Bostrycin and 4-deoxybostrycin: two nonspecific phytotoxins produced by Alternaria eichhorniae.

Two crystalline red pigments with phytotoxic activity were isolated from culture filtrates of Alternaria eichhorniae, a pathogen of the water hyacinth Eichhornia crassipes. The pigments were present in the ratio of 4:1 and were identified as bostrycin and 4-deoxybostrycin, respectively. This is the first isolation of 4-deoxybostrycin from a natural source. Bostrycin, 4-deoxybostrycin, and their isopropylidene derivatives induced necrosis on tested plant leaves comparable to the A. eichhorniae-induced necrosis on water hyacinth. The lowest phytotoxic concentrations of crystalline bostrycin and 4-deoxybostrycin on water hyacinth leaves were about 7 and 30 microgram/ml, respectively. Both substances were inhibitory to Bacillus subtilis but were inactive against the fungus Geotrichum candidum.     

Activation of polyvinyl chloride sheet surface for covalent immobilization of oxalate oxidase and its evaluation as inert support in urinary oxalate determination

Polyvinyl chloride (PVC) sheets are a promising material for enzyme immobilization owing to the PVC’s properties such as being chemically inert, corrosion free, weather resistant, tough, lightweight, and maintenance free and having a high strength-to-weight ratio. In this study, this attractive material surface was chemically modified and exploited for covalent immobilization of oxalate oxidase using glutaraldehyde as a coupling agent. The enzyme was immobilized on activated PVC surface with a conjugation yield of 360 μg/cm². The scanning electron micrographs showed the microstructures on the PVC sheet surface revealing the successful immobilization of oxalate oxidase. A colorimetric method was adopted in evaluating enzymatic activity of immobilized and native oxalate oxidase. The immobilized enzyme retained 65% of specific activity of free enzyme. Slight changes were observed in the optimal pH, incubation temperature, and time for maximum activity of immobilized oxalate oxidase. PVC support showed no interference when immobilized oxalate oxidase was used for estimation of oxalic acid concentration in urine samples and showed a correlation of 0.998 with the values estimated with a commercially available Sigma kit. The overall results strengthen our view that PVC sheet can be used as a solid support for immobilization of enzymes and in the field of clinical diagnostics, environmental monitoring and remediation.     

Immobilization of thermolysin to polyamide nonwoven materials

In the last few years, an increasing number of biotechnological techniques have been applied to the restoration and conservation of works of art, paintings, old maps, and papers or books. Enzymes can solve problems that give restorers difficulties, although for many applications it is not possible to use soluble enzymes; therefore, it is necessary to look for suitable carriers for immobilization. Different methods for covalent immobilization of enzymes to polyamide nonwovens were tested, using thermolysin as an example. Two distinct strategies were pursued: (1). controlled, partial hydrolysis of the polymer and subsequent binding of the enzyme to the released amino and carboxy groups; and (2). attachment of reactive groups directly to the polyamide without disintegrating the polymeric structure (O-alkylation). Different spacers were used for covalent fixation of the enzyme in both cases. The enzyme was fixed to the released amino groups by glutaraldehyde, either with or without a spacer. Either way, active enzyme could be immobilized to the matrix. However, intense treatment caused severe damage to the stability of the nonwoven fabric, and reduced the mechanical strength. Conditions were investigated to conserve the nonwoven fabric structure while obtaining near-maximum immobilized enzyme activity. Immobilization of the enzyme to the released carboxy group after acid hydrolysis was performed using dicyclohexylcarbodiimide. In comparison to the enzyme bound via the amino group, the yield of immobilized enzyme activity was slightly lower when benzidine was taken as spacer and still lower with a 1,6-hexanediamine spacer. O-alkylation performed with dimethylsulfate caused severe damage to the nonwoven fabric structure. Considerably better results were obtained with triethyloxonium tetrafluoroborate. As the spacers 1,6-hexanediamine and adipic acid dihydrazide were used, activation for immobilizing thermolysin was performed with glutaraldehyde, adipimidate, and azide. With the exception of azide, all combinations of spacers and activation reagents gave high yields of immobilized enzyme activity. Thermolysin immobilized by this technique showed a remarkably improved stability with respect to elevated temperature, extreme pH values, and reduced polarity. The nonwoven fabric can be stored for weeks without loss of enzyme activity by washing with distilled water and drying.     

Immobilization of glucose oxidase on polymer membranes treated by low-temperature plasma

Low-temperature plasma was employed for activation of polymer membranes as a carrier for enzyme immobilization. Glucose oxidase was immobilized on polypropylene (PP), polyvinylidene fluoride (PVDF), or polytetrafluoroethylene (PTFE) membrane surfaces treated by nitrogen or ammonia gas plasma using glutaraldehyde as a linking agent. Enzyme activity was evaluated by the response of glucose sensor composed of the immobilized enzyme membrane and a dissolved oxygen electrode. The sensor response was found to depend on the kind of carrier membrane and to become maximum at suitable conditions of plasma treatment.     

Antibacterial functionalization of wool fabric via immobilizing lysozymes

Greater attention has been given to enzymatic processes of textiles as effective alternatives to conventional chemical treatments because of the non-toxic and eco-friendly characteristics of enzymes as well as the increasingly important requirement for reducing pollution in textile production. A new functionalization method for wool fabrics based on immobilization of lysozymes was investigated in this paper. Wool fabric was first activated with glutaraldehyde, and then employed to covalently immobilize lysozymes. Main immobilization parameters were optimized in terms of the activity of immobilized enzyme. A high activity of the immobilized enzyme was obtained when the fabric was activated at 25 °C for 6 h in a bath containing with 0.2% of glutaraldehyde followed by the immobilization at 4 °C and pH 7.0 for 6 h with 5 g l⁻¹ lysozyme. The scanning electron microscopy and staining tests based on modified Coomassie protein assay (Bradford method) revealed that the lysozyme was fixed covalently on the wool fabric. Wool fabrics immobilizing lysozymes presented a higher ratio of bacteriostasis to Staphylococcus aureus. The durability of antibacterial wool was assessed and the lysozyme immobilized on wool fabric retained ca. 43% of its activity after five cycles of use when taking the activity of the immobilized lysozyme before using as reference.     

Development of an eco-friendly antibacterial textile: lysozyme immobilization on wool fabric

Lysozyme, a type of natural enzyme, has been widely used for bacteriostatic functionalization of various materials due to its efficient and selective antibacterial properties. Herein, we report the preparation and characterization of an eco-friendly antibacterial textile based on the immobilization of lysozyme from chicken egg white onto wool fibers. Tris(hydroxymethyl)phosphine (THP) was employed as the cross-linker for the immobilization of lysozyme on the surface of wool fiber. The mechanism of THP cross-linking was investigated via phosphorus test, energy-dispersive spectroscopy (EDX) and Fourier transform infrared spectroscopy (FT-IR). The surface staining, optimization of immobilization parameters, morphology, antibacterial properties, and durability of wool fibers with immobilized lysozyme were also assessed. The results show that hydroxymethyl groups of THP reacted with amino groups of wool fiber and lysozyme through Mannich reaction, which successfully immobilized lysozyme on the wool fiber. The wool fibers incorporated with lysozyme had better antibacterial properties and durability compared with the untreated wool fabric. This facile immobilization approach of lysozyme provides an effective strategy for environmentally benign modification and functionalization of keratin and keratin-containing materials.

     

Detoxification of organophosphate nerve agents by immobilized Escherichia coli with surface-expressed organophosphorus hydrolase

An improved whole-cell technology for detoxifying organophosphate nerve agents was recently developed based on genetically engineered Escherichia coli with organophosphorus hydrolase anchored on the surface. This article reports the immobilization of these novel biocatalysts on nonwoven polypropylene fabric and their applications in detoxifying contaminated wastewaters. The best cell loading (256 mg cell dry weight/g of support or 50 mg cell dry weight/cm2 of support) and subsequent hydrolysis of organophosphate nerve agents were achieved by immobilizing nongrowing cells in a pH 8, 150 mM citrate-phosphate buffer supplemented with 1 mM Co2+ for 48 h via simple adsorption, followed by organophosphate hydrolysis in a pH 8, 50 mM citrate-phosphate buffer supplemented with 0.05 mM Co2+ and 20% methanol at 37 degrees C. In batch operations, the immobilized cells degraded 100% of 0.8 mM paraoxon, a model organophosphate nerve agent, in approximately 100 min, at a specific rate of 0.160 mM min-1 (g cell dry wt)-1. The immobilized cells retained almost 100% activity during the initial six repeated cycles and close to 90% activity even after 12 repeated cycles, extending over a period of 19 days without any nutrient supplementation. In addition to paraoxon, other commonly used organophosphates, such as diazinon, coumaphos, and methylparathion were hydrolyzed efficiently. The cell immobilization technology developed here paves the way for an efficient, simple, and cost-effective method for detoxification of organophosphate nerve agents.     

Amino silicones finished fabrics for lipase immobilization: Fabrics finishing and catalytic performance of immobilized lipase

Finishing of silk fabric was achieved by using amino-functional polydimethylsiloxane (PDMS) and lipase from Candida sp. 99-125 was immobilized on the treated silk fabrics. Hydrophobic fabrics were obtained by dipping the native fabric in 0.125–0.25% (w/v) PDMS solution and dried at 70 °C. The direct adsorption on PDMS-treated fabric was verified to be a better strategy for lipase immobilization than that by covalent binding. Compared to unfinished fabrics, the hydrolytic activity of immobilized enzyme on the finished fabric was improved by 1.6 times. Moreover, the activity of immobilized enzymes on hydrophobic fabrics was significantly improved in different concentrations of strong polar solvents such as methanol and ethanol, and in common organic solvents with different octanol–water partition coefficients (Log P). Enzymatic activity and stability in 15% water content system (added water accounted for the total reaction mixtures, v/v) showed more than 30% improvement in each batch. The amino–silicone finished fabric surface was investigated by scanning electron microscopy and X-ray photoelectron spectroscopy. The hydrophobic fabric immobilized enzyme could be recycled for more than 80 times with no significant decrease in esterification activity. PDMS-treated woven silk fabrics could be a potential support for lipase immobilization in catalytic esterification processes.     

Covalent immobilization of glucose oxidase onto poly(styrene-co-glycidyl methacrylate) monodisperse fluorescent microspheres synthesized by dispersion polymerization

In this study, micron-sized poly(styrene-co-glycidyl methacrylate) (PSt-GMA) fluorescent microspheres of 5.1microm in diameter were synthesized via dispersion polymerization of styrene and glycidyl methacrylate in the presence of 1,4-bis(5-phenyloxazol-2-yl) benzene (POPOP), which provided surface functional groups for covalent immobilization of enzymes. In an effort to study the biocompatibility of the microspheres' surface, glucose oxidase and beta-d-(+)-glucose were selected as a catalytic system for enzymatic assays. A colorimetric method was adopted in evaluating enzymatic activity by introducing horseradish peroxidase (HRP). Both the immobilization amount and the apparent activity of immobilized glucose oxidase from Aspergillus niger (GOD) were determined at different conditions. The results show that the immobilized enzymes retained approximately 28 to 34% activity, as compared with free enzymes, without pronounced alteration of the optimum pH and temperature. Kinetics studies show that the corresponding values of K(m) and V(max) are 23.2944 mM and 21.6450M/min.mg GOD for free enzymes and 35.1780 mM and 15.4799M/min.mg GOD for immobilized enzymes. The operational stability studies show that immobilized GOD could retain nearly 50% initial activity after being washed 20 times. The results suggest that the resultant PSt-GMA fluorescent microspheres provide a suitable surface for covalent immobilizing biomolecules; therefore, they have the potential of being used in fluorescence-based immunoassays in high-throughput screening or biosensors.     

Effect of the state of graphite surfaces on the activity of immobilized glucose oxidase

The work deals with immobilization of glucose oxidase on modified graphite. The effect of the carrier surface composition on the major properties of the immobilized enzyme was examined. The level of activity of the immobilized enzyme is shown to depend on the quantity of Ti and Zr oxides applied on the graphite surface. The high activity of the enzyme immobilized on graphite will enable it to be used for construction of enzyme electrodes.     

Understanding the pH-dependent immobilization efficacy of feruloyl esterase-C on mesoporous silica and its structure–activity changes

The purpose of the present investigation was to study the pH dependence of both the immobilization process and the enzyme activity of a feruloyl esterase (FoFaeC from Fusarium oxysporum) immobilized in mesoporous silica. This was done by interpreting experimental results with theoretical molecular modeling of the enzyme structure. Modeling of the 3D structure of the enzyme together with calculations of the electrostatic surface potential showed that changes in the electrostatic potential of the protein surface were correlated with the pH dependence of the immobilization process. High immobilization yields were associated with an increase in pH. The transesterification activity of both immobilized and free enzyme was studied at different values of pH and the optimal pH of the immobilized enzyme was found to be one unit lower than that for the free enzyme. The surface charge distribution around the binding pocket was identified as being a crucial factor for the accessibility of the active site of the immobilized enzyme, indicating that the orientation of the enzyme inside the pores is pH dependent. Interestingly, it was observed that the immobilization pH affects the specific activity, irrespective of the changes in reaction pH. This was identified as a pH memory effect for the immobilized enzyme. On the other hand, a change in product selectivity of the immobilized enzyme was also observed when the transesterification reaction was run in MOPS buffer instead of citrate phosphate buffer. Molecular docking studies revealed that the MOPS buffer molecule can bind to the enzyme binding pocket, and can therefore be assumed to modulate the product selectivity of the immobilized enzyme toward transesterification.     

Kinetics and stability of immobilized chicken liver xanthine dehydrogenase.

Xanthine dehydrogenase (EC 1.2.1.37) was isolated from chicken livers and immobilized by adsorption to a Sepharose derivative, prepared by reaction of n-octylamine with CNBr-activated Sepharose 4B. Using a crude preparation of enzyme for immobilization it was observed that relatively more activity was adsorbed than protein, but the yield of immobilized activity increased as a purer enzyme preparation was used. As more activity and protein were bound, relatively less immobilized activity was recovered. This effect was probably due to blocking of active xanthine dehydrogenase by protein impurities. The kinetics of free and immobilized xanthine dehydrogenase were studied in the pH range 7.5-9.1. The Km and V values estimated for free xanthine dehydrogenase increase as the pH increase; the K'm and V values for the immobilized enzyme go through a minimum at pH 8.1. By varying the amount of enzyme activity bound per unit volume of gel, it was shown that K'm is larger than Km are result of substrate diffusion limitation in the pores of the support material. Both free and immobilized xanthine dehydrogenase showed substrate activation at low concentrations (up to 2 microM xanthine). Immobilized xanthine dehydrogenase was more stable than the free enzyme during storage in the temperature range of 4-50 degrees C. The operational stability of immobilized xanthine dehydrogenase at 30 degrees C was two orders of magnitude smaller than the storage stability, t 1/2 was 9 and 800 hr, respectively. The operational stability was, however, better than than of immobilized milk xanthine oxidase (t 1/2 = 1 hr). In addition, the amount of product formed per unit initial activity in one half-life, was higher for immobilized xanthine dehydrogenase than for immobilized xanthine oxidase. Unless immobilized milk xanthine oxidase can be considerable stabilized, immobilized chicken liver xanthine dehydrogenase is more promising for application in organic synthesis.     

The effect of cell immobilization on the antibacterial activity of Lactobacillus reuteri DPC16 cells during passage through a simulated gastrointestinal tract system

Cell immobilization has the ability to influence the survival and functional characteristics of probiotic bacterial strains in harsh environments. This study investigated the effect of cell immobilization and passage through a simulated gastrointestinal tract (GI) on the antibacterial activity of Lactobacillus reuteri DPC16. Antibacterial activity, reuterin production and diol dehydratase activity were assayed in recovered isolates of L. reuteri that had been immobilized in Ca alginate-skim milk, and incubated in simulated GI fluids. Among all the recovered isolates tested, any that had undergone immobilization followed by immediate recovery of the cells without subsequent incubation in any fluids demonstrated the highest reuterin production, antimicrobial activity and diol dehydratase enzyme activity. L. reuteri DPC16 cells that had been immobilized, incubated in simulated GI fluids, and subsequently recovered from the beads often showed some loss of antimicrobial activity compared to the immobilized cells. The data confirm that the process of immobilization of L. reuteri in Ca alginate-skim milk, rather than the passage through simulated GI fluids, resulted in enhanced antibacterial activity. This is attributed to increased diol dehydratase activity, resulting in increased reuterin production.     

Immobilization of DNA by UV irradiation and its utilization as functional materials.

The water-insoluble DNA film was successfully prepared by UV irradiation. The DNA film was stable in water. It could effectively accumulated the DNA-binding intercalating materials, such as ethidium bromide, dibenzo-p-dioxin and benzo[a]pyrene, in their aqueous solutions. On the other hand, DNA was immobilized onto nonwoven cellulose fabrics, also by the UV irradiation. The DNA immobilized cloth was found to bind silver ions. The DNA-cloth containing silver ion showed antibacterial activity. The water-insoluble DNA prepared by UV irradiation has a potential ability to serve as biomaterials for medical, engineering and environmental objects.     

Studies on the stability of immobilized xanthine oxidase and urate oxidase.

The stability of immobilized preparations of xanthine oxidase and urate oxidase was studied, and optimized, because of the potential joint use of both enzymes in clinical analysis. Xanthine oxidase was immobilized on cellulose, Sepharose, hornblende, Enzacryl-TIO, and porous glass. Thehalf-lives of these preparations at 30 degree C ranged from 40 min to 5.0 hr. In this respect immobilized enzyme resembled soluble enzyme in dilute solution (0.11 mg/ml), when the half-live was about 3.5 hr. More concentrated enzyme solution (1 mg/ml) had a half-life of 64 hr, and was, therefore, considerably more stable than the untreated immobilized xanthine oxidase preparations. Inclusion of albumen in storage and assay buffer increased the half-life of bound xanthine oxidase. So also did treatment with glutaraldehyde: in the case of xanthine oxidase bound to Enzarcyl-TIO such treatment increased the half-life at 30 degree C from 3 hr to about 100 hr. Immobilized xanthine dehydrogenase was more stable than immobilized xanthine oxidase: the dehydrogenase lost no activity during continuous assay for 5 hr at 30 degree C. The stability of immobilized urate oxidase depended on the quantity of enzyme used and on the time of stirring during immobilization: thus a preparation was made (by stirring urate oxidase (48 mg/g support) with Enzacryl-TIO for 24 hr) which lost no activity during 350 hr at 30 degree C.     

Immobilization of enzymes in porous supports: Effects of support-enzyme solution contacting

Proteins have been immobilized in porous support particles held in a fixed-bed reactor through which protein solution is continuously circulated. Changing the recirculation flow rate alters the observed immobilization kinetics and the maximum enzyme loading which can be achieved for glucose oxidase and glucoamylase on carbodiimide-treated activated carbon and for glucoamylase immobilized on CNBr-Sepharose 4B. Direct microscopic examination of FITC-labelled protein in sectioned Sepharose particles and indirect activity-loading studies with activated carbon-enzyme conjugates all indicate that immobilized enzyme is increasingly localized near the outer surface of the support particles at larger recirculation flow rates. Restricted diffusion of enzymes may be implicated in this phenomenon. These contacting effects may be significant considerations in the scaleup of processes for protein impregnation in porous supports, since apparent activity and stability of the final preparation depend on internal protein distribution.     

烟草多酚氧化酶的分离与固定化技术研究

多酚氧化酶属于氧化还原酶类,国际酶学委员会推荐名为儿茶酚氧化酶（ＥＣ１．１０．３．１ｐｏｌｙｐｈｅｎｏｌｏｘｉｄａｓｅ,ＰＰＯ）．该酶与食品工业、三废处理、医药卫生关系较为密切,因而研究较多．如近年来鸭梨［１］、蘑菇［２］、香蕉果肉组织［３］、荔枝果皮［４］等等中的多酚氧化酶均有研究报道．目前研究用固定化多酚氧化酶检测废水中酚类物质含量,进行环境检测;及其从工业废水中除去酚类,达到治理三废的目的．Ｍｏｓｂａｃｎ［５］（１９７６）研制成多酚氧化酶固定化酶柱,与氧电极检测器组合联用,可检测水中２０…     

Covalent immobilization of enzymes within micro-aqueous organic media

Traditional covalent immobilization of enzymes was mostly operated within water phase. However, most of enzymes are flexible when they are in water environment, and the covalent reactions generally lead to complete or partial activity losing due to the protein conformational changes.This paper examined enzyme covalent immobilization operated in micro-aqueous organic media, to display the differences between two environments of immobilization within water and micro-aqueous organic solvent by activity and stability determination of the resulting immobilized enzymes. Catalase, trypsin, horseradish peroxidase, laccase and glucose oxidase have been employed as model enzymes. Results showed the thermal, pH and reusable stabilities of the micro-aqueous organic covalently immobilized enzymes were improved when compared with the immobilized enzymes within water. Micro-aqueous covalent immobilization showed a remarkable advantage in remaining the enzymes catalytic activity for all the five enzymes compared with the traditional water phase immobilization. And the optimum pH values for both immobilization within water and micro-aqueous organic media shifted slightly.     

Preparation and characterization of alkaline phosphatase,horseradish peroxidase,and glucose oxidase conjugates with carboxylated carbon nano-onions

Carbon nanomaterials have emerged as suitable supports for enzyme immobilization and stabilization due to their inherently large surface area, high electrical conductivity, chemical stability, and mechanical strength. In this paper, carbon nano-onions (CNOs) were used as supports to immobilize alkaline phosphatase, horseradish peroxidase, and glucose oxidase. CNOs were first functionalized by oxidation to generate carboxylic groups on the surface followed by the covalent linking of using a soluble carbodiimide as coupling agent. The CNO–enzyme conjugates were characterized by transmission electron microscopy and Raman spectroscopy. Thermogravimetric analysis revealed a specific enzyme load of ～0.5?mg of protein per milligram of CNO. The immobilized enzymes showed enhanced storage stability without altering the optimum pH and temperatures. These properties make the prepared nanobiocatalyst of potential interest in biosensing and other biotechnological applications.     

Pulmonary xanthine oxidase activity of rats exposed to prolonged immobilization stress

This study was designed to study xanthine oxidase (XO) and xanthine dehydrogenase (XD) activity in the lung of rats exposed to prolonged restraining immobilization stress. Immobilization caused more than twofold increase of xanthine oxidase activity in the rat lung. The activity of xanthine oxidase decreased in lung homogenates incubated at -20 degrees C for 24 h. The same incubation of homogenates from control rats caused a non-significant increase of the activity. No measurable NAD(+)-dependent xanthine dehydrogenase activity could be established in the lungs of both control rats and rats subjected to immobilization. All rats revealed methylene blue-dependent xanthine dehydrogenase activity which was more than two-times higher in the immobilized animals. Incubation at -20 degrees C for 24 h increased the methylene blue-dependent xanthine dehydrogenase activity in homogenates from control rats and decreased the enzyme activity in homogenates from immobilized rats. A working hypothesis was proposed for the sequence of events explaining the results obtained: XO-catalyzed generation of activated oxygen species may take place in the initiation of lipid peroxidation in the lung of rats immobilized for prolonged periods of time.