Immobilization of microbial cells on cellulose–polymer surfaces by radiation polymerization

Streptomyces phaeochromogenes cells were immobilized on cellulose–polymer surfaces by radiation polymerization using hydrophilic monomers and paper. The enzyme activity of immobilized cell sheets was higher than that of immobilized cell composites obtained by the usual radiation polymerization technique. The enzyme activity of the sheets was affected by monomer concentration, the thickness of paper, and the degree of polymerization of paper. The copolymerization of hydroxyethyl methacrylate and methoxytetraethyleneglycol methacrylate in the sheets led to a further increase of the enzyme activity due to the increase of the hydrophilicity of the polymer matrix. The Michaelis constant of the sheets from low monomer concentration was close to that of intact cells.     

Selective Immobilization of His-Tagged Phosphomannose Isomerase on Ni Chelated Nanoparticles with Good Reusability and Activity

Self-stable precipitation polymerization was used to prepare an enzyme-immobilized microsphere composite. Phosphomannose isomerase (PMI) with His-tag was successfully immobilized on Ni²⁺ charged pyridine-derived particles. The maximum amount of PMI immobilized on such particles was ∼184 mg/g. Compared with free enzyme, the activity of the immobilized enzymes was significantly improved. In addition, the immobilized enzymes showed a much better thermostability than free enzymes. At the same time, the immobilized enzymes can be reused for multiple reaction cycles. We observed that the enzyme activity did not decrease significantly after six cycles. We conclude that the pyridine-derived particles can be used to selectively immobilize His-tagged enzymes, which can couple the enzyme purification and catalysis steps and improve the efficiency of enzyme-catalyzed industrial processes.     

Preparation and characterization of polymer-coated mesoporous silica nanoparticles and their application in Subtilisin immobilization

The preparation and characterization of polymer-coated mesoporous silica nanoparticles (MSNs) and their application in Subtilisin (Alcalase®) immobilization were investigated. For the synthesis of polymer-coated MSNs, acrylic acid (AA) and chitosan (CS) mixture were blended as poly(acrylic acid) (PAA) and CS polymer layer onto MSNs via in-situ polymerization technique. Then, both uncoated MSNs and polymer-coated mesoporous silica nanoparticles (CS-PAA/MSNs) were characterized by taking into account properties such as morphologic pattern, size distribution, surface charge of the particles as well as thermogravimetric stability with SEM, TEM, Zetasizer and TGA analyses. Subtilisin was immobilized onto polymer-coated mesoporous silica nanoparticles via adsorption technique. For optimizing the enzyme immobilization process, the percent enzyme loading depending on the matrix amount, immobilization time and pH were investigated. Then, the activity values of immobilized enzyme and free enzyme were compared at various pH and temperature values. The maximum enzyme activity was achieved at pH 9.0 for both immobilized and free enzyme. Immobilized enzyme showed more stability at higher temperatures compared with free enzyme. Furthermore, the operational and storage stability of immobilized enzyme were determined. The activity of immobilized enzyme was reduced from 100% to 45.83% after five repeated uses. The storage stability of immobilized enzyme was found to be higher than that of free enzyme. The activity of immobilized enzyme was reduced from 100% to 60% after 28 days of storage time. We concluded that the polymer-coated MSNs were a suitable matrix for Subtilisin immobilization compared to uncoated MSNs.     

Immobilization of catalase in poly(isopropylacrylamide-co-hydroxyethylmethacrylate) thermally reversible hydrogels

Catalase was entrapped in thermally reversible poly(isopropylacrylamide-co-hydroxyethylmethacrylate) (pNIPAM/HEMA) copolymer hydrogels. The thermoresponsive hydrogels, in cylindrical geometry, were prepared in an aqueous buffer by redox polymerization. It was observed that upon entrapment, the activity retention of catalase was decreased between 47 and 14%, and that increasing the catalase loading of hydrogel adversely affected the activity. The kinetic behaviour of the entrapped enzyme was investigated in a batch reactor. The apparent kinetic constant of the entrapped enzyme was determined by the application of Michaelis–Menten model and indicated that the overall reaction rate was controlled by the substrate diffusion rate through the hydrogel matrix. Due to the thermoresponsive character of the hydrogel matrix, the maximum activity was achieved at 25 °C with the immobilized enzyme. The K_m value for immobilized catalase (28.6 mM) was higher than that of free enzyme (16.5 mM). Optimum pH was the same for both free and immobilized enzyme. Operational, thermal and storage stabilities of the enzyme were found to increase with immobilization. © 1999 Society of Chemical Industry     

A polyacrylic acid as a carrier for immobilization of penicillin acylase

A copolymer of acrylic acid with divinylbenzene was synthesized by suspension polymerization. This polymer is an effective carrier. Penicillin acylase was immobilized on this carrier to convert benzylpenicillin to 6‐aminopenicillanic acid, which may be employed in the manufacture of semisynthetic penicillins. Factors that affect the activity of immobilized penicillin acrylase, such as temperature, pH, and amount of native enzyme, were studied. Under suitable conditions, the activity and activity recovery of the immobilized enzyme were 3100 U/g (dry carrier, p‐dimethylaminobenzaldehyde method) and 59.7%, respectively. The immobilized penicillin acylase shows a remarkable increase in stability. At 40°C and pH 8.0 the value of the kinetic Michaelis–Menten constant (K_m) of the immobilized enzyme is 2.8 × 10^?3 mol/L, and the value of activation energy of enzyme catalysis is 71.5 kJ/mol. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 2067–2069, 2002     

Stability and activity of chymotrypsin immobilized on magnetic nanogels covered with carboxyl groups

α‐chymotrypsin as model enzyme was anchored to the carboxyl‐functionalized magnetic nanogels prepared by in situ photochemical polymerization. Furthermore, to explore the optimum immobilization, the effects of immobilization time, pH of the reaction mixture, and proportion of enzyme to the magnetic nanogels were studied. The immobilized enzyme was stable in the presence of enzyme denaturation surfactants, and maintained their activity against protein‐digesting enzyme. The immobilized enzyme exhibited hydrolysis activity against the substrate of casein, and cleaved the substrate into small fragments. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2009     

Bio- and bioelectro-catalytic properties of polyaniline/poly(acrylic acid) composite films bearing covalently-immobilized acid phosphatase

Conducting films composed of polyaniline (PANI) and poly(acrylic acid) (PAA) were prepared by electrochemical polymerization of aniline in the presence of various concentrations of PAA. The content of PAA moiety on the surface of the composite films (PANI/PAA films) was estimated by determination of carboxyl groups and found to be controlled by the concentration of PAA in polymerization solution. Acid phosphatase (ACP) was immobilized covalently on the PANI/PAA films by the condensation reaction with the carboxyl groups on the films. It was confirmed that the enzyme activity of the ACP-immobilized PANI/PAA film increased with increasing content of PAA moiety on the surface of the film, accompanying an increase in the amount of the immobilized ACP. The activity of the covalently immobilized ACP was significantly higher than that of the ACP adsorbed on the PANI/PAA film. By use of the ACP-immobilized PANI/PAA film as an enzyme electrode, bioelectrocatalytic oxidation of L-ascorbic acid 2-phosphate (ASA2P) was examined. The enzyme electrode gave the current due to the oxidation of ASA2P in proportion to the content of PAA moiety on the surface of the PANI/PAA film used, which was relevant to the activity of the covalently immobilized ACP.     

触角状环氧链结构对固定化脂肪酶(YCJ01)酶活的影响

为了改善酶的刚性固定化对酶蛋白构象的负面影响,以原子转移自由基聚合法（ATRP）合成了亲水性、柔性、触角状环氧化酶固定化载体PS-acyl-P（AM-co-GMA）,通过改变单体总量与引发剂量比例得到不同链长触角状环氧化酶固定化载体,并将其用于耐有机溶剂脂肪酶（YCJ01）的共价柔性固定化,重点考察了链长对固定化酶酶活的影响。结果表明,链长（增重率不超过3200%）越长,酶活越高。     

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     

Porous substances immobilizing enzymes with polymer matrix

The preparation of porous substances immobilizing enzymes with polymer matrix having various properties have been studied by radiation polymerization method. The enzyme (cellulase) was immobilized on the surface parts of porous substances such as activated carbon, molecular sieve, silica gel, and coating with hydrophilic and hydrophobic monomers. As substrate of enzyme reaction, lignocellulosic wastes such as chaff pretreated by radiation irradiation (100 Mrad) were used. The enzyme activity of the immobilized enzyme substances was markedly affected by hydrophilicity of monomer and the copolymerization of methoxypoly(ethyle glycol) methacrylate (75%) and poly(ethylene glycol) dimethacrylate (25%) monomer gave the most highest enzyme activity. The immobilized enzyme substances was able to hydrolyze chaff pretreated by radiation irradiation.     

Immobilization of β-galactosidase onto magnetic poly(GMA–MMA) beads for hydrolysis of lactose in bed reactor

In the present study, novel magnetic beads were prepared from glycidylmethacrylate and methylmethacrylate via suspension polymerization in the presence of a cross-linker (i.e. ethylenedimethylmethacrylate). The magnetic poly(GMA–MMA) beads were characterized with scanning electron microscope, FT-IR and ESR spectrophotometers. The reactive character of the epoxy groups allowed the attachment of the amino groups. The aminated magnetic beads were used for the covalent immobilization of β-galactosidase via glutaric dialdehyde activation. The maximum amount of immobilized β-galactosidase on the magnetic poly(GMA–MMA) beads was 9.87 mg/g support. The values of Michaelis constants K_m for immobilized β-galactosidase was significant larger, indicating decreased affinity by the enzyme for its substrate, whereas V_max values were smaller for the immobilized β-galactosidase. However, the β-galactosidase immobilized on the magnetic poly(GMA–MMA) beads resulted in an increase in enzyme stability with time. Optimum operational temperature for immobilized enzyme was 5 °C higher than that of the free enzyme and was significantly broader. Finally, a bed reactor with β-galactosidase immobilized was used for hydrolysis of lactose. The enzyme reactor operated continuously at 35 °C for 60 h and the immobilized enzyme lost about 12% of its initial activity after this period.     

Cinnamic ester of D‐sorbitol for immobilization of mushroom tyrosinase

Mushroom tyrosinase was immobilized by adsorption onto the totally cinnamoylated derivative of D ‐sorbitol. The polymerization and cross‐linking of the derivative initially obtained was achieved by irradiation in the ultraviolet region, where this prepolymer shows maximum sensitivity. Immobilization of tyrosinase on this support involves a process of physical adsorption and intense hydrophobic interactions between the cinnamoyl groups of the support and related groups of the enzyme. The pH value, enzyme concentration and immobilization time were all important parameters affecting immobilization efficiency; also, enzyme immobilization efficiency correlated well with the tyrosinase isoelectric point. The immobilized enzyme showed an optimum measuring pH of 3.5 and greater activity at acid and neutral pH values than the soluble enzyme. The optimal reaction temperature was 35 °C and the temperature profile was broader than that of the free enzyme or of the enzyme immobilized on other supports. The apparent Michaelis constant of mushroom tyrosinase immobilized on the SOTCN derivative acting on 4‐tert‐butylcatechol (TBC) was 0.40 ± 0.02 mmol dm^?3, which was lower than for the soluble enzyme, suggesting that the affinity of this enzyme for this substrate was greater when immobilized than when in solution. Immobilization stabilized the enzyme and made it less susceptible to activity loss during storage at pH values in the range 4–5.5, and the suicide inactivation of the immobilized tyrosinase was null or negligible in a reaction medium with 4‐tert‐butylcatechol at a concentration of 0.4 mmol dm^?3. The results show that cinnamic carbohydrate esters of D ‐sorbitol are an appropriate support for tyrosinase immobilization and could be of use for several tyrosinase applications. Copyright © 2005 Society of Chemical Industry     

Effect of the polymer matrix on the immobilization of lipase by radiation polymerization

Summary Effect of the hydrophobicity of the polymer matrix on the immobilization of lipase by radiation polymerization was studied. The enzymatic activity of the immobilized enzyme composites was affected by hydrophobicity, and optimum hydrophobicity existed for the enzyme reaction at the surface of the immobilized enzyme composites. Furthermore, the enzymatic activity was affected by monomer concentration. In the immobilization using polyethyleneglycole diacrylate monomers, the enzymatic activity increased with increasing number of ethyleneglycole unit in the monomer molecule.     

Preparation of clay–poly(glycidyl methacrylate) composite support for immobilization of cellulase

A composite material was synthesized by grafting of glycidyl methacrylate onto clay using surface initiation atom transfer radical polymerization (SI-ATRP) technique. Epoxy group of the grafted p(GMA) chains was reacted with hexamethylenediamine (HMDA). The composite material was characterized using scanning electron microscopy (SEM) and FTIR. Cellulase from Trichoderma reesei was immobilized on the aminated composite particles via adsorption and covalent binding methods. The amounts of adsorbed enzyme on the aminated composite particles were 43.4 mg/g. The recovered activities of the adsorbed and covalently immobilized cellulase were found to be 87.7% and 73.2% for the substrate, carboxymethyl cellulose (CMC, 1.0 g/L). The pH stabilities and thermo-stabilities, repeated use and storage stabilities of both immobilized cellulase preparations were evaluated. The immobilized cellulase preparations have better stabilities and higher retained activities with respect to pH, temperature and storage than those of the free enzyme. Operational stability of the covalently immobilized cellulase was tested in a continuous flow system, and the activity loss was about 4% at the end of 48 h operation period.     

Immobilization of glucoamylase on the plain and on the spacer arm‐attached poly(HEMA‐EGDMA) microspheres

Immobilization glucoamylase onto plain and a six‐carbon spacer arm (i.e., hexamethylene diamine, HMDA) attached poly(2‐hydroxyethylmethacrylate‐ethyleneglycol dimethacrylate) [poly(HEMA‐EGDMA] microspheres was studied. The microspheres were prepared by suspension polymerization and the spacer arm was attached covalently by the reaction of carbonyl groups of poly(HEMA‐EGDMA). Glucoamylase was then covalently immobilized either on the plain of microspheres via CNBr activation or on the spacer arm‐attached microspheres via CNBr activation and/or using carbodiimide (CDI) as a coupling agent. Incorporation of the spacer arm resulted an increase in the apparent activity of the immobilized enzyme with respect to enzyme immobilized on the plain of the microspheres. The activity yield of the immobilized glucoamylase on the spacer arm‐attached poly(HEMA‐EGDMA) microspheres was 63% for CDI coupling and 82% for CNBr coupling. This was 44% for the enzyme, which was immobilized on the plain of the unmodified poly(HEMA‐EGDMA) microspheres via CNBr coupling. The K_m values for the immobilized glucoamylase preparations (on the spacer arm‐attached microspheres) via CDI coupling 0.9% dextrin (w/v) and CNBr coupling 0.6% dextrin (w/v) were higher than that of the free enzyme 0.2% dextrin (w/v).The temperature profiles were broader for both immobilized preparations than that of the free enzyme. The operational inactivation rate constants (k_iop) of immobilized enzymes were found to be 1.42 × 10^?5 min^?1 for CNBr coupled and 3.23 × 10^?5 min^?1 for CDI coupled glucoamylase. © 2001 John Wiley & Sons, Inc. J Appl Polym Sci 81: 2702–2710, 2001     

Immobilization of mortierella vinacea cells by radiation polymerization

Immobilization of Mortierella vinacea cells, which contain active α-galactosidase, by radiation polymerization at low temperatures was studied. The durability of the enzymatic activity of the immobilized cells was examined by repeating the batch enzyme reaction. The enzymatic activities of the immobilized cells obtained with hydrophilic monomers was affected by the concentrations of the cells and monomer in which optimum conditions were observed. The enzymatic activity of the immobilized cells obtained with hydrophilic monomer was compared to that of hydrophobic monomers. Michaelis constants of the immobilized cells varied with monomer concentration. The effect of addition of porous solid substances on the immobilization of the cells was studied.     

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     

Synthesis of magnetic polymer microspheres and application for immobilization of proteinase of balillus sublitis

Core–shell composite magnetic polymer microspheres, containing a magnetic core and a polymer shell, were synthesized by dispersion copolymerization of styrene (St) and 2-hydroxyethyl methacrylate (HEMA) in the presence of magnetic oxide (Fe₃O₄) powder. The Fe₃O₄ powder was ultrasonically disperesed in poly(ethylene glycol) (PEG) and the affinity between the obtained superfine powder and the monomer and initator was improved. It shows that the dispersion medium and stabilizer system have a great effect on the diameter and dispersion parameter of microspheres. In the condition of controlling polymerization, the magnetic polymer microspheres containing surface ? OH groups, having 50–500 μm diameter and with better magnetic induction, were synthesized. The proteinase of Balillus sublitis was immobilized on magnetic polymer microspheres with an average diameter of 50–60 μm by covalent coupling. The magnetic immobilized proteinase shows an enzyme activity of 1000 U/g, the enzyme yields are usually 20–30 mg/g of carriers, and the activity retention is about 40%. The stability of the immobilized enzyme was obviously improved. © 1995 John Wiley & Sons, Inc.     

Fabrication of bioanode by using electrically conducting polythiophene via entrapment technique

A glassy carbon electrode (GCE) was tailored with conducting polymer polythiophene and further immobilized by an enzyme glucose oxidase (GOx). A thin film of polymer was developed by electrochemical polymerization of thiophene monomer. During electrochemical polymerization of the monomer the enzyme GOx and the redox active mediator ferritin (Frt) were entrapped within this polymer matrix. In this novel approach, the entrapment of enzyme and mediators within a polymer matrix occurs without chemical reaction that could affect their activity. The entrapment of enzyme and mediator within the conducting polymer matrices increases the surface area of the electrode. The tailored GCE/Ptp/Frt/GOx electrode showed a high catalytic activity. The increased surface area causes a high rate of electron transfer between the electrode and Frt engaged as an electron transfer mediator. The electrochemical properties of the electrode were determined by cyclic voltammetry (CV) and linear sweep voltammetry (LSV). The fabricated bioanode showed a current density of 3.9mA cm^?2 at 1.0 V vs. Ag/AgCl in a 45 mM glucose solution and suggests proficient chances in biofuel cells (BFCs) applications.     

Zwitterionic poly(carboxybetaine) microgels for enzyme (chymotrypsin) covalent immobilization with extended stability and activity

There is emerging evidence that biocompatible zwitterionic materials can prevent nonspecific interactions within protein systems and increase protein stability. Here, a zwitterionic microgel was synthesized from poly (carboxybetaine methyl methacrylate) (pCB) using an inverse emulsion, free radical polymerization reaction technique. The microgel was loaded with a model enzyme, α-chymotrypsin (ChT), using a post-fabrication loading technique. A reaction scheme was developed and studied for covalent immobilization of ChT within the microgel. Confocal laser microscopy studies showed that immobilized ChT (i-ChT) was distributed within the hydrogel. The enzyme-immobilized microgels showed excellent reusability (72% of its initial activity after 10 uses) and could undergo several freezing/drying/rehydration cycles while retaining enzymatic activity. The i-ChT activity, half-life, and conformational stability were studied at varying pH and temperatures with results compared to free ChT in buffer. ChT immobilized within pCB hydrogel showed increased enzymatic stability as observed by a 13°C increase in the temperature at which i-ChT loses activity compared to free ChT. Furthermore, enzyme half-life increased up to seven-fold for the pCB immobilized ChT, and the increased stability resulted in higher activity at elevated pH. The i-ChT was most active at pH of 8.5 and was partially active up to the pH of 10.2.