Immobilization of β-Galactosidase by Polyacrylamide in the Presence of Protective Agents

β-Galactosidase from Aspergillus oryzae was immobilized in crosslinked polyacrylamide gel beads. The presence of the enzyme inhibitor, such as glucono-δ-lactone or galactono-γ-lactone, during polymerization procedure enhanced the residual enzymatic activity in the polymer beads, and activity yield attained up to 45%. Such enhancement effect was also observed when bovine serum albumin, dithiothreitol or glutathione was added during polymerization. Temperature and pH optima were not affected by the immobilization. The Michaelis constants for free and immobilized β-galactosidase were comparable. Lyophilized beads exhibited good stability without loss of enzymatic activity when stored at 4°C for 47 days.     

Stabilization of immobilized cathepsin L in non-aqueous medium

A lysosomal cysteine protease cathepsin L (3.4.22.15) purified from goat brain has been immobilized in calcium alginate beads in the presence of BSA through entrapment. Most favorable conditions for the entrapment were standardized as 3.0%(w/v) alginate and 1.5%(w/v) calcium chloride. Comparing the properties of free and immobilized enzyme using Z-Phe-Arg-4mβNA as chromogenic substrate, it was found that the immobilized enzyme could retain～70% of the original activity after five successive batch reactions. Vis-à-vis the free enzyme, immobilization conferred high stability to the enzyme both in the acidic and alkaline range, the enzyme lost no activity up to 60°C (Temperature stability for free enzyme is only up to 50°C). The pH optima for the enzyme shifted from 6.2 to 6.6 on entrapment. The increase in activity and stability of the enzyme in immobilized form even in the presence of high concentration of DMSO and ethanol is surprising and may make it useful for catalyzing organic reactions like trans-esterification and trans-amidation.     

Immobilization of laccase from Streptomyces psammoticus and its application in phenol removal using packed bed reactor

Laccase was produced from Streptomyces psammoticus under solid-state fermentation. The enzyme was partially purified by ammonium sulphate precipitation and was immobilized in alginate beads by entrapment method. Calcium alginate beads retained 42.5% laccase activity, while copper alginate beads proved a better support for laccase immobilization by retaining 61% of the activity. Phenol and colour removal from a phenol model solution was carried out using immobilized laccase. Batch experiments were performed using packed bed bioreactor, containing immobilized beads. Reusability of the immobilized matrix was studied for up to 8 successive runs, each run with duration of 6 h. The system removed 72% of the colour and 69.9% of total phenolics from the phenol model solution after the initial run. The immobilized system maintained 50% of its efficiency after eight successive runs. The degradation of phenolic compounds by immobilized laccase was evaluated and confirmed by Thin layer chromatography and nuclear magnetic resonance spectroscopy.     

Production of acrylamide using immobilized cells of<Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> M33

The cellsof Rhodococcus rhodochrous M33, which produce a nitrile hydratase enzyme, were immobilized in acrylamide-based polymer gels. The optimum pH and temperature for the activity of nitrile hydratase in both the free and immobilized cells were 7.4 and 45°C, respectively, yet the optinum temperature for acrylamide production by the immobilized cells was 20°C. The nitrile hydratase of the immobilized cells was more stable with acrylamide than that of the free cells. Under optimal conditions, the final acrylamide concentration reached about 400 g/L with a conversion yield of almost 100% after 8 h of reaction when using 150 g/L of immobilized cells corresponding to a 1.91 g-dry cell weight/L. The enzyme activity of the immobilized cells rapidly decreased with repeated use. However, the quality of the acrylamide produced by the immobilized cells was much better than that produced by the free cells in terms of color, salt content, turbidity, and foam formation. The quality of the aqueous acrylamide solution obtained was found to be of commercial use without further purification.     

Enzyme electrode formed by evaporative concentration and its performance characterization

A highly concentrated immobilized enzyme layer was formed on a small working electrode, and the behavior of the electrode as an amperometric sensor was examined. To this end, a super-hydrophobic layer was formed in an area other than the sensitive area by using polytetrafluoroethylene (PTFE) beads. A small droplet of an enzyme solution containing glucose oxidase (GOD) and bovine serum albumin (BSA) was placed on the sensitive area, concentrated by evaporation, and crosslinked with glutaraldehyde. With the same enzyme activity per unit area, the current density increased with smaller working electrodes. Also, the current density increased with higher enzyme loadings up to a limiting value. In addition, the linear range of the calibration plot was expanded to higher glucose concentrations. The enzyme electrode fabricated by the novel method was incorporated in a micro-flow channel. Compared with large enzyme electrodes with the same enzyme activity per unit area, smaller electrodes showed a significant increase in the current density and a decrease in the flow dependence. The conversion efficiency could be improved by narrowing the flow channel and increasing the number of electrodes, which was comparable with a large electrode placed in a shallow flow channel.     

Immobilization of β-galactosidases from Thermus aquaticus YT-1 for oligosaccharides synthesis

Summary -galactosidases of Thermus aquaticus YT-1, exhibiting a galactosyl transferase activity, were immobilized using different techniques. Entrapment in agarose or gellan gum beads was unsuitable for enzyme immobilization due to enzyme leakage. A technique that efficiently immobilized the enzymes was developed using glutaraldehyde co-crosslinking of -galactosidases with bovine serum albumin, followed by entrapment in agarose beads.     

Immobilization of enzymes on Spheron: 2. β-Amylase — The development of a column reactor—separator

The titanium-chelation method has been used to immobilize β-amylase (1,4-α-d-glucan maltohydrolase, EC 3.2.1.2) on to Spheron. On various grades of Spheron, protein coupling yields of 56–76% were obtained with barley and sweet-potato β-amylases. The specific enzymic activities of the immobilized enzymes fell in the range 3.7–7.6% of those of the soluble enzymes. The immobilized enzymes were more stable than the soluble, especially in the presence of l-cysteine and serum albumin. The presence of cysteine and serum albumin brought about increases in activity in the preparations, presumably by regenerating essential thiol groups in the enzyme which had been oxidized during the operations. Maltose could be separated from amylopectin and other large polysaccharides by chromatography on Spheron P100, and a system was developed in which maltose, produced by hydrolysis of amylopectin applied in pulses to a column of immobilized β-amylase, was separated from starting material and by-products on a second column of Spheron P100.     

Immobilization of Β-D-galactosidase fromLactobacillus bulgaricus on polyacrylamide in the presence of protective agents and properties of the immobilized enzyme

Β-D-Galactosidase (EC 3.2.1.23) fromLactobacillus bulgaricus (1373) was immobilized in a Polyacrylamide gel lattice in the presence of dithiothreitol, glutathione, cysteine, bovine serum albumin, casein, lactose and glucono-δ-lactone. Cysteine, bovine serum albumin, and lactose were found very effective in preserving the activity. With cysteine, bovine serum albumin and lactose, the activity yields were 61, 60 and 66% respectively, as compared to 31% without protective agents. The yield improved upto 85% when all the three protective agents, cysteine, bovine serum albumin and lactose were added during immobilization. The addition of protective agents did not have any effect on optimum pH, optimum temperature, kinetic constants and pH stability when compared with Β-galactosidase immobilized without the use of protective agents; however the heat and storage stabilities were found to increase.     

Some properties of a papain–agarose reactor provided with a pH monitor

A small reactor of immobilized papain was used to gain some knowledge about the effect of immobilization upon the reactivity of the enzyme towards one substrate and various types of inhibitors. A buffer solution containing benzoyl–arginine ethyl ester as substrate was run through a small column of papain immobilized by attachment to agarose beads. The pH of the effluent was measured continuously and provided the data used to calculate the substrate conversion during passage through the reactor. The operation of the system was checked by determining the substrate conversion as a function of flow rate. It proved to operate as theory demanded. The rate and extent of inhibition were measured after addition of various inhibitors to the buffer–substrate solution. The following quantities of immobilized papain were found to be equal within ±20% to those of the free enzyme in solution: the overall activity, the K_m of benzoyl–arginine ethyl ester, the K_i of the competitive inhibitor benzoylamino-acetonitrile, the rate of inactivation by chloroacetic acid and by chloroacetamide, the rate of activation by cysteine of the mixed disulfide of papain and cysteine, and the rate of spontaneous reactivation of the KCNO–papain adduct. The inactivation by KCNO proved to be strongly pH dependent. This may explain why the rate of the latter reaction is only 66% of the rate with free enzyme. It is concluded that the rates and equilibrium constants measured in the present reactor system are within ±20% of the values of the dissolved enzyme, provided that the reactions are not strongly pH dependent. Calculation showed there was no diffusion limitation.     

Lipase production by free and immobilized protoplasts of Sporotrichum (Chrysosporium) thermophile Apinis

Summary Production of lipase by free and alginate-entrapped protoplasts was studied in batch culture. Cell-wall-degrading enzymes Novozym 234 and cellulase CP improved lipase secretion of normal mycelium by 25%–100%. The protoplast-regenerated mycelium exhibited several-fold higher lipase activity in batch replacements in TRIS buffer over normal spore-derived mycelium. The specific lipase activity of immobilized protoplasts was about four times higher than normal mycelial beads. Protoplasts beads were stable and retained high enzyme activity even after three buffer replacements lasting 120 h; TRIS buffer was better than acetate or normal glucose medium. A minimum of 8 h regeneration period was necessary for lipase synthesis. Triolein, olive oil, tributyrin and oleic acid butylester were able to induce lipase in immobilized protoplasts. Tween 80 enhanced lipase activity of the immobilized protoplasts. Partially degraded immobilized mycelium was nearly as effective as normal immobilized protoplasts for lipase secretion. Both free and immobilized protoplasts could be reused for up to 200 h with some loss in enzyme activity.     

Immobilization of fructosyltransferase by chitosan and alginate for efficient production of fructooligosaccharides

The effective system of reusing mycelial fructosyltransferase (FTase) immobilized with two polymers, chitosan and alginate were evaluated for continuous production of fructooligosaccharides (FOS). The alginate beads were successfully developed by maintaining spherical conformation of using 0.3% (w/v) sodium alginate with 0.1% (w/v) of CaCl₂ solution for highest transfructosylating activity. The characteristics of free and immobilized FTase were investigated and results showed that optimum pH and temperature of FTase activity were altered by immobilized materials. A successive production of FOS by FTase entrapped alginate beads was observed at an average of 62.96% (w/w) up to 7 days without much losing its activity. The data revealed by HPLC analysis culminate 67.75% (w/w) of FOS formation by FTase entrapped alginate beads and 42.79% (w/w) by chitosan beads in 36 h of enzyme substrate reaction.     

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.     

Preparation and characterization of immobilized phospholipase A2 on chitosan beads for lowering serum cholesterol concentration

Phospholipase A₂ (PLA₂) from cobra venom, which can hydrolyze the S_N2 ester bond of 1,2-diacylphosphatides, was immobilized by covalent binding to porous chitosan beads. Immobilization has to be carried out by using the carboxylic groups instead of the amine groups of the enzyme to get reasonable activity retention (higher than 50%). The effects of amount of activating reagent EDC and enzyme loading during the immobilization step were investigated. Since EDC could modify important Asp groups in the enzyme, the EDC/enzyme weight ratio should be less than 10. Although the activity retention of immobilized enzyme increased with enzyme/bead weight ratio, this ratio should be kept to a minimum at 1×10⁻³ to optimize coupling yield of enzyme activity and reduce internal diffusion resistance. The kinetic properties and stability of the immobilized enzyme were determined. The immobilized PLA₂ was packed into a column to hydrolyze phospholipid in a circulating packed-bed reactor. The flow rate of the substrate solution should be set at 37.5 cm/min (superficial velocity) to eliminate external diffusion resistance, under which condition the column reactor could be reused up to 10 times with less than 20% loss of activity. Since enzymatic hydrolysis of phospholipid on low density lipoprotein (LDL) particle surface with PLA₂ could result in faster plasma clearance of the modified LDL particles, an in vitro bioreactor containing immobilized PLA₂ should be able to lower serum cholesterol concentration. A significant decrease in total serum cholesterol concentration in hypercholesterolemic rabbits was observed after 90-min treatment.     

Controlled layer-by-layer immobilization of horseradish peroxidase.

Horseradish peroxidase (HRP) was biotinylated with biotinamidocaproate N-hydroxysuccinimide ester (BcapNHS) in a controlled manner to obtain biotinylated horseradish peroxidase (Bcap-HRP) with two biotin moieties per enzyme molecule. Avidin-mediated immobilization of HRP was achieved by first coupling avidin on carboxy-derivatized polystyrene beads using a carbodiimide, followed by the attachment of the disubstituted biotinylated horseradish peroxidase from one of the two biotin moieties through the avidin-biotin interaction (controlled immobilization). Another layer of avidin can be attached to the second biotin on Bcap-HRP, which can serve as a protein linker with additional Bcap-HRP, leading to a layer-by-layer protein assembly of the enzyme. Horseradish peroxidase was also immobilized directly on carboxy-derivatized polystyrene beads by carbodiimide chemistry (conventional method). The reaction kinetics of the native horseradish peroxidase, immobilized horseradish peroxidase (conventional method), controlled immobilized biotinylated horseradish peroxidase on avidin-coated beads, and biotinylated horseradish peroxidase crosslinked to avidin-coated polystyrene beads were all compared. It was observed that in solution the biotinylated horseradish peroxidase retained 81% of the unconjugated enzyme's activity. Also, in solution, horseradish peroxidase and Bcap-HRP were inhibited by high concentrations of the substrate hydrogen peroxide. The controlled immobilized horseradish peroxidase could tolerate much higher concentrations of hydrogen peroxide and, thus, it demonstrates reduced substrate inhibition. Because of this, the activity of controlled immobilized horseradish peroxidase was higher than the activity of Bcap-HRP in solution. It is shown that a layer-by-layer assembly of the immobilized enzyme yields HRP of higher activity per unit surface area of the immobilization support compared to conventionally immobilized enzyme.     

Preparation and characterization of urease immobilized onto porous chitosan beads for urea hydrolysis

Urease was covalently immobilized onto porous chitosan beads via primary amine groups connected to the backbone via a six-carbon linear alkyl spacer. The optimum conditions for enzyme immobilization are activating the beads with 1%(w/w) glutaraldehyde, reacting the activated beads in pH 7 buffer with the enzyme, using an enzyme to bead weight ratio of 25, and without lyophilization. Chitosan-bound urease was found to fully retain its specific activity. Properties of the immobilized urease were characterized under batch and flow conditions. Increased optimum reaction temperature, enhanced thermal stability and storage stability, and excellent reusability were found after enzyme immobilization. Continuous hydrolysis of urea solution was studied in a column packed with the enzyme-containing beads for its possible application in regenerating dialysate solution during hemodialysis.     

Photooxidation of methionine with immobilized methylene blue photooxidizer

Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37°C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84% acetic acid at 4°C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94% while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21%. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.     

Photooxidation of methionine with immobilized methylene blue as photooxidizer.

Methylene blue immobilized on porous glass beads was used to catalyze the photooxidation of methionine alone and the methionine residues of lysozyme. A solution of 2 mM methionine in 50% acetic acid was oxidized to methionine sulfoxide in the presence of immobilized methylene blue after 6 h of photooxidation at 37 degrees C. Selective photooxidation of the methionyl residues in lysozyme was achieved after 26 h of reaction in 84% acetic acid at 4 degrees C. The specific activity of lysozyme exposed to light in the presence of methylene blue decreased by 94%, while that of a lysozyme solution in the presence of methylene blue not exposed to light decreased by 21%. The lysozyme solution exposed to light but not containing the methylene blue beads lost 33% of its specific activity after the same period of photooxidation. It was shown that the decrease in enzyme activity was not caused by adsorption of the enzyme onto the beads.     

Immobilization of tyrosinase on chitosan-clay composite beads

Tyrosinase was immobilized on glutaraldehyde crosslinked chitosan-clay composite beads and used for phenol removal. Immobilization yield, loading efficiency and activity of tyrosinase immobilized beads were found as 67%, 25% and 1400 U/g beads respectively. Optimum pH of the free and immobilized enzyme was found as pH 7.0. Optimum temperature of the free and immobilized enzyme was determined as 25-30 °C and 25 °C respectively. The kinetic parameters of free and immobilized tyrosinase were calculated using l-catechol as a substrate and K(m) value for free and immobilized tyrosinase were found as 0.93 mM and 1.7 mM respectively. After seven times of repeated tests, each over 150 min, the efficiency of phenol removal using same immobilized tyrosinase beads were decreased to 43%.     

Fibrous polymer-grafted chitosan/clay composite beads as a carrier for immobilization of papain and its usability for mercury elimination

Papain, which is an industrially important enzyme, has been immobilized on fibrous polymer-modified composite beads, namely poly(methacrylic acid)-grafted chitosan/clay. Characterization studies have been done using FTIR and SEM analysis. Operating parameters such as pH and initial concentration of papain have been varied to obtain the finest papain immobilized polymer-modified composite beads. The immobilization capacity of composite beads has been determined as 34.47 ± 1.18 (n = 3) mg/g. The proteolytic activity of immobilized papain was operated using bovine serum albumin (BSA) and maximum velocity (V _max) and Michaelis–Menten constant (K_m) values of the free and immobilized enzymes were determined using Lineweaver–Burk and Eadie–Hofstee equations. Usability of papain immobilized polymer-modified composite beads as adsorbents for the elimination of mercury was investigated. The maximum removal capacity of PIPMC beads has been found to be 4.88 ± 0.21 mg Hg/g when the initial metal concentration and weight of polymer-modified composite beads were 50 mg/L and 0.04 g at pH 7, respectively. Mercury removal performance of the papain immobilized polymer-modified composite beads was investigated in conjunction with Cu (II), Zn (II) and Cd (II) ions. The mercury adsorption capacity of papain immobilized polymer-modified composite beads was a slight reduction from 1.15 to 0.89 mg/g in presence of multiple metal salts.     

Stability and reactivity of acid phosphatase immobilized on composite beads of chitosan and ZrO2 powders

Equal weights of chitosan and ZrO2 powders were mixed in acetic acid solution to prepare the composite beads. They were then cross-linked with glutaraldehyde and stored with and without freeze-drying before use. The physicochemical properties of acid phosphatase immobilized on four types of the supports (wet/dried pure chitosan beads, wet/dried chitosan-ZrO2 composite beads) were compared. Various parameters including glutaraldehyde concentration, cross-linking time, enzyme concentration, temperature, and pH on enzyme activity were studied. It was shown that the activity yield of enzyme immobilized on the dried chitosan-ZrO2 beads was the highest, and the relative activity remained above 83.2% within pH 2.9-5.8. Regardless of wet or dried beads, the Michaelis constant KM and maximum rate of reaction Vmax of acid phosphatase immobilized on chitosan-ZrO2 composite beads were 1.8 times larger than those on pure chitosan beads. Of the four immobilized enzymes, the use of wet chitosan-ZrO2 bead as the support showed the lowest thermal deactivation energy (78 kJ mol(-1)).