catalyze the transesterification of soybean oil with methanol for the production of biodiesel. With this magnetic biocatalyst, the biodiesel yield of 92.8% could be obtained at a temperature of 40 °C by three-step addition of methanol in a shaking water bath. The immobilized lipase could be easily recovered by using an external magnetic field, allowing for recycling of the biocatalyst five times without significant loss of its activity.

Immobilization of Thermomyces lanuginosus lipase on multi-walled carbon nanotubes and its application in the hydrolysis of fish oil

In this study, lipase from Thermomyces lanuginosus (TLL) was immobilized on carbon nanotubes (MWCNTs) by physical adsorption, and the immobilizing conditions were optimized for maximum activity. The effects of enzyme loading, pH, temperature and time on the immobilization efficiency and specific activity were evaluated. The highest enzyme activity and immobilization efficiency of 90.66% and 110.5%, respectively, were achieved when the immobilized pH was 8, and a high rate of recovery activity of 111.3% occurred at 45 °C with 30–60 min providing a good result. When the immobilization efficiency was 95.8%, the recovery activity was 112.4%. The immobilization time had little effect on the immobilization efficiency, and 6 mg g−1 of lipase provided the highest immobilization efficiency of 97.78% and recovery activity of 112.8%. When the immobilized lipase was utilized to enrich docosahexaenoic acid (DHA) from fish oil, the DHA content increased with increasing amount of lipase up to 9 mg g−1. The water content had a clear effect when of 50% water was used at 45 °C and at a pH of 7 after 10 h. The DHA contents were 4.2-fold and 2.5-fold greater than the initial content of DHA fish oil for TLL-MWCNTs and free lipase, respectively. The degrees of hydrolysis after 6 cycles of successive use were over 80% and 62% for the immobilized TLL and free TLL, respectively, indicating the system recyclability and the ease of use of the immobilized TLL in industrial applications, especially in the fields of food and medicine.

Optimal direct electron transfer between MWCNTs@COOH/BOD/chitosan layer and porous carbon felt for dioxygen reduction

We present the effect of thermal treatment of carbon felt by gas flow containing 1% of oxygen at high temperature on the direct electron transfer and electrocatalytic oxygen reduction currents by a bilirubin oxidase (BOD) film directly adsorbed in the presence of carbon nanotubes on to a porous carbon felt (PCF). The upgraded properties (surface area, pore volume and hydrophilicity) of the resulting porous carbon felt (PCF) in comparison to commercial carbon felt (raw CF), creates a suitable support for the entrapment of MWCNTs bearing negative charges at neutral pH and BOD enzymes, all the components being entrapped in chitosan layer reticulated with glutaraldehyde. Since functional MWCNTs are usually used to facilitate DET, we introduce MWCNTs@COOH, bearing negative charges at neutral pH, in the enzyme layer to evaluate their impact on the electron transfer properties with BOD. The enzyme immobilization efficiency is examined by varying the amount of the components and the immobilization procedure. Linear sweep voltammetry (LSV) and chronoamperometry measurements are used to evaluate the electrochemical behavior of the enzymatic biocathodes. Based on the experimental results, we show that the optimized bioelectrode delivers a current density of 3.70mAcm−2 at 0.15V vs Ag/AgCl and could retain above 55% of its initial response after 4 months, proving its outstanding performance. This new bioelectrode allows for optimal DET-type bioelectrocatalytic activity toward O2 reduction and is a very promising candidate for the construction of 3-dimensional cathodes in (bio)-electrochemical devices needing high current output.

Bioinspired Multifunctional Membrane for Aquatic Micropollutants Removal.

Micropollutants present in water have many detrimental effects on the ecosystem. Membrane technology plays an important role in the removal of micropollutants, but there remain significant challenges such as concentration polarization, membrane fouling, and variable permeate quality. The work reported here uses a multifunctional membrane with rejection, adsorption, and catalysis functions to solve these problems. On the basis of mussel-inspired chemistry and biological membrane properties, a multifunctional membrane was prepared by applying "reverse filtration" of a laccase solution and subsequent "dopamine coating" on a nanofiltration (NF) membrane support, which was tested on bisphenol A (BPA) removal. Three NF membranes were chosen for the preparation of the multifunctional membranes on the basis of the membrane properties and enzyme immobilization efficiency. Compared with the pristine membrane, the multifunctional membrane exhibited significant improvement of BPA removal (78.21 ± 1.95%, 84.27 ± 7.30%, and 97.04 ± 0.33% for NT103, NF270, and NF90, respectively), all of which are clearly superior to the conventional Fenton treatment (55.0%) under similar conditions and comparable to soluble laccase coupled with NF270 membrane filtration (89.0%). The improvement would appear to be due to a combination of separation (reducing the enzymatic burden), adsorption (enriching the substrate concentration as well as prolonging the residence time), and lastly, catalysis (oxidizing the pollutants and breaking the "adsorption saturation limits"). Furthermore, the synergistic effect of the polydopamine (PDA) layer on the enzymatic oxidation of BPA was confirmed, which was due to its enhanced adsorption and electron transfer performance. The multifunctional membrane could be reused for at least seven cycles with an acceptable activity loss, demonstrating good potential for removal of micropollutants.

Covalent immobilization of Candida rugosa lipase on a novel functionalized Fe3O4@SiO2 dip-coated nanocomposite membrane

Membrane-immobilized enzymes have received increasing attention in a variety of fields such as biosensors, enzymatic reactors, and membrane bioreactors. In this study, Candida rugosa lipase (CRL) was covalently immobilized on nanocomposite membrane. Initially, the Fe3O4@SiO2 nano-particles were dip-coated onto the ultrafiltration membrane surface through a low temperature hydrothermal (LTH) process, and then, reacted with 3-aminopropyletriethoxysilane (APTS). Glutaraldehyde was used as a coupling agent to covalently immobilize lipase on the nanocomposite membrane surface. Various techniques such as SEM, TEM, XRD, FTIR, AFM, contact angle goniometry and surface free energy measurement were applied to characterize the nanocomposite membrane. The results showed that the immobilization process was successful in terms of enzyme activity and immobilization efficiency. It was found that the activated nanocomposite membrane greatly improved the relative activity and loading capacity in comparison to unmodified UF membrane. The investigation of kinetic parameters of enzymatic reaction shows the decreasing of Km and vmax due to immobilization which represents the increasing of substrate affinity and decreasing of catalytic activity of immobilized enzyme. The higher thermal, storage, and operational stability of immobilized enzyme make it a suitable candidate for to bio-catalytic processes.

An easily regenerable enzyme reactor prepared from polymerized high internal phase emulsions

A large-scale high-efficient enzyme reactor based on polymerized high internal phase emulsion monolith (polyHIPE) was prepared. First, a porous cross-linked polyHIPE monolith was prepared by in-situ thermal polymerization of a high internal phase emulsion containing styrene, divinylbenzene and polyglutaraldehyde. The enzyme of TPCK-Trypsin was then immobilized on the monolithic polyHIPE. The performance of the resultant enzyme reactor was assessed according to the conversion ability of Nα-benzoyl-l-arginine ethyl ester to Nα-benzoyl-l-arginine, and the protein digestibility of bovine serum albumin (BSA) and cytochrome (Cyt-C). The results showed that the prepared enzyme reactor exhibited high enzyme immobilization efficiency and fast and easy-control protein digestibility. BSA and Cyt-C could be digested in 10 min with sequence coverage of 59% and 78%, respectively. The peptides and residual protein could be easily rinsed out from reactor and the reactor could be regenerated easily with 4 M HCl without any structure destruction. Properties of multiple interconnected chambers with good permeability, fast digestion facility and easily reproducibility indicated that the polyHIPE enzyme reactor was a good selector potentially applied in proteomics and catalysis areas.

Covalent immobilization of α-amylase on magnetic particles as catalyst for hydrolysis of high-amylose starch

Enzyme immobilized on magnetic particles can be used as efficient recoverable biocatalysts under strong magnetic response. To enable re-use of enzyme, modified Fe3O4 particles were used as carrier to immobilize α-amylase in this paper. Firstly, the surface of Fe3O4 particles were coated with amino groups by direct using TEOS (tetraethoxysilane) followed by treatment with APTES (3-aminopropyltriethoxysilane) and then carboxylated by reacting it with succinic anhydride. In addition, the effect of the immobilization condition on enzyme activity recovery and immobilization efficiency were investigated. The results showed that the optimal immobilization occurred under following conditions: pH 5.5, 40°C, enzyme concentration of 20mgmL−1, reaction time for 36h. Using immobilized α-amylase as biocatalyst, the optimum pH and temperature for hydrolysis were observed to be 6.5 and 60°C. The kinetics of hydrolysis reaction were studied using Michaelis–Menten equation. The affinity constant (Km) and maximum reaction rate (vmax) of magnetic particles immobilization α-amylase (MPIA) was 0.543mgmL−1 and 1.321mgmin−1 compared to those of 0.377mgmL−1 and 6.859mgmin−1 of free enzyme. After immobilization, enzymatic activity, storage stability, thermo-stability, and reusability of MPIA were found superior to those of the free one. MPIA maintained 86% enzyme activity after 30 days and maintained 78% enzyme activity after recycling six times.

Covalent immobilization of microbial naringinase using novel thermally stable biopolymer for hydrolysis of naringin.

Naringinase induced from the fermented broth of marine-derived fungus Aspergillus niger was immobilized into grafted gel beads, to obtain biocatalytically active beads. The support for enzyme immobilization was characterized by ART-FTIR and TGA techniques. TGA revealed a significant improvement in the grafted gel’s thermal stability from 200 to 300 °C. Optimization of the enzyme loading capacity increased gradually by 28-fold from 32 U/g gel to 899 U/g gel beads, retaining 99 % of the enzyme immobilization efficiency and 88 % of the immobilization yield. The immobilization process highly improved the enzyme’s thermal stability from 50 to 70 °C, which is favored in food industries, and reusability test retained 100 % of the immobilized enzyme activity after 20 cycles. These results are very useful on the marketing and industrial levels.

Performance Improvement of Ca-Alginate Bead Cross- Linked Laccase from Trametes versicolor IBL-04

Extracellular laccase was produced by Trametes versicolor IBL-04 using corn cobs as a substrate under pre-optimized culture conditions. A 64-kDa laccase enzyme was purified and immobilized on calcium alginate beads using glutaraldehyde as a cross-linking reagent. Maximum enzyme immobilization efficiency (89%) was observed with 2-mm calcium-alginate beads that were developed using 4% (w/v) sodium alginate in 2% (w/v) calcium chloride solution. Immobilization of laccase enhanced the optimum temperature but caused an acidic shift in the optimum pH of the enzyme. The immobilized enzyme showed optimum activity at pH 3.0 and 60 °C as compared to pH 4.5 and 45 °C for free laccase. The kinetic constants Km and Vmax of laccase were significantly altered by immobilization. The affinity of enzyme toward its substrate increased (Km decreased), leading to enhanced catalytic efficiency (Vmax increased). Scanning electron microscopy (SEM) was performed to characterize the free and enzyme-bound immobilization matrix. Free and immobilized enzymes also were used for decolorization of the Reactive T Blue dye (030905 GWF) for three days. The free and immobilized laccases decolorized the dye by 65% and 92%, respectively, in 72 h. The immobilized enzyme retained 68% of its original activity after three cycles of repeated reuse for dye decolorization, indicating the usefulness of immobilized laccase in repeated industrial batch operations.

An enzymatic biosensor based on three-dimensional ZnO nanotetrapods spatial net modified AlGaAs/GaAs high electron mobility transistors

We designed and constructed three dimensional (3D) zinc oxide Nanotetrapods (T-ZnOs) modified AlGaAs/GaAs high electron mobility transistors (HEMTs) for enzymatic uric acid (UA) detection. The chemical vapor deposition synthesized T-ZnOs was distributed on the gate areas of HEMTs in order to immobilize uricase and improve the sensitivity of the HEMTs. Combining with the high efficiency of enzyme immobilization by T-ZnOs and high sensitivity from HEMT, the as-constructed uricase/T-ZnOs/HEMTs biosensor showed fast response towards UA at ∼1 s, wide linear range from 0.2 nM to 0.2 mM and the low detect limit at 0.2 nM. The results point out an avenue to design electronic device as miniaturized lab-on-chip device for high sensitive and specific in biomedical and clinical diagnosis applications.

Nanostructured porous Si optical biosensors: effect of thermal oxidation on their performance and properties.

The influence of thermal oxidation conditions on the performance of porous Si optical biosensors used for label-free and real-time monitoring of enzymatic activity is studied. We compare three oxidation temperatures (400, 600, and 800 °C) and their effect on the enzyme immobilization efficiency and the intrinsic stability of the resulting oxidized porous Si (PSiO2), Fabry-Pérot thin films. Importantly, we show that the thermal oxidation profoundly affects the biosensing performance in terms of greater optical sensitivity, by monitoring the catalytic activity of horseradish peroxidase and trypsin-immobilized PSiO2. Despite the significant decrease in porous volume and specific surface area (confirmed by nitrogen gas adsorption-desorption studies) with elevating the oxidation temperature, higher content and surface coverage of the immobilized enzymes is attained. This in turn leads to greater optical stability and sensitivity of PSiO2 nanostructures. Specifically, films produced at 800 °C exhibit stable optical readout in aqueous buffers combined with superior biosensing performance. Thus, by proper control of the oxide layer formation, we can eliminate the aging effect, thus achieving efficient immobilization of different biomolecules, optical signal stability, and sensitivity.

Immobilization of cellulase enzyme on functionalized multiwall carbon nanotubes

Abstract For the past decades, the global trends in the demand of cellulase has been arisen due to its extensive range of applications in food and agriculture industry, and its potential use in the fermentation of biomass into biofuels. However, the instability, highly solubility in water, low catalytic efficiency and high cost of enzyme has become the main obstacles for the development of large scale operations and applications. In this study, cellulase enzyme was immobilized onto functionalized multiwalled carbon nanotubes (MWCNTs) via physical adsorption method to yield a stable and ease of separate enzyme. Fourier transform infrared (FTIR) spectroscopy and field emission scanning electron microscopy (FESEM) are used to confirm the successful immobilization of cellulase enzyme. In this approach, the efficiency of enzyme immobilization reaches an optimal value when 4 mg/mL enzyme concentration is used in which approximately 97% enzyme loading can be attained. Based on the UV–visible spectroscopy analysis, the optimum reaction conditions for immobilized cellulase are at pH 5 and a temperature of 50 °C. Results have revealed that MWCNT–cellulase composite still retained 52% of its cellulase activity after six cycles of the CMC analysis. This feature is beneficial to the industrial applications because of its potential to be easily separated from the end product at the end of the reaction, reuse for multiple times and allow the development of multiple enzyme reaction system.

Method for the stabilization and immobilization of enzymatic extracts and its application to the decolorization of textile dyes.

Peroxidases from Pleurotus eryngii have been investigated for their ability to degrade recalcitrant, phenolic pollutants. The use of crude enzymatic extracts can reduce the high costs associated with enzyme purification, and enzyme immobilization can enhance enzyme stability and recovery. The present study tests the effectiveness of various conditions for crude enzyme stabilization in polyethylene glycol and glycine solutions, and immobilization on monofunctional and heterofunctional agarose solid supports. Glycine at 0.5 M at 4 °C and pH 4 was most effective stabilization agent for the crude enzymatic extracts, and enzyme immobilization efficiency was greatest for heterofunctional supports. MANA-glyoxyl heterofunctional supports were demonstrated to have the greatest enhancement of decolorization (1.3-fold) and velocity of substrate consumption (fivefold). Therefore, the application of crude enzymatic extracts to industrial processes, such as dye decolorization, represents a cost-effective alternative to purified enzymes.