Chemoselective hydrolysis of methyl 2‐acetoxybenzoate using free and entrapped esterase in K‐carrageenan beads

Porcine liver esterase was entrapped in natural polysaccharides K‐carrageenan and retention of its activity was determined using p‐nitrophenyl acetate as the substrate. The optimum pH for esterase activity of entrapped enzyme showed a little shift towards acidic side. Immobilized enzyme showed improved thermal and storage stability. The entrapped esterase retained 50% of its activity after eight repetitive cycles. Michaelis constant K_m for the free and entrapped enzymes was almost same indicting no conformational change during immobilization. Maximum velocity V_max was observed to decrease on immobilization. The free and entrapped esterase was used for selective hydrolysis of methyl 2‐acetoxybenzoate to methyl 2‐hydroxybenzoate in batch process as well as in a fixed bed reactor. The hydrolysis was observed to be 99% within 2 h for free as well as immobilized enzyme in batch process. The rate of hydrolysis was found to depend on pH. The turn over number of selective hydrolysis in batch and fixed bed reactor was 3.08 × 10⁶ and 1.19 × 10⁷, respectively. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008     

Chitosaneous hydrogel beads for immobilizing neutral protease for application in the preparation of low molecular weight chitosan and chito‐oligomers

Enzyme hydrolysis with immobilized neutral protease was carried out to produce low molecular weight chitosan (LMWC) and chito‐oligomers. Neutral protease was immobilized on (CS), carboxymethyl chitosan (CMCS), and N‐succinyl chitosan (NSCS) hydrogel beads. The properties of free and immobilized neutral proteases on chitosaneous hydrogel beads were investigated and compared. Immobilization enhanced enzyme stability against changes in pH and temperature. When the three different enzyme supports were compared, the neutral protease immobilized on CS hydrogel beads had the highest thermal stability and storage stability, and the enzyme immobilized on NSCS hydrogel beads had the highest activity compared to those immobilized on the other supports, despite its lower protein loading. Immobilized neutral protease on all the three supports had a higher K_m (Michaelis‐Menten constant) than free enzyme. The V_max (maximum reaction velocity) value of neutral protease immobilized on CS hydrogel beads was lower than the free enzyme, whereas the V_max values of enzyme immobilized on CMCS and NSCS hydrogel beads were higher than that of the free enzyme. Immobilized neutral protease on CS, CMCS, and NSCS hydrogel beads retained 70.4, 78.2, and 82.5% of its initial activity after 10 batch hydrolytic cycles. The activation energy decreased for the immobilization of neutral protease on chitosaneous hydrogel beads. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 3743–3750, 2006     

Catalytic potential of a poly(AAc‐co‐HPMA‐cl MBAm)‐matrix‐immobilized lipase from a thermotolerant Pseudomonas aeruginosa MTCC‐4713

A purified alkaline thermo‐tolerant lipase from Pseudomonas aeruginosa MTCC‐4713 was immobilized on a series of five noble weakly hydrophilic poly(AAc‐co‐HPMA‐cl MBAm) hydrogels. The hydrogel synthesized by copolymerizing acrylic acid and 2‐hydroxy propyl methacrylate in a ratio of 5 : 1 (HG_5:1 matrix) showed maximum binding efficiency for lipase (95.3%, specific activity 1.96 IU mg^?1 of protein). The HG_5:1 immobilized lipase was evaluated for its hydrolytic potential towards p‐NPP by studying the effect of various physical parameters and salt‐ions. The immobilized lipase was highly stable and retained ～92% of its original hydrolytic activity after fifth cycle of reuse for hydrolysis of p‐nitrophenyl palmitate at pH 7.5 and temperature 55°C. However, when the effect of pH and temperature was studied on free and bound lipase, the HG_5:1 immobilized lipase exhibited a shift in optima for pH and temperature from pH 7.5 and 55°C to 8.5 and 65°C in free and immobilized lipase, respectively. At 1 mM concentration, Fe³⁺, Hg²⁺, NH₄⁺, and Al³⁺ ions promoted and Co²⁺ ions inhibited the hydrolytic activities of free as well as immobilized lipase. However, exposure of either free or immobilized lipase to any of these ions at 5 mM concentration strongly increased the hydrolysis of p‐NPP (by ～3–4 times) in comparison to the biocatalysts not exposed to any of the salt ions. The study concluded that HG_5:1 matrix efficiently immobilized lipase of P. aeruginosa MTCC‐4713, improved the stability of the immobilized biocatalyst towards a higher pH and temperature than the free enzyme and interacted with Fe³⁺, Hg²⁺, NH₄⁺, and Al³⁺ ions to promote rapid hydrolysis of the substrate (p‐NPP). © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4252–4259, 2006     

Immobilization of invertase onto crosslinked poly(p‐chloromethylstyrene) beads

Invertase was immobilized onto poly(p‐chloromethylstyrene) (PCMS) beads that were produced by a suspension polymerization with an average size of 186 μm. The beads had a nonporous but reasonably rough surface. Because of this, a reasonably large external surface area (i.e., 14.1 m²/g) could be achieved with the proposed carrier. A two‐step functionalization protocol was followed for the covalent attachment of invertase onto the bead surface. For this purpose, a polymeric ligand that carried amine groups, polyethylenimine (PEI), was covalently attached onto the bead surface by a direct chemical reaction. Next, the free amine groups of PEI were activated by glutaraldehyde. Invertase was covalently attached onto the bead surface via the direct chemical reaction between aldehyde and amine groups. The appropriate enzyme binding conditions and the batch‐reactor performance of the immobilized enzyme system were investigated. Under optimum immobilization conditions, 19 mg of invertase was immobilized onto each gram of beads with 80% retained activity after immobilization. The effects of pH and temperature on the immobilized invertase activity were determined and compared with the free enzyme. The kinetic parameters K_M and V_M were determined with the Michealis–Menten model. K_M of immobilized invertase was 1.75 folds higher than that of the free invertase. The immobilization caused a significant improvement in the thermal stability of invertase, especially in the range of 55–65°C. No significant internal diffusion limitation was detected in the immobilized enzyme system, probably due to the surface morphology of the selected carrier. This result was confirmed by the determination of the activation energies of both free and immobilized invertases. The activity half‐life of the immobilized invertase was approximately 5 times longer than that of the free enzyme. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 1268–1279, 2002     

Immobilization of a nonspecific chitosan hydrolytic enzyme for application in preparation of water‐soluble low‐molecular‐weight chitosan

A nonspecific chitosan hydrolytic enzyme, cellulase, was immobilized onto magnetic chitosan microspheres, which was prepared in a well spherical shape by the suspension crosslinking technique. The morphology characterization of the microspheres was carried out with scanning electron microscope and the homogeneity of the magnetic materials (Fe₃O₄) in the microspheres was determined from optical micrograph. Factors affecting the immobilization, and the properties and stabilities of the immobilized enzyme were studied. The optimum concentration of the crosslinker and cellulase solution for the immobilization was 4% (v/v) and 6 mg/mL, respectively. The immobilized enzyme had a broader pH range of high activity and the loss of the activity of immobilized cellulase was lower than that of the free cellulase at high temperatures. This immobilized cellulase has higher apparent Michaelis–Menten constant K_m (1.28 mg/mL) than that of free cellulase (0.78 mg/mL), and the maximum apparent initial catalytic rate V_max of immobilized cellulase (0.39 mg mL^?1 h^?1) was lower than free enzyme (0.48 mg mL^?1 h^?1). Storage stability was enhanced after immobilization. The residual activity of the immobilized enzyme was 78% of original after 10 batch hydrolytic cycles, and the morphology of carrier was not changed. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 1334–1339, 2006     

Immobilization of α‐chymotrypsin for use in batch and continuous reactors

α‐Chymotrypsin from bovine pancrease (EC 3.4.21.1) was entrapped in Ca‐alginate gel particles to carry out hydrolysis of N‐acetyl‐L ‐phenylalanine methyl ester (APME) in batch as well as continuous fixed bed reactor. The enzyme was covalently modified with homo‐bifunctional polyethylene glycol derivatives in order to reduce its leakage from the beads; 85% modification of the ∈‐NH₂ groups of lysine residues caused reduction in the enzyme activity by 50%. However, this modification was helpful in a long run because it reduced both enzyme leakage and deactivation. Effective diffusivities and the distribution coefficients of the substrate and the product were determined experimentally, and later used in simulation of a batch experiment employing the beads. A continuous fixed bed reactor with the gel beads was operated to study the deactivation of the enzyme. During a 15‐day period, the enzyme showed about 15% loss in the conversion which occurred only during the first 5 days. After that the enzyme did not deactivate further which demonstrates that this method can be applied for continuous reactions. © 2000 Society of Chemical Industry     

Urease immobilization on an ion‐exchange textile for urea hydrolysis

Ion‐exchange textiles are used as organic supports for urease immobilization with the aim of developing reactive fibrous materials able to promote urea removal. A non‐woven, polypropylene‐based cation‐exchange textile was prepared using UV‐induced graft polymerization. Urease was covalently immobilized onto the cation‐exchange textile using three different coupling agents: N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC), N‐cyclohexyl‐N′‐(b‐[N‐methylmorpholino]ethyl)carbodiimide p‐toluenesulfonate (CMC), and glutaraldehyde (GA). The immobilized biocatalyst was characterized by means of FT‐IR spectrometry, SEM micrographs, dependence of the enzyme activity on pH and temperature, and according to the kinetic constants of the free and immobilized ureases. The biotextile prepared with EDC in the presence of N‐hydroxysuccinimide performs best. The optimum pH was 7.2 for the free urease and 7.6 for the immobilized ureases. The reactivity was maximal at 45 °C for free urease, 50 °C for biotextiles prepared using EDC or CMC, and 55 °C for biotextiles prepared with GA. The activation energy for the immobilized ureases was 4.73–5.67 kcal mol^?1, which is somewhat higher than 4.3 kcal mol^?1 for free urease. The urea conversion for a continuous‐flow immobilized urease reactor is nearly as good as a continuously stirred tank reactor having a much longer residence time, suggesting that the packed bed reactor had sufficient diffusive mixing and residence time to reach nearly optimal results. Urease immobilized on a biotextile using EDC has good storage and operational stability. Copyright © 2006 Society of Chemical Industry     

Stability of β‐galactosidase immobilized on composite microspheres of artemisia seed gum and chitosan

In this work, composite microspheres were prepared by using artemisia seed gum and chitosan as a source. The composite microspheres have activated aldehyde groups by using glutaraldehyde. β‐Galactosidase was covalently bound on these activated microspheres. The properties of the immobilized enzyme were investigated and compared with those of the free enzyme, for which o‐nitrophenol β‐D ‐galactopyranoside (ONPG) was chosen as a substrate. The results showed that the pH and thermal stability of the immobilized β‐galactosidase were higher than those of the soluble one. Apart from these, the Michaelis constant K_m was evaluated for the immobilized β‐galactosidase and the soluble enzyme. The immobilized β‐galactosidase exhibited better environmental adaptability and reusability than the soluble one. POLYM. COMPOS., 29:9–14, 2008. © 2007 Society of Plastics Engineers     

Immobilization of invertase onto dimer acid‐co‐alkyl polyamine

Invertase was immobilized onto the dimer acid‐co‐alkyl polyamine after activation with 1,2‐diamine ethane and 1,3‐diamine propane. The effects of pH, temperature, substrate concentration, and storage stability on free and immobilized invertase were investigated. Kinetic parameters were calculated as 18.2 mM for K_m and 6.43 × 10^?5 mol dm^?3 min^?1 for V_max of free enzyme and in the range of 23.8–35.3 mM for K_m and 7.97–11.71 × 10^?5 mol dm^?3 min^?1 for V_max of immobilized enzyme. After storage at 4°C for 1 month, the enzyme activities were 21.0 and 60.0–70.0% of the initial activity for free and immobilized enzyme, respectively. The optimum pH values for free and immobilized enzymes were determined as 4.5. The optimum temperatures for free and immobilized enzymes were 45 and 50°C, respectively. After using immobilized enzyme in 3 days for 43 times, it showed 76–80% of its original activity. As a result of immobilization, thermal and storage stabilities were increased. The aim of this study was to increase the storage stability and reuse number of the immobilized enzyme and also to compare this immobilization method with others with respect to storage stability and reuse number. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1526–1530, 2004     

Immobilization of β-galactosidase onto polymeric supports

Poly(vinyl alcohol) cross-linked with para-formaldehyde (PVA–F) and natural polysaccharide–chitosan in bead form and salicylic acid–resorcinol–formaldehyde polymeric resin (SRF) in powder form were used for immobilization of β-galactosidase through covalent linkages. Various activation processes and conditions were optimized. Immobilized enzyme showed very good storage stability at room temperature. Durability of the enzyme was also improved on immobilization. On repeated use of enzyme immobilized on chitosan beads, no loss was observed in enzyme activity even after 10 batches. Michaelis constant K_m and maximum reaction velocity V_m were calculated for free and immobilized enzyme systems. Effect of pH and temperature on enzyme activity was estimated and energy of activation (E_a) and inactivation constant (K_i) for free and immobilized enzyme were calculated. © 1995 John Wiley & Sons, Inc.     

Characteristics of poly(AAc5‐co‐HPMA3‐cl‐EGDMA15) hydrogel‐immobilized lipase of Pseudomonas aeruginosa MTCC‐4713

Four series of noble networks were synthesized with acrylic acid (AAc) copolymerized with varying amount of 2‐hydroxy propyl methacrylate or dodecyl methacrylate (AAc/HPMA or AAc/DMA; 5:1 to 5:5, w/w) in the presence of ethylene glycol dimethacrylate (EGDMA; 1, 5, 10, 15, and 20%, w/w) as a crosslinker and ammonium per sulfate (APS) as an initiator. Each of the networks was used to immobilize a purified lipase from Pseudomonas aeruginosa MTCC‐4713. The lipase was purified by successive salting out with (NH₄)₂SO₄, dialysis, and DEAE anion exchange chromatography. Two of the matrices, E_15a, i.e. [poly (AAc₅‐co‐DMA₁‐cl‐EGDMA₁₅)] and I_15c, i.e. [poly (AAc₅‐co‐HPMA₃‐cl‐EGDMA₁₅)], that showed relatively higher binding efficiency for lipase were selected for further studies. I_15c‐hydrogel retained 58.3% of its initial activity after 10th cycle of repetitive hydrolysis of p‐NPP, and I_15c was thus catalytically more stable and efficient than the other matrix. The I_15c‐hydrogel‐immobilized enzyme showed maximum activity at 65°C and pH 9.5. The hydrolytic activity of free and I_15c‐hydrogel‐immobilized enzyme increased profoundly in the presence of 5 mM chloride salts of Hg²⁺, NH₄⁺, Al³⁺, K⁺, and Fe³⁺. The immobilized lipase was preferentially active on medium chain length p‐nitrophenyl acyl ester (C:8, p‐nitrophenyl caprylate). © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4636–4644, 2006     

Production of L‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor

Production of L ‐methionine by immobilized pellets of Aspergillus oryzae in a packed bed reactor was investigated. Based on the determination of relative enzymatic activity in the immobilized pellets, the optimum pH and temperature for the resolution reaction were 8.0 and 60 °C, respectively. The effects of substrate concentration on the resolution reaction were also investigated and the kinetic constants (K_m and V_m) of immobilized pellets were found to be 7.99 mmol dm^?3 and 1.38 mmol dm^?3 h^?1, respectively. The maximum substrate concentration for the resolution reaction without inhibition was 0.2 mol dm^?3. The L ‐methionine conversion rate reached 94% and 78% when substrate concentrations were 0.2 and 0.4 mol dm^?3, respectively, at a flow rate of 7.5 cm³ h^?1 using the small‐scale packed bed reactor developed. The half‐life of the L ‐aminoacylase in immobilized pellets was 70 days in continuous operation. All the results obtained in this paper exhibit a practical potential of using immobilized pellets of Aspergillus oryzae in the production of L ‐methionine. © 2002 Society of Chemical Industry     

Immobilization of pepsin on an acrylamide/2‐hydroxyethyl methacrylate copolymer and its use in casein hydrolysis

Pepsin was immobilized through covalent bonding on a copolymer of acrylamide and 2‐hydroxyethyl methacrylate via the individual and simultaneous activation of both groups. The extent of enzyme coupling upon the activation of both the amino and hydroxyl groups of the copolymer resulted in a synergistic effect. However, the order of activation of the support was critical. The covalently bound enzyme retained more than 50% of its activity even after six cycles. The storage stability of the covalently bound enzyme was 60% after storage for 1 month, whereas the free enzyme lost all of its activity within 10 days of storage at 35°C. The Michaelis constant (K_m) and maximum reaction velocity (V_max) were 1.1 × 10^?6 and 0.87 for the free enzyme and 1.2 × 10^?6 and 0.98 for the covalently bound enzyme when the enzyme concentration was kept constant and the substrate concentration was varied. Similarly, K_m and V_max were 6.73 × 10^?11 and 0.47 for the free enzyme and 7.59 × 10^?11 and 0.545 for the covalently bound enzyme when the substrate concentration was kept constant and the enzyme concentration was varied; this indicated no conformational change during coupling, but the reaction was concentration‐dependent. The hydrolysis of casein was carried out with a fixed‐bed reactor (17 cm × 1 cm). Maximum hydrolysis (90%) was obtained at a 2 cm³/min flow rate at 35°C with a 1 mM casein solution. © 2005 Wiley Periodicals, Inc. J Appl Polym Sci 96: 1544–1549, 2005     

Protease immobilization onto porous chitosan beads

Water-insoluble proteases were prepared by immobilizing papain, ficin, and bromelain onto the surface of porous chitosan beads with any length of spacer by covalently fixation. The activity of the immobilized proteases was found to be still high toward small ester substrate, N-benzyl-L -arginine ethyl ester (BAEE), but rather low toward casein, a high-molecular-weight substrate. The relative activity of the immobilized proteases with spacer gave an almost constant value for the substrate hydrolysis within the surface concentration region studied. The values of the Michaelis constant K_m and the maximum reaction velocity V_m for free and immobilized proteases on the porous chitosan beads are estimated. The apparent K_m values were larger for immobilized proteases than for the free ones, while V_m values were smaller for the immobilized proteases. The pH, thermal, and storage stability of the immobilized proteases were higher than those of the free ones. The initial enzymatic activity of the immobilized protease maintained almost unchanged without any elimination and inactivation of proteases, when the batch enzyme reaction was performed repeatedly, indicating the excellent durability.     

In situ entrapment of α‐chymotrypsin in the network of acrylamide and 2‐hydroxyethyl methacrylate copolymers

A mild and reproducible method has been developed for the entrapment of α‐chymotrypsin into a crosslinked copolymer. A porous copolymer was synthesized at 293 K by solution copolymerization of acrylamide and 2‐hydroxyethyl methacrylate. α‐Chymotrypsin was entrapped during copolymerization at different polymerization stages. The effect of crosslinking on enzyme loading and retention of its activity was examined. Copolymer with 2% crosslinking could entrap >90% of the enzyme. The activity of free and immobilized α‐chymotrypsin was determined by using N‐benzoyl‐L ‐tyrosine ethyl ester and casein as low and high molecular weight substrates respectively. Storage as well as thermal stability of the immobilized enzyme was superior to that of the free one. Effect of calcium and heavy metal ions was studied on immobilized enzyme activity. The immobilized enzyme showed little variation in activity with pH and retained 50% activity after nine cycles. The Michaelis constant K_m of the free and immobilized enzyme was estimated to be 2.7 and 4.2 × 10⁻³ mM, respectively, indicating no conformational changes during entrapment. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 77: 2996–3002, 2000     

Influence of TiO2‐Layer Thickness of Spray‐Coated Glass Beads on Their Photocatalytic Performance

TiO₂ is a suitable catalyst for potential photocatalytic processes, e.g., in wastewater treatment. For a technical realization of such processes, the application of immobilized TiO₂ in a continuous process would be desirable. However, since UV radiation has a limited penetration depth into a packed bed of pure TiO₂, supporting it on UV‐transparent glass beads offers the possibility to implement continuous photocatalytic processes in a fixed‐bed reactor. Considering this fact, glass beads were coated with TiO₂ powder in a fluidized‐bed reactor. The coated glass beads with varying TiO₂ layer thickness were tested in the photocatalytic degradation of methylene blue, and the influence of an addition of methyl cellulose during the coating process on the photocatalytic performance was investigated.     

Preparation of chitosan‐coated magnetite nanoparticles and application for immobilization of laccase

In this study, immobilization of laccase (L) enzyme on magnetite (Fe₃O₄) nanoparticles was achieved, so that the immobilized enzyme could be used repeatedly. For this purpose, Fe₃O₄ nanoparticles were coated and functionalized with chitosan (CS) and laccase from Trametes versicolor was immobilized onto chitosan‐coated magnetic nanoparticles (Fe₃O₄‐CS) by adsorption or covalent binding after activating the hydroxyl groups of chitosan with carbodiimide (EDAC) or cyanuric chloride (CC). For chitosan‐coated magnetic nanoparticles, the thickness of CS layer was estimated as 1.0–4.8 nm by TEM, isoelectric point was detected as 6.86 by zeta (ζ)‐potential measurements, and the saturation magnetization was determined as 25.2 emu g^?1 by VSM, indicating that these nanoparticles were almost superparamagnetic. For free laccase and immobilized laccase systems, the optimum pH, temperature, and kinetic parameters were investigated; and the change of the activity against repeated use of the immobilized systems were examined. The results indicated that all immobilized systems retained more than 71% of their initial activity at the end of 30 batch uses. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Use of chitosan for removal of bisphenol A and bisphenol derivatives through tyrosinase‐catalyzed quinone oxidation

In this study, the availability of chitosan was systematically investigated for removal of bisphenol A (BPA, 2,2‐bis(hydroxyphenyl)propane) through the tyrosinase‐catalyzed quinone oxidation and subsequent quinone adsorption on chitosan beads. In particular, the process parameters, such as the hydrogen peroxide (H₂O₂)‐to‐BPA ratio, pH value, temperature, and tyrosinase dose, were discussed in detail for the enzymatic quinone oxidation. Tyrosinase‐catalyzed quinone oxidation of BPA was effectively enhanced by adding H₂O₂ and the optimum conditions for BPA at 0.3 mM were determined to be pH 7.0 and 40°C in the presence of H₂O₂ at 0.3 mM ([H₂O₂]/[BPA] = 1.0). Removal of BPA from aqueous solutions was accomplished by adsorption of enzymatically generated quinone derivatives on chitosan beads. The use of chitosan in the form of beads was found to be more effective because heterogeneous removal of BPA with chitosan beads was much faster than homogeneous removal of BPA with chitosan solutions, and the removal efficiency was enhanced by increasing the amount of chitosan beads dispersed in the BPA solutions and BPA was completely removed by quinone adsorption in the presence of chitosan beads more than 0.10 cm³/cm³. In addition, a variety of bisphenol derivatives were completely or effectively removed by the procedure constructed in this study, although the enzyme dose or the amount of chitosan beads was further increased as necessary for some of the bisphenol derivatives used. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2010     

Vinyl triazole carrying metal‐chelated beads for the reversible immobilization of glucoamylase

Poly(ethylene glycol dimethacrylate–1‐vinyl‐1,2,4‐triazole) [poly(EGDMA–VTAZ)] beads with an average diameter of 100–200 μm were obtained by the copolymerization of ethylene glycol dimethacrylate (EGDMA) with 1‐vinyl‐1,2,4‐triazole (VTAZ). The copolymer hydrogel bead composition was determined by elemental analysis and was found to contain 5 EGDMA monomer units for each VTAZ monomer unit. The poly(EGDMA–VTAZ) beads were characterized by swelling studies and scanning electron microscopy (SEM). The specific surface area of the poly(EGDMA–VTAZ) beads was found 65.8 m²/g. Cu²⁺ ions were chelated on the poly(EGDMA–VTAZ) beads. The Cu²⁺ loading was 82.6 μmol/g of support. Cu²⁺‐chelated poly(EGDMA–VTAZ) beads with a swelling ratio of 84% were used in the immobilization of Aspergillus niger glucoamylase in a batch system. The maximum glucoamylase adsorption capacity of the poly(EGDMA–VTAZ)–Cu²⁺ beads was 104 mg/g at pH 6.5. The adsorption isotherm of the poly(EGDMA–VTAZ)–Cu²⁺ beads fitted well with the Langmuir model. Adsorption kinetics data were tested with pseudo‐first‐ and second‐order models. The kinetic studies showed that the adsorption followed a pseudo‐second‐order reaction model. The Michaelis constant value for the immobilized glucoamylase (1.15 mg/mL) was higher than that for free glucoamylase (1.00 mg/mL). The maximum initial rate of the reaction values were 42.9 U/mg for the free enzyme and 33.3 U/mg for the immobilized enzyme. The optimum temperature for the immobilized preparation of poly(EGDMA–VTAZ)–Cu²⁺–glucoamylase was 65°C; this was 5°C higher than that of the free enzyme at 60°C. The glucoamylase adsorption capacity and adsorbed enzyme activity slightly decreased after 10 batch successive reactions; this demonstrated the usefulness of the enzyme‐loaded beads in biocatalytic applications. The storage stability was found to increase with immobilization. © 2010 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Application of tris(hydroxymethyl)phosphine as a coupling agent for β‐galactosidase immobilized on chitosan to produce galactooligosaccharides

β‐Galactosidase was immobilized on chitosan using tris(hydroxymethyl)phosphine (THP) as a coupling agent to produce galactooligosaccharides (GOS) from lactose. Both the THP‐immobilized and the free enzymes were maximally achieved at pH 5.0 and the optimal temperature was 55 °C. The residual activities for the THP‐immobilized enzyme and the free enzyme were 75 and 25%, respectively, after being incubated in 0.1 mol dm^?3 sodium acetate buffer (pH 5.0) at 55 °C for 13 days. The formation of GOS was catalyzed by free and THP‐immobilized β‐galactosidase from lactose. The yield of GOS produced by the free enzyme from the lactose solution (36%, w/v) at 55 °C was 43% on a dry weight basis, which was similar to the 41% GOS yield produced by the THP‐immobilized enzyme system. Copyright © 2005 Society of Chemical Industry