IMMOBILIZATION OF BOVINE CATALASE ONTO MAGNETIC NANOPARTICLES

The scope of this study is to achieve carrier-bound immobilization of catalase onto magnetic particles (Fe₃O₄ and Fe₂O₃NiO₂ · H₂O) to specify the optimum conditions of immobilization. Removal of H₂O₂ and the properties of immobilized sets were also investigated. To that end, adsorption and then cross-linking methods onto magnetic particles were performed. The optimum immobilization conditions were found for catalase: immobilization time (15 min for Fe₃O₄; 10 min for Fe₂O₃NiO₂ · H₂O), the initial enzyme concentration (1 mg/mL), amount of magnetic particles (25 mg), and glutaraldehyde concentration (3%). The activity reaction conditions (optimum temperature, optimum pH, pH stability, thermal stability, operational stability, and reusability) were characterized. Also kinetic parameters were calculated by Lineweaver–Burk plots. The optimum pH values were found to be 7.0, 7.0, and 8.0 for free enzyme, Fe₃O₄-immobilized catalases, and Fe₂O₃NiO₂ · H₂O-immobilized catalases, respectively. All immobilized catalase systems displayed the optimum temperature between 25 and 35°C. Reusability studies showed that Fe₃O₄-immobilized catalase can be used 11 times with 50% loss in original activity, while Fe₂O₃NiO₂ · H₂O-immobilized catalase lost 67% of activity after the same number of uses. Furthermore, immobilized catalase systems exhibited improved thermal and pH stability. The results transparently indicate that it is possible to have binding between enzyme and magnetic nanoparticles.     

Optimization of covalent immobilization of pectinase on sodium alginate support

Pectinase was immobilized on a sodium alginate support using glutaraldehyde and retained 66% activity. The optimal pH for activity shifted from 3.0 to 3.5 after immobilization; however, the optimum temperature remained unchanged at 40°C. The immobilized enzyme also had a higher thermal stability and reusability than the free enzyme, and retained 80% of initial activity after 11 batch reactions.     

Immobilization and characterization of lipase from an indigenous Bacillus aryabhattai SE3-PB isolated from lipid-rich wastewater

Abstract

Extracellular lipase from an indigenous Bacillus aryabhattai SE3-PB was immobilized in alginate beads by entrapment method. After optimization of immobilization conditions, maximum immobilization efficiencies of 77%?±?1.53% and 75.99%?±?3.49% were recorded at optimum concentrations of 2% (w/v) sodium alginate and 0.2?M calcium chloride, respectively, for the entrapped enzyme. Biochemical properties of both free and immobilized lipase revealed no change in the optimum temperature and pH of both enzyme preparations, with maximum activity attained at 60?°C and 9.5, respectively. In comparison to free lipase, the immobilized enzyme exhibited improved stability over the studied pH range (8.5–9.5) and temperature (55–65?°C) when incubated for 3?h. Furthermore, the immobilized lipase showed enhanced enzyme-substrate affinity and higher catalytic efficiency when compared to soluble enzyme. The entrapped enzyme was also found to be more stable, retaining 61.51% and 49.44% of its original activity after being stored for 30 days at 4?°C and 25?°C, respectively. In addition, the insolubilized enzyme exhibited good reusability with 18.46% relative activity after being repeatedly used for six times. These findings suggest the efficient and sustainable use of the developed immobilized lipase for various biotechnological applications.     

Effect of calcium on immobilization of rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.) peroxidase for bioassays in sodium alginate and Agarose gel

The direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium alginate beads and in calcium agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in presence of hydrogen peroxide. The maximum specific activity and immobilization yield of the calcium agarose immobilized peroxidase reached 2,200 U mg⁻¹ protein (540 mU cm⁻³ gel) and 82%, respectively. In calcium alginate the maximum activity of peroxidase upon immobilization was 210 mU g⁻¹ bead with 46% yield. The optimal pH for agarose immobilized peroxidase was 7.0 which differed from the pH 6.0 for soluble peroxidase. The optimum temperature for the agarose immobilized peroxidase however was 30°C, which was similar to that of soluble peroxidase. The thermal stability of calcium agarose immobilized peroxidase significantly enhanced over a temperature range of 30∼60°C upon immobilization. The operational stability of peroxidase was examined with repeated hydrogen peroxide oxidation at varying time intervals. Based on 50% conversion of hydrogen peroxide and four times reuse of immobilized gel, the specific degradation of guaiacol for the agarose immobilized peroxidase increased three folds compared to that of soluble peroxidase. Nearly 165% increase in the enzyme protein binding to agarose in presence of calcium was noted. The results suggest that the presence of calcium, ions help in the immobilization process of peroxidase from rice shoots and mediates the direct binding of the enzyme to the agarose gel and that agarose seems to be a better immobilization matrix for peroxidase compared to sodium alginate.     

Stabilization of Aspergillus parasiticus cytosine deaminase by immobilization on calcium alginate beads improved enzyme operational stability

Cytosine deaminase (CD) from Aspergillus parasiticus, which has half-life of 1.10?h at 37°C, was stabilized by immobilization on calcium alginate beads. The immobilized CD had pH and temperature optimum of 5 and 50°C respectively. The immobilized enzyme also stoichiometrically deaminated Cytosine and 5-fluorocytosine (5-FC) with the apparent K_M values of 0.60?mM and 0.65?mM respectively, displaying activation energy of 10.72 KJ/mol. The immobilization of native CD on calcium alginate beads gave the highest yield of apparent enzymatic activity of 51.60% of the original activity and the enzymatic activity was lost exponentially at 37°C over 12?h with a half-life of 5.80?h. Hence, the operational stability of native CD can be improved by immobilization on calcium alginate beads.     

Phytate degradation by immobilized<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> phytase in soybean-curd whey

Saccharomyces cerevisiae CY phytase-producing cells were immobilized in calcium alginate beads and used for the degradation of phylate. The maximum activity and immobilization yield of the immobilized phytase reached 280 mU/g-bead and 43%, respectively. The optimal pH of the immobilized cell phytase was not different from that of the free cells. However, the optimum temperature for the immobilized phytase was 50°C, which was 10°C higher than that of the free cells; pH and thermal stability were enhanced as a consequence of immobilization. Using the immobilized phytase, phytate was degraded in a stirred tank bioreactor. Phytate degradation, both in a buffer solution and in soybean-curd whey mixture, showed very similar trends. At an enzyme dosage of 93.9 mU/g-phytate, half of the phytate was degraded after 1 h of hydrolysis. The operational stability of the immobilized beads was examined with repeated batchwise operations. Based on 50% conversion of the phytate and five times of reuse of the immobilized beads, the specific degradation (g phytate/g dry cell weight) for the immobilized phytase increased 170% compared to that of the free phytase.     

A new method for immobilization of acetylcholinesterase

A new method for immobilization of acetylcholinesterase (AChE) to alginate gel beads by activating the carbonyl groups of alginate using carbodiimide coupling agent has been successfully developed. Maximum reaction rate (V _max) and Michaelis–Menten constant (K _m) were determined for the free and binary immobilized enzyme. The effects of pH, temperature, storage stability, reuse number and thermal stability on the free and immobilized AChE were also investigated. For the free and binary immobilized enzyme on the Ca–alginate gel beads, optimum pH values were found to be 7 and 8, respectively. Optimum temperatures for the free and immobilized enzyme were observed to be 30 and 35 °C, respectively. Upon 60 days of storage the preserved activity of free and immobilized enzyme were found as 4 and 68%, respectively. In addition, reuse number, and thermal stability of the free AChE were increased by as a result of binary immobilization.     

IMMOBILIZATION OF CARBONIC ANHYDRASE ENZYME PURIFIED FROM Bacillus subtilis VSG-4 AND ITS APPLICATION AS CO2 SEQUESTERER

The purification, immobilization, and characterization of carbonic anhydrase (CA) secreted by Bacillus subtilis VSG-4 isolated from tropical soil have been investigated in this work. Carbonic anhydrase was purified using ammonium sulfate precipitation, Sephadex-G-75 column chromatography, and DEAE-cellulose chromatography, achieving a 24.6-fold purification. The apparent molecular mass of purified CA obtained by SDS-PAGE was found to be 37 kD. The purified CA was entrapped within a chitosan–alginate polyelectrolyte complex (C-A PEC) hydrogel for potential use as an immobilized enzyme. The optimum pH and temperature for both free and immobilized enzymes were 8.2 and 37°C, respectively. The immobilized enzyme had a much higher storage stability than the free enzyme. Certain metal ions, namely, Co²⁺, Cu²⁺, and Fe³⁺, increased the enzyme activity, whereas CA activity was inhibited by Pb²⁺, Hg²⁺, ethylenediamine tetraacetic acid (EDTA), 5,5′-dithiobis-(2-nitrobenzoic acid (DTNB), and acetazolamide. Free and immobilized CAs were tested further for the targeted application of the carbonation reaction to convert CO₂ to CaCO₃. The maximum CO₂ sequestration potential was achieved with immobilized CA (480 mg CaCO₃/mg protein). These properties suggest that immobilized VSG-4 carbonic anhydrase has the potential to be used for biomimetic CO₂ sequestration.     

Immobilization and characterization of cellulase on concanavalin A (Con A)-layered calcium alginate beads

Cellulase has been immobilized on hybrid concanavalin A (Con A)-layered calcium alginate–starch beads. Immobilized cellulase retained about 82% of its activity. Con A was extracted from jack bean and the obtained crude protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The immobilized beads showed high mechanical and storage stability; immobilized cellulase retained 100% and 85% activity at 4°C and 30°C, respectively, over one month. The immobilized cellulase retained about 70% of its activity after five cycles of use. The immobilized cellulase retained 70% activity after 120-min exposure to 60°C, whereas the soluble form only retained about 20%, showing that immobilization improved thermal stability. Surface morphology and elemental analysis of immobilized cellulase were examined using scanning electron microscope equipped with energy-dispersive X-ray. Based on the enzyme stability and reuse, this method of immobilization is both convenient and cheap.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

Recycling of textile bleaching effluents for dyeing using immobilized catalase

Catalase was immobilized on alumina carrier and crosslinked with glutaraldehyde. Storing stability, temperature and pH profiles of enzyme activity were studied in a column reactor with recirculation and in a batch stirred-tank reactor. The immobilized enzyme retained 44% of its activity at pH 11, 30 °C and 90% at 80 °C, pH 7. The half-life time of the immobilized catalase was increased to 2 h at pH 12, and 60 °C. Acceptable results were achieved when the residual water from the washing process of H₂O₂-bleached fabrics was treated with the immobilized enzyme and then reused for dyeing.     

Immobilization of catalase into chemically crosslinked chitosan beads

Bovine liver catalase was immobilized into chitosan beads prepared in crosslinking solution. Various characteristics of immobilized catalase such as the pH–activity curve, the temperature–activity curve, thermal stability, operational stability, and storage stability were evaluated. Among them the pH optimum and temperature optimum of free and immobilized catalase were found to be pH 7.0 and 35 °C. The K_m value of immobilized catalase (77.5 mM) was higher than that of free enzyme (35 mM). Immobilization decreased in V_max value from 32,000 to 122 μmol (min mg protein)⁻¹. It was observed that operational, thermal and storage stabilities of the enzyme were increased with immobilization.     

Enhanced bioconversion of colchicine to regiospecific 3-demethylated colchicine (3-DMC) by whole cell immobilization of recombinant E. coli harboring P450 BM-3 gene

Biotransformation of colchicine into regiospecific 3-demethylated colchicine (3-DMC) which is pharmacologically active and a potent anti-cancer drug, mediated by immobilization of recombinant microbial monooxygenases is a novel and promising strategy for its production. In the present study, recombinant Escherichia coli expressing P450 BM-3 was immobilized in calcium-alginate beads and its ability to catalyze colchicine into 3-DMC was investigated. Characteristics of immobilized system showed that optimum conditions for activity of microbial cells were not affected due to immobilization. The optimum pH and temperature for both free and immobilized cells were found to be 7.5 and 37.5 °C, respectively. Experimental variables under consideration such as Ca²⁺ concentration, alginate concentration, P450 BM-3 enzyme activity and colchicine concentration were optimized using response surface methodology. The immobilized cells exhibited a markedly improved thermal stability as compared to free cells. The yield of 3-DMC with immobilized microbial cells was found to be an average of 69%, with 82, 73 and 52% across three independent batches in succession as against bioconversion by free cells, which indicated improved operational stability and reusability of immobilized cells in batch processes. Additionally, a packed bed reactor has been proposed for the immobilized biocatalytic system for bioconversion of colchicine and other biochemicals.     

Immobilization of catalase on cotton fabric oxidized by sodium periodate

Cotton fabric was first oxidized with sodium periodate, and then employed to immobilize catalase. Optimization studies for oxidation of the fabric and immobilization of the enzyme were performed. The properties of the immobilized catalase were examined and compared with those of the free enzyme. A high activity of the immobilized enzyme was obtained when the fabric was oxidized at 40°C and pH 6.0 for 8h in a bath containing 0.20 mol L^?1 sodium periodate and the enzyme was immobilized at 4°C for 24h with a catalase dosage of 120.0 U mL^?1. The immobilized enzyme exhibited optimum activity at 40°C, while the free enzyme had optimal temperature of 30°C, suggesting that the immobilized catalase could be used in a broader temperature range. Both the immobilized and free enzyme had pH optima of 7.0. The staining test and reusability showed that the catalase was fixed covalently on the oxidized cotton fabric.     

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

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

Production,partial characterization,and immobilization in alginate beads of an alkaline protease from a new thermophilic fungus <Emphasis Type="Italic">Myceliophthora</Emphasis> sp.

Thermophilic fungi produce thermostable enzymes which have a number of applications, mainly in biotechnological processes. In this work, we describe the characterization of a protease produced in solidstate (SSF) and submerged (SmF) fermentations by a newly isolated thermophilic fungus identified as a putative new species in the genus Myceliophthora. Enzyme-production rate was evaluated for both fermentation processes, and in SSF, using a medium composed of a mixture of wheat bran and casein, the proteolytic output was 4.5-fold larger than that obtained in SmF. Additionally, the peak of proteolytic activity was obtained after 3 days for SSF whereas for SmF it was after 4 days. The crude enzyme obtained by both SSF and SmF displayed similar optimum temperature at 50°C, but the optimum pH shifted from 7 (SmF) to 9(SSF). The alkaline protease produced through solid-state fermentation (SSF), was immobilized on beads of calcium alginate, allowing comparative analyses of free and immobilized proteases to be carried out. It was observed that both optimum temperature and thermal stability of the immobilized enzyme were higher than for the free enzyme. Moreover, the immobilized enzyme showed considerable stability for up to 7 reuses.     

Improved Properties of Bacillus coagulans β‐Galactosidase through Immobilization

Thermostable β‐galactosidase from Bacillus coagulans RCS3 was purified by successive column chromatography using DEAE‐cellulose and Sephadex G‐50. Immobilization of the purified enzyme was studied with DEAE‐cellulose and calcium alginate. The efficiency of β‐galactosidase retention was 87 % with DEAE‐cellulose (17 mg protein/mL of matrix) and 80 % with calcium alginate (2.2 mg protein/g bead). Comparative studies of immobilization displayed a shift in the optimum temperature from 65 °C to 70 °C provoked by DEAE‐cellulose, although no effect was observed with calcium alginate. The heat inactivation curve revealed an improvement in the stability (t_1/2 of 14.5 h for the immobilized enzyme as compared to 2 h for the free enzyme at 65 °C) in a calcium alginate system. This immobilized enzyme has a wide pH stability range (6.5–11). β‐Galactosidase immobilized by DEAE‐cellulose and calcium alginate allowed a 57 and 70 % lactose hydrolysis, respectively, to be achieved within 48 h after repeated use for twenty times.     

Improved stability and catalytic activity of graphene oxide/chitosan hybrid beads loaded with porcine liver esterase

Graphene oxide/chitosan and reduced graphene oxide/chitosan (GO/CS and RGO/CS) beads were prepared by precipitation with NaOH. Porcine liver esterase was immobilized on these beads to give GO/CS/E and RGO/CS/E beads. The optimum conditions for the maximum activity of RGO/CS/E beads were pH 8 and 50°C. The stability of the enzyme immobilized on GO/CS/E and RGO/CS/E was high in the pH range of 5–8. The GO/CS/E beads showed superior stability compared to that of the free enzyme and CS/E beads between 20 and 50°C. Kinetic analysis showed that GO/CS/E was a better catalyst than the RGO/CS/E beads with a lower K_m value of 0.9?mM. The hybrid beads also retained more than 95% activity after 10 consecutive cycles. The GO/CS/E and RGO/CS/E beads retained 84% and 87% activity after 40 days at 4°C. The GO/CS/E beads were used for the successful hydrolysis of methyl 4-hydroxy benzoate.     

Preparation and properties of immobilized amyloglucosidase

Amyloglucosidase was immobilized on a copolymer of methyl methacrylate and 2-dimethylaminoethyl methacrylate. The resulting immobilized amyloglucosidase has 19% of the soluble enzyme specific activity. The pH optimum of immobilized amyloglucosidase is shifted towards acidity by 1.9 units. The temperature optimum of immobilized enzyme is shifted upward by 5°C. The immobilized amyloglucosidase has the maximum stability at pH 4.6, whereas the soluble enzyme has maximum stability at pH 5.5. While soluble amyloglucosidase has a maximum thermal stability at 50°C, the stability of the immobilized amyloglucosidase steadily decreases with the increase in temperature.     

Immobilized Sclerotinia sclerotiorum invertase to produce invert sugar syrup from industrial beet molasses by product

The fungus Sclerotinia sclerotiorum produces invertase activity during cultivation on many agroindustrial residues. The molasses induced invertase was purified by DEAE-cellulose chromatography. The molecular mass of the purified enzyme was estimated at 48 kDa. Optimal temperature was determined at 60 °C and thermal stability up to 65 °C. The enzyme was stable between pH 2.0 and 8.0; optimum pH was about 5.5. Apparent K_m and V_max for sucrose were estimated to be respectively 5.8 mM and 0.11 μmol/min. The invertase was activated by β-mercaptoethanol. Free enzyme exhibited 80 % of its original activity after two month’s storage at 4 °C and 50 % after 1 week at 25 °C. In order to investigate an industrial application, the enzyme was immobilized on alginate and examined for invert sugar production by molasses hydrolysis in a continuous bioreactor. The yield of immobilized invertase was about 78 % and the activity yield was 59 %. Interestingly the immobilized enzyme hydrolyzed beet molasses consuming nearly all sucrose. It retained all of its initial activity after being used for 4 cycles and about 65 % at the sixth cycle. Regarding productivity; 20 g/l of molasses by-product gave the best invert sugar production 46.21 g/day/100 g substrate related to optimal sucrose conversion of 41.6 %.