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.     

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.     

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.     

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 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.     

Changes in morphology and activity of transglutaminase following cross-linking and immobilization on a polypropylene microporous membrane

Transglutaminase (TGase) was cross-linked with glutaraldehyde, and cross-linked crystalline transglutaminase was immobilized on a polypropylene microporous membrane by UV-induced grafting. Immobilized enzyme activity were calculated to be 0.128 U/cm2 polypropylene microporous membrane. The microstructure and enzyme characteristics of free, cross-linked and immobilized transglutaminase were compared. The optimum temperature of free transglutaminase was determined to be approximately 40 °C, while cross-linking and immobilization resulted in an increase to approximately 45 °C and 50 °C. At 60 °C, immobilized, cross-linked and free transglutaminase retained 91.7 ± 1.20%, 63.2 ± 1.05% and 37.9 ± 0.98% maximum activity, respectively. The optimum pH was unaffected by the state of transglutaminase. However, the thermal and pH stabilities of cross-linked and immobilized transglutaminase were shown to increase.     

Design and Properties of an Immobilization Enzyme System for Inulin Conversion

A commercial inulinase could convert inulin into fructose, which was optimized to be entrapped in the calcium alginate-gelatin beads with the immobilization yield of 86% for free inulinase activities. The optimum pH values and temperatures were 4.5 and 40 °C for the free enzyme and 5.0–5.5 and 45–50 °C for the immobilized enzyme. The kinetic parameters of V _max and K _m were 5.24 μmol/min and 57.6 mg/mL for the free inulinase and 4.32 μmol/min and 65.8 mg/mL for the immobilized inulinase, respectively. The immobilized enzyme retained 80% of its initial activities at 45 °C for 4 days, which could exhibit better thermal stability. The reuse of immobilized inulinase throughout the continuous batch operations was explored, which had better reusability of the immobilized biocatalyst. At the same time, the stability of immobilized enzyme in the continuous packed-bed bioreactor was estimated, which showed the better results and had its potential scale-up fructose production for inulin conversion.     

Entrapment of plant invertase within novel composite of agarose-guar gum biopolymer membrane

Invertase or β-d-Fructofuranosidase (E.C.3.2.1.26) was extracted from Cucumis melo. L. fruit (Family-Cucurbitaceae). Soluble, plant invertase enzyme was immobilized in novel composite of agarose-guar gum biopolymer matrix in the form of hydrophilic, porous membranes. The immobilized invertase was characterized for sucrose hydrolytic activity and leakage from the matrix support. The efficiency of immobilization was found to be 91% with negligible leaching. The kinetic parameters K_m and V_max for free and immobilized invertase were also determined. Immobilized invertase was optimally active in the wide pH range of 4.5-6.5. The immobilization process also enhanced the thermal stability of enzyme up to 65 °C. Immobilized invertase membranes showed excellent storage stability with shelf life of 110 days. Entrapped invertase showed better operational stability and reusability up to 12 cycles. The fluorescence spectra of the composite membranes were studied and compared with that of soluble enzyme. All these characteristics of the immobilized invertase membranes make them suitable for the fabrication of biosensors.     

Immobilization of α-amylase from Bacillus circulans GRS 313 on coconut fiber

A simple and inexpensive method for immobilizing alpha-amylase from Bacillus circulans GRS 313 on coconut fiber was developed. The immobilization conditions for highest efficiency were optimized with respect to immobilization pH of 5.5, 30 degrees C, contact time of 4 h, and enzyme to support a ratio of 1:1 containing 0.12 mg/mL of protein. The catalytic properties of the immobilized enzyme were compared with that of the free enzyme. The activity of amylase adsorbed on coconut fiber was 38.7 U/g of fiber at its optimum pH of 5.7 and 48 degrees C, compared with the maximum activity of 40.2 U/mL of free enzyme at the optimum pH of 4.9 and 48 degrees C. The reutilization capacity of the immobilized enzyme was up to three cycles.     

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%.     

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.     

Screening of Dowex® anion-exchange resins for invertase immobilization

Commercial yeast invertase (Bioinvert^®) was immobilized by adsorption on anion-exchange resins, collectively named Dowex^® (1×8:50–400, 1×4:50–400, and 1×2:100–400). Optimal binding was obtained at pH 5.5 and 32°C. Among different polystyrene beads, the complex Dowex-1×4–200/invertase showed a yield coupling and an immobilization coefficient equal to 100%. The thermodynamic and kinetic parameters for sucrose hydrolysis for both soluble and insoluble enzyme were evaluated. The complex Dowex/invertase was stable without any desorption of enzyme from the support during the reaction, and it had thermodynamic parameters equal to the soluble form. The stability against pH presented by the soluble invertase was between 4.0 and 5.0, whereas for insoluble enzyme it was between 5.0 and 6.0. In both cases, the optimal pH values were found in the range of the stability interval. The K _m and V _max for the immobilized invertase were 38.2 mM and 0.0489 U/mL, and for the soluble enzyme were 40.3 mM and 0.0320 U/mL.     

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.     

一种简化的重氮化法制备固定化酶的载体合成方法

重氮化法是固定化酶时常用的一种方法。用多孔玻璃等无机物作载体时,一般是先用g-氨丙基三乙氧基硅烷与多孔玻璃等载体反应[1,2],生成烷基胺玻璃,然后与对硝基苯酰氯反应,产物经过还原,生成带有芳胺的衍生物,最后进行重氮化。本文通过烷基胺与对氨基苯甲酸反应,直接生成芳胺的衍生物,比常用的方法缩短了一步。通过在新合成的载体上对木瓜蛋白酶进行固定化,研究了固定化条件对酶活力回收的影响,最适固定化条件如下:pH为7.0,时间为6h,酶量为240mg/g载体,并比较了固定化酶和溶液酶的有关性质,考察了固定化酶的操作稳定性。结果表明,用这种方法合成的载体固定化酶,其对热稳定性、操作稳定性及产率都比较理想。     

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.     

Preparation and characterization of lipase immobilized on reversibly soluble-insoluble N-(2-carboxylbenzoyl) chitosan

N-(2-carboxylbenzoyl) chitosan (CBC), a reversibly soluble-insoluble polymer with pH change, was prepared by modifying chitosan backbone with phthalic anhydride and employed as carrier for lipase immobilization. The obtained CBC exhibited reversible solubility in aqueous solution; it was soluble at pH above 3.8 and precipitated at pH below 3.4. The porcine pancreatic lipase was covalently immobilized on CBC with glutaraldehyde as the crosslinking agent. Under the optimal immobilization condition, the retention activity of the immobilized lipase was found to be 69.8 %. The maximum activity of lipase immobilized on CBC was observed at 40 °C, pH 8.0, while the free lipase presented maximum activity at 37 °C, pH 7.5. The lipase immobilized on CBC exhibited improved thermal and storage stabilities and retained 58.7 % of its initial activity after 9 times of repeated use.     

Improved Thermal and Reusability Properties of Xylanase by Genipin Cross-Linking to Magnetic Chitosan Particles

Enzymes are gradually increasingly preferred over chemical processes, but commercial enzyme applications remain limited due to their low stability and low product recovery, so the application of an immobilization technique is required for repeated use. The aims of this work were to produce stable enzyme complexes of cross-linked xylanase on magnetic chitosan, to describe some characteristics of these complexes, and to evaluate the thermal stability of the immobilized enzyme and its reusability. A xylanase was cross-linked to magnetite particles prepared by in situ co-precipitation of iron salts in a chitosan template. The effect of temperature, pH, kinetic parameters, and reusability on free and immobilized xylanase was evaluated. Magnetization, morphology, size, structural change, and thermal behavior of immobilized enzyme were described. 1.0?±?0.1 μg of xylanase was immobilized per milligram of superparamagnetic chitosan nanoparticles via covalent bonds formed with genipin. Immobilized xylanase showed thermal, pH, and catalytic velocity improvement compared to the free enzyme and can be reused three times. Heterogeneous aggregates of 254 nm were obtained after enzyme immobilization. The immobilization protocol used in this work was successful in retaining enzyme thermal stability and could be important in using natural compounds such as Fe₃O₄@Chitosan@Xylanase in the harsh temperature condition of relevant industries.

     

Immobilization of Candida antarctica lipase B on the surface of modified sol–gel matrix

The use of modified sol–gel matrix to immobilize the enzyme Candida antartica lipase B (CALB) was investigated. Free hydroxyl groups on the matrix surface were exploited to covalently immobilize the enzyme. Based from the results, incorporating hydrophobic sol–gel precursor (ethyltrimethoxysilane) enhanced enzyme activity. An enzyme activity of 192.02 U/g beads with 80.88 % attachment was obtained. At alkaline pH, immobilization yield of enzyme increased. The attachment of enzyme on the surface of the matrix was confirmed by scanning electron microscope images. Covalently immobilized CALB on sol–gel supports has higher thermal stability with 2.7 times higher half-life compared to soluble enzymes at 60 °C. This enzyme immobilization system retains the enzyme residual activity even for repetitive use. Hence, the immobilization approach developed recommends its further application.     

Enzymatic Decolorization of Anthraquinone and Diazo Dyes Using Horseradish Peroxidase Enzyme Immobilized onto Various Polysulfone Supports

In this study, covalent immobilization of the horseradish peroxidase (HRP) onto various polysulfone supports was investigated. For this purpose, different polysulfones were methacrylated with methacryloyl chloride, and then, nonwoven fabric samples were coated by using solutions of these methacrylated polysulfones. Finally, support materials were immersed into aquatic solution of HRP enzyme for covalent immobilization. Structural analysis of enzyme immobilization onto various polysulfones was confirmed with Fourier transform infrared spectroscopy, atomic force microscopy, and proton nuclear magnetic resonance spectroscopy. Decolorization of textile diazo (Acid Black 1) and anthraquinone (Reactive Blue 19) dyes was investigated by UV–visible spectrophotometer. Covalently immobilized enzyme has been used seven times in freshly prepared dye solutions through 63 days. Dye decolorization performance of the immobilized systems was observed that still remained high (70 %) after reusing three times. Enzyme activities of immobilized systems were determined and compared to free enzyme activity at different conditions (pH, temperature, thermal stability, storage stability). Enzyme activities of immobilized systems and free enzyme were also investigated at the different temperatures and effects of temperature and thermal resistance for different incubation time at 50 °C. In addition, storage activity of free and immobilized enzymes was determined at 4 °C at different incubation days.     

Immobilization of invertase through its carbohydrate moiety onOcimum basilicum seed

Yeast invertase, a glycoprotein, was covalently coupled toOcimum basilicum seeds either through its protein or carbohydrate moiety. Of the various methods investigated, binding of the enzyme through its carbohydrate moiety resulted in the retention of considerably higher amounts of enzyme activity. Immobilized invertase showed a shift in the pH optimum toward the alkaline side without appreciable change in temperature optimum. However, the immobilized preparation was more thermostable than the free enzyme. Invertase bound to the seeds could be used repeatedly for the hydrolysis of sucrose syrups in a batch process without appreciable loss in activity. The seeds could serve as an inexpensive, ready-to-use, natural pellicular polysaccharide support for immobilizing enzymes.