Immobilization of Rhizomucor miehei lipase onto montmorillonite K-10 and polyvinyl alcohol gel

Abstract

Immobilization of enzymes from different sources on various supports in designed systems increases enzymes’ stability by protecting the active site of it from undesired effect of reaction environment. Also, immobilization decreases the cost of separation and facilities the reuse of the enzymes. Therefore, the design of new immobilization enzyme preparations has been an inevitable area of modern biotechnology. Herein, Rhizomucor miehei lipase (RML) was immobilized on montmorillonite K-10 (MMT-RML) by adsorption and in polyvinyl alcohol (PVA-RML) by entrapment to obtain a more stable and active lipase preparation. The free and immobilized lipase preparations were characterized for p-nitrophenyl palmitate hydrolysis. The apparent Michaelis–Menten (K_mapp) constant was almost the same for the free RML and PVA-RML, whereas the corresponding value was 17.7-fold lower for MMT-RML. PVA-RML and MMT-RML have shown a 1.1 and 23.8 folds higher catalytic efficiency, respectively, than that of the free RML. The half-lives of PVA-RML and MMT-RML were found to be 7.4 and 3.4 times longer than the free RML at 35?°C, respectively. PVA-RML and MMT-RML maintained 65% and 87% of their initial activities after four reuses. These results showed that the catalytic performance of RML has improved significantly by immobilization.     

Immobilization of cellulase on magnetoresponsive graphene nano-supports

In this study, we report the preparation of pH tunable, temperature sensitive magnetoresponsive graphene-based nano-bio carriers for cellulase immobilization. We discuss a simple route to overcome the geometric disadvantage imposed by most 2D immobilization supports and make them capable of closely mimicking free enzymes (FE) operating under similar reaction conditions. The supramolecular assembly of oppositely charged quenched polyelectrolytes and maghemite–magnetite nanoparticles on 2D graphene supports followed by covalent immobilization of cellulase shows a marked improvement in the bio-receptivity of graphene supports. The incorporation of magnetic nanoparticles opens up the possibility of recovery and reuse of the enzyme over multiple cycles. The immobilized enzymes retained about 55% of the original specific activity even after four cycles of reuse. Cellulase immobilization is achieved by a combination of annealed polyelectrolyte brushes and zero-length spacer molecules. The swelling behavior of annealed polyelectrolyte brushes is a strong function of the environmental conditions. The degree of polyelectrolyte swelling can be easily tweaked by manipulating the pH and temperature, providing us an effective tool to control the activity of immobilized enzymes. At a pH of 5.1 and a temperature of 50 °C, the immobilized enzymes with the annealed polyelectrolyte brushes displayed close to 1.5-fold improvement in the activity as compared to immobilized enzymes without the brushes. Activity of immobilized cellulase is evaluated using both soluble as well as insoluble substrates like 2% (w/v) CMC and avicel respectively.     

Recent advances in nano-carrier immobilized enzymes and their applications

Enzymes have been widely used because of their catalytic properties, and immobilization is a promising technique to improve their catalytic activity and stability. Due to their large specific surface areas, exceptional chemical, mechanical, thermal and cost effective characteristics, nanomaterials should be ideal carriers for the immobilization of enzymes. Enzymes immobilized on nano-carriers are more robust and stable, and can be recycled and reused. This review focuses on the nanomaterial immobilized enzymes and their applications. The introduction addresses the advantages of immobilized enzymes and the features of enzyme immobilization nanocarriers. The next section covers carbonaceous nanomaterials used in enzymes immobilization, with subsections on carbon nanotube, graphene, graphene oxide and reduced graphene oxide. The third section treats metallic nanomaterials for enzymes immobilization, with subsections on metal (gold), metal oxide (titanium dioxide, zinc oxide) and metal hydroxide (layered double hydroxide) nanomaterials. Then, the next section summarizes the applications of nanomaterial immobilized enzymes. A concluding section discusses the challenges and prospects of nanomaterial immobilized enzymes.     

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.     

Efficient immobilization technique for enhancement of cellobiose dehydrogenase activity on silica gel

In this study, cellobiose dehydrogenase (CDH) of Phanerochaete chrysosporium ATCC 32629 was immobilized on silica gel for the further application of CDH in the saccharification process of biomass. To prevent the loss of enzyme activity during enzyme immobilization, the pretreatment of CDH was performed by various pretreatment materials before immobilization. When pretreated enzymes were used in immobilization, the activities of immobilized CDH were higher than non-pretreated CDH even in same amounts of immobilized protein. The specific activity of pretreated immobilized CDH with lactose was about two times higher than that of non-pretreated immobilized CDH. Moreover, the pretreated immobilized CDH showed better reusability than non-pretreated immobilized CDH, with 67.3% of its original activity being retained after 9 reuses.     

Comparative studies on soluble and immobilized yeast hexokinase

Comparative studies have been carried out on soluble and immobilized yeast hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1). The enzyme was immobilized by covalent attachment to a polyacrylamide type support containing carboxylic functional groups. The effects of immobilization on the catalytic properties and stability of hexokinase were studied. As a result of immobilization, the pH optimum for catalytic activity was shifted in the alkaline direction to ～pH 9.7. The apparent optimum temperature of the immobilized enzyme was higher than that of the soluble enzyme. The apparent K_m value with D-glucose as substrate increased, while that with ATP as substrate decreased, compared with the data for the soluble enzyme. Differences were found in the thermal inactivation processes and stabilities of the soluble and immobilized enzymes. The resistance to urea of the soluble enzyme was higher at alkaline pH values, while that for the immobilized enzyme was greatest at ～pH 6.0.     

Immobilization and characterization of horseradish peroxidase into chitosan and chitosan/PEG nanoparticles: A comparative study

Chitosan (CS) is considered a suitable biomaterial for enzyme immobilization. CS combination with polyethylene glycol (PEG) can improve the biocompatibility and the properties of the immobilized system. Thus, the present work investigated the effect of the PEG in the horseradish peroxidase (HRP) immobilization into chitosan nanoparticles from the morphological, physicochemical, and biochemical perspectives. CS and CS/PEG nanoparticles were obtained by ionotropic gelation and provided immobilization efficiencies (IE) of 65.8 % and 51.7 % and activity recovery (AR) of 76.4 % and 60.4 %, respectively. The particles were characterized by DLS, ZP, SEM, FTIR, TGA and DSC analysis. Chitosan nanoparticles showed size around 135 nm and increased to 229 nm after PEG addition and HRP immobilization. All particles showed positive surface charges (20−28 mV). Characterizations suggest nanoparticles formation and effective immobilization process. Similar values for optimum temperature and pH for immobilized HRP into both nanoparticles were found (45 °C, 7.0). V_max value decreased by 5.07 to 3.82 and 4.11 mM/min and K_M increased by 17.78 to 18.28 and 19.92 mM for free and immobilized HRP into chitosan and chitosan/PEG nanoparticles, respectively. Another biochemical parameters (K_cat, K_e, and K_α) evaluated showed a slight reduction for the immobilized enzyme in both nanoparticles compared to the free enzyme.     

Enhancing oxidation activity and stability of iso-1-cytochrome c and chloroperoxidase by immobilization in nanostructured supports

The immobilization of enzymes in inorganic materials has been widely used because it can produce an enhancement of the catalytic stability and enzymatic activity. In this article, the effect of the immobilization of iso-1-cytochrome c (CYC-Sc) from Saccharomyces cerevisiae and chloroperoxidase (CPO) from Caldariomyces fumago on the enzyme stability and catalytic oxidation of styrene was studied. The immobilization was carried out in three silica nanostructured supports with different pore size MCM-41 (3.3 nm), SBA-15 (6.4 nm) and MCF (12.1 nm). The adsorption parameters and leaching degree of immobilized enzymes were determined. Catalytic parameters of immobilized and free enzymes were determined at different temperatures (20–60 °C) and in different acetonitrile/water mixtures (15–85% of acetonitrile). The results show that there is low leaching of the enzymes in the three supports assayed and the adsorption capacity (q_max) was higher as the pore size of the support increased. The pore size also produces the enhancement of peroxidase activities on the styrene oxidation. Thus, CPO adsorption into SBA-15 and MCF showed remarkable thermal and solvent stabilities at 40 °C showing a total turnover numbers of 48,000 and 54,000 times higher than free CPO, respectively. The enhancement of activity and stability doubtless is interesting for the potential industrial use of peroxidases.     

Immobilization/stabilization of acid urease on Eupergit® supports

The adsorption capacity and immobilization rate of two Eupergit® supports for acid urease was studied by varying the ionic strength and enzyme preparation concentration in the immobilizing solution at pH 7. Eupergit® C250 L yielded a series of derivatives with enzyme loadings (Y_P/B) ranging from 48 to 171 mg of bovine serum albumin equivalent (BSAE) per gram of dry support (ds). Use of drastic postimmobilization conditions at pH 9 for 3–9 days yielded a slight decrease (8–14%) in the initial activity of immobilized enzymes and a limited increase in the stabilization factor (1.1–1.5), as assessed by accelerated aging tests at 65°C. Further storage tests at 4°C in the wet state showed that the activity of several derivatives either stabilized or not was practically constant for as long as 547 days. Both free enzyme and immobilized acid urease derivatives exhibited a kinetic pattern of the Michaelis–Menten type. Using the Eadie–Hofstee diagram, the specific ammonia formation rate constant for free (k_cat) or immobilized (k′_cat) enzyme resulted to be little affected by immobilization (k_cat ≈ k′_cat ≈ 18.86 ± 0.34 IU/mg BSAE), whereas the apparent Michaelis constant for immobilized enzymes exhibited a statistically significant increase at P < 0.05 from the intrinsic value (2.55 ± 0.14 mM) for free enzyme to 5.38 ± 0.87 mM as Y_P/B increased to 171 mg BSAE/g ds. By estimating the observable Thiele modulus (?_obs), the activity of the biocatalyst with the greatest enzyme loading at the lowest urea concentrations tested (0.833 mM) was reduced by a factor of about 2 due to internal diffusional limitations. By operating in the pseudofirst‐order regime with immobilized derivatives at Y_P/B about 126 mg BSAE/g ds, their activity after grinding was no more limited by intraparticle diffusion and approached the value for free enzyme. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Model of the regulation of activity of immobilized enzymes (amylases) in soil

The preservation of activity of extracellular enzymes in soil is presently associated with their immobilization on organic or inorganic carriers. Enzyme immobilization results, however, in a significant decrease in enzymatic activity. In the present work, the mechanism responsible for promotion of the catalytic activity was revealed, as well as the favorable effect of low-molecular alkylhydrozybenzenes of the class of alkylresorcinols, which are common in soil organic matter, on stability of immobilized enzymes (exemplified by amylases) by their post-translational modification. Optimal conditions (enzyme to sorbent ratio, pH optimum, CaCl₂ concentration, and sorption time) for amylase sorption on a biological sorbent (yeast cell walls) were determined and decreased activity of the immobilized enzyme compared to its dissolved state was confirmed. Alkylresorcinols (C₇AHB) at concentrations of 1.6 to 80 mM were found to cause an increase of amylase activity both in the case of already sorbed enzymes (by 30%) and in the case of a free dissolved enzyme with its subsequent immobilization (by 50–60%). In both cases, the optimal C₇AHB concentration was 16 mM. Amylase stability was determined for C7AHB-modified and unmodified enzymes immobilized on the biological sorbent after two cycles of freezing (–20°C) and thawing (4°C). Inverse dependence was revealed between increasing stability of C₇AHB-modified enzymes and an increase in their activity, as well as higher stability of immobilized modified amylases than of the dissolved modified enzyme. Investigation of the effect of C₇HOB-modification in the preservation of activity in immobilized amylases after four freeze–thaw cycles revealed: (1) better preservation of activity by the modified immobilized enzymes compared to immobilized ones; (2) differences in the dynamics of activity loss within compared pairs, with activity of immobilized amylases decreasing after the second cycle to a lower level (42%) than activity of the modified immobilized enzymes after the fourth cycle (48%). These results demonstrate that in the preservation of activity of extracellular enzymes in soil both stabilization mechanisms are of importance: immobilization on organic carriers and modification of the enzyme conformation by low-molecular compounds with the functions of chemical chaperones.     

Improvement of the functional properties of a thermostable lipase from alcaligenes sp. via strong adsorption on hydrophobic supports

Lipase QL from Alcaligenes sp. is a quite thermostable enzyme. For example, it retains 75% of catalytic activity after incubation for 100 h at 55 °C and pH 7.0. Nevertheless, an improvement of the enzyme properties was intended via immobilization by covalent attachment to different activated supports and by adsorption on hydrophobic supports (octadecyl-sepabeads). This latter immobilization technique promotes the most interesting improvement of enzyme properties: (a) the enzyme is hyperactivated after immobilization: the immobilized preparation exhibits a 135% of catalytic activity for the hydrolysis of p-nitrophenyl propionate as compared to the soluble enzyme; (b) the thermal stability of the immobilized enzyme is highly improved: the immobilized preparation exhibits a half-life time of 12 h when incubated at 80 °C, pH 8.5 (a 25-fold stabilizing factor regarding to the soluble enzyme); (c) the optimal temperature was increased from 50 °C (soluble enzyme) up to 70 °C (hydrophobic support enzyme immobilized preparations); (d) the enantioselectivity of the enzyme for the hydrolysis of glycidyl butyrate and its dependence on the experimental conditions was significantly altered. Moreover, because the enzyme becomes reversibly but very strongly adsorbed on these highly hydrophobic supports, the lipase may be desorbed after its inactivation and the support may be reused. Very likely, adsorption occurs via interfacial activation of the lipase on the hydrophobic supports at very low ionic strength. On the other hand, all the covalent immobilization protocols used to immobilize the enzyme hardly improved the properties of the lipase.     

Improving reuse cycles of Thermomyces lanuginosus lipase (NS-40116) by immobilization in flexible polyurethane

Enzyme immobilization using a low-cost support that allows increasing operational stability and reutilization arise as a great economic advantage for the industry. In this work, it was explored different methods of Thermomyces lanuginosus lipase (NS-40116) immobilization in flexible polyurethane foam (PU). PU polymer was synthesized using polyether and toluene diisocyanate as monomers. PU-NS-40116 immobilized was evaluated in terms of stability in a range of pH (7.0 and 9.0), temperature (24, 50 and 60?°C) for 24?h, and storage stability (room temperature and 4?°C) for 30?days. The results showed that after 30?days of storage immobilized enzyme kept 80% of initial enzyme activity. PU support before and after immobilization process was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Free and immobilized enzymes were compared in terms of hydrolysis of soybean oil. Immobilized enzyme by entrapment was evaluated in successive cycles of reuse showing catalytic activity above 50% even after 5 successive cycles of reuse, confirming the efficiency of immobilization process.     

Immobilization of fungal peroxidase on alkylaminated organic and inorganic porous supports

Several alkylaminated porous silica gels and acrylic type porous polymers have been used for covalent binding of fungal peroxidase from Trametes versicolor. The immobilization efficiency expressed in terms of the bound protein content, specific enzyme activity, and enzyme storage stability have been determined for both types of supports used. The results indicate a better immobilization ability of organic polymers in comparison with silica gels.     

Novel polymeric microspheres: Synthesis,enzyme immobilization,antimutagenic activity,and antimicrobial evaluation against pathogenic microorganisms

New polymeric microspheres containing azomethine ( 1a ‐ 1c and 2a ‐ 2c ) were synthesized by condensation to compare the enzymatic properties of the enzyme glucose oxidase (GOx) and to investigate antimutagenic and antimicrobial activities. The polymeric microspheres were characterized by elemental analysis, infrared spectra (FT‐IR), proton nuclear magnetic resonance spectra, thermal gravimetric analysis, and scanning electron microscopy analysis. The catalytic activity of the glucose oxidase enzyme follows Michaelis‐Menten kinetics. Influence of temperature, reusability, and storage capacity of the free and immobilized glucose oxidase enzyme were investigated. It is determined that immobilized enzymes exhibit good storage stability and reusability. After immobilization of GOx in polymeric supports, the thermal stability of the enzyme increased and the maximum reaction rate (V_max) decreased. The activity of the immobilized enzymes was preserved even after 5 months. The antibacterial and antifungal activity of the polymeric microspheres were evaluated by well‐diffusion method against some selected pathogenic microorganisms. The antimutagenic properties of all compounds were also examined against sodium azide in human lymphocyte cells by micronuclei and sister chromatid exchange tests.     

Studies on Pectolytic Enzymes of Molds

Experiments have been made on fractionation of the pectolytic enzymes produced by Coniothyrium diplodiella. It has been observed that 30 to 35% of the polygalacturonase (PG) activity of the pectolytic enzymes of the said microorganism is salted out with ammonium sulfate, and this portion contains cndo-PG I, endo-PG II and pectin esterase (PE) (with a trace of exo-PG). The endo-PG I accounts for 60 to 65% of the total PG activity, and the endo-PG II, 25 to 30%. Both types of endo-PG scarcely act on pectin, and hydrolyze pectic acid to the extent of 65 to 70%.     

Active site titration as a tool for the evaluation of immobilization procedures of penicillin acylase

Native and immobilized preparations of penicillin acylase from Escherichia coli and Alcaligenes faecalis were studied using an active site titration technique. Knowledge of the number of active sites allowed the calculation of the average turnover rate of the enzyme in the various preparations and allowed us to quantify the contribution of irreversible inactivation of the enzyme to the loss of catalytic activity during the immobilization procedure. In most cases a loss of active sites as well as a decrease of catalytic activity per active site (turnover rate) was observed upon immobilization. Immobilization techniques affected the enzymes differently. The effect of increased loading of penicillin acylase on the average turnover rate was determined by active site titration to assess diffusion limitations in the carrier.     

Potential applications of enzymes immobilized on/in nano materials: A review

Several new types of carriers and technologies have been implemented in the recent past to improve traditional enzyme immobilization which aimed to enhance enzyme loading, activity and stability to decrease the enzyme biocatalyst cost in industrial biotechnology. These include cross-linked enzyme aggregates, microwave-assisted immobilization, click chemistry technology, mesoporous supports and most recently nanoparticle-based immobilization of enzymes. The union of the specific physical, chemical, optical and electrical properties of nanoparticles with the specific recognition or catalytic properties of biomolecules has led to their appearance in myriad novel biotechnological applications. They have been applied time and again for immobilization of industrially important enzymes with improved characteristics. The high surface-to-volume ratio offered by nanoparticles resulted in the concentration of the immobilized entity being considerably higher than that afforded by experimental protocols based on immobilization on planar 2-D surfaces. Enzymes immobilized on nanoparticles showed a broader working pH and temperature range and higher thermal stability than the native enzymes. Compared with the conventional immobilization methods, nanoparticle based immobilization served three important features; (i) nano-enzyme particles are easy to synthesize in high solid content without using surfactants and toxic reagents, (ii) homogeneous and well defined core-shell nanoparticles with a thick enzyme shell can be obtained, and (iii) particle size can be conveniently tailored within utility limits. In addition, with the growing attention paid to cascade enzymatic reaction and in vitro synthetic biology, it is possible that co-immobilization of multi-enzymes could be achieved on these nanoparticles.     

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.     

Reversible immobilization of glucoamylase onto magnetic chitosan nanocarriers

A simple preparation process for the monodispersed pH-sensitive core-shell magnetic microspheres was carried out consisting of chitosan self-assembled on magnetic iron oxide nanoparticles. Meanwhile, glucoamylase was immobilized as a model enzyme on this carrier of Fe₃O₄/CS microspheres by ionic adsorption. The morphology, inner structure, and high magnetic sensitivity of the resulting magnetic chitosan microspheres were studied, respectively, with a field emission scanning electron microscope (SEM), transmission electron microscope (TEM), FT-IR spectroscopy, thermogravimetric analysis (TGA), and a vibrating sample magnetometer (VSM). Subsequently, the properties of glucoamylase immobilized on the regenerated supports were also investigated by determining storage stability, pH stability, reusability, magnetic response, and regeneration of supports. The results from characterization and determination remarkably indicated that the immobilized glucoamylase obtained presents excellent storage stability, pH stability, reusability, magnetic response, and regeneration of supports. Therefore, this kind of magnetic Fe₃O₄/CS microspheres with perfect monodispersity should be an ideal support for enzyme immobilization.     

Immobilization of <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis> penicillin G acylase on epoxy-type supports

Alcaligenes faecalis penicillin G acylase has several desired features over other penicillin G acylases and its use in industry requires immobilization. In this work, two novel supports ZH-EP (epoxy type) and ZH-HA (epoxy-amino type) were used to immobilize Alcaligenes faecalis penicillin G acylase (AfPGA) with Eupergit C as reference. The saturation of immobilized protein on ZH-EP (269 mg/g, 116 h) and ZH-HA (296 mg/g, 15 h) was obtained more rapidly than Eupergit C (197 mg/g, 260 h). And the activity of immobilized AfPGA on ZH-EP (520 U/g) and ZH-HA (2200 U/g) was higher than that on Eupergit C (310 U/g). The properties of three immobilized enzymes were compared and no obvious difference was observed, which indicated that ZH-EP and ZH-HA were promised in industry.