重氮盐制备间甲酚的水解动力学研究

对间甲苯胺重氮盐的水解反应动力学进行了研究,考察了重氮盐浓度及反应温度对反应速率的影响,测定了反应级数及表观活化能。结果表明,间甲苯胺重氮盐的水解反应近似于一级反应,温度对反应速率的影响符合阿伦尼乌斯方程,表观活化能为98.3 kJ/mol。     

Evaluation of nanogels as supports for enzyme immobilization

Nanogels are hydrophilic polymers made up of crosslinked nano‐sized particles. These nanogels have large surface area that offers several functional groups as reserves for binding drugs, generating biosensors and as supports for enzyme immobilization. This mini‐review is an attempt to evaluate the recent developments in the use of nanogels as supports in enzyme immobilization. Emphasis is laid on the effect of nanogel structure and immobilization protocol on the property profile of the immobilized enzymes as compared with their free counterparts. The prospective applications of the nanogel‐immobilized enzymes are also evaluated. © 2014 Society of Chemical Industry     

Preparation of porous polymeric structures for enzyme immobilization

Porous polymethacrylic acid‐co‐triethylene glycol dimethacrylate (MAA‐co‐3G) and polyacrylic acid‐co‐triethylene glycol dimethacrylate (AA‐co‐3G) were prepared by four different polymerization techniques, namely, suspension, dispersion, seed, and microemulsion polymerization using an inert diluent (n‐hexane and polystyrene). The morphology and porosity of the obtained polymers were examined by means of a scanning electron microscope. The surface areas of the obtained polymers were determined colorimetrically. The copolymers were modified by hydroximation and chlorination using hydroxyl amine and thionyl chloride, respectively. The effect of polymerization type, surface area, modification, and pH on the protease enzyme immobilization over poly(MAA‐co‐3G) and poly(AA‐co‐3G) was examined. The enzyme activity was measured by means of a spectrophotometer. The reactivity ratios of the two monomers MAA and 3G were determined by means of the elemental analysis method. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 76: 594–601, 2000     

Crosslinked poly(vinyl alcohol) supports for the immobilization of a lipolytic enzyme

Different esters of crosslinked poly(vinyl alcohol) (PVA) were synthetized. They were developed for protein fractionation and immobilization. PVA was crosslinked with epichlorohydrin (CL‐PVA) and esterified with linear fatty acids of different length (Cn‐CL‐PVA). A characterization of the obtained products was made. The swelling behavior, the solubility, and the percentage of esterification were examined. Values of equilibrium water content of about 81% were reached for CL‐PVA samples. The polymers' stability and morpholgy were also investigated. Thermal analysis showed an increase in matrices stability, while SEM data showed the superficial development due to crosslinking and esterification reactions. Moreover, evident morphological inhomogeneities, mainly in the commercial and crosslinked products rather than in the final polymer, were present. Finally, immobilization experiments with a commercial crude of Candida rugosa were performed. Experiments showed a greater affinity of the protein for carbon chain length ranging from 8 to 12. Data indicated that compared to Celite 545, C8‐CL‐PVA was a better support for enzyme immobilization by physical adsorption, confirming the fact that microbial lipases prefer hydrophobic supports. © 1999 John Wiley & Sons, Inc. J Appl Polym Sci 74: 1881–1889, 1999     

Characterization and application of immobilized lipase enzyme on different radiation grafted polymeric films: Assessment of the immobilization process using spectroscopic analysis

Lipase has been immobilized onto different films, polypropylene and poly(tetrafluoroethylene‐perfluroro‐propyl vinyl ether) using glutalaradehyde as a crosslinker. Differential scanning calorimetery, Fourier transform infrared spectroscopic, x‐ray diffraction, and scanning electron microscopy measurements were carried out to confirm the structure of the polymer films as well as the immobilization process of the enzyme onto the polymeric carrier. The activity and stability of the resulting biopolymers produced by lipase have been compared to those for the native lipase. The experimental results showed that the optimum temperature and pH were 40°C and 8.0, respectively. The activity of the immobilized lipases varied with lipase concentration and with the yield of grafting. Subjecting the immobilized enzymes to a dose of γ‐radiation of (0.5–10 Mrad) showed complete loss in the activity of the free enzyme at a dose of 5 Mrad. A leakage of the enzyme from the irradiated membranes was not observed in the repeated batch enzyme reactions. The operational stability of the free and immobilized lipase in n‐hexane showed that the immobilized enzyme was much more stable than the free one. © 2003 Wiley Periodicals, Inc. J Appl Polym Sci 90: 155–167, 2003     

新法合成三氟甲基苯乙酮

提出了利用三氟甲基苯胺重氮盐与乙醛肟合三氟甲基苯乙酮的新方法。     

Enzyme immobilization on smart polymers: Catalysis on demand

A new approach for the synthesis of hydrogel films with thermo-sensitive enzymatic activity is reported. Pepsin (PEP) was covalently immobilized on thermo-responsive hydrogels by radical polymerization in the presence of N-isopropylacrylamide and poly-(ethylene glycol) dimethacrylate 750, acting as functional monomer and crosslinking agent, respectively. Hydrogels showing lower critical solution temperatures between 32.9 and 36.1 °C were synthesized by UV-irradiation of reaction batches differing in the PEP/monomers ratio. The derivatization degree of the hydrogels was expressed as mg of PEP per gram of matrix and found to be in the range of 6 to 11% as assessed by Lowry method. Scanning electron microscopy analysis and water affinity evaluation allowed to highlight the porous morphology and thermo-responsivity of hydrogels as a function of temperature. Using bovine serum albumin as a substrate, kinetics parameters were determined by Lineweaver–Burk plots and the catalyst efficiency evaluated. The influence of temperature on enzyme activity, as well as the thermal stability and reusability of devices, were also investigated.     

Pectinlyase immobilization on epoxy supports for application in the food processing industry

The immobilization of a pectinlyase (PL, EC 4.2.2.3) contained in a commercial enzymic preparation was studied in view of its use for fruit pulp and juice processing. Two epoxy supports were tested for immobilization. These included Eupergit C (Rohm), a synthetic polymer, and γ-alumina functionalized with γ-glycidoxypropyltrimethoxysilane. In both biocatalysts, the catalytic response was not found to be high. The highest response in terms of immobilization yield and immobilized PL activity, however, was reported for Eupergit C (approx. 115 unit g^?1 at optimum pH).     

One-dimensional crosslinked enzyme aggregates in SBA-15: Superior catalytic behavior to conventional enzyme immobilization

α-Chymotrypsin (CT) and lipase (LP) were immobilized in SBA-15 mesoporous silica by crosslinking adsorbed enzymes. This simple approach resulted in one-dimensional crosslinked enzyme aggregates (CLEAs) in the linear pore channels of SBA-15, which was very effective in preventing the enzyme leaching and consequently improving the enzyme stability. Both CLEAs of CT and LP showed negligible activity decrease under harsh shaking condition for one week while the conventional approaches including adsorption and covalent attachment resulted in more than 50–90% enzyme inactivation under the same condition. This effective stabilization results from the bent pore structure of SBA-15 with a high aspect ratio, which prevents the leaching of one-dimensional CLEAs and thereby achieves the higher enzyme loading capacity. Along with the higher specific activity than that of adsorbed enzymes, this CLEA approach is much simpler than that of covalent attachment by obviating the tedious processes for silica functionalization and enzyme attachment.     

New supports for enzyme immobilization based on copolymers of vinylene carbonate and β‐hydroxyethylene acrylate

Vinylene carbonate (VCA), β‐hydroxyethylene acrylate, and N,N′‐methylene bisacrylamide were dissolved in water, and the aqueous solutions were copolymerized via reverse‐phase suspension copolymerization in paraffin oil. A series of hydrophilic and beaded supports containing reactive cyclic carbonate groups for enzyme immobilization were obtained. The supports were examined by coupling with trypsin, and the results showed that the amount of enzymes coupled to the supports and the specific activity of the immobilized trypsin were related to the content of VCA structure units and the reaction time. Meanwhile, the optimal pH and temperature, as well as the Michaelis–Menten constant K_m, for both native and immobilized trypsin were measured. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 83: 94–102, 2002     

Particulate precipitation polymerization: A convenient procedure for the synthesis of crosslinked polymers useful as polymeric supports

Suitable choice of monomer/nonsolvent and monomer plus solvent/nonsolvent ratios affords a very simple and convenient laboratory scale method for the synthesis of functionalized crosslinked polymers suitable as polymeric supports in a wide variety of applications. The method involves an initially homogeneous solution polymerization which, because of the presence of nonsolvents ultimately produces insoluble, particulate (i.e., nonswollen) resin. Advantages over the usual suspension polymerization processes include the absence of stabilizers and suspension agents and complete freedom to utilize water soluble or water insoluble monomers, or mixtures of the two. Reaction conditions must be determined experimentally for individual systems and a number of representative examples are given.     

Electrochemical grafting of organic coating onto gold surfaces: Influence of the electrochemical conditions on the grafting of nitrobenzene diazonium salt

This paper aims at studying the influence of the following parameters on the electrodeposition, at both constant potential and potential sweeping, of paranitrobenzene diazonium (PNBD) onto gold electrodes: applied potential, number of cycles or reduction time. Quartz Crystal Microbalance, IR spectroscopy and Atomic Force Microscopy were used to bring evidence of a multilayer architecture favoured either by lower reduction potentials or higher cycle numbers but also showed a delamination phenomenon due to over-reductive potentials.     

A simple rate model for the dynamics of immobilization of anaerobic bacteria on a plastic support

The dynamics of biofilm formation on polyethylene bioparticles in mesophilic anaerobic conditions, using an inverse fluidized bed as immobilization system, have been studied. The immobilization process was carried out using acetate and glucose as carbon sources. Scanning electron microscopy (SEM) showed cocci cells and Methanosaeta rods embedded into an exopolimeric substances (ESP) matrix. The acetoclastic activity was found to increase as soon as the lag biomass accumulation phase ended. The rate of immobilization was found to conform to a pseudo first order kinetics with good correlation. This kinetic model was used to estimate rate and equilibrium constants for the immobilization process. Rate properties have been explained in terms of attachment and detachment processes. © 2000 Society of Chemical Industry     

A low,particle‐sized,nonporous support for enzyme immobilization: Uniform poly(glycidyl methacrylate) latex particles

The uniform and nonporous poly(glycidyl methacrylate) (poly(GMA)) latex particles, 1.7 μm in size, were first tried as a support in enzyme immobilization. For this purpose, α‐chymotrypsin (CT) was selected as the model enzyme. The low particle size and nonporous character of the selected support allowed to carry out the enzyme–subtrate interaction on a sufficiently large surface area (3.36 m²/g) and in the absence of intraparticular diffusion limitations. This property is particularly important when the immobilized CT is used for the substrates with high molecular weights (i.e., proteins). The latex particles were synthesized by dispersion polymerization of GMA. The reactive character of poly(GMA) allowed the direct attachment of primary amine groups onto the particles. Confocal laser scanning microscopy (CLSM) showed that primary amine groups were preferentially located on the particle's surface. Hence, the selected enzyme, CT was immobilized on the surface of nonporous particles via glutaraldehyde activation. For CT‐immobilized poly(GMA) particles, the maximum activity (r_m) and Michealis constant (K_m) were found to be 17.2 μmol/mg CT min and 121.6 μm, respectively. No significant loss was observed in the activity of immobilized CT, during the course of experiments. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 818–824, 2006     

Soybean oil based resin: A new tool for improved immobilization of α‐amylase

Acrylated epoxidized soybean resin has been utilized to immobilize the α‐amylase via UV‐curing technique. Among the numerous methods that exist for enzyme immobilization, entrapment and covalent binding are the focus of this study. The properties of immobilized enzyme were investigated and compared with those of the free enzyme. Upon immobilization by the two methods, the catalytic properties of the enzyme were not considerably changed as compared with that of nonimmobilized form; only the pH profile was broadened for the immobilized enzyme. The free enzyme lost its activity completely in 20 days, where as storage and repeated usage capability experiments demonstrated higher stability for the immobilized form. Immobilized enzyme prepared by attachment method possesses relatively higher activity compared with the activity of those obtained by entrapment method. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 100: 4757–4761, 2006     

Response of NIPAAm‐Ch gel to temperature changes and its effectiveness on nitrification as medium for immobilization

Increase in mass transport resistance is one of the major difficulties in immobilization by the use of gels. Functional gels can change their volume and inner structure depending on ambient conditions as stimuli and may reduce the difficulty as a novel immobilization matrix. The response of poly N‐isopropylacrylamide‐co‐chlorophyllin (NIPAAm‐Ch) gel to temperature changes and its performance on nitrification with nitrifiers immobilized by the gel were investigated in a continuous‐flow stirred‐tank reactor (CFSTR) with cyclic temperature changes. The gel with an immobilized nitrifier swelled and shrank alternately with 1.2–1.6‐fold volume change under a cyclic temperature change of 32–36°C with a period of 2 or 4 h in the reactor. Volume changes of the gel brought periodic changes of its structure that accelerated dissolved oxygen transfer and concentrated ammonium into, and as a result, promoted nitrification compared with that at constant temperature. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 103: 681–686, 2007     

An electrochemical immunosensor for ochratoxin A based on immobilization of antibodies on diazonium-functionalized gold electrode

A novel electrochemical immunosensor for sensitive detection of ochratoxin A (OTA) was reported. An electrochemical and chemical reaction protocol was elaborated to modify the gold electrode. A screen-printed gold electrode (SPGE) was modified with a layer of 4-nitrophenyl, assembled from 4-nitrophenyl diazonium salt synthesized in situ in acidic aqueous solution. Next, the nitro groups were electrochemically reduced to amines followed by activation with glutaraldehyde to give a stable intermediate derivative that covalently binds antibodies against OTA during the second step, thereby tailoring an immunosensor for ochratoxin A. The utility of the electrochemical immunosensor for a competitive immunoassay was demonstrated. A competition between OTA and fixed concentration of a horseradish peroxidase-labeled OTA (OTA-HRP) for the immobilized antibodies was realized. The activity of the bound OTA-HRP was electrochemically measured by chronoamperometry (CA) using 3,3′,5,5′-tetramethylbenzidine (TMB) as substrate. The immunosensor obtained using this novel approach enabled a detection limit of 12 ng mL⁻¹ and a dynamic range up to 60 ng mL⁻¹ of OTA. Precision, accuracy and stability studies were satisfactory for the developed immunosensor.