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.     

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.     

Production of lipase in a fermentor using a mutant strain of Corynebacterium species: its partial purification and immobilization

Extracellular Corynebacterium lipase was produced using a 2.5 L Chemap fermentor using 1300 ml fermentation medium at temperature 33 degrees C, agitator speed 50 rpm, aeration rate 1 VVM having KLa 16.21 hr(-1). Crude lipase was purified by salting out method followed by dialysis and immobilized using calcium alginate gel matrix followed by glutaraldehyde cross linking Purification process increased specific activity of enzyme from 2.76 to 114.7 IU/mg. Activity of immobilized enzyme was 107.31 IU/mg. Optimum temperature for purified and immobilized enzyme activity were 65 degrees and 50 degrees C respectively. Optimum pH was 8.0 in both the cases, Km and Vmax value for purified lipase were 111.1 micromol/min and 14.7% respectively. Ca2+ (5 mM) was found to be stimulator for enzyme activity. Immobilized lipase retained 68.18% of the original activity when stored for 40 days.     

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

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

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.     

Characterization of microbial endo-β-glucanase immobilized in alginate beads

Endo-β-glucanase (endo-β-1,4-glucano-glucanase EC 3.2.1.4), isolated from Trichoderma reesei, was immobilized in calcium alginate beads, retaining 75% of its original activity. The polyanionic moiety surrounding the immobilized enzyme displaced the pH-activity profile to alkaline regions with respect to that of the free enzyme. The enzyme was inhibited by carboxymethylcellulose, but this inhibition appeared to be decreased by immobilizatíon. The enzyme immobilized in alginate beads showed a K_m value (1.02% w/v) lower than that of the enzyme (1.31%). The apparent V_max of immobilized cellulase preparations (238.3 μmol glucose/ml × h) decreased by a factor of 0.59 with respect to that of the soluble enzyme. The optimum temperature (60°C) of the free and entrapped enzymes remained unaltered. In contrast, the half-life of the endoglucanase immobilized in calciumalginate beads was 4.6 h at 55°C and 5.4 h at 60°C, while that of the free enzyme was 3.0 h at 55°C and 1.2 h at 60°C. A technological application of the immobilized enzymes was tested using wheat straw as a source of fermentable sugars. The hydrolytic degradation of straw, by means of a crude extract of free and immobilized cellulases and β-glucosidase, released a large amount of reducing sugars from wheat straw after 48 h (between 250–720 mg glucose/g straw), carrying out more than a 90% saccharification. A mixture of immobilized β-glucosidase and free cellulases maintained 80% of the activity of the soluble counterparts, and the co-immobilization of both types of enzymes reduced by hydrolytic efficiency to half.     

Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes

The extracellular amylolytic enzymes of Schwanniomyces alluvius were studied to determine future optimization of this yeast for the production of industrial ethanol from starch. Both alpha-amylase and glucoamylase were isolated and purified. alpha-Amylase had an optimum pH of 6.3 and was stable from pH 4.5 to 7.5. The optimum temperature for the enzyme was 40 degrees C, but it was quickly inactivated at temperatures above 40 degrees C. The Km for soluble starch was 0.364 mg/ml. The molecular weight was calculated to be 61,900 +/- 700. alpha-Amylase was capable of releasing glucose from starch, but not from pullulan. Glucoamylase had an optimum pH of 5.0 and was stable from pH 4.0 to greater than 8.0. The optimum temperature for the enzyme was 50 degrees C, and although less heat sensitive than alpha-amylase, it was quickly inactivated at 60 degrees C. Km values were 12.67 mg/ml for soluble starch and 0.72 mM for maltose. The molecular weight was calculated to be 155,000 +/- 3,000. Glucoamylase released only glucose from both soluble starch and pullulan. S. alluvius is one of the very few yeasts to possess both alpha-amylase and glucoamylase as well as some fermentative capacity to produce ethanol.     

Isolation and characterization of Schwanniomyces alluvius amylolytic enzymes.

The extracellular amylolytic enzymes of Schwanniomyces alluvius were studied to determine future optimization of this yeast for the production of industrial ethanol from starch. Both alpha-amylase and glucoamylase were isolated and purified. alpha-Amylase had an optimum pH of 6.3 and was stable from pH 4.5 to 7.5. The optimum temperature for the enzyme was 40 degrees C, but it was quickly inactivated at temperatures above 40 degrees C. The Km for soluble starch was 0.364 mg/ml. The molecular weight was calculated to be 61,900 +/- 700. alpha-Amylase was capable of releasing glucose from starch, but not from pullulan. Glucoamylase had an optimum pH of 5.0 and was stable from pH 4.0 to greater than 8.0. The optimum temperature for the enzyme was 50 degrees C, and although less heat sensitive than alpha-amylase, it was quickly inactivated at 60 degrees C. Km values were 12.67 mg/ml for soluble starch and 0.72 mM for maltose. The molecular weight was calculated to be 155,000 +/- 3,000. Glucoamylase released only glucose from both soluble starch and pullulan. S. alluvius is one of the very few yeasts to possess both alpha-amylase and glucoamylase as well as some fermentative capacity to produce ethanol.     

Statistical optimization of alpha-amylase production by Streptomyces erumpens MTCC 7317 cells in calcium alginate beads using response surface methodology.

Alpha-amylase has a wide range of applications in starch industries, i.e. baking, brewing, distillery, etc. The alpha-amylase production from Streptomyces erumpens MTCC 7317 immobilized cells was compared with that of free cells. The immobilized cells of S. erumpens in calcium alginate beads were more effective for production of alpha-amylase (12.2% more yield) than free cells. Response surface methodology (RSM) was used to evaluate the effect of main variables, i.e. incubation period, pH and temperature on enzyme production with immobilized cells. A full factorial Central Composite Design (CCD) was applied to study these main factors that affected alpha-amylase production. The experimental results showed that the optimum incubation period, pH and temperature were 36 h, 6.0 and 50 degrees C, respectively for immobilized cells. Repeated batch fermentation of immobilized cells in shake flasks carried out in starch-beef extract medium showed that S. erumpens cells were physiologically active on the support even after four cycles of fermentation.     

Optimization of alpha-amylase immobilization in calcium alginate beads

alpha-Amylase enzyme was produced by Aspergillus sclerotiorum under SSF conditions, and immobilized in calcium alginate beads. Effects of immobilization conditions, such as alginate concentration, CaCl(2) concentration, amount of loading enzyme, bead size, and amount of beads, on enzymatic activity were investigated. Optimum alginate and CaCl(2) concentration were found to be 3% (w/v). Using a loading enzyme concentration of 140 U mL(-1), and bead (diameter 3 mm) amount of 0.5 g, maximum enzyme activity was observed. Beads prepared at optimum immobilization conditions were suitable for up to 7 repeated uses, losing only 35% of their initial activity. Among the various starches tested, the highest enzyme activity (96.2%) was determined in soluble potato starch hydrolysis for 120 min at 40 degrees C.     

Improved properties of bilirubin oxidase by entrapment in alginate-silicate sol-gel matrix

Bilirubin oxidase from Myrothecium verrucaria was immobilized by immersing enzyme-containing calcium alginate beads in a hexane solution of tetramethoxy-ortho-silicate (TMOS). Products from TMOS hydrolysis permeated and co-polymerized with the alginate gel and formed a colloid within the beads that entraps the enzyme. Bilirubin oxidase in the composite alginate-silicate-protein gel showed twice the activity compared to that of the free enzyme and improved thermal stability and excellent reusability. © Rapid Science Ltd. 1998     

Enzymatic detection of mercuric ions in ground-water from vegetable wastes by immobilizing pumpkin (Cucumis melo) urease in calcium alginate beads

Present report describes a quick and simple test based on enzyme inhibition for the detection of mercury in aqueous medium by urease immobilized in alginate beads. Urease was extracted from the discarded seeds of pumpkin (Cucumis melo) and was purified to apparent homogeneity (5.2-fold) by heat treatment at 48+/-0.1 degrees C and gel filtration through Sephadex G-200. The homogeneous enzyme preparation (Sp activity 353 U/mg protein, A(280)/A(260)=1.12) was immobilized in 3.5% alginate leading to 86% immobilization. Effect of mercuric ion on the activity of soluble as well as immobilized enzyme was investigated. Hg(2+) exhibited a concentration-dependent inhibition both in the presence and absence of the substrate. The alginate immobilized enzyme showed less inhibition. There was no leaching of the enzyme over a period of 15 days at 4 degrees C. The inhibition was non-competitive and the K(i) was found to be 1.26x10(-1)microM. Time-dependent interaction of urease with Hg(2+) exhibited a biphasic inhibition behavior in which approximately half of the initial activity was lost rapidly (within 10 min) and reminder in a slow phase. Binding of Hg(2+) with the enzyme was largely irreversible, as the activity could not be restored by dialysis. The significance of the observations is discussed.     

Thermostability of soluble and immobilized alpha-amylase from Bacillus licheniformis

In view of a possible application of the alpha-amylase from Bacillus licheniformis as a time-temperature integrator for evaluation of heat processes,(11) thermal inactivation kinetics of the dissolved and covalently immobilized enzyme were studied in the temperature range 90-108 degrees C. The D-values (95 degrees C) for inactivation of alpha-amylase, dissolved in tris-HCl buffer, ranged from 6 to 157 min, depending on pH, ionic strength, and Ca(2+) and enzyme concentration. The z-value fluctuated between 6.2 and 7.6 degrees C. On immobilization of the alpha-amylase by covalent coupling with glutaraldehyde to porous glass beads, the thermoinactivation kinetics became biphasic under certain circumstances. For immobilized enzyme, the D-values (95 degrees C) ranged between 17 and 620 min, depending largely on certain environmental conditions. The z-value fluctuated between 8.1 and 12.9 degrees C. In each case of biphasic inactivation, the z-value of the stable fraction (with the higher D-values) was lower than the z-value of the labile fraction. (c) 1992 John Wiley & Sons, Inc.     

Preparation and properties of an immobilized pectinlyase for the treatment of fruit juices

Pectinlyase, present in different commercial pectinases used in juice technology, was immobilized on alginate beads. The optimal conditions were: 0.17 g alginate ml(-1), 1.2% (w/v or v/v) enzyme concentration and acetic-HCl/glycine-HCl buffer at pH 3.6 or tris-HCl/imidazole buffer at pH 6.4. Maximum percentage of immobilization (10.6%) was obtained with Rapidase C80. Kinetic parameters of free and immobilized pectinlyase were also determined. The pH and temperature at which activity of soluble and immobilized enzyme was maximum were 7.2 and 55 degrees C. Thermal stability was not significantly altered by immobilization, especially at 40 degrees C, showing two periods of different stability. Free and immobilized preparation reduced the viscosity of highly esterified pectin from 1.09 to 0.70 and 0.72 mm(2) s(-1), respectively, after 30 min at 40 degrees C. Furthermore, the immobilized enzyme could be re-used through 4 cycles and the efficiency loss in viscosity reduction was found to be only 9.2%.     

Immobilized enzyme system for determination of sialic acid in serum or urine.

An immobilized enzyme system has been developed and employed to determine the concentration of sialic acid (N-acetylneuraminic acid) in human serum and urine. Two enzyme pairs, neuramindiase-Neu-5-Ac lyase and pyruvate oxidase-peroxidase, have been respectively co-immobilized onto 1,12-aminododecane-agarose with glutaraldehyde. The relative specific activity of the co-immobilized neuraminidase and Neu-5-Ac lyase were 60% and 78%, and those of pyruvate oxidase and peroxidase were 50% and 95% of the corresponding soluble enzymes, respectively. The optimal reaction pH at 37 degrees C for each of the co-immobilized enzymes was about one pH unit higher than that of the corresponding soluble enzyme. The optimal reaction temperature of each enzyme was increased as a result of immobilization. The thermal stability at 45 degrees C of the immobilized neuraminidase, Neu-5-Ac lyase, pyruvate oxidase, and peroxidase were increased 80-, 83-, 115-, and 147-fold, respectively. Km and Vm of each immobilized and co-immobilized enzyme have also been determined. The system provided a convenient and rapid method to determine the concentration of total sialic acid without pretreatment of the sample. The results correlated satisfactorily with those obtained by using a soluble enzyme system. The co-immobilized enzymes were stable for at least 1 year of 500 tests when used repeatedly. The system is thus a reproducible and reliable novel assay method for sialic acid in the serum or urine sample.     

Utilization of an exopolysaccharide produced by Chryseomonas luteola TEM05 in alginate beads for adsorption of cadmium and cobalt ions

Cadmium and cobalt adsorption from aqueous solution onto calcium alginate, sodium alginate with an extracellular polysaccharide (EPS) produced by the activated sludge bacterium Chryseomonas luteola TEM05 and immobilized C. luteola TEM05 was studied. In addition, solutions containing both of these ions were prepared and partial competitive adsorption of these mixtures was investigated. Metal adsorption onto gel beads was carried out at pH 6.0 and 25 degrees C. The maximum adsorption capacities determined by fitting Langmuir isotherms to the data for calcium alginate, calcium alginate+EPS, calcium alginate + C. luteola TEM05 and calcium alginate + EPS + C. luteola TEM05 were 45.87, 55.25, 49.26, 51.81 mg g(-1) for Co(II) and 52.91, 64.10, 62.5, 61.73 mg g(-1) for Cd(II), respectively. The biosorption capacity of the carrier for both metal ions together in competition was lower than those obtained when each was present alone.     

A novel matrix for the immobilization of acetylcholinesterase

In this study, a new matrix for immobilization of acetylcholinesterase was investigated by using alginate and kappa-carrageenan. The effects of pH, temperature, storage and thermal stability on the free and immobilized acetylcholinesterase activity were examined. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) was also investigated for free and immobilized enzymes. For free and immobilized enzymes into Ca-alginate and alginate/kappa-carrageenan polymer blends, optimum pH and temperature was found to be 7 and 30 degrees C, respectively. For free enzyme, maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) values were found to be 6.35 mM and 50 mM min(-1), respectively, the same values for immobilized enzymes were determined as 8.68, 12.7 mM and 39.7, 52.9 mM min(-1), respectively. Storage and thermal stability of acetylcholinesterase was increased by as a result of immobilization.     

Purification and characterization of the extracellular alpha-amylase from Clostridium acetobutylicum ATCC 824.

The extracellular alpha-amylase (1,4-alpha-D-glucanglucanohydrolase; EC 3.2.1.1) from Clostridium acetobutylicum ATCC 824 was purified to homogeneity by anion-exchange chromatography (mono Q) and gel filtration (Superose 12). The enzyme had an isoelectric point of 4.7 and a molecular weight of 84,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It was a monomeric protein, the 19-amino-acid N terminus of which displayed 42% homology with the Bacillus subtilis saccharifying alpha-amylase. The amino acid composition of the enzyme showed a high number of acidic and hydrophobic residues and only one cysteine residue per mole. The activity of the alpha-amylase was not stimulated by calcium ions (or other metal ions) or inhibited by EDTA, although the enzyme contained seven calcium atoms per molecule. alpha-Amylase activity on soluble starch was optimal at pH 5.6 and 45 degrees C. The alpha-amylase was stable at an acidic pH but very sensitive to thermal inactivation. It hydrolyzed soluble starch, with a Km of 3.6 g . liter-1 and a Kcat of 122 mol of reducing sugars . s-1 . mol-1. The alpha-amylase showed greater activity with high-molecular-weight substrates than with low-molecular-weight maltooligosaccharides, hydrolyzed glycogen and pullulan slowly, but did not hydrolyze dextran or cyclodextrins. The major end products of maltohexaose degradation were glucose, maltose, and maltotriose; maltotetraose and maltopentaose were formed as intermediate products. Twenty seven percent of the glucoamylase activity generally detected in the culture supernatant of C. acetobutylicum can be attributed to the alpha-amylase.     

Production and immobilization of D-aminoacylase of Alcaligenes faecalis DA1 for optical resolution of N-acyl-DL-amino acids.

The production of D-aminoacylase by Alcaligenes faecalis DA1 was induced 5- to 50-fold by N-acetyl-D-amino acids. This strain produced about 443 units of D-aminoacylase and 52 units of L-aminoacylase per gram of cells (wet weight) when cultivated in a medium containing 1% N-acetyl-DL-leucine as the carbon source. The D-aminoacylase was partially purified by Fractogel DEAE 650 column chromatography and then immobilized on another Fractogel DEAE 650 column. The catalytic activity of the immobilized D-aminoacylase was 2,650 units per milliliter of gel. The Km values for the free and the immobilized enzymes were found to be 1.00 and 0.22 mM, respectively, using N-acetyl-D-methionine as a substrate. The optimal reaction pH and temperature for both soluble and immobilized enzyme were around 8.0 and 45 degrees C, respectively. The free enzyme was stable in the pH range from 5.0 to 11.0, whereas the immobilized enzyme tended to detach from the gel at pH values higher than 9.0. Both forms of enzyme were stable up to 40 degrees C. When used for the optical resolution of N-acetyl-DL-methionine, the immobilized enzyme maintained 90% initial activity after 17 days of continuous operation at 45 degrees C. The process of purification and immobilization of D-aminoacylase described in this report is very effective and easy to scale up.     

Immobilization of thermoalkalophilic recombinant esterase enzyme by entrapment in silicate coated Ca-alginate beads and its hydrolytic properties

Thermoalkalophilic esterase enzyme from Bal?ova (Agamemnon) geothermal site were aimed to be immobilized effectively via a simple and cost-effective protocol in silicate coated Calcium alginate (Ca-alginate) beads by entrapment. The optimal immobilization conditions of enzyme in Ca-alginate beads were investigated and obtained with 2% alginate using 0.5mg/ml enzyme and 0.7 M CaCl(2) solution. In order to prevent enzyme from leaking out of the gel beads, Ca-alginate beads were then coated with silicate. Enzyme loading efficiency and immobilization yield for silicate coated beads was determined as 98.1% and 71.27%, respectively and compared with non-coated ones which were 68.5% and 45.80%, respectively. Surface morphologies, structure and elemental analysis of both silicate coated and non-coated alginate beads were also compared using Fourier Transform Infrared Spectroscopy (FT-IR) and Scanning Electron Microscope (SEM) equipped with Energy-dispersive X-ray spectroscopy (EDX). Moreover, silicate coated alginate beads enhanced reusability of esterase in continuous processes compared to non-coated beads. The hydrolytic properties of free and immobilized enzyme in terms of storage and thermal stability as well as the effects of the temperature and pH were determined. It was observed that operational, thermal and storage stabilities of the esterase were increased with immobilization.