Magnetite Nanoparticles Immobilized Pectinase: Preparation,Characterization and Application for the Fruit Juices Clarification

In this work, pectinase was immobilized on the surface of silica‐coated magnetite nanoparticles via covalent attachment. The magnetite‐immobilized enzyme was characterized by Fourier transform infrared spectroscopy, X‐ray powder diffraction, scanning electron microscopy and vibrating sample magnetometery techniques. Response Surface Methodology using Minitab Software was applied for statistical designing of operating conditions in order to immobilize pectinase on magnetic nanoparticles. The optimal conditions were obtained at 30 °C and pH 5.5 with 42.97 μl pectinase for 2 h. The immobilization yield was 50.6% at optimized conditions. Compared to the free pectinase, the immobilized pectinase was found to exhibit enhanced enzyme activity, better tolerance to the variation of pH and temperature, and improved storage stability. Both free and immobilized samples reduced the viscosity of apple juice from 1.12 to 0.88 and 0.92 mm²s^?1, respectively, after 30 min at their optimum temperature. Furthermore, the immobilized enzyme could be reused six consecutive cycles and the efficiency loss in viscosity reduction was found to be only 8.16%.     

Glucose oxidase–magnetite nanoparticle bioconjugate for glucose sensing

Immobilization of bioactive molecules on the surface of magnetic nanoparticles is of great interest, because the magnetic properties of these bioconjugates promise to greatly improve the delivery and recovery of biomolecules in biomedical applications. Here we present the preparation and functionalization of magnetite (Fe₃O₄) nanoparticles 20 nm in diameter and the successful covalent conjugation of the enzyme glucose oxidase to the amino-modified nanoparticle surface. Functionalization of the magnetic nanoparticle surface with amino groups greatly increased the amount and activity of the immobilized enzyme compared with immobilization procedures involving physical adsorption. The enzymatic activity of the glucose oxidase-coated magnetic nanoparticles was investigated by monitoring oxygen consumption during the enzymatic oxidation of glucose using a ruthenium phenanthroline fluorescent complex for oxygen sensing. The glucose oxidase-coated magnetite nanoparticles could function as nanometric glucose sensors in glucose solutions of concentrations up to 20 mmol L^–1. Immobilization of glucose oxidase on the nanoparticles also increased the stability of the enzyme. When stored at 4°C the nanoparticle suspensions maintained their bioactivity for up to 3 months.     

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.     

Analytical use of immobilized glucose oxidase

With the aim of immobilizing glucose oxidase (GO) for routine determination of glucose, a covalent bond immobilization method on titanium (IV) chloride activated silica supports was used (1). Several parameters were studied in order to optimize the residual activity upon immobilization and during operation. The immobilized enzyme can be reutilized at 25°C for several h a day alternating with storage (4°C) for at least 3,300 h.     

Covalent Immobilization of α-Galactosidase from <Emphasis Type="Italic">Penicillium griseoroseum</Emphasis> and its Application in Oligosaccharides Hydrolysis

Partially purified α-Galactosidase from Penicillium griseoroseum was immobilized onto modified silica using glutaraldehyde linkages. The effective activity of immobilized enzyme was 33%. Free and immobilized α-galactosidase showed optimal activity at 45 °C and pH values of 5 and 4, respectively. Immobilized α-galactosidase was more stable at higher temperatures and pH values. Immobilized α-galactosidase from P. griseoroseum maintained 100% activity after 24 h of incubation at 40 °C, while free enzyme showed only 32% activity under the same incubation conditions. Defatted soybean flour was treated with free and immobilized α-galactosidase in batch reactors. After 8 h of incubation, stachyose was completely hydrolyzed in both treatments. After 8 h of incubation, 39% and 70% of raffinose was hydrolyzed with free and immobilized α-galactosidase respectively. Immobilized α-galactosidase was reutilized eight times without any decrease in its activity.     

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.     

Evaluation of supports and methods for immobilization of enzyme cyclodextringlycosyltransferase

An experimental design with factorial planning was used for the immobilization of the enzyme cyclodextringlycosyltransferase (CGTase) from Bacillus firmus (strain no.37) to select the best combination of support, method of immobilization, and conditions that gives primarily higher average values for the specific immobilized enzyme activity, and secondarily, higher average values for the percentage of protein fixation. The experimental design factors were as follows: supports—controlled-pore silica, chitosan, and alumina; immobilization methods—adsorption, and two covalent bonding methods, either with γ-aminopropyltriethoxysilane or hexamethylenediamine (HEMDA); conditions—7°C without agitation and 26°C with stirring. The best combination of factors that lead to higher average values of the response variables was obtained with immobilization of CGTase in silica with HEMDA at 7°C. However, immobilization in chitosan at 7°C gave the highest immobilized CGTase specific activity, 0.25 μmole of β-CD/(min·mg protein). Physical adsorption gave low specific enzyme activities, and, in general, a high load of enzyme leads to lower specific enzyme activity.     

Aldehyde Oxidase Activity and Stability in Water-Miscible Organic Solvents

In this study, the catalytic activity of aldehyde oxidase was investigated in various water-miscible organic solvents for the first time. The enzyme was partially purified from rabbit liver, and its activity was determined in the absence/presence of nine hydro-organic mixtures. The effects of pH and temperature on the enzyme activity were also investigated. The activity was reduced in the presence of all nine organic solvents. However, in some cases, the residual activity remained almost unchanged throughout the incubation of enzyme at 35 °C for 24 h. Considering potential advantages of doing reactions in the presence of organic solvents, these results could be of value. The enzyme showed different behavior in the reaction solutions making it difficult to formulate results in a single comprehensive model. The results indicated that binding and cleavage of the substrate are influenced in the presence of organic solvents.     

Preparation,characterization, and potential application of an immobilized glucose oxidase

A simple, one-step process, using 0.25Mp-benzoquinone dissolved in 20% dioxane at 50°C for 24 h was applied to the activation of polyacrylamide beads. The activated beads were reacted with glucose oxidase isolated fromAspergillus niger. The coupling reaction was performed in 0.1M potassium phosphate at pH 8.5 and 0–4°C for 24 h. The protein concentration was 50 mg/mL. In such conditions, the highest activity achieved was about 100 U/g solid. The optimum pH for the catalytic activity was shifted by about 1 pH unit in the acidic direction to pH 5.5. Between 35 and 50°C, the activity of the immobilized form depends on the temperature to a smaller extent than that of the soluble form. Above 50°C, the activity of immobilized glucose oxidase shows a sharper heat dependence. The enzyme-substrate interaction was not profoundly altered by the immobilization of the enzyme. The heat resistance of the immobilized enzyme was enhanced. The immobilized glucose oxidase is most stable at pH 5.5. The practical use of the immobilized glucose oxidase was tested in preliminary experiments for determination of the glucose concentration in blood sera.     

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.     

氨基化树枝状介孔二氧化硅固定葡萄糖氧化酶用于检测葡萄糖

以三乙胺为碱源合成了树枝状介孔二氧化硅纳米粒子（DMSNs），并用3-氨基丙基三乙氧基硅烷（APTES）进行氨基修饰合成了氨基化树枝状介孔二氧化硅纳米粒子（DMSNs-NH₂），将其用于葡萄糖氧化酶（GOD）的固定化研究.采用扫描电子显微镜、透射电子显微镜、红外光谱仪、X射线衍射仪、氮气吸附仪及热重分析仪对固定化GOD（DMSNs-NH₂-GOD）进行了表征，测定了其活性及蛋白载量.结果表明，固定化GOD的直径约为200 nm，形状均一，呈分散的球形微粒；在最佳固定条件下，蛋白载量达225 mg/g，酶活性达215 U/mg；固定化GOD检测葡萄糖的最低检测限为0.0014 mg/mL.利用固定化GOD检测了血清和饮料中的葡萄糖，重复使用36次以上其相对酶活性仍剩余80%.该方法操作方便、准确度高，提高了酶的pH稳定性、热稳定性及重复使用性，降低了检测成本.     

Inulin Hydrolysis by Immobilized Inulinase on Functionalized Magnetic Nanoparticles Using Soy Protein Isolate and Bovine Serum Albumin

Inulin hydrolysis was performed by inulinase from Aspergillus niger covalently immobilized on magnetite nanoparticles (Fe₃O₄) covered with soy protein isolate (Fe₃O₄/SPI) functionalized by bovine serum albumin (Fe₃O₄/SPI/BSA) nanoparticles as a new bio‐functional carrier. The specific activity and protein content of the immobilized enzyme were 25.99 U/mg and 3.52 mg/mL, respectively, with 80% enzyme loading. The immobilized inulinase showed maximum activity at 45 °C, which is 5 °C higher than the optimum temperature of the free enzyme. Also, the optimum pH of the immobilized enzyme shifted from 6 to 5.5, which is more acidic compared to that of the free enzyme. The K_m value of immobilized inulinase decreased to 2.03 mg/mL. Thermal stability increased considerably at 65 and 75 °C, and a 5.13‐fold rise was detected in the enzyme half‐life at 75 °C after immobilization. Moreover, 80% of initial activity of immobilized inulinase remained after 10 cycles of hydrolysis.     

Fabrication of poly(ethylene glycol)‐based hydrogels entrapping enzyme‐immobilized silica nanoparticles

In this study, we immobilized enzymes by combining covalent surface immobilization and hydrogel entrapment. A model enzyme, glucose oxidase (GOX), was first covalently immobilized on the surface of silica nanoparticles (SNPs) via 3‐aminopropyltriethoxysilane (APTES), and the resultant SNP‐immobilized enzyme was physically entrapped within photopolymerized hydrogels prepared from two different molecular weights (MWs) (575 and 8000 Da) of poly(ethylene glycol)(PEG). The hydrogel entrapment resulted in a decrease in reaction rate and an increase in apparent K_m of SNP‐immobilized GOX, but these negative effects could be minimized by using hydrogel with a higher MW PEG, which provides higher water content and larger mesh size. The catalytic rate of the PEG 8000 hydrogel was about ten times faster than that of the PEG 575 hydrogel because of enhanced mass transfer. Long‐term stability test demonstrated that SNP‐immobilized GOX entrapped within hydrogel maintained more than 60% of its initial activity after a week, whereas non‐entrapped SNP‐immobilized GOX and entrapped GOX without SNP immobilization maintained less than 20% of their initial activity. Incorporation of SNPs into hydrogel enhanced the mechanical strength of the hydrogel six‐fold relative to bare hydrogels. Finally, a hydrogel microarray entrapping SNP‐immobilized GOX was fabricated using photolithography and successfully used for quantitative glucose detection. Copyright © 2009 John Wiley & Sons, Ltd.     

基于DNA链置换反应的新型固定化酶反应器制备及应用

建立了一种新型的酶固定化方法,采用DNA链置换反应成功地在单链DNA标记的磁性纳米粒子上实现了酶的链置换无损更替。该技术可实现目标酶的再利用,节约了生产成本。制备的固定化胰蛋白酶微反应器具有较好的重复利用性和高酶切效率,重复使用10次后仍可保持原酶活性的86%;利用链置换反应制备的MNPs@DNATrypsin酶切马心肌红蛋白5 min后,即可获得95%±0%(n=3)的氨基酸序列覆盖率,远超过相同条件下自由酶酶切12 h的结果。实验表明,发展的固定化酶技术具有高磁响应性,便于从反应体系中回收固定化酶和重复使用,同时此技术可显著提高酶活性,因此可用于固定各种重要的酶,同时可将其广泛应用于各种酶促反应中。     

Stabilization of the Cellulase Enzyme Complex as Enzyme Nanoparticle

The native Celluclast BG cellulase enzyme complex consists of different enzymes which can also degrade great substrate molecules as native celluloses. This enzyme complex has been covered by a very thin, a few nanometers thick, polymer layer, in order to improve its stability. It has been proved that the polymer layer around the enzyme molecules does not hinder the digestion as great substrates as crystalline cellulose polymer. The stability of the prepared enzyme nanoparticles (PE) could significantly be increased comparing to that of the native one what was proved by results of the total cellulose activity measured. The pretreated enzyme complex holds its activity often a few magnitudes of orders longer in time than that of the native enzyme complex (enzyme without pretreatment). It retains its activity at least ten times longer than that of the native one, at a temperature range between 20 and 37?°C. The pretreated enzyme complex can have about 50?% of its original activity during 12?h of incubation at even 80?°C, while the native cellulase one totally lost it during 6?h incubation time. The activity of PE has not been significantly reduced even at extreme pH values, namely in the pH range of 1.5 to 12.     

Glucose Sensor Based On an Amorphous Silicon Isfet

Abstract

An amorphous silicon ion sensitive field effect transistor (a-ISFET) was first applied to glucose sensors. When glucose oxidase was immobilized on the membrane, the sensor gave a linear relationship between the initial rate of the gate output voltage change and the logarithmic value of glucose concentration between 0.1 and 1 mg/ml at pH 7.0, 37°C. Determination of glucose was possible within 1 min. the system can be used for three weeks with only slight loss of enzymatic activity.     

Rapid and Efficient Proteolysis for Protein Analysis by an Aptamer-Based Immobilized Chymotrypsin Microreactor

Efficient protein digestion is a key step for successful mass spectrometry identification. However, traditional in-solution digestion suffers some drawbacks, such as autolysis of protease, long analysis times and lack of control. Recently, specific single-stranded nucleic acids, aptamers, screened from random sequence pools, have been performed high affinity for targets. In this paper, we have developed a novel enzyme reactor, which immobilized chymotrypsin based on aptamer-grafted silica beads. Mixed proteins, which consist of bovine serum albumin, myoglobin, and cytochrome c, were used as samples, to evaluate the digestion performance of the enzymatic reactor. With the use of this novel tool, proteins were digested in 40 min to an extent similar to that achieved with soluble enzyme at 37°C after 16 h. Moreover, enzymatic reactor regeneration was carried out through chymotrypsin elution and re-immobilization. The advanced characteristics of the aptamer-based chymotrypsin reactor demonstrated that aptamers could serve as novel materials for rapid and efficient enzyme immobilization and application in protein studies.     

Immobilization of a lactase onto a magnetic support by covalent attachment to polyethyleneimine-glutaraldehyde-activated magnetite

A magnetic immobilized lactase has been prepared using magnetite as the magnetic material. Magnetite was functionalized by treatment with polyethyleneimine and crosslinked with glutaraldehyde. Lactase was then covalently coupled to the activated magnetic matrix via the aldehyde groups. The conditions for optimal immobilization of enzyme are described. Eighty percent of the lactase activity was lost on immobilization and is thought to be owing to the orientation of enzyme binding to the matrix. The amount of protein coupled was 80% of that applied. The maximum lactase activity retained on the matrix following immobilization was 360 U/g matrix. The immobilized lactase showed optimal activity at pH 4.5 and 65 degrees C. The immobilized lactase was more heat stable than the free enzyme, and retained 83% of its original activity after 14 d at 55 degrees C. Galactose competitively inhibited the immobilized lactase preparation (Ki 20 m/M). The presence of high initial concentrations of galactose (10% w/v) did not prevent total hydrolysis of lactose. Glucose and calcium ions were activators of the immobilized enzyme. The immobilized enzyme hydrolyzed high concentrations of lactose (up to 25% w/v) to completion within 4-6 h in a stirred batch reactor at 55 degrees C. There was no evidence of substrate inhibition at high substrate concentrations. The efficiency of hydrolysis of lactose by the immobilized lactase was better than that of the free enzyme. The magnetic immobilized lactase was demonstrated to be suitable for use in the enzymatic hydrolysis of both pure, and cheese whey permeate, lactose.     

Immobilization of β-Galactosidase onto Magnetic Beads

A study of the cross-linking of β-galactosidase on magnetic beads is reported here. The magnetic beads were prepared from artemisia seed gum, chitosan, and magnetic fluid in the presence of a cross-linking regent (i.e., glutaraldehyde). The reactive aldehyde groups of the magnetic beads allowed the reaction of the amino groups of the enzymes. The animated magnetic beads were used for the covalent immobilization of β-galactosidase. The effect of various preparation conditions on the activity of the immobilized β-galactosidase, such as immobilizing time, amount of enzyme, and the concentration of glutaraldehyde, were investigated. The influence of pH and temperature on the activity and the stability of the enzyme, both free and immobilized, have been studied. And o-nitrophenyl-β-d-galactopyranoside (ONPG) was chosen as a substrate. The β-galactosidase immobilized on the magnetic beads resulted in an increase in enzyme stability. Optimum operational temperature for immobilized enzyme was 10 °C higher than that of free enzyme and was significantly broader.     

Preparation of zirconium diboride ultrafine hollow spheres by a combined sol–gel and boro/carbothermal reduction technique

In this work, we introduce a modified novel silica sol–gel process to synthesize hexagonal close-packed (hcp) and face-centered cubic (fcc) nickel (Ni) nanoparticles supported on amorphous carbon and silica matrix. The supporting of amorphous carbon and silica can prevent the Ni nanoparticles from aggregating and being oxided which would result in the loss of their magnetism and dispersibility. The phase structure of the Ni nanoparticles which were obtained from the gels pyrolyzed from 250 to 350 °C is hcp structure, whereas that of the Ni nanoparticles pyrolyzed at 750 °C is fcc structure. The grain sizes of the hcp Ni nanoparticles calcined at 250 °C range from 5 to 20 nm in diameter, and that of the fcc Ni nanoparticles calcined at 750 °C range in 7–35 nm. The studies of magnetic properties of the hcp and fcc Ni nanoparticles show that both have quite different magnetic behaviors.