Construction of an acetylcholinesterase-choline oxidase biosensor for aldicarb determination

In this study, acetylcholinesterase and choline oxidase were co-immobilized on poly(2-hydroxyethyl methacrylate) membranes and the change in oxygen consumption upon aldicarb introduction was measured. Immobilization of the enzymes was achieved either by entrapment or by surface attachment via a hybrid immobilization method including epichlorohydrin and Cibacron Blue F36A activation. Immobilized enzymes had a long-storage stability (only 15% activity decrease in 2 months in wet storage and no activity loss in dry storage). Aldicarb detection studies showed that a linear working range of 10-500 and 10-250 ppb aldicarb could be achieved by entrapped and surface immobilized enzymes, respectively. Enzymes immobilized on membrane surfaces responded to aldicarb presence more quickly than entrapped enzymes. Aldicarb concentrations as low as 23 and 12 ppb could be detected by entrapped and surface immobilized enzymes, respectively, in 25 min.     

Preparation and application of poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan hydrogels for immobilization of lipase

In the present of this study, two novel polymeric matrixes that are poly(N,N-dimethylacrylamide-co-acrylamide) and poly(N-isopropylacrylamide-co-acrylamide)/kappa-Carrageenan was synthesized and applied for immobilization of lipase. For the immobilization of enzyme, two different immobilization procedures have been carried out via covalently binding and entrapment methods. On the free and immobilized enzymes activities, optimum pH, temperature, storage and thermal stability was investigated. The optimum temperature for free, covalently immobilized and entrapped enzymes was found to be 30, 35 and 30 degrees C, respectively. Optimum pH for both free and immobilized enzymes was also observed at pH 8. Maximum reaction rate (Vmax) and Michaelis-Menten constant (Km) were determined for free and immobilized lipases. Furthermore, the reuse numbers of immobilized enzymes also studied. It was observed that after 40th use in 5 days, the retained activities for covalently immobilized and entrapped lipases were found as 39% and 22%, respectively. Storage and thermal stability of enzyme was also increased by as a result of immobilization procedures.     

Improving reuse cycles of Thermomyces lanuginosus lipase (NS-40116) by immobilization in flexible polyurethane

Enzyme immobilization using a low-cost support that allows increasing operational stability and reutilization arise as a great economic advantage for the industry. In this work, it was explored different methods of Thermomyces lanuginosus lipase (NS-40116) immobilization in flexible polyurethane foam (PU). PU polymer was synthesized using polyether and toluene diisocyanate as monomers. PU-NS-40116 immobilized was evaluated in terms of stability in a range of pH (7.0 and 9.0), temperature (24, 50 and 60?°C) for 24?h, and storage stability (room temperature and 4?°C) for 30?days. The results showed that after 30?days of storage immobilized enzyme kept 80% of initial enzyme activity. PU support before and after immobilization process was characterized by scanning electron microscopy and Fourier transform infrared spectroscopy. Free and immobilized enzymes were compared in terms of hydrolysis of soybean oil. Immobilized enzyme by entrapment was evaluated in successive cycles of reuse showing catalytic activity above 50% even after 5 successive cycles of reuse, confirming the efficiency of immobilization process.     

Immobilization of proteases in composite hydrogel based on poly(N-vinyl caprolactam)

Summary A novel one-step method was developed for entrapment of proteolytic enzymes, namely carboxypeptidase B and trypsin, in polymer composite gel based on poly(N-vinyl caprolactam). Native proteases and enzymes previously stabilized by covalent attachment to poly(vinylpyrrolidone-co-acrolein) were immobilized in gels. After immobilization, about 90% and 75% of original trypsin and CPB activities, respectively, were retained. The immobilized enzymes were active within a wide pH range. The optimum temperature of the entrapped enzymes was approximately 25°C higher than that of the soluble enzymes. The entrapped enzymes were successfully used to obtain human insulin from recombinant proinsulin.     

桦褶孔菌漆酶固定化及其对染料的降解

采用壳聚糖交联法和海藻酸钠-壳聚糖包埋交联法固定化桦褶孔菌产生的漆酶,探讨最佳固定化条件,固定化漆酶的温度,pH稳定性及操作稳定性,并以两种固定化酶分别对4种染料进行了降解.结果表明:(1)壳聚糖交联法固定化漆酶的最佳条件为:壳聚糖2.5%,戊二醛7%,交联时间2h,固定化时间5h,给酶量1g壳聚糖小球:1mL酶液(1U/mL),固定化效率56%;(2)海藻酸钠-壳聚糖包埋交联法固定化漆酶的最佳条件为:海藻酸钠浓度4%,壳聚糖浓度0.7%,氯化钙浓度5%,戊二醛浓度0.6%,给酶量4mL 4%海藻酸钠:1mL酶液(1U/mL),固定化效率高达86%;(3)固定化的漆酶相比游离漆酶有更好的温度和pH稳定性;(4)比较两种固定化漆酶,海藻酸钠-壳聚糖包埋交联法固定化酶的温度及酸度稳定性要优于壳聚糖固定化酶,但可重复操作性要弱于后者,两者重复使用8次后的剩余酶活比率分别为71%及64%;(5)两种固定化酶对所选的4种不同结构的合成染料均有较好的降解效果,其中壳聚糖固定化酶对茜素红的降解效果及重复使用性极佳,重复降解40mg/L的茜素红10次,降解率仍保持在100%.     

A new generation approach in enzyme immobilization: Organic-inorganic hybrid nanoflowers with enhanced catalytic activity and stability

Many different micro and nano sized materials have been used for enzymes immobilization in order to increase their catalytic activity and stability. Generally, immobilized enzymes with conventional immobilization techniques exhibit improved stability while their activity is lowered compared to free enzymes. Recently, an elegant immobilization approach was discovered in synthesis of flower-like organic-inorganic hybrid nanostructures with extraordinary catalytic activity and stability. In this novel immobilization strategy, proteins (enzymes) and metal ions acted as organic and inorganic components, respectively to form hybrid nanoflowers (hNFs). It is demonstrated that the hNFs highly enhanced catalytic activities and stability in a wide range of experimental conditions (pHs, temperatures and salt concentration, etc.) compared to free and conventionally immobilized enzymes. This review mainly discussed the synthesis, characterization, development and applications of organic-inorganic hybrid nanoflowers formed of various enzymes and metal ions and explained potential mechanism underlying enhanced catalytic activity and stability.     

A microbial biosensor for hydrogen sulfide monitoring based on potentiometry

In this study, a microbial biosensor for hydrogen sulfide (H₂S) detection based on Thiobacillus thioparus immobilized in a gelatin matrix was developed. The T. thioparus was immobilized via either surface adsorption on the gelatin matrix or entrapment in the matrix. The bacterial and gelatin concentration in the support were then varied in order to optimize the sensor response time and detection limit for both methods. The optimization was conducted by a statistical analysis of the measured biosensor response with various bacterial and polymer concentrations. According to our experiments with both immobilization methods, the optimized conditions for the entrapment method were found to be a gelatin concentration of 1% and an optical density of 82. For the surface adsorption method, 0.6% gelatin and an optical density of 88 were selected as the optimal conditions. A statistical model was developed based on the extent of the biosensor response in both methods of immobilization. The maximum change in the potential of the solution was 23.16 mV for the entrapment method and 34.34 mV for the surface absorption method. The response times for the entrapment and adsorption methods were 160 s and 105 s, respectively. The adsorption method is more advantageous for the development of a gas biosensor due to its shorter response time.     

Evaluation of biodiesel production using lipase immobilized on magnetic silica nanocomposite particles of various structures

Nonporous and mesoporous silica-coated magnetite cluster nanocomposites particles were fabricated with various silica structures in order to develop a desired carrier for the lipase immobilization and subsequent biodiesel production. Lipase from Pseudomonas cepacia was covalently bound to the amino-functionalized particles using glutaraldehyde as a coupling agent. The hybrid systems that were obtained exhibited high stability and easy recovery regardless of the silica structure, following the application of an external magnetic field. The immobilized lipases were then used as the recoverable biocatalyst in a transesterification reaction to convert the soybean oil to biodiesel with methanol. Enzyme immobilization led to higher stabilities and conversion values as compared to what was obtained by the free enzyme. Furthermore, the silica structure had a significant effect on stability and catalytic performance of immobilized enzymes. In examining the reusability of the biocatalysts, the immobilized lipases still retained approximately 55% of their initial conversion capability following 5 times of reuse.     

Effects of relative humidity on enzyme activity immobilized in sol–gel-derived silica nanocomposites

Enzyme immobilization has attracted great interest in biotechnology processes. Herein we report the immobilization of urease from Canavalia ensiformis (jack bean) in sol–gel-derived silica nanocomposites. Urease activity, differential scanning calorimetry (DSC), nitrogen and water adsorption isotherms were used to characterize the effect of storage at various relative humidities on enzyme activity immobilized in sol–gel-derived silica nanocomposites. In this study, the nanocomposites consist of tetraethoxysilane, as inorganic silicate precursor, in combination with glycerol or trehalose as organic additives. Entrapped urease was more stable for all the formulations aged with a relative humidity of 80%. However, significant differences (p < 0.05) in enzyme activity recovered at this relative humidity were observed between samples with different formulations, reflecting the effect of additives during the immobilization process. The applications of biocompatible sol–gel-derived matrices can be further extended and utilized in the development of biosensors with immobilized biomolecules that can be used for long time periods by taking into account different factors, among which the storage relative humidity has permitted to greatly improve the stability of the immobilized urease.     

Artificial cell-immobilization: a model simulating immobilization in natural environments?

A study of the marine diatom Haslea ostrearia was performed in controlled culture conditions. Biofilm formation by cells grown in liquid medium was studied through macroscopic events and extracellular polysaccharides (EPS) production. Alcian blue staining and image analysis methods were used to quantify EPS production in regard to the usual pattern (lag, exponential and stationary phases) of microalgal growth. Simultaneously, an artificial immobilization of H. ostrearia cells was carried out using an agar layer. Samples from liquid medium and artificially immobilized cultures were studied in scanning electron microscopy. They revealed the structure of the entrapping material and progressive changes of the natural biofilm along culture ageing. The use of artificially immobilized cultures for solute diffusivity investigations is discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Improving the activity and stability of Yarrowia lipolytica lipase Lip2 by immobilization on polyethyleneimine-coated polyurethane foam

In this study, polyurethane foam (PUF) was used for immobilization of Yarrowia lipolytica lipase Lip2 via polyethyleneimine (PEI) coating and glutaraldehyde (GA) coupling. The activity of immobilized lipases was found to depend upon the size of the PEI polymers and the way of GA treatment, with best results obtained for covalent-bind enzyme on glutaraldehyde activated PEI-PUF (MW 70,000 Da), which was 1.7 time greater activity compared to the same enzyme immobilized without PEI and GA. Kinetic analysis shows the hydrolytic activity of both free and immobilized lipases on triolein substrate can be described by Michaelis–Menten model. The K_m for the immobilized and free lipases on PEI-coated PUF was 58.9 and 9.73 mM, respectively. The V_max values of free and immobilized enzymes on PEI-coated PUF were calculated as 102 and 48.6 U/mg enzyme, respectively. Thermal stability for the immobilization preparations was enhanced compared with that for free preparations. At 50 °C, the free enzyme lost most of its initial activity after a 30 min of heat treatment, while the immobilized enzymes showed significant resistance to thermal inactivation (retaining about 70% of its initial activity). Finally, the immobilized lipase was used for the production of lauryl laurate in hexane medium. Lipase immobilization on the PEI support exhibited a significantly improved operational stability in esterification system. After re-use in 30 successive batches, a high ester yield (88%) was maintained. These results indicate that PEI, a polymeric bed, could not only bridge support and immobilized enzymes but also create a favorable micro-environment for lipase. This study provides a simple, efficient protocol for the immobilization of Y. lipolytica lipase Lip2 using PUF as a cheap and effective material.     

A water-soluble galactomannan from the seeds of Phoenix dactylifera L

A water-soluble polysaccharide, isolated from the seeds of dates, has been investigated using methylation, periodate and CrO(3) oxidation, NMR spectroscopy, and reaction with Bandeiraea simplicifolia lectin and alpha-D-galactosidase. The polysaccharide consists of a backbone composed of (1-->4)-beta-D-mannopyranosyl residues and carries a single (1-->6)-alpha-linked D-galactopyranosyl residue.     

Immobilization of pneumococcal polysaccharide vaccine on silicon oxide wafer for an acoustical biosensor.

One the most important aspects of a biosensor is related to immobilization and maintenance of specific reference compounds on sensing surfaces. A method for the immobilization of polysaccharides to a silicon oxide surface intended for Surface Acoustical Waves (SAW) sensors is described. Silicon oxide is a hydrophobic inorganic support used for the fabrication of many electronic devices. The pneumococcal polysaccharide (PPS) vaccine is immobilized via Protein A after pre-treatment of the surface with hydrochloric acid. The effects of non-specific binding are discussed. The results indicate that the immobilization of PPS via Protein A increases the sensitivity of detecting Streptococcus pneumoniae antibodies in human sera and offers greater reproducibility of response compared with ELISA methods. The principles of this technique are simple and are applicable to the immobilization of many capsular polysaccharides.     

Ultrasound‐assisted swelling of bacterial cellulose

Bacterial cellulose (BC) was obtained by static cultivation using commercial BC gel from scoby. BC membranes (oven dried and freeze‐dried) were swelled with 8% NaOH, in the absence and in the presence of ultrasound (US), for 30, 60, and 90 min. The influence of swelling conditions on both physico‐chemical properties and molecules entrapment was evaluated. Considering the highest levels of entrapment, an optimum swelling procedure was established: 8% NaOH for 30 min at room temperature in the presence of US. Native and PEGylated laccase from Myceliophthora thermophila was immobilized on BC membranes and a different catalytic behaviour was observed after immobilization. Native laccase presented activity values similar to published reports (5–7 U/gBC) after immobilization whereas PEGylated enzymes showed much lower activity (1–2 U/gBC). BC swelled membranes are presented herein as a potential support for the preparation of immobilized enzymes for industrial applications, like phenolics polymerization.     

酶固化技术最新研究进展(英文)

酶的本质是一种具有催化功能的蛋白质,能影响化学反应。然而,与传统的天然酶分子比较,固化酶相对更为脆弱,而传统的有机或无机催化剂其活性则比较固定。固化酶对于优化产业生产过程非常重要,近几十年来已开发出多种新型固化酶。本文在回顾酶固定化技术最新发展的同时。着重将其最新技术分别从吸附于载体,诱惑侦查及交联等三个方面进行综述。     

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.     

Metabolites of marine organisms as regulators of O-glycosylhydrolases

The ability of metabolites contained in culture liquid of 62 strains of marine fungi to affect the activity of two digestive enzymes of marine mollusks--endo-1,3-beta-D-glucanase of Spisula sachalinensis and beta-D-glucosidase of Littorina kurila--was studied. It was found that 66 and 71% of specimens activated, 18 and 7% inhibited, and 16 and 22% did not affect the activity of endo-1,3-beta-D-glucanase and beta-D-glucosidase, respectively. It is demonstrated that the metabolites of brown algae and marine sponges can be used for a targeted regulation of enzyme biosynthesis by marine fungi. The protein inhibitor of endo-1,3-beta-D-glucanases isolated from the brown alga Laminaria cichorioides blocked the biosynthesis of almost all O-glycosylhydrolases in five strains of marine fungi studied. The presence in culture medium of halistanol sulfate from the marine sponge of the family Halichondriidae either did not affect or activated the biosynthesis of enzymes involved in carbohydrate metabolism by marine fungi.     

Partial nitrification to nitrite for treating ammonium-rich organic wastewater by immobilized biomass system

This study focused on the characteristics of the partial nitrification and degradation of organics with immobilized biomass beads in the treatment of ammonium-rich organic wastewater. Sodium alginate (SA) was selected as the best entrapment support after comparing partial nitrification rate and adsorption efficiency. The immobilization methods were optimized by an orthogonal experiment. Zeta position and BET surface area were used to explain the adsorption behavior of SA immobilized beads. FT-IR revealed that a SA immobilized biomass bead was not a simply physical mixture of SA and biomass. The porous structure of SA immobilized biomass beads were observed by scanning electron microscopy (SEM), which confirmed the porosity of the beads. According to the experimental data, the effects of pH and temperature on partial nitrification and COD removal were evidently weakened in SA immobilized biomass beads due to the “protective” effect of immobilization, whereas the effects of HRT and DO were enhanced.     

Enzymatic polymerization to novel polysaccharides having a glucose-N-acetylglucosamine repeating unit, a cellulose-chitin hybrid polysaccharide

A cellulose-chitin hybrid polysaccharide having alternatingly beta(1-->4)-linked D-glucose (Glc) and N-acetyl-d-glucosamine (GlcNAc) was synthesized via two modes of enzymatic polymerization. First, a sugar oxazoline monomer of Glcbeta(1-->4)GlcNAc (1) was designed as a transition-state analogue substrate (TSAS) monomer for chitinase catalysis. Monomer 1 was recognized by chitinase from Bacillus sp., giving rise to a cellulose-chitin hybrid polysaccharide (2) via ring-opening polyaddition with perfect regioselectivity and stereochemistry. Molecular weight (M(n)) of 2 reached 4030, which corresponds to 22 saccharide units. Second, a sugar fluoride monomer of GlcNAcbeta(1-->4)Glc (3) was synthesized for the catalysis of cellulase from Trichoderma viride. The enzyme catalyzed polycondensation of 3, providing a cellulose-chitin hybrid polysaccharide (4) in regio- and stereoselective manner. M(n) of 4 reached 2840, which corresponds to 16 saccharide units. X-ray diffraction measurements revealed that these hybrid polysaccharides did not form any characteristic crystalline structures. Furthermore, these unnatural hybrids of 2 and 4 were successfully digested by lysozyme from human neutrophils.     

Recent developments and applications of immobilized laccase

Laccase is a promising biocatalyst with many possible applications, including bioremediation, chemical synthesis, biobleaching of paper pulp, biosensing, textile finishing and wine stabilization. The immobilization of enzymes offers several improvements for enzyme applications because the storage and operational stabilities are frequently enhanced. Moreover, the reusability of immobilized enzymes represents a great advantage compared with free enzymes. In this work, we discuss the different methodologies of enzyme immobilization that have been reported for laccases, such as adsorption, entrapment, encapsulation, covalent binding and self-immobilization. The applications of laccase immobilized by the aforementioned methodologies are presented, paying special attention to recent approaches regarding environmental applications and electrobiochemistry.