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.     

α‐Amylase immobilization on functionalized nano CaCO3 by covalent attachment

In this study, α‐amylase was immobilized on glutaraldehyde activated silanized calcium carbonate nanoparticles by a using covalent binding method. The surface modified nano calcium carbonate (CaCO₃) were characterized using FTIR and SEM. Immobilization yield was found as 199.43 mg/g of calcium carbonate nanoparticles. The maximum activity was observed at pH 6.5. The immobilized enzyme had a higher activity at elevated temperature (50–90°C) than the free one. Reuse studies demonstrated that the immobilized enzyme could reuse 25 times while retaining 18.2% of its activity. Free enzyme lost its activity completely within 15 days. V_max values for the free and immobilized enzymes were calculated as 10 and 0.35 mg/mL/min, respectively.     

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.     

Immobilization and characterization of naringinase for the hydrolysis of naringin

The degree of hydrolysis of naringin was investigated at various temperatures (40, 50, 60 °C), enzyme concentrations (0.01–0.30 mg ml⁻¹), and pH values (2.5–5.5) for naringinase enzyme. Naringinase was immobilized on celite by simple adsorption. Naringin content was determined by HPLC method. The degree of hydrolysis of naringin showed a linear increase up to an enzyme concentration of 0.2 mg ml⁻¹ that corresponds to 82% hydrolysis. The optimum values of pH for the hydrolysis of naringin were 4.0 for free and 3.5 for immobilized enzymes. Maximum enzyme activities were found to be 70 and 60 °C for free and immobilized enzymes, respectively. The values of K _m,app and V _max,app calculated were 1.22 mM and 0.45 μmol min⁻¹ mg enzyme⁻¹ for free and 2.16 mM and 0.3 μmol min⁻¹ mg enzyme⁻¹ for immobilized enzyme, respectively. The mathematical modelling was applied to the experimental data for hydrolysis of naringin as a function of time at 30, 40 and 50 °C. The increase in temperature from 30 to 50 °C increased the rate constant 3.09 times for free enzyme. However, the rate constants found for immobilized enzyme applications did not increase in a similar trend as a function of temperature. The retained activity of celite-adsorbed naringinase was found to be 83% at their optimum conditions. The retained activity of immobilized enzyme was followed up to the fifth run and was found to be almost unchanged after the third use at optimum reaction conditions (pH 3.5, 60 °C).     

Properties of <Emphasis Type="Italic">Aspergillus subolivaceus</Emphasis> free and immobilized dextranase

Aspergillus subolivaceus dextranase is immobilized on several carriers by entrapment and covalent binding with cross-linking. Dextranase immobilized on BSA with a cross-linking agent shows the highest activity and considerable immobilization yield (66.7%). The optimum pH of the immobilized enzyme is shifted to pH 6.0 as compared with the free enzyme (pH 5.5). The optimum temperature of the reaction is resulted at 60 °C for both free and immobilized enzyme. Thermal and pH stability are significantly improved by the immobilization process. The calculated K _m of the immobilized dextranase (14.24 mg mL⁻¹) is higher than that of the free dextranase (11.47 mg mL⁻¹), while V _max of the immobilized enzyme (2.80 U μg protein⁻¹) is lower than that of the free dextranase (11.75 U μg protein⁻¹). The immobilized enzyme was able to retain 76% of the initial catalytic activity after 5.0 cycles.     

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.     

Immobilized alcalase alkaline protease on the magnetic chitosan nanoparticles used for soy protein isolate hydrolysis

An efficient immobilization of Alcalase 2.4L alkaline protease has been developed by using chitosan-coated magnetic nanoparticles as support via glutaraldehyde cross-linking reaction. The Fe₃O₄ nanoparticles, Fe₃O₄-chitosan, and immobilized Alcalase 2.4L alkaline protease were characterized by X-ray diffraction, transmission electron microscope, Fourier transform infrared spectroscopy, electron spin resonance, and vibrating sample magnetometry. Results showed that the binding of chitosan and Alcalase 2.4L alkaline protease on Fe₃O₄ through cross-linking was successful. In addition, the Alcalase 2.4L alkaline protease immobilized with chitosan-coated magnetic nanoparticles enhanced the activity, the optimum reaction temperature and pH value for the immobilized enzyme were 55 °C and 10, respectively, compared with the free enzyme, and the optimal temperature and pH profile range were considerably broadened. Similarly, the thermal stability was enhanced by immobilization, and the kinetic parameters of free and immobilized Alcalase 2.4L alkaline protease were determined. Then, from our hydrolysis experiments, we found that immobilized Alcalase 2.4L alkaline protease uses Fe₃O₄-chitosan had a greatest hydrolytic activity, and the DH of soy protein isolate (SPI) can reach to 18.38 %, against 17.50 % with the free enzyme after 140 min. Furthermore, the immobilized Alcalase 2.4L alkaline protease could maintain about 86 % of its original activity after ten consecutive operations. Thus, Fe₃O₄-chitosan immobilized Alcalase 2.4L alkaline protease a good candidate for the continuous hydrolysis of SPI.     

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.     

Stability and Kinetic Properties of a Magnetic Immobilized alpha-Amylase K

The properties of α-amylase K immobilized on hydrous titanium(IV)oxide coated magnetic iron oxide are reported and compared with the previously reported properties of the soluble form of the enzyme. The optimum pH was increased on immobilization but the addition calcium chloride caused a decrease. Compared to the soluble form the immobilized enzyme is less stable at 60 °C in calcium enriched buffers but more stable in calcium free buffers. The temperature-activity profile has a plateau between 40 °C and 60 °C attributable to a comformational change above 60 °C to a more active form. The action pattern in the hydrolysis of soluble starch was found to be unaffected by immobilization. Parameters affecting the amount of bound activity were studied. The magnetic recovery of the immobilized enzyme was complete.     

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.     

Preparation and Properties of Rhizopus oryzae Lipase Immobilized Using an Adsorption-Crosslinking Method

The aim was to develop an enzyme immobilization method for Rhizopus oryzae lipase to improve its acidolysis activity and stability. Lipase was adsorbed into NKA-9 resin and then crosslinked with glutaraldehyde as a crosslinker. The optimum conditions obtained using the response surface methodology were as follows: 34.6 mg lipase/100 mg support, 4.1 h of adsorption time, 45°C, pH 6.8, 0.5% glutaraldehyde concentration, and 2.5 h of crosslinking time. The acidolysis activity of the immobilized lipase was 31.78%, the free lipase activity was only 11.02%. The immobilized lipase showed better performance, such as higher acidolysis activity, better pH tolerance and temperature stability, enhanced storage stability, and improved reusability.     

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.     

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.     

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 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.     

Activity of diastase α-amylase immobilized on polyanilines (PANIs)

Starch hydrolysing enzyme α-amylase (EC 3.2.1.1) was immobilized by physical adsorption and covalent binding onto chemically synthesised polymer, polyanilines (PANIs) in two different forms, emeraldine salt and emeraldine base powder. The immobilization efficiency was affected by the pH of the immobilization medium, contact time and amount of enzyme. The kinetic parameters, reusability and storage stability of the enzyme were studied under free and immobilized condition at optimum pH and temperature. The immobilized enzyme showed enhanced storage stability over a period of 6 months, while free enzyme in solution lost all of its activity within a period of 8 days. Reusability of the enzyme was improved by immobilization. The K_m values for starch hydrolysis were found to be high for the immobilized enzymes.     

LINAMARASE: IMMOBILIZATION OF A CRUDE ENZYME

ABSTRACT Linamarase from cassava cortex was immobilized on polyacrylamide gel. A crude extract of the enzyme containing most of the intracellular proteins was used for the technique to simulate the enzyme in cassava. Immobilization of the enzyme increased the Michaelis constants for all substrates when compared with the native enzyme. Both free and immobilized linamarase hydrolyzed p-nitrophenylβ D-glucoside, linamarin and p-nitrophenylβ-D-galactoside. The immobilized enzyme was more stable than the free enzyme at room temperature (25C) and 41C. Both forms gave a bell-shaped curve with maximal activity at pH 6.0; these data indicated two catalytic ionizable residues with pK_a 5.7 and pK_a 6.5.     

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 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.     

接枝淀粉作为固定化酶载体研究进展

该文综述接枝淀粉用作固定化酶载体应用情况,通过接枝不同类型单体,淀粉将具有不同理化性质。以接枝淀粉为固定化酶载体主要方法有:共价结合法、物理吸附法或包埋法;以接枝淀粉为载体固定化酶主要有:糖化酶、葡萄糖异构酶、葡萄糖氧化酶、α-淀粉酶、α-胰凝乳蛋白酶和脲酶等。