Immobilization of tyrosinase and alcohol oxidase in conducting copolymers of thiophene functionalized poly(vinyl alcohol) with pyrrole

Immobilization of tyrosinase and alcohol oxidase is achieved in the copolymer of pyrrole with vinyl alcohol with thiophene side groups (PVATh-co-PPy) which is a newly synthesized conducting polymer. PVATh-co-PPy/alcohol oxidase and PVATh-co-PPy/tyrosinase electrodes are constructed by the entrapment of enzyme in conducting copolymer matrix during electrochemical copolymerization. For tyrosinase and alcohol oxidase enzymes, catechol and ethanol are used as the substrates, respectively. Kinetic parameters: maximum reaction rates (V(max)) and Michaelis-Menten constants (K(m)) are obtained. V(max) and K(m) are found as 2.75 micromol/(minelectrode) and 18 mM, respectively, for PVATh-co-PPy/alcohol oxidase electrode and as 0.0091micromol/(minelectrode) and 40 mM, respectively, for PVATh-co-PPy/tyrosinase electrode. Maximum temperature and pH values are investigated and found that both electrodes have a wide working range with respect to both temperature and pH. Operational and storage stabilities show that although they have limited storage stabilities, the enzyme electrodes are useful with respect to operational stabilities.     

A kinetic study on the binding of monomeric and polymeric derivatives of NAD+ to yeast alcohol dehydrogenase

The binding to yeast alcohol dehydrogenase of NAD+ and its five derivatives (N6-[2-[N-[2-[N-(2-methacrylamidoethyl)carbamoyl]ethyl] carbamoyl]ethyl]-NAD (I), N6-[N-[2-[N-(2-methacrylamidoethyl) carbamoyl]ethyl]carbamoylmethyl]-NAD (II), copolymer of I with acrylamide (PA-I), copolymer of II with acrylamide (PA-II), and copolymer of I with N,N-dimethylacrylamide (PDMA-I] were studied statically and kinetically by the stopped-flow method by using the quenching of the enzyme fluorescence in the presence of pyrazole. Apparent dissociation constants and apparent rate constants were determined therefrom. It was concluded that (1) the N6-CH2CH2CO group (of I) is effective in making the derivative bind more strongly as well as faster than NAD+, while the N6-CH2CO group (of II) is not; and (2) the binding of the polymer derivatives of NAD+ to the enzyme is not essentially weaker and slower than that of native NAD+, but is even faster in some cases. The coenzymic activities of the above compounds were also determined with yeast alcohol dehydrogenase, pig heart malate dehydrogenase, and rabbit muscle lactate dehydrogenase.     

Immobilization of Bacillus licheniformis α-amylase onto reactive polymer films

Alpha-amylase was covalently immobilized onto maleic anhydride copolymer films preserving activity. The initial activity of the immobilized layers strongly depended on the immobilization solution, and on the physicochemical properties of the copolymer film. Higher enzyme loading (quantified by amino acid analysis using HPLC) and activity (measured by following starch hydrolysis) were attainable onto hydrophilic, highly swelling 3-D poly(ethylene-alt-maleic anhydride) (PEMA) copolymer films, while immobilization onto hydrophobic poly(octadecene-alt-maleic anhydride) (POMA) copolymer films resulted in low content enzyme layers and lower activity. No significant activity was lost upon dehydration/re-hydration or storage of enzyme containing PEMA copolymer layers in deionised water for up to 48 h. In contrast, α-amylase decorated POMA films suffered a significant activity loss under those conditions. The distinct behaviours may be attributed to the different intrinsic physicochemical properties of the copolymer films. The compact, hydrophobic POMA films possibly favours hydrophobic interactions between the hydrophobic moieties of the protein and the surface, which may result in conformational changes, and consequent loss of activity. Surprisingly, residual activity was found after harsh treatments of active α-amylase PEMA based layers revealing that immobilization onto the hydrophilic polymer films improved the stability of the enzyme.     

Immobilization of tyrosinase in polysiloxane/polypyrrole copolymer matrices

Immobilization of tyrosinase in conducting copolymer matrices of pyrrole functionalized polydimethylsiloxane/polypyrrole (PDMS/PPy) was achieved by electrochemical polymerization. The polysiloxane/polypyrrole/tyrosinase electrode was constructed by the entrapment of enzyme in conducting matrices during electrochemical copolymerization. Maximum reaction rate (V(max)) and Michaelis-Menten constant (K(m)) were investigated for immobilized enzyme. Enzyme electrodes were prepared in two different electrolyte/solvent systems. The effect of supporting electrolytes, p-toluene sulfonic acid and sodium dodecyl sulfate on the enzyme activity and film morphology were determined. Temperature and pH optimization, operational stability and shelf-life of enzyme electrodes were also examined. Phenolic contents of green and black tea were determined by using enzyme electrodes.     

Preparation, characterization, and properties of chitosan-g-poly(vinyl alcohol) copolymer

The graft copolymer chitosan-g-poly(vinyl alcohol), with nontoxicity, biodegradability, and biocompatibility, was prepared by a novel method. The copolymer with porous net structure was observed by scanning electron microscopy (SEM). It is a potential method to combine chitosan with the synthetic polymers. The grafting reactions were conducted with various poly(vinyl alcohol) (PVA)/6-O-succinate-N-phthaloyl-chitosan (PHCSSA) feed ratios to obtain chitosan-g-poly(vinyl alcohol) copolymers with various PVA contents. The chemical structure of the chitosan-g-poly(vinyl alcohol) was characterized by Fourier transform infrared and nuclear magnetic resonance (NMR) spectroscopy. Differential scanning calorimetry (DSC), X-ray diffraction (XRD), and SEM were also detected to characterize the copolymer.     

Synthesis and properties of carrageenan grafted copolymer with poly(vinyl alcohol)

The aim of this work was to prepare a carrageenan-g-poly(vinyl alcohol) (CG-g-PVA) polymer using potassium persulphate as an initiator. The effect of different ratios of the polymer blends on the parameters of the grafted polymer was investigated. The grafting ratio decreased with an increase of the CG content in the graft copolymer. The resulting CG-g-PVA was characterized by ATR-FTIR, tensile strength, elongation at break, swelling ratio, contact angle and biodegradation in soil. From the ATR-FTIR the 3,6-anhydride-galactose of the CG showed a peak at 927 cm⁻¹ that was absent in the CG-g-PVA and the ether linkage of PVA-g-CG between the hydroxyl group of PVA and the 3,6-anhydride-galactose of CG showed a peak at 1089 cm⁻¹ in the graft copolymer. The tensile strength and elongation at break decreased with an increase of the CG due to its phase separation. The highest tensile strength was observed at 2:8 CG/PVA. In addition, the swelling ratio decreased and the contact angle increased as a function of the increase of the CG in the grafted copolymer. The best ratio of CG-g-PVA was 2:8 CG/PVA. This graft copolymer was easily biodegraded in natural soil.     

Electrogenerated chemiluminescence biosensor with alcohol dehydrogenase and tris(2,2'-bipyridyl)ruthenium (II) immobilized in sol-gel hybrid material

An ethanol biosensor based on electrogenerated chemiluminescence detection was developed. Electrogenerated chemiluminescence reagent tris(2,2'-bipyridyl)ruthenium (II) and alcohol dehydrogenase were immobilized in the same sol-gel hybrid film. The copolymer poly(vinyl alcohol) with 4-vinylpyridine and cation exchanger Nafion were incorporated into sol-gel film to provide the microenvironment for retaining the activity of enzyme and immobilize tris(2,2'-bipyridyl)ruthenium (II). The design was simpler than the previous two-layer format. The experimental conditions, such as scan rate, pH and concentration of the cofactor were investigated. The intensity of electrogenerated chemiluminescence increased linearly with ethanol concentration from 2.5x10(-5) to 5.0x10(-2) M and detection limit was 1.0x10(-5) M. The prepared biosensor exhibited high sensitivity, wide linear range and good stability.     

Wood-polyethylene composites using ethylene-vinyl alcohol copolymer as adhesion promoter

Saw dust-reinforced linear low density polyethylene (LLDPE) (1:1) composites were prepared by using ethylene-vinyl alcohol copolymer (EVAL) as adhesion promoter to improve mechanical strength. To evaluate the optimum vinylalcohol (VA) content in EVAL, various EVAL samples containing different contents of VA were used. The tensile properties of saw dust-LLDPE composites were improved by using EVAL as adhesion promoter in place of ethylene-vinyl acetate copolymer (EVAc). The saw dust-LLDPE composites prepared with EVAL containing 15 mol% VA showed the maximum yield stress and modulus. The tensile stress increased with addition of EVAL up to 3wt% on the wood filler, and then leveled off in the range of 3-10 wt%. However, the elongation was decreased with increasing VA content. Hydrogen bonding interaction between saw dust and EVAL was detected by FT-IR spectra. When EVAL consisting with 15 mol% VA was used, good adhesion between saw dust and LLDPE matrix was confirmed by SEM fractography.     

Effect of alcohol feeding mode on the biosynthesis of poly (3-hydroxybutyrate-co-3-hyroxyvalerate)

An alcohol utilizing Alcaligenes eutrophus produced poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) copolymer under phosphate limitation. Fermentation was performed for 42-46 h in a computer-controlled 5-L working volume fed-batch fermentor using ethanol and propanol as carbon sources. The culture experienced phosphate limitation in approximately 19 h. When propanol was used as a sole carbon source, 24 g/L of copolymer with 36.5 mol % of hydroxyvalerate (HV) was produced at a polymer yield of 0.41 g polymer/g alcohol (g/g) and an average polymer production rate of 0.08 g polymer/g residual biomass-h (g/g-h). Two experiments switching alcohol after phosphate exhaustion resulted in better polymer production (g/L), polymer yield (g/g) on alcohol, HV yield (g/g) on propanol, and average polymer production rate (g/g-h) as compared to propanol run without alcohol switching. One switching experiment was from a mixture of 50% ethanol and 50% propanol to 100% propanol and the other experiment was from 100% ethanol to a mixture of 65% ethanol and 35% propanol. Polymer yield for these two experiments was 0.51 g/g and 0.46 g/g, respectively. However, HV mol % in the copolymer for these two runs (30.8 mol % 12.6 mol % respectively) was lower compared to propanol run without alcohol switching (3605 mol %). Direct switch from ethanol to propanol did not support cell growth and polymer production. Polymer production rate and polymer yield changed with time, and the pattern was dependent upon the alcohol feeding mode. (c) 1994 John Wiley & Sons, Inc.     

Sol-gel-derived titanium oxide/copolymer composite based glucose biosensor

A new type of sol-gel-derived titanium oxide/copolymer composite material was developed and used for the construction of glucose biosensor. The composite material merged the best properties of the inorganic species, titanium oxide and the organic copolymer, poly(vinyl alcohol) grafting 4-vinylpyridine (PVA-g-PVP). The glucose oxidase entrapped in the composite matrix retained its bioactivity. Morphologies of the composite-modified electrode and the enzyme electrode were characterized with a scanning electron microscope. The dependence of the current responses on enzyme-loading and pH was studied. The response time of the biosensor was < 20 s and the linear range was up to 9 microM with a sensitivity of 405 nA/microM. The biosensor was stable for at least 1 month. In addition, the tetrathiafulvalene-mediated enzyme electrode was constructed for the decrease of detection potential and the effect of three common physiological sources that might interfere was also investigated.     

Immobilization of horse liver alcohol dehydrogenase on copolymers of glycidyl methacrylate and ethylene dimethacrylate

Alcohol dehydrogenase has been immobilized to the basic copolymer and its several derivatives using various techniques. Enzyme coupling to the supports with amino groups by means of glutaraldehyde was found the most suitable. Activity of alcohol dehydrogenase coupled to these amino supports was comparable to that of the enzyme bound to Sepharose. Thermal and pH stability of alcohol dehydrogenase increased essentially upon immobilization. Kinetic properties of the immobilized enzyme differed from those of free alcohol dehydrogenase, pH optimum shifted to alkaline range, and apparent Michaelis constants for substrates and coenzymes increased. Curvatures observed in Lineweaver-Burk plots for coenzymes suggest an involvement of diffusion effects in the reaction catalyzed by alcohol dehydrogenase linked to these polymers.     

Rat liver alcohol dehydrogenase. I. Purification and characterization

Alcohol dehydrogenase was purified in 14 h from male Fischer-344 rat livers by differential centrifugation, (NH4)2SO4 precipitation, and chromatography over DEAE-Affi-Gel Blue, Affi-Gel Blue, and AMP-agarose. Following HPLC more than 240-fold purification was obtained. Under denaturing conditions, the enzyme migrated as a single protein band (Mr congruent to 40,000) on 10% sodium dodecyl sulfate-polyacrylamide gels. Under nondenaturing conditions, the protein eluted from an HPLC I-125 column as a symmetrical peak with a constant enzyme specific activity. When examined by analytical isoelectric focusing, two protein and two enzyme activity bands comigrated closely together (broad band) between pH 8.8 and 8.9. The pure enzyme showed pH optima for activity between 8.3 and 8.8 in buffers of 0.5 M Tris-HCl, 50 mM 2-(N-cyclohexylamino)ethanesulfonic acid (CHES), and 50 mM 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS), and above pH 9.0 in 50 mM glycyl-glycine. Kinetic studies with the pure enzyme, in 0.5 M Tris-HCl under varying pH conditions, revealed three characteristic ionization constants for activity: 7.4 (pK1); 8.0-8.1 (pK2), and 9.1 (pK3). The latter two probably represent functional groups in the free enzyme; pK1 may represent a functional group in the enzyme-NAD+ complex. Pure enzyme also was used to determine kinetic constants at 37 degrees C in 0.5 M Tris-HCl buffer, pH 7.4 (I = 0.2). The values obtained were Vmax = 2.21 microM/min/mg enzyme, Km for ethanol = 0.156 mM, Km for NAD+ = 0.176 mM, and a dissociation constant for NAD+ = 0.306 mM. These values were used to extrapolate the forward rate of ethanol oxidation by alcohol dehydrogenase in vivo. At pH 7.4 and 10 mM ethanol, the rate was calculated to be 2.4 microM/min/g liver.     

A new amperometric enzyme electrode for alcohol determination

A new enzyme electrode for the determination of alcohols was developed by immobilizing alcohol oxidase in polvinylferrocenium matrix coated on a Pt electrode surface. The amperometric response due to the electrooxidation of enzymatically generated H(2)O(2) was measured at a constant potential of +0.70 V versus SCE. The effects of substrate, buffer and enzyme concentrations, pH and temperature on the response of the electrode were investigated. The optimum pH was found to be pH 8.0 at 30 degrees C. The steady-state current of this enzyme electrode was reproducible within +/-5.0% of the relative error. The sensitivity of the enzyme electrode decreased in the following order: methanol>ethanol>n-butanol>benzyl alcohol. The linear response was observed up to 3.7 mM for methanol, 3.0 mM for ethanol, 6.2 mM for n-butanol, and 5.2 mM for benzyl alcohol. The apparent Michaelis-Menten constant (K(Mapp)) value and the activation energy, E(a), of this immobilized enzyme system were found to be 5.78 mM and 38.07 kJ/mol for methanol, respectively.     

纳米磁性壳聚糖微球固定化酵母醇脱氢酶的研究

建立了以纳米级磁性壳聚糖微球(magnetic chitosan microspheres , M-CS)为载体固定化酵母醇脱氢酶(yeast alcohol dehydrogenase,YADH)的方法,优化了YADH的固定化条件,考察了固定化酶的性质。结果表明,M-CS 呈规则的圆球形,粒径在30nm 左右,具有较好的磁响应性。酵母醇脱氢酶固定化适宜条件为:50 mg 磁性壳聚糖微球,加入20mL 0.25 mg/mL 酵母醇脱氢酶(蛋白质含量)磷酸盐缓冲液(0.05 mol/L ,pH 7.0) ,在4 ℃固定2h。M-CS 容易吸附酵母醇脱氢酶,但吸附的酶量受载体与酶的比例、溶液的离子浓度、溶液pH的影响明显,而温度对吸附的酶量的影响则相对较弱。相对于游离的酵母醇脱氢酶,固定化酶的最适温度略有升高,可明显改善其热稳定性、酸碱稳定性、操作稳定性和贮存稳定性。     

Silica nanotubes-doped alginate gel for yeast alcohol dehydrogenase immobilization

A novel alginate–silica nanotubes (ALG–SiNTs) composite was prepared through the incorporation of silica nanotubes (SiNTs) into the alginate (ALG) gel followed by Ca²⁺ cross-linking for encapsulating yeast alcohol dehydrogenase (YADH, EC 1.1.1.1) from Saccharomyces cerevisiae. Pre-adsorption of YADH onto the surface of SiNTs before encapsulating in alginate gel was adopted to circumvent the enzyme leakage. AFM and SEM characterization confirmed that YADH molecules were substantially adsorbed on the SiNTs. SEM and EDX studies showed that the SiNTs homogenously distributed in alginate matrix. The enzyme leakage from ALG–SiNTs–YADH composite was remarkably reduced about 50% compared to that of ALG–YADH composite. Meanwhile, the optimum reaction condition, catalytic activity and kinetic parameters of immobilized YADH in ALG–SiNTs composite were studied. The results showed that stronger affinity between substrates and enzyme, higher activity retention, improved storage and operational stability were achieved when YADH was immobilized in ALG–SiNTs composite instead of ALG–YADH composite.     

Regulating the molar fraction of 4-hydroxybutyrate in Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) by biological fermentation and enzymatic degradation

The regulation of 4-hydroxybutyrate (4HB) molar fraction in the poly(3-hydroxybutyrate-co-4-hydroxybutyrate) [P(3HB-co-4HB)] of a local isolate Cupriavidus sp. USMAA1020 was attempted by employing a feeding strategy through fed-batch fermentation in 100-L fermenter. The growth of Cupriavidus sp. USMAA1020 was enhanced by frequently feeding carbon and nitrogen at a ratio of 5 (C/N 5) using a DO-stat with cascade mode at 20% (v/v) dissolved oxygen (DO). The feeding of C/N 5 and the use of the DO-stat mode were able to regulate the 4HB composition from 0–67 mol% by sequential feeding of γ-butyrolactone and supplementing oleic acid. A high 4HB molar fraction of 67 mol% with a PHA concentration of 5.2 g/L was successfully obtained by employing this feeding strategy. Notably, enzymatic degradation carried out enhanced the 4HB composition of the copolymer synthesized. PHB depolymerase enzyme from Acidovorax sp. was used to degrade this P(3HB-co-70-mol%4HB) copolymer and the 4HB composition could be increased up to 83 mol%. The degradation process was observed by monitoring the time-dependent change in the weight loss of copolymer films. The percentage of weight loss of solvent-cast film increased proportionally up to 19% within 3 h, whereas salt-leached films showed 90% of weight loss within 3 h of incubation and were completely degraded by 4 h. The molecular weight (M _n ) of the films treated with enzyme demonstrated a slight decrease. SEM observation exhibited a rough surface morphology of the copolymer degraded with depolymerase enzyme.     

聚乙烯醇脱氢酶的提取及检测

为解决结合在细胞上的可溶性蛋白聚乙烯醇脱氢酶（PVADH）的检测困难问题,从提取及检测两方面对该酶进行研究,并对检测方法进行改进。结果表明,非离子型表面活性剂Triton X-100对可溶性蛋白PVADH的提取效果优于离子型表面活性剂炕基苯磺酸钠（LAS）和溴化十六烷基吡啶（CPB）,酶活力比LAS和CPB提取后所得酶活力分别提高246．5％和831．3％。而非离子型表面活性剂中,Triton X-100与Tween80相比,所得最高酶活提高了101．4％。Triton X—100浓度和提取时间对测定有明显影响,以1％Triton X-100提取18h为宜,最高比酶活达14．9U／g。在PVADH检测体系中,加入电子受体启动反应比加入酶液与底物启动反应可使酶活性分别提高60．6％和126．5％;酶液与吡咯喹啉醌（PQQ）预先保温对检测该酶活性是十分重要的,可使酶活性提高59．1％.在检测系统中加入的KCN、CaCl2和PQQ的适宜浓度分别为1．ommol／L、0．5mmol／L和2μmol／L,可使测定酶活分别提高37．1％、38．7％和214．0％．     

Immobilization of porcine pancreatic lipase on glycidyl methacrylate grafted poly vinyl alcohol

Graft copolymerization of glycidyl methacrylate (GMA) on to polyvinyl alcohol (PVA) using benzophenone (BP) as initiator was carried out. Grafted PVA was used as carrier for pancreatic lipase immobilization. The effects of GMA and BP concentrations as well as grafting reaction times on grafting yields and activities of the immobilized lipase were determined. The influence of enzyme concentrations was also studied. The optimal conditions for the grafting reaction were: 1 h at 15 mM BP and 2.3 M GMA, the optimum enzyme concentration for immobilization was 1 mg/ml. After optimization of the immobilization process a physical and chemical characterization of the immobilized enzyme was performed. Furthermore, the thermal, pH, storage and operational stability of the immobilized enzyme in comparison to the free form was tested.     

Immobilization of glucose oxidase in polypyrrole/polytetrahydrofuran graft copolymers

Glucose oxidase (GOD) was immobilized in four different conducting polymer matrices, namely: polypyrrole, (PPy), poly(pyrrole-graft-polytetrahydrofuran), (1) and (3); and poly(pyrrole-graft-polystyrene/polytetrahydrofuran), (2). The kinetic parameters V(max) and K(m), and the optimum temperature were determined for both immobilized and native enzymes. The effect of electrolysis time and several supporting electrolytes, p-toluenesulfonic acid, p-toluene sulfonic acid (PTSA), sodium p-toluene sulfonate, sodium p-toluene sulfonate (NaPTS), and sodium dodecyl sulfate, sodium dodecyl sulfate (SDS), on enzyme immobilization were investigated. The high K(m) value (59.9 mM) of enzyme immobilized in PPy was decreased via immobilization in graft copolymer matrices of pyrrole. V(max), which was 2.25 mM/min for pure PPy, was found as 4.71 mM/min for compound (3).     

磁性纳米颗粒固定化乙醇脱氢酶的研究

本研究采用3-丙氨基三乙氧基硅烷(APTES)和戊二醛修饰包裹有SiO2磁性Fe3O4纳米颗粒表面,将其作为固定化载体固定化乙醇脱氢酶,研究固定化条件对固定化效率的影响,并对固定化酶性质进行分析。研究发现,当Fe3O4@SiO2纳米颗粒修饰上氨基和醛基后依然具有良好的水分散性和胶体稳定性,适合作为固定化载体。通过单因素优化,发现当最适给酶量为11. 3U/100 mg,搅拌转速为150 r/min,固定化p H和固定化温度分别控制在6. 5和5℃～15℃,固定化时长为45 min时,具有较好的固定化效果,固定化率可达到60. 2%。在此条件下制备得到的固定化酶与游离酶相比,固定化酶具有良好的耐高温和耐碱性。所得固定化乙醇脱氢酶在连续使用8次后,固定化率仍保留在57%左右,表明该固定化酶具有较好的操作稳定性,可为连续生产NADH提供技术依据。