Glucoamylase Covalently Bound to Acrylic Carriers

Glucoamylase from Aspergillus niger was covalantly bound to three acrylic carriers differing in the content of amino groups, particle size, porosity, etc. Yield of immobilization ranged from 58.7% to 87.0%. Enzyme immobilized on Vinylaff 818 was the most stable. That carrier was a copolymer of butyl acrylate and ethylene dimethacrylate, containing 0.44 mmol/g of amino groups. The immobilized catalyst used for hydrolysis of 40% maltodextrin solution (DE 18.3) retained its initial activity at 50°C for about 33 d. The pretreatment of soluble glucoamylase with low concentrated glutaraldehyde solution (0.13%, 1.4 mg/100 mg of protein) increased the operational stability of immobilized enzyme to about 50 d and its half-life to 60 d. The immobilization of enzyme on Vinylaff 818 resulted in the slight shift of pH optimum to acid side, though it did not influence the temperature optimum. Thermostability of immobilized glucoamylase in the range of temperature between 50 and 75°C was higher than that of soluble enzyme. Maximum concentrations of glucose in hydrolyzates were obtained in relatively short time. The prolonged hydrolysis of concentrated starch substrate by enzyme immobilized on the porous carrier caused, however, the accumulation of disaccharides with simultaneous reduction of glucose content.     

Immobilization of <Emphasis Type="BoldItalic">Aspergillus oryzae</Emphasis> β-Galactosidase onto Duolite A568 Resin via Simple Adsorption Mechanism

In this study, a rapid, simple and economic method of enzyme immobilization was developed to hydrolyze lactose. Duolite A568 resin was used for the immobilization of β-galactosidase via simple adsorption mechanism. The effects of immobilization parameters such as time, pH, and temperature were studied. Immobilization parameters for maximum enzyme activity were estimated at 35 °C temperature, pH 4.5, 5 mg/mL enzyme concentration, and approximately 60 min immobilization time. A significant amount of enzyme was immobilized with high catalytic activity. Enzyme immobilization procedure explained in this study slightly affected the enzyme kinetic. The value of Michaelis constant K _m for immobilized enzyme was significantly larger, indicating decreased affinity by the enzyme for its substrate. It was observed that both free and immobilized enzyme showed maximum activity at 65 °C reaction temperature. Immobilized β-galactosidase was significantly more active at all temperatures as compared to its free form. However, optimal pH of immobilized enzyme was slightly affected by immobilization procedure. The optimum pH of immobilized enzyme was shifted up 0.5 unit to a more alkaline value of 6.0 compared to the free enzyme.     

Characterization of glucoamylase immobilized on magnetic nanoparticles

Magnetic support was prepared by precipitation from an alkaline solution of divalent and trivalent iron ions and subsequently was modified with 3‐aminopropyltriethoxysilane. FTIR analysis showed existence of a new Si–O–Fe bond in obtained particles. Scanning electronic microscopy images shows that the nanoparticles of all samples have particle size below 30 nm. Glucoamylase AMG 300L was immobilized onto the modified magnetic support using glutaraldehyde as a coupling agent. Obtained preparations had specific activity of 148 U/g of the support when measured at 55°C using maltose as substrate. The immobilized enzyme exhibited mass transfer limitation as reflected by a higher apparent K_m value and a lower energy of activation. The immobilization was almost completely terminated after 30 min of the reaction at 30°C. The highest immobilization yield of the enzyme was achieved at glutaraldehyde concentration of 10 mM. The immobilization did not influence considerably on optimum pH and temperature of substrate hydrolysis catalyzed by investigated enzyme (55°C, pH 4.5). Moreover, immobilized glucoamylase was easily separated from the reaction medium by an external magnetic field and retained about 60% of initial activity after nine repeated cycles of enzyme reaction followed by magnetic separation.     

Thermostability of Soluble and Immobilized Horseradish Peroxidase

Thermal inactivation kinetics of soluble horseradish peroxidase and the enzyme covalently immobilized onto glass beads were studied in phosphate buffer and organic solvents in a temperature range of 65° to 98°C. The z value of the heat-stable fraction of peroxidase was changed from 26.3°C to 14.1°C by the method of enzyme immobilization. Further, the z value of 14.1°C of the immobilized peroxidase was lowered to 11.1°C using an organic solvent in order to modify the environment of the enzyme.     

Immobilization of inulinase for sucrose hydrolysis

A commercial inulinase preparation immobilized on various supports was used for sucrose hydrolysis. Entrapment and encapsulation in Ca-alginate and entrapment in an alginate–silicate sol–gel matrix were evaluated. Best results were obtained with Ca-alginate beads. The influence of sodium-alginate concentration on the immobilization yield was assessed. Inulinase entrapped in Ca-alginate beads displayed high activity in the range 50–60 °C, whereas the optimum for the free enzyme was 60 °C. The optimum pH of the immobilized enzyme was slightly more acidic (4.0) than the one observed for the free form (4.5). The apparent K_M for sucrose of the immobilized inulinase was 184 mM, as compared to 82 mM for free inulinase, as a result of diffusion resistances.     

Immobilization of invertase on celite and on polyacrylamide by an absorption procedure

Invertase from Saccharomyces cerevisiae was immobilized on celite and on polyacrylamide by an absorption procedure. The properties of the immobilized invertase were characterized and compared with those of soluble invertase. The activity yield for immobilized invertase on celite and on polyacrylamide was 92% and 81% respectively. The optimum pH and temperature for both soluble and immobilized invertase activity were 4.6 and 60 °C respectively. The activity of immobilized invertase is stable in the range pH 4.0–6.5. The immobilized invertase was thermostable when incubated at temperatures ranging from 40 to 60 °C. Immobilized invertase had a high stability when stored at room temperature for 90 days and had an excellent operational stability when used 20 times repeatedly. The invertase immobilized on celite was more stable than invertase immobilized on polyacrylamide. The invertase preparations immobilised by absorption procedures exhibited marked stability towards temperature, pH changes and had high storage and operational stability, suggesting their excellent potential for use as supports. Copyright © 2003 Society of Chemical Industry     

Immobilization of Glucose Isomerase on Radiation-Modified Gelatine Gel

Whole-cell glucose isomerase immobilization on radiation-modified gelatine gel was demonstrated. The enzyme was stabilized within the cells by heat-treatment at 70°C. The heat-treated cells were mixed with radiation-modified gelatine gel and cross-linked by glutaraldehyde. Immobilized enzyme exhibited 70–75% activity as compared to the free cells. Several properties of immobilized glucose isomerase were studied under various reaction conditions. A continuous isomerization was performed using a column packed with cell granules. It has been found, that the half-life of the immobilized glucose isomerase amounted to 650 h at 60°C.     

Immobilization of amyloglucosidase on polystyrene anion exchange resin I. preparation and properties

Amyloglucosidase (exo‐1,4‐ α‐D‐glucosidase, E C 3.2. 1.3) was coupled to glutaraldehyde activated Indion 48‐R (a cross‐linked macroporous anion exchanger) by Schiff base reaction. The bound enzyme exhibited 60–70% activity of the free enzyme. Substrate concentrations as high as 32% (w/w) liquefied tapioca starch could be quantitatively converted into 96–98% (w/w) dextrose in 24 h at 50°C and pH 4.5. Though immobilization lowered the temperature optimum to 50–60°C from 65°C for the free enzyme, it increased the temperature stability. However, there was no change either in the pH optimum or pH stability after immobilization. In batch operations, the immobilized preparation showed a half life of 32 and 12 days at 50°C and 60°C respectively.     

Immobilization of urease on dialdehyde porous starch

Dialdehyde porous starch (DPS) was prepared by enzymatic hydrolysis and periodate oxidation. Urease was immobilized on DPS by several techniques including physical adsorption and covalent bonding. Optimum urease immobilization conditions for maximum enzyme activity were shown as following: immobilization pH 6.0, processing time of immobilization 25 h, immobilization temperature 25°C, aldehydic groups content 79.6% and ratio of supporter to urease 1:2 (w/v in g/mL). Temperature optimum of the free and immobilized urease were 60 and 70°C, respectively. The temperature curve of the immoblized urease was wider than that registered for the free urease. After being stored at ambient temperature for 42 days, the immobilized urease retained almost all of the original activity. DPS was found capable of holding the urease enzyme during a repeated cyclic test for ten times. An increase of the K_m value for the immobilized urease was found. The foregoing data indicated that DPS is a fine supporter for urease and provided a basis for biosensor or bioreactor development with reduced costs and improved shelf life.     

Hydrolysis of sucrose by invertase immobilized onto novel magnetic polyvinylalcohol microspheres

The magnetic polyvinylalcohol (PVAL) microspheres were prepared by crosslinking glutaraldehyde. 1,1′-Carbonyldiimidazole (CDI), a carbonylating agent was used for the activation of hydroxyl groups of polyvinylalcohol, and invertase immobilized onto the magnetic PVAL microspheres by covalent bonding through the amino group. The retained activity of the immobilized invertase was 74%. Kinetic parameters were determined for immobilized invertase, as well as for the free enzyme. The K_m values for immobilized invertase (55 mM sucrose) were higher than that of the free enzyme (24 mM sucrose), whereas V_max values were smaller for the immobilized invertase. The optimum operational temperature was 5°C higher for immobilized enzyme than that of the free enzyme. The operational inactivation rate constant (k_opi) of the immobilized invertase at 35°C with 200 mM sucrose was 5.83×10⁻⁵ min⁻¹. Thermal and storage stabilities were found to increase with immobilization.     

Immobilized Glucose Isomerase on DEAE Cellulose Beads

Purified glucose isomerase from Actinoplanes missouriensis was immobilized on porous DEAE-cellulose beads by simple adsorption. The immobilized glucose isomerase retained over 70% of its original activity. The hinderance of immobilized enzyme activity due to pore diffusion and film diffusion was insignificant with the bead size at 35 mesh or smaller. The relative substrate flow rate can be kept at 0.04 cm/s or higher. The optimum pH of the imobilized enzyme did not change, however, the optimum pH range became broader. The broader pH profile indicated that immobilized enzymes are less sensitive to pH change in the substrate. The half life of the immobilized enzyme was at around 1,000 h at 60°C. Cobalt ions are not required for enzyme stability. The cost of using immobilized enzyme on DEAE cellulose beads should be less be than that of the whole cell immobilization system due primarily to the fact that DEAE-cellulose beads are reusable for immobilization as well as for enzyme purification.     

Studies on the Combined Action of Amylases and Glucose Isomerase on Starch and Its Hydrolysates Part II. Immobilization of Glucoamylase

Amberlite IR-45 resin was used for immobilization of glucoamylase and the factors affecting the binding power of enzyme to resin, pH, buffer concentration and methods used for immobilization were studied and discussed. Also the factors affecting the reaction velocity of immobilized enzyme were in vestigated. The immobilized enzyme has a higher Km value (0.072) and narrow range of optimum temperature (65–70°C) than free enzyme. Optimum pH for immobilized glucoamylase was 5.5–6.5 while it was 4.5 for free enzyme.     

Optimization of Covalent Immobilization of Extracellular Ice Nucleators from Erwinia herbicola on Magnetic Fe3O4/Chitosan Nanoparticles for Potential Application in Freeze Concentration

A magnetically separable extracellular ice nucleators (ECINs) immobilized system was constructed, ECINs from Erwinia herbicola were covalently immobilized on magnetic Fe₃O₄/chitosan nanoparticles by coupling via glutaraldehyde (GA) or epichlorohydrin (ECH). The immobilization process parameters were optimized, and under optimized conditions, ice nucleation activity (INA) of GA and ECH immobilized ECINs reached at 1.56?×?10⁶?±?1.54?×?10⁵ Units/mg and 1.62?×?10⁶?±?1.91?×?10⁵ Units/mg, respectively. The stability, reusability and cytotoxicity of GA and ECH immobilized ECINs were characterized and compared; both GA and ECH immobilized ECINs demonstrated good reusability performance after 15 freeze–thaw cycles and low cytotoxicity, while GA immobilized ECINs were recovered more easily and more stable at different pH compared to ECH immobilized ECINs. Application of the immobilized ECINs in freeze concentration was investigated, and an increase of ice nucleation temperature for water, seawater and apple juice (6.83?±?0.18 °C, 8.08?±?0.50 °C and 9.38?±?1.12 °C) was achieved by using GA immobilized ECINs. The results collected so far show that there are extensively promising applications for GA immobilized ECINs in freeze concentration industry fields.     

Stabilization of an Amylase from Neurospora crassa by Immobilization on Highly Activated Supports

The amylase from Neurospora crassa is an interesting enzyme, having higher stability than amylase from Aspergillus oryzea under a broad range of pH values. Moreover, the N. crassa enzyme may be immobilized on different supports with good retention of enzyme activity. The best stabilizations were achieved using Eupergit C 250 L or glyoxyl agarose, with which the enzyme remained fully active at 60°C for 24 h while the soluble enzyme remained about 17%. The glyoxyl agarose immobilized enzyme had high thermostability, high optimal temperature (65°C) and broad pH/activity profile, suggesting that this enzyme has potential for food and industrial applications for starch modification.     

Fabrication of Oleic Acid Grafted Starch-based Hybrid Carriers for l-Asparaginase Encapsulation

In this study, artificial red blood cells (RBCs) for immobilization of l -asparaginase are developed. The artificial red blood cells are prepared via a self-assembly method and characterized by various methods. The prepared material shows a small hydrodynamic size of nearly 120 nm and a zeta potential of -7.2 ± 0.2 mV. After l -asparaginase encapsulation, the particle size is nearly 295 nm and the zeta potential value is -9.8 ± 0.1. As expected, the immobilized enzyme shows better pH stability and thermal stability than the free enzyme. For instance, at 60 °C, the immobilized enzyme maintains 63.5% of the original activity, while the free enzyme retains only 27.6% of the original activity. Furthermore, the immobilized enzyme exhibits excellent reusability and storage stability, retaining 46.1% of its initial activity after 15 reuses. Overall, the developed artificial RBCs can potentially be applied as a novel support for immobilization of l -asparaginase.     

Immobilized bacterial α-amylase for effective hydrolysis of raw and soluble starch

The major concern in an enzymatic process is the instability of the enzyme under repetitive or prolonged use and inhibition by high substrate and product concentration. Immobilization is a very effective alternative in overcoming problems of instability and repetitive use of enzymes. Entrapment method of immobilization is advantageous over other methods as they do not involve chemical modification of the enzyme. α-Amylase produced by Bacillus amyloliquefaciens ATCC 23842 was immobilized in calcium alginate beads and used for the effective hydrolysis of soluble and raw potato starch which was comparable to the free enzyme. The levels of parameters (sodium alginate, calcium chloride and curing time) that significantly influence the immobilization of α-amylase in calcium alginate were analyzed and optimized using response surface methodology. Reactor studies were performed to study the reusability and operational stability of the beads. The alginate beads retained more than 60% of their initial efficiency after five batches of successive use and 40% of efficiency was exhibited in the 6th and 7th batch run of 6 h duration.     

Immobilization and characterization of C‐S lyase from onion (Allium cepa) bulbs

Cysteine sulfoxide lyase (C‐S lyase; EC 4.4.1.4), the enzyme responsible for flavor potentiation in minced Allium tissues, was immobilized by entrapment within an alginate gel. Both the free and immobilized C‐S lyase had an optimum pH of 7.5 for activity. Also, similar Km values were observed for both forms of the enzyme using (±)‐S‐methyl‐L‐cysteine sulfoxide (± MCSO; 14.2–19.2 mM) and an alk(en)yl‐L‐cysteine sulfoxide extract (0.9–1.8 mM) prepared from onion bulbs. Both forms of the enzyme were stable for 6–8 weeks at 4°C. At 25°C, the immobilized enzyme was fully stable for 5 weeks whereas the free enzyme lost 50% of its activity within 2 weeks. As the alginate content in the gel was increased from 0.25 to 3%, the % yield of activity of the immobilized enzyme system decreased from 65 to 44%. However, as % alginate content was increased, the yield of active enzyme immobilized in the gel increased from 62 to 97%. Reaction of both the free and alginate‐entrapped C‐S lyase with (±) MCSO resulted in the production of a distinctive cabbage‐like aroma. Onion aroma was liberated when either form of the enzyme was recombined with an odorless alk(en)yl‐L‐cysteine sulfoxide extract from onion.     

Immobilization of β-galactosidase by bioaffinity adsorption on concanavalin A layered calcium alginate–starch hybrid beads for the hydrolysis of lactose from whey/milk

Aspergillus oryzae β-galactosidase was immobilized on the surface of a novel bioaffinity support: concanavalin A layered calcium alginate–starch beads. The maximum activity of the immobilized β-galactosidase was obtained at 60 °C, approximately 10 degrees higher than that of the free enzyme. The immobilized β-galactosidase exhibited significantly higher stability to heat, urea, MgCl₂, and CaCl₂ than the free enzyme. An enhancement of the activity of immobilized β-galactosidase by up to 5.0% MgCl₂ was seen, whereas the activity of the free enzyme decreased above 3.0% MgCl₂. Immobilized β-galactosidase retained 61%, 50% and 43% activity in the presence of 5% CaCl₂, 5% galactose and 4 m urea, respectively, when incubated for 1 h at 37 °C. The immobilized β-galactosidase had a much higher K_iapp value than the free enzyme, which indicated less susceptibility to product inhibition by galactose. The immobilized β-galactosidase preparation was superior to the free enzyme in hydrolysing lactose in whey or milk in a batch process: it hydrolyzed 89% of the lactose in whey in 3 h and 79% of the lactose in milk in 4 h. The immobilized β-galactosidase retained 61% of its original activity after 2 months storage at 4 °C, while the soluble enzyme showed only 37% of the initial activity under identical conditions.     

Preparation and characterization of immobilized beta-lactamase for destruction of penicillin in milk

beta-Lactamase I (Bacillus cereus) was covalently bound to cyanogen bromide-activated, crosslinked agarose. An initial 5.00 mg of soluble beta-lactamase were used in the immobilization reaction for each preparation, and average coupling yield was 80.5%. Of the enzyme immobilized on the matrix, an average 53.4% remained active. To minimize diffusional effects on immobilized enzyme activity, reaction mixtures were rotated at 250 rpm throughout the study. The shape of the pH activity curve of the immobilized enzyme was identical to that of the soluble enzyme; both exhibited optimum pH around 7.0. In general, only 2-fold differences in Michaelis constant and maximum volume were observed between native and immobilized enzyme when penicillin G was used as the substrate. However, the Michaelis constant of the immobilized enzyme increased up to 22-fold that of the native enzyme when cephaloridine was used as the substrate. The immobilized enzyme exhibited enhanced stability in the acidic pH region in contrast to the native enzyme, which had superior stability in the alkaline pH region. The heat stability of the immobilized enzyme was about twice that of native enzyme after heat treatment at 60 degrees C for 30 min. Approximately a 10% increase of storage stability on immobilization of beta-lactamase was observed when stored at room temperature (23 +/- 1 degree C) for up to 6 d in the absence of antimicrobial agents. Little loss of activity (less than 2%) was noted after repeated use of the immobilized enzyme up to seven times each in 10.0 ml of skim milk containing .5 U/ml penicillin G.(ABSTRACT TRUNCATED AT 250 WORDS)     

Maltose hydrolysis kinetics with glucoamylase immobilized in porous glass fibers in a tubular flow reactor

Glucoamylase has been covalently immobilized in controlled pore glass fibers packed parallel to the axis of a tubular reactor. Flow kinetic studies have been carried out for a range of flow rates and substrate concentrations at 50°C and pH 4.5. Diffusion control has been found negligible with high flow rates and substrate concentrations. The apparent Michaelis constant was three orders of magnitude higher than that of the free enzyme. As flow rate and substrate concentration decrease, the extent of diffusion control increased up to a moderate degree. The immobilized glucoamylase was more stable than the free enzyme when incubated at 50°C.