Physical and Covalent Immobilization of <Emphasis Type="Italic">Lipase</Emphasis> onto Amine Groups Bearing Thiol-Ene Photocured Coatings

In this study, amine groups containing thiol-ene photocurable coating material for lipase immobilization were prepared. Lipase (EC 3.1.1.3) from Candida rugosa was immobilized onto the photocured coatings by physical adsorption and glutaraldehyde-activated covalent bonding methods, respectively. The catalytic efficiency of the immobilized and free enzymes was determined for the hydrolysis of p-nitrophenyl palmitate and also for the synthesis of p-nitrophenyl linoleate. The storage stability and the reusability of the immobilized enzyme and the effect of temperature and pH on the catalytic activities were also investigated. The optimum pH for free lipase and physically immobilized lipase was determined as 7.0, while it was found as 7.5 for the covalent immobilization. After immobilization, the optimum temperature increased from 37 °C (free lipase) to 50–55 °C. In the end of 15 repeated cycles, covalently bounded enzyme retained 60 and 70 % of its initial activities for hydrolytic and synthetic assays, respectively. While the physically bounded enzyme retained only 56 % of its hydrolytic activity and 67 % of its synthetic activity in the same cycle period. In the case of hydrolysis V _max values slightly decreased after immobilization. For synthetic assay, the V _max value for the covalently immobilized lipase was found as same as free lipase while it decreased dramatically for the physically immobilized lipase. Physically immobilized enzyme was found to be superior over covalent bonding in terms of enzyme loading capacity and optimum temperature and exhibited comparable re-use values and storage stability. Thus, a fast, easy, and less laborious method for lipase immobilization was developed.     

Efficient Immobilization of Porcine Pancreatic α-Amylase on Amino-Functionalized Magnetite Nanoparticles: Characterization and Stability Evaluation of the Immobilized Enzyme

The potential of the modified magnetic nanoparticles for covalent immobilization of porcine pancreatic α-amylase has been investigated. The synthesis and immobilization processes were simple and fast. The co-precipitation method was used for synthesis of magnetic iron oxide (Fe₃O₄) nanoparticles (NPs) which were subsequently coated with silica through sol–gel reaction. The amino-functionalized NPs were prepared by treating silica-coated NPs with 3-aminopropyltriethoxysilane followed by covalent immobilization of α-amylase by glutaraldehyde. The optimum enzyme concentration and incubation time for immobilization reaction were 150 mg and 4 h, respectively. Upon this immobilization, the α-amylase retained more than 50 % of its initial specific activity. The optimum pH for maximal catalytic activity of the immobilized enzyme was 6.5 at 45 °C. The kinetic studies on the immobilized enzyme and its free counterpart revealed an acceptable change of K_m and V_max. The Km values were found as 4 and 2.5 mM for free and immobilized enzymes, respectively. The V_max values for the free and immobilized enzymes were calculated as 1.75 and 1.03 μmol mg^?1 min^?1, in order, when starch was used as the substrate. A quick separation of immobilized amylase from reaction mixture was achieved when a magnetically active support was applied. In comparison to the free enzyme, the immobilized enzyme was thermally stable and was reusable for 9 cycles while retaining 68 % of its initial activity.     

Immobilization of Horseradish Peroxidase on Nonwoven Polyester Fabric Coated with Chitosan

The immobilization of horseradish peroxidase (HRP) on composite membrane has been investigated. This membrane was prepared by coating nonwoven polyester fabric with chitosan glutamate in the presence of glutraldehyde as a crosslinking agent. The physico-chemical properties of soluble and immobilized HRP were evaluated. The soluble HRP lost 90% of its activity after 4 weeks of storage at 4°C, whereas the immobilized enzyme retained 85% of its original activity at the same time. A reusability study of immobilized HRP showed that the enzyme retained 54% of its activity after 10 cycles of reuse. Soluble and immobilized HRP showed the same pH optima at pH 5.5. The immobilized enzyme had significant stability at different pH values, where it had maximum stability at pH 3.0 and 6.0. The kinetic properties indicated that the immobilized enzyme had more affinity toward substrates than soluble enzyme. The soluble and immobilized enzymes had temperature optima at 30 and 40°C and were stable up to 40 and 50°C, respectively. The stability of HRP against metal ion inactivation was improved after immobilization. Immobilized HRP exhibited high resistance to proteolysis by trypsin. The immobilized HRP was more resistant to inactivation induced by urea, Triton X-100, and organic solvents compared to its soluble counterpart. The immobilized HRP showed very high yield of immobilization and markedly high stabilization against several forms of denaturants that offer potential for several applications.     

Improved Covalent Immobilization of Horseradish Peroxidase on Macroporous Glycidyl Methacrylate-Based Copolymers

A macroporous copolymer of glycidyl methacrylate and ethylene glycol dimethacrylate, poly(GMA-co-EGDMA), with various surface characteristics and mean pore size diameters ranging from 44 to 200 nm was synthesized, modified with 1,2-diaminoethane, and tested as a carrier for immobilization of horseradish peroxidase (HRP) by two covalent methods, glutaraldehyde and periodate. The highest specific activity of around 35 U g^?1 dry weight of carrier was achieved on poly(GMA-co-EGDMA) copolymers with mean pore diameters of 200 and 120 nm by the periodate method. A study of deactivation kinetics at 65 °C and in 80 % dioxane revealed that periodate immobilization also produced an appreciable stabilization of the biocatalyst, while stabilization factor depended strongly on the surface characteristics of the copolymers. HRP immobilized on copolymer with a mean pore diameter of 120 nm by periodate method showing not only the highest specific activity but also good stability was further characterized. It appeared that the immobilization resulted in the stabilization of enzyme over a broader pH range while the Michaelis constant value (K _m) of the immobilized HRP was 10.8 mM, approximately 5.6 times higher than that of the free enzyme. After 6 cycles of repeated use in a batch reactor for pyrogallol oxidation, the immobilized HRP retained 45 % of its original activity.     

Affinity Covalent Immobilization of Glucoamylase onto ρ-Benzoquinone-Activated Alginate Beads: II. Enzyme Immobilization and Characterization

A novel affinity covalent immobilization technique of glucoamylase enzyme onto ρ-benzoquinone-activated alginate beads was presented and compared with traditional entrapment one. Factors affecting the immobilization process such as enzyme concentration, alginate concentration, calcium chloride concentration, cross-linking time, and temperature were studied. No shift in the optimum temperature and pH of immobilized enzymes was observed. In addition, K _m values of free and entrapped glucoamylase were found to be almost identical, while the covalently immobilized enzyme shows the lowest affinity for substrate. In accordance, V _m value of covalently immobilized enzyme was found lowest among free and immobilized counter parts. On the other hand, the retained activity of covalently immobilized glucoamylase has been improved and was found higher than that of entrapped one. Finally, the industrial applicability of covalently immobilized glucoamylase has been investigated through monitoring both shelf and operational stability characters. The covalently immobilized enzyme kept its activity over 36 days of shelf storage and after 30 repeated use runs. Drying the catalytic beads greatly reduced its activity in the beginning but recovered its lost part during use. In general, the newly developed affinity covalent immobilization technique of glucoamylase onto ρ-benzoquinone-activated alginate carrier is simple yet effective and could be used for the immobilization of some other enzymes especially amylases.     

Covalent Immobilization of Human Placental 17β-Hydroxysteroid Dehydrogenase Type 1 onto Glutaraldehyde Activated Silica Coupled with LC-TOF/MS for Anti-Cancer Drug Screening Applications

Human 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1), a potential target in breast cancer prevention and therapy, was extracted from human placenta and immobilized on nonporous silica (～5 μm) with a covalent method for the first time. The optimum initial enzyme concentration and immobilization time during the immobilization process were 0.42 mg mL^?1 and 12 h, repectively. The binding was confirmed by scanning electron microscope (SEM) and infrared spectroscopy (FT-IR). It could improve the pH, thermal and storage stability compared to free enzyme. Moreover, the immobilized enzyme could be reused at least four times. A screening method based on it coupled with liquid chromatography–time-of-flight mass spectrometer (LC-TOF/MS) was established, and the half-maximal inhibitory concentration (IC 50) of apigenin for the immobilized enzyme was 291 nM. Subsequently, 10 natural products were evaluated leading to inhibition of the activity of 17β-HSD1 at the concentration of 25 μM, and six of them inhibit the activity over 50%.     

Direct introduction of amine groups into cellulosic paper for covalent immobilization of tyrosinase: support characterization and enzyme properties

Tyrosinase is used to eliminate phenolic compounds from wastewater. Therefore, its immobilization is important to enhance catalytic efficiency. Papery materials are of particular interest for use as support for enzyme immobilization since the porous microstructure of fiber networks in papers can provide a suitable reaction environment, especially in flow-type catalytic reactions. However, immobilization of protein onto papery structure needs chemical modifications in severe conditions. To overcome this challenge, a cellulosic paper was directly amine-functionalized in moderate conditions and used for tyrosinase immobilization. The support was pretreated with HCl (0.5 N) solution and then sequentially immersed in ethylenediamine (EDA), glutaraldehyde solution (2% v/v) and the crude enzyme. In comparison with the untreated one, the immobilized enzyme on the EDA-treated support offered a 3.7-fold increase in activity. The FTIR spectra as well as EDX analysis proved the presence of amine groups in the cellulosic paper and also covalent immobilization of tyrosinase on the modified support. When considering the effect of pH on the activity at 25 °C, a maximum relative activity of 134% at pH 6 was revealed. Similarly, evaluating the effect of temperature on the activity at pH 7 displayed a maximum relative activity of 152% at 35 °C. The immobilized enzyme was suitable for use for more than four cycles to degrade a phenolic compound at severe pH and temperature conditions. Additionally, the immobilized enzyme was active after treatment of the surface at different pHs and temperatures for 105 min. The chemically modified cellulosic paper can be used as a support for enzyme immobilization.     

Immobilization and Kinetics of Catalase on Calcium Carbonate Nanoparticles Attached Epoxy Support

A novel hybrid epoxy/nano CaCO₃ composite matrix for catalase immobilization was prepared by polymerizing epoxy resin in the presence of CaCO₃ nanoparticles. The hybrid support was characterized using scanning electron microscopy and Fourier transform infrared spectroscopy. Catalase was successfully immobilized onto epoxy/nano CaCO₃ support with a conjugation yield of 0.67?±?0.01 mg/cm² and 92.63?±?0.80 % retention of activity. Optimum pH and optimum temperature of free and immobilized catalases were found to be 7.0 and 35 °C. The value of K _m for H₂O₂ was higher for immobilized enzyme (31.42 mM) than native enzyme (27.73 mM). A decrease in V _max value from 1,500 to 421.10 μmol (min mg protein)^?1 was observed after immobilization. Thermal and storage stabilities of catalase improved immensely after immobilization. Immobilized enzyme retained three times than the activity of free enzyme when kept at 75 °C for 1 h and the half-life of enzyme increased five times when stored in phosphate buffer (0.01 M, pH 7.0) at 5 °C. The enzyme could be reused 30 times without any significant loss of its initial activity. Desorption of catalase from the hybrid support was minimum at pH 7.0.     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.     

Highly Efficient Method Towards In Situ Immobilization of Invertase Using Cryogelation

A novel method was developed for the immobilization of Saccharomyces cerevisiae invertase within supermacroporous polyacrylamide cryogel and was used to produce invert sugar. First, the cross-linking of invertase with soluble polyglutaraldehyde (PGA) was carried out prior to immobilization in order to increase the bulkiness of invertase and thus preventing the leakage of the cross-linked enzyme after immobilization by entrapment. And then, in situ immobilization of PGA cross-linked invertase within cryogel synthesis was achieved by free radical polymerization in semi-frozen state. The method resulted in 100 % immobilization and 74 % activity yields. The immobilized invertase retained all the initial activity for 30 days and 30 batch reactions. Immobilization had no effect on optimum temperature and it was 60 °C for both free and immobilized enzyme. However, optimum pH was affected upon immobilization. Optimum pH values for free and immobilized enzyme were 4.5 and 5.0, respectively. The immobilized enzyme was more stable than the free enzyme at high pH and temperatures. The kinetic parameters for free and immobilized invertase were also determined. The newly developed method is simple yet effective and could be used for the immobilization of some other enzymes and microorganisms.     

超顺磁性氧化石墨烯复合材料固定辣根过氧化物酶催化去除氯酚

采用超声辅助共沉淀法成功地将磁性Fe3O4纳米颗粒沉积在氧化石墨烯表面,利用透射电镜、磁滞回归曲线和X射线光电子能谱对材料进行了表征。将该材料作为载体固定辣根过氧化物酶,考察了固定化酶催化2-氯酚、4-氯酚和2,4-二氯酚降解反应,研究了溶液pH值、反应温度、反应时间、H2O2和氯酚浓度以及固定化酶用量对酚类物质去除率的影响。基于取代基数量和位置不同,去除率排序为2-氯酚<4-氯酚<2,4-二氯酚。另外,采用GC-MS研究了降解过程中的氧化产物。固定化酶的生化性质研究表明,固定化酶比游离酶具有更好的储存稳定性、pH稳定性和热稳定性。经过4次循环利用,固定化酶仍保留66%的活性,说明磁性纳米材料可以分离回收并重复利用,在污水处理领域具有应用前景。     

Characterization and performance of immobilized amylase and cellulase

The performance of cellulase and amylase immobilized on siliceous supports was investigated. Enzyme uptake onto the support depended on the enzyme source and immobilization conditions. For amylase, the uptake ranged between 20 and 60%, and for cellulase, 7–10%. Immobilized amylase performance was assessed by batch kinetics in 100–300 g/L of corn flour at 65°C. Depending on the substrate and enzyme loading, between 40 and 60% starch conversion was obtained. Immobilized amylase was more stable than soluble amylase. Enzyme samples were preincubated in a water bath at various temperatures, then tested for activity. At 105°C, soluble amylase lost ∼55% of its activity, compared with ∼30% loss for immobilized amylase. The performance of immobilized cellulase was evaluated from batch kinetics in 10 g/L of substrate (shredded wastepaper) at 55°C. Significant hydrolysis of the wastepaper was also observed, indicating that immobilization does not preclude access to and hydrolysis of insoluble cellulose.     

Production of Xylooligosaccharides by Immobilized His-tagged Recombinant Xylanase from Penicillium occitanis on Nickel-Chelate Eupergit C

Penicillium occitanis xylanase 2 expressed with a His-tag in Pichia pastoris, termed PoXyn2, was immobilized on nickel-chelate Eupergit C by covalent coupling reaction with a high immobilization yield up to 93.49 %. Characterization of the immobilized PoXyn2 was further evaluated. The optimum pH was not affected by immobilization, but the immobilized PoXyn2 exhibited more acidic and large optimum pH range (pH 2.0–4.0) than that of the free PoXyn2 (pH 3.0). The free PoXyn2 had an optimum temperature of 50 °C, whereas that of the immobilized enzyme was shifted to 65 °C. Immobilization increased both pH stability and thermostability when compared with the free enzyme. Time courses of the xylooligosaccharides (XOS) produced from corncob xylan indicated that the immobilized enzyme tends to use shorter xylan chains and to produce more xylobiose and xylotriose initially. At the end of 24-h reaction, XOS mixture contained a total of 21.3 and 34.2 % (w/w) of xylobiose and xylotriose with immobilized xylanase and free xylanase, respectively. The resulting XOS could be used as a special nutrient for lactic bacteria.     

Immobilization of Fungal β-Glucosidase on Silica Gel and Kaolin Carriers

β-Glucosidase is a key enzyme in the hydrolysis of cellulose for producing feedstock glucose for various industrial processes. Reuse of enzyme through immobilization can significantly improve the economic characteristics of the process. Immobilization of the fungal β-glucosidase by covalent binding and physical adsorption on silica gel and kaolin was conducted for consequent application of these procedures in large-scale industrial processes. Different immobilization parameters (incubation time, ionic strength, pH, enzyme/support ratio, glutaric aldehyde concentration, etc.) were evaluated for their effect on the thermal stability of the immobilized enzyme. It was shown that the immobilized enzyme activity is stable at 50 °C over 8 days. It has also been shown that in the case of immobilization on kaolin, approximately 95% of the initial enzyme was immobilized onto support, and loss of activity was not observed. However, covalent binding of the enzyme to silica gel brings significant loss of enzyme activity, and only 35% of activity was preserved. In the case of physical adsorption on kaolin, gradual desorption of enzyme takes place. To prevent this process, we have carried out chemical modification of the protein. As a result, after repeated washings, enzyme desorption from kaolin has been reduced from 75 to 20–25% loss.     

Photochemical activation of a polycarbonate surface for covalent immobilization of a protein ligand

Polycarbonate—a thermostable polymer is activated by a simple and rapid method using a photolinker, 1-fluoro-2-nitro-4-azidobenzene (FNAB) for covalent immobilization of a biomolecule. Horseradish peroxidase (HRP) is used as a model enzyme to check the efficacy of the activated surface. HRP is immobilized on the activated polycarbonate surface without addition of any reagent or catalyst and is found to give 2-2.5-fold increase in absorbance with the substrate as compared to the directly adsorbed enzyme. Photochemical attachment of FNAB to the PC surface is confirmed by X-ray photoelectron spectroscopy (XPS), which shows the presence of nitrogen and fluorine in the ratio of 2:1 in the activated polycarbonate. Disappearance of fluorine peak in the XP spectra of PC bound enzyme further confirms the covalent binding of HRP, through displacement of fluorine moiety of the activated PC by the amino group of the protein. Optimized concentration of the photolinker is found as 6 μmol of FNAB per well and time of photo irradiation is 8 min for activation of a PCR polycarbonate plate. PC bound HRP has shown enhanced thermal and storage stability. Kinetic studies of the immobilized HRP shows improved catalytic activity. The potential application of activated polycarbonate surface includes immobilization of biomolecules for biosensors, immunoassays, and protein and DNA micro-arrays. Due to the stability of the polycarbonate at high temperature, the activated polycarbonate has an advantage for immobilization of thermostable biomolecule such as thermostable enzyme for reaction at elevated temperature.     

Immobilization of peroxidase on SiO2 surfaces with the help of a dendronized polymer and the avidin-biotin system

Horseradish peroxidase (HRP) is immobilized in three easy steps on SiO(2) surfaces with the help of a polycationic second generation dendronized polymer (denpol) and the biotin-avidin system. This stepwise immobilization process is monitored and quantitatively analyzed with the transmission interferometric adsorption sensor. Partially biotinylated denpol is first adsorbed onto SiO(2) , followed by addition of avidin and then of biotinylated HRP. Denpols in their molecular structure combine properties of polymers as well as dendrimers which are found to be of clear advantage for this type of non-covalent enzyme immobilization. With respect to the reproducibility of the adsorption process and with respect to the stability of the adsorbed polymer layer, the denpol is superior to α-poly-D-lysine which is used as a reference polymer. Furthermore, HRP immobilized with the denpol on commercial glass slides remains considerably more active upon storage as compared to HRP immobilized with the help of α-poly-D-lysine with a similar number of repeating units. The ease of the denpol-mediated HRP immobilization and the high stability of the immobilized enzyme are promising for bioanalytical applications.     

Synthesis and characterization of carboxymethyl cellulose-silver nanoparticle (AgNp)-silica hybrid for amylase immobilization

Carboxymethyl cellulose-silver nanoparticle (AgNp)-silica hybrids have been synthesized in a modified Stöber process. The hybrid synthesis was optimized to obtain an efficient immobilization matrix for diastase alpha amylase, a multimeric enzyme of high technological significance. The synthesized hybrids were characterized using FTIR, XRD, SEM, TGA and BET studies. The enzyme immobilization was done by adsorption and using the immobilized enzyme, the hydrolysis of soluble starch has been optimized in comparison to free enzyme. The optimum usable pH for the immobilized enzyme ranged from pH 4 to 5, while pH 5 was optimum pH for the free enzyme activity. The kinetic parameters for the immobilized, (K _M = 3.4610 mg ml^?1; V _max = 6.3540 mg ml^?1 min^?1) and free enzyme (K _M = 4.1664 mg ml^?1; V _max = 4.291 mg ml^?1 min^?1) hydrolysis indicated that the immobilization at the nanohybrid has significantly improved the catalytic property of the enzyme. In the immobilized state, the enzyme remained usable for many repeated cycles like our previous material, gum acacia-gelatin-AgNp-silica. Storage experiments indicated that the immobilization has increased the stability of the enzyme and also that AgNps play a role in stabilizing the immobilized enzyme.     

Attachment of horseradish peroxidase (HRP) onto the poly(styrene/acrolein) latexes and onto their derivatives with amino groups on the surface; activity of immobilized enzyme

The polystyrene (P(S)), poly(styrene/acrolein) (P(SA)), and polyacrolein (P(A)) latexes, with varied fraction of polyacrolein in the surface layer (f _A=0, 0.50, 0.63, 0.84, 1.00), were used for the attachment of horseradish peroxidase. Surfaces of latexes were modified by reaction with ethylenediamine. In this step the aldehyde groups from polyacrolein were blocked and the primary amino groups were introduced. The carbohydrate portion of HRP was oxidized in the reaction leading to formation of aldehyde groups. The adsorption and covalent immobilization of HRP onto the P(S), P(SA), and P(A) latexes and of the oxidized HRP (HRP-OX) onto the modified latex particles, with amino groups on the surface (P(SA)-M and P(A)-M), were investigated. The activities of parent and oxidized HRP were compared with activities of the corresponding enzymes in solution. It has been found that whereas HRP is not suitable for the covalent immobilization on P(SA) latex and loses its activity after adsorption onto P(S) latex, HRP-OX can be adsorbed onto P(S) latex and is readily immobilized covalently onto the ethylenediamine modified P(SA) and P(A) latexes, retaining much of its former enzymatic reactivity.This work was supported by the KBN Grant 2 0624 91 01     

Effect of the Presence of Surfactants and Immobilization Conditions on Catalysts’ Properties of <Emphasis Type="Italic">Rhizomucor miehei</Emphasis> Lipase onto Chitosan

Lipase from Rhizomucor miehei (RML) was immobilized onto chitosan support in the presence of some surfactants added at low levels using two different strategies. In the first approach, the enzyme was immobilized in the presence of surfactants on chitosan supports previously functionalized with glutaraldehyde. In the second one, after prior enzyme adsorption on chitosan beads in the presence of surfactants, the complex chitosan beads-enzyme was then cross-linked with glutaraldehyde. The effects of surfactant concentrations on the activities of free and immobilized RML were evaluated. Hexadecyltrimethylammonium bromide (CTAB) promoted an inhibition of enzyme activity while the nonionic surfactant Triton X-100 caused a slight increase in the catalytic activity of the free enzyme and the derivatives produced in both methods of immobilization. The best derivatives were achieved when the lipase was firstly adsorbed on chitosan beads at 4 °C for 1 h, 220 rpm followed by cross-link the complex chitosan beads-enzyme with glutaraldehyde 0.6% v.v^?1 at pH 7. The derivatives obtained under these conditions showed high catalytic activity and excellent thermal stability at 60° and 37 °C. The best derivative was also evaluated in the synthesis of two flavor esters namely methyl and ethyl butyrate. At non-optimized conditions, the maximum conversion yield for methyl butyrate was 89%, and for ethyl butyrate, the esterification yield was 92%. The results for both esterifications were similar to those obtained when the commercial enzyme Lipozyme® and free enzyme were used in the same reaction conditions and higher than the one achieved in the absence of the selected surfactant.     

Immobilized Laccase on Activated Poly(Vinyl Alcohol) Microspheres For Enzyme Thermistor Application

Poly(vinyl alcohol) (PVA) microspheres were prepared by inverse suspension crosslinked method, with glutaraldehyde as a crosslinking agent. PVA microspheres activated with aldehyde groups were employed for Trametes versicolor laccase immobilization. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to characterize the activated PVA microspheres and PVA microspheres with immobilized laccase (Lac/PVA microspheres), which show that laccase was successfully immobilized on the PVA microspheres. The optimum pH and temperature coupling conditions for the immobilized laccase were determined to be 3.3 and 30 °C, respectively. Residual activity was also investigated by soaking the immobilized laccase in organic solvents at different concentrations, proving it chemically stable. Immobilized laccase exhibited good storage stability at 4 °C. The enzyme biosensor showed good performance in 2,2-azinobis(3-ethylthiazoline-6-sulfonate) and bisphenol A, with concentration ranges of 2 to 8 mM and 0.05 to 0.25 mM, respectively. Therefore, PVA microspheres may have high potential as support for enzyme thermistor applications.