Immobilization of biocatalysts with bombyx mori silk fibroin by several kinds of physical treatment and its application to glucose sensors

Biocatalyst-immobilized Bombyx mori silk fibroin membrane was prepared. The insolubilization of the water-soluble membranes was performed by physical treatments only, i.e. stretching, compressing and standing under high humidity and methanol-immersion treatment, without any use ofcovalently binding reagent. All physical treatments performed were effective for the purpose of the immobilization of the enzymes in the membranes. The structural characterization of the glucose oxidase (GOD) immobilized membrane was performed in detail. The permeability of the substrate depends on the crystalline structure, i.e. the fraction of Silk I and Silk II of the membrane. The activity yield of the immobilized GOD was more than 80% of the value of free enzyme when 0–002% of the enzyme was entrapped in the membrane, but it decreased with increasing the concentration of the GOD in the membrane. This seems to result from diffusion limitation of the substrate. The pH and thermal stabilities of the immobilized enzyme were much improved, and were essentially independent of the methods of the immobilization. Development of the GOD or microorganism, Pseudomonas fluorescens immobilized silk fibroin membranes as glucose sensors are described.     

固定化过氧化物酶丝素膜的制备及其性质

家蚕丝素经高浓度的中性盐氯化钙溶解后,制成了固定化过氧化物酶丝素膜,对这种酶膜的活性和理化特性作了分析,结果表明这种酶膜的活性高,酶促反应温度范围宽,最适pH5.0-7.0,热稳定性也较游离酶好,这与用溴化锂溶解丝素后制成的固定化过氧化物酶膜相仿.因此,用这种方法制成的丝素膜同样是一种良好的固定化酶的生物材料.     

An ESR study of spin-labeled silk fibroin membranes and spin-labeled glucose oxidase immobilized in silk fibroin membranes

This article describes the characteristics of silk fibroin membranes and glucose oxidase, immobilized in membranes as determined by a variety of physical methods, mainly the spin-label electron spin resonance (ESR) method. The properties of membranes insolubilized by different methods, i. e., immersion in 80% methanol aqueous solution, uniaxially drawing by placing on a stretcher, and hydration by placing in a desiccator of 96% relative humidity (RH) for 17 h, are compared. The results are also analyzed in relation to ESR spectra of spin-labeled immobilized glucose oxidase and 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy as a model of the substrate. It is concluded that the heterogeneous structures of the swollen membranes in water differ locally among membranes insolubilized by different methods, but the immobilized state of the enzyme in such membranes is mostly similar. This is correlated to the fact that the thermal or pH stabilities are essentially same among glucose-oxidase-immobilized silk fibroin membranes insolubilized by different methods.     

Glucose oxidase immobilization on a novel cellulose acetate-polymethylmethacrylate membrane

Glucose oxidase (GOD) was immobilized on cellulose acetate-polymethylmethacrylate (CA-PMMA) membrane. The immobilized GOD showed better performance as compared to the free enzyme in terms of thermal stability retaining 46% of the original activity at 70 degrees C where the original activity corresponded to that obtained at 20 degrees C. FT-IR and SEM were employed to study the membrane morphology and structure after treatment at 70 degrees C. The pH profile of the immobilized and the free enzyme was found to be similar. A 2.4-fold increase in Km value was observed after immobilization whereas Vmax value was lower for the immobilized GOD. Immobilized glucose oxidase showed improved operational stability by maintaining 33% of the initial activity after 35 cycles of repeated use and was found to retain 94% of activity after 1 month storage period. Improved resistance against urea denaturation was achieved and the immobilized glucose oxidase retained 50% of the activity without urea in the presence of 5M urea whereas free enzyme retained only 8% activity.     

丝素蛋白膜固定β-葡萄糖苷酶及其改良食品风味的研究

从黑曲霉发酵液中提取β-葡萄糖苷酶酶液,用丝素蛋白将其固定,探讨酶固定化的影响因素及固定化酶的性质。β-葡萄糖苷酶的固定化条件为:取0.8 Uβ-葡萄糖苷酶与4.0%戊二醛和10%牛血清白蛋白混合(体积比为5:3:2),涂布于1cm2丝素蛋白膜上交联作用8h。在此条件下获得的固定化酶性质为:最适温度为60℃,比游离酶提高10℃;最适pH为5.0;t1/2为75℃,热稳定性比游离酶有明显改善;最佳反应时间为15 min;与游离酶相比,与底物亲和力降低。将固定化酶膜应用于果汁、果酒、茶汁等食品的增香,经感官鉴评,样品间存在显著差异,进一步经色谱一质谱联用仪分析,发现酶解后的样品,原有香气物质有不同程度的增加,4-萜品醇增加了107%、紫苏醇增加了42%,还有三种未知的香气组分分别增加了251%、79%和33%;并有新风味物质——芳樟醇、香叶醇和2-羟基-5-甲基苯乙酮产生,显示了较好增香效果。     

Immobilization of glucose oxidase on silica-based supports

Glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) was covalently coupled to silica-based supports containing aldehyde functional groups. The activity of the immobilized enzyme was about 1000 U/g support. The optimum pH of the catalytic activity was 5.5 for the soluble enzyme and 6.0 for the immobilized enzyme. With glucose as a substrate the Km value of the immobilized enzyme was higher than in case of the soluble enzyme. The immobilized enzyme was found to be more thermostable than the soluble one. The immobilization did not affect the stability of glucose oxidase against the denaturing effect of urea.     

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.     

Mechanical control of the activity of glucose oxidase immobilized on porous polyvinylchloride membrane

Glucose Oxidase was immobilized on a porous polyvinylchloride (PVC) membrane. The activity of the glucose oxidase-PVC membrane decreased when the membrane was mechanically stretch. A linear relationship was observed between the stress and the relative logarithmic activity of the glucose oxidase-PVC membrane. Apparent Michaelis constant (K_m′) and maximum velocity (V_m′) of the glucose oxidase-PVC membrane under stretched conditions were 1.4 and 0.71 times those without stretching, respectively. The decrease of the membrane activity with stress was reproducible. Therefore, the glucose oxidase activity of the membrane can be controlled with stress.     

Immobilization of glucose oxidase in conducting graft copolymers and determination of glucose amount in orange juices with enzyme electrodes

Glucose oxidase was immobilized in conducting copolymers of three different types of poly(methyl methacrylate-co-thienyl methacrylate). Immobilization of enzyme was carried out by the entrapment in conducting polymers during electrochemical polymerization of pyrrole on the copolymer electrodes. Maximum reaction rate, Michaelis-Menten constants, temperature, pH and operational stabilities were determined for immobilized enzyme. The amount of glucose in orange juices of Turkey was investigated by using enzyme electrodes.     

Effect of enzyme-matrix composition on potentiometric response to glucose using glucose oxidase immobilized on platinum

Glucose oxidase (beta-D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) was immobilized in a crosslinked matrix of bovine serum albumin, catalase, glucose oxidase and glutaraldehyde on platinum foil. When placed in glucose solution, this enzyme-electrode elicited a potentiometric response that varied with the changes in glucose concentration. The immobilized glucose oxidase was present at 7.4-10.1 micrograms enzyme protein/ml of matrix, as determined with 125I-labelled enzyme. The coupled enzyme activity was stable over 120 h; however, the apparent activity of the immobilized glucose oxidase was markedly less than that for the same amount of enzyme free in solution. This indicated a significant level of diffusional resistance within the enzyme-matrix. The potentiometric response to glucose increased significantly as either the thickness of the enzyme-matrix or the glutaraldehyde content was reduced; this also was attributed to diffusional effects. Several enzyme-electrodes, constructed without exogenous catalase and with different amounts of glucose oxidase, showed greater sensitivity in potentiometric response at low glucose oxidase loadings. These results are consistent with the hypothesis that the potentiometric response arises from an interfacial reaction involving a hydrogen peroxide redox couple at a platinum surface. The data also suggest that an optimum range of hydrogen peroxide concentration exists for maximum electrode sensitivity.     

Performance evaluation of a silk protein-based matrix for the enzymatic conversion of tyrosine to L-DOPA

L-DOPA (3,4-dihydroxyphenyl-L-alanine), one of the most important intermediates in the melanin biosynthesis pathway, is used for the treatment of Parkinson's disease. With a view of developing a cheaper and more effective method for the bioconversion of tyrosine to L-DOPA, the potential and performance of a novel fibrous matrix prepared from Bombyx mori silk protein fibroin were evaluated for the immobilization of tyrosinase. Cross-linkage between fibroin and tyrosinase using glutaraldehyde was evident from Fourier transform infra red spectroscopy. Maximum product formation occurred when 1000 U enzyme was immobilized on 20 mg fibroin. The optimum conditions for maximal L-DOPA production using immobilized tyrosinase were 40 degrees C and pH 5.5, conditions that caused a 50% loss of free enzyme activity. Immobilized tyrosinase also showed to have a higher degree of stability during storage and it retained 80% of its original activity after repeated reuses. The efficiency of this immobilized tyrosinase system to produce L-DOPA was high, as evident from a high effectiveness factor, between 0.7 and 0.8, thereby making this method feasible for the large-scale production of L-DOPA.     

Glucose oxidase immobilized polyethylene-g-acrylic acid membrane for glucose oxidase sensor

A polyethylene-g-acrylic acid (PE-g-AA) graft copolymer was prepared via gamma-ray-irradiation-induced postirradiation procedures, and was used as support material for the immobilization of glucose oxidase. Soluble carbodiimides were used as the coupling agent. Reasonable yields were obtained with CMC but not with EDAC, EEDQ, or WRK. A number of factors were studied. (1) The use of water-soluble carbodiimides as condensing agent was attempted and the optimum condition for coupling glucose oxidase to PE-g-AA was established; (2) the effect of pH and temperature on the reactivity of native and immobilized glucose oxidase was studied. When exposed to temperatures in excess of 60 degrees C, the immobilized glucose oxidase was less sensitive to thermal inactivation than the native enzyme. The optimum pH value for the performance of the enzyme-immobilized membrane was 5. 6. For 200 tests, the response error of glucose sensor was less than 4% and its linear detected range was 0-1000 ppm. The obtained glucose oxidase-immobilized PE-g-AA membranes were kept in pH 5. 6 acetate buffer solution at 4 degrees C. The glucose oxidase activity of the membrane was determined at sevenday intervals. The membranes still have 92% glucose oxidase activity even after eight weeks of storage.     

Immobilization of β-galactosidase on modified polypropilene membranes

A new immobilized system: β-galactosidase-modified polypropylene membrane was created. It was obtained 13 different carriers by chemical modification of polypropylene membranes by two stages. The first stage is treatment with K(2)Cr(2)O(7) to receive carboxylic groups on membrane surface. The second stage is treatment with different modified agents ethylendiamine, hexamethylenediamine, hydrazine dihydrochloride, hydroxylamine, o-phenylenediamine, p-phenylenediamine, N,N'-dibenzyl ethylenediamine diacetate to receive amino groups. The quantity of the amino groups, carboxylic groups and the degree of hydrophilicity of unmodified and modified polypropilene membranes were determined. β-Galactosidase was chemically immobilized on the obtained carries by glutaraldehyde. The highest relative activity of immobilized enzyme was recorded at membrane modified with 10% hexamethylenediamine (Membrane 5) - 92.77%. The properties of immobilized β-galactosidase on different modified membranes - pH optimum, temperature optimum, pH stability and thermal stability were investigated and compared with those of free enzyme. The storage stability of all immobilized systems was studied. It was found that the most stable system is immobilized enzyme on Membrane 5. The system has kept 90% of its initial activity at 300th day (pH=6.8; 4°C). The stability of the free and immobilized β-galactosidase on the modified membrane 5 with 10% HMDA in aqueous solutions of alcohols - mono-, diol and triol was studied. The kinetics of enzymatic reaction of free and immobilized β-galactosidase on the modified membrane 5 at 20°C and 40°C and at the optimal pH for both forms of the enzyme were investigated. It was concluded that the modified agent - hexamethylenediamine, with long aliphatic chain ensures the best immobilized β-galactosidase system.     

Entrapment of phenylalanine ammonia-lyase in silk fibroin for protection from proteolytic attack

Phenylalanine ammonia-lyase was entrapped in silk fibroin. The entrapped enzyme showed a similar Km for Phe and pH optimum to the free enzyme. It was resistant against chymotrypsin and trypsin in vitro. To assess the activity in vivo, the free or entrapped enzymes and then Phe were injected into rat duodenum, and cinnamate, a product, in plasma was determined as the most direct evidence of the enzyme activity. The entrapped enzyme but not the free form caused a marked raise of plasma cinnamate. It declined with a half life of about 45 min, which was significantly longer than that (10-15 min) observed upon i.v. administration of cinnamate. These results indicated that the entrapped enzyme was actively degrading Phe in the intestinal tract. Entrapment of phenylalanine ammonia-lyase in fibroin thus provides a new prospect for oral enzyme therapy of phenylketonuria.     

Immobilization of horseradish peroxidase with a regenerated silk fibroin membrane and its application to a tetrathiafulvalene-mediating H2O2 sensor

Horseradish peroxidase (HRP) was immobilized onto a membrane of the regenerated silk fibroin (RSF) from waste milk. The structure of the blend membrane of RSF and HRP was characterized by the use of IR spectra. A second generation of H₂O₂ sensor on the basis of the immobilized HRP was fabricated, in which tetrathiafulvalene acts as mediating electron transfer between the immobilized enzyme and a glassy carbon electrode. Dependencies of pH and temperature on the H₂O₂ biosensor were checked by utilizing cyclic voltammetry. The sensor exhibits high sensitivity, good reproducibility and storage stability.     

Inner epidermis of onion bulb scale: As natural support for immobilization of glucose oxidase and its application in dissolved oxygen based biosensor

Inner epidermal membrane of the onion bulb scales was studied as a natural polymer support for immobilization of the glucose oxidase (GOD) enzyme for biosensor application. Onion epidermal membrane was used for immobilization of glucose oxidase and was associated with dissolved oxygen (DO) probe for biosensor reading. Glucose was detected on the basis of depletion of oxygen, when immobilized GOD oxidizes glucose into gluconolactone. A wide detection range between 22.5 and 450 mg/dl was estimated from calibration plot. A single membrane was reused for 127 reactions with retention of approximately 90% of its initial enzyme activity. Membrane was stable for 45 days ( approximately 90% activity) when stored in buffer at 4 degrees C. Surface structure studies of the immobilized membranes were carried under SEM. To our knowledge, this is the first report on employing inner epidermal membrane of onion bulb scales as the solid support for immobilization of enzyme.     

固定化尿酸酶丝素膜的性质及其尿酸传感器

应用电化学分析法对固定化酶丝素膜的性质进行了分析,结果表明这种酶经丝素膜固定后,活性得率高、性能稳定、能长期存放.用这种酶膜和氧电极等组成的流动注射分析式尿酸传感器对生物样品进行的百次重复分析结果表明,这种传感器的重现性良好,每小时能分析60个人血清样品.     

Various applications of immobilized glucose oxidase and polyphenol oxidase in a conducting polymer matrix

In this study, glucose oxidase and polyphenol oxidase were immobilized in conducting polymer matrices; polypyrrole and poly(N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide-co-pyrrole) via electrochemical method. Fourier transform infrared and scanning electron microscope were employed to characterize the copolymer of (N-(4-(3-thienyl methylene)-oxycarbonyl phenyl) maleimide) with pyrrole. Kinetic parameters, maximum reaction rate and Michealis-Menten constant, were determined. Effects of temperature and pH were examined for immobilized enzymes. Also, storage and operational stabilities of enzyme electrodes were investigated. Glucose and polyphenol oxidase enzyme electrodes were used for determination of the glucose amount in orange juices and human serum and phenolic amount in red wines, respectively.     

Immobilization of glucose oxidase and lactate dehydrogenase onto magnetic nanoparticles for bioprocess monitoring system

Glucose oxidase (GOD) and lactate dehydrogenase (LDH) were immobilized onto magnetic nanoparticles, viz. Fe₃O₄, via carbodiimide and glutaraldehyde. The immobilization efficiency was largely dependent upon the immobilization time and concentration of glutaraldehyde. The magnetic nanoparticles had a mean diameter of 9.3 nm and were superparamagnetic. The immobilization of GOD and LDH on the nanoparticles slightly decreased their saturation magnetization. However, the FT-IR spectra showed that GOD and LDH were immobilized onto the nanoparticles by different binding mechanisms, the reason for which was not well explained. The optimum pH values of the immobilized GOD and LDH were changed to 8 and 10, respectively. The free and immobilized enzyme kinetic parameters (K_m and V_max) were determined by Michaelis-Menten enzyme kinetics. The Km values for free and immobilized GOD were 0.168 and 0.324 mM, respectively, while those for free and immobilized LDH were 0.19 and 0.163 mM for NAD, and 2.976 and 4.785 mM for lactate, respectively. High operational stability was observed, with more than 80% of the initial enzyme activity being retained for the immobilized GOD up to 12 h and for the immobilized LDH up to 24 h. The immobilized GOD was applied to a sequential injection analysis system for the application of bioprocess monitoring.     

Characterization and optimization of β-galactosidase immobilization process on a mixed-matrix membrane

β-Galactosidase is an important enzyme catalyzing not only the hydrolysis of lactose to the monosaccharides glucose and galactose but also the transgalactosylation reaction to produce galacto-oligosaccharides (GOS). In this study, β-galactosidase was immobilized by adsorption on a mixed-matrix membrane containing zirconium dioxide. The maximum β-galactosidase adsorbed on these membranes was 1.6 g/m2, however, maximal activity was achieved at an enzyme concentration of around 0.5 g/m2. The tests conducted to investigate the optimal immobilization parameters suggested that higher immobilization can be achieved under extreme parameters (pH and temperature) but the activity was not retained at such extreme operational parameters. The investigations on immobilized enzymes indicated that no real shift occurred in its optimal temperature after immobilization though the activity in case of immobilized enzyme was better retained at lower temperature (5 °C). A shift of 0.5 unit was observed in optimal pH after immobilization (pH 6.5 to 7). Perhaps the most striking results are the kinetic parameters of the immobilized enzyme; while the Michaelis constant (K(m)) value increased almost eight times compared to the free enzyme, the maximum enzyme velocity (V(max)) remained almost constant.