金属框架结构材料MOF-199对漆酶的固定化及其性质

以金属框架结构材料MOF-199为载体对漆酶进行固定化,并对固定化酶的性质进行初步研究。首先,以3-氨基丙基三乙氧基硅烷对载体MOF-199进行表面氨基化修饰,再用戊二醛对载体进行活化,最后对漆酶进行固定化。固定化条件优化结果表明:在漆酶质量浓度0.3 g/L,戊二醛用量1%(体积分数),pH 4.8下固定7 h,制得固定化酶活性最高。对固定化酶的研究发现:最适反应温度为40℃,最适pH为5.2,在连续操作7次后,固定化酶的活力仍能保持在51%。固定化漆酶热稳定性,pH耐受性,贮存稳定性均明显高于游离漆酶。     

Laccase stabilization by covalent binding immobilization on activated polyvinyl alcohol carrier

AIMS: Attempts were made to immobilize laccase from Panus conchatus. METHODS AND RESULTS: The laccase was immobilized on carboxylated polyvinyl alcohol (PVA) activated by N-hydroxysuccinimide (N-HSI) in aqueous solution at different pHs, temperatures, and with different reaction times. An optimum condition for laccase immobilization is at pH 3.2, 40 degrees C and 12 h, respectively. Immobilization of laccase increased optimal pH for reaction with 2, 2'-azinobis (3-ethylbenzthiazoline-6-solfonate) (ABTS) and pH stability. Immobilized laccase proved to be reacted consecutively 17 times with only a 50% decrease on activity and be used in removal of 2,4,6-trichlorophenol (TCP). CONCLUSIONS: It was possible to immobilize the laccase on carboxylated polyvinyl alcohol by activation with N-hydroxysuccinimide in HAc-NaAc buffer. The immobilized laccase is both stable and reusable. SIGNIFICANCE IMPACT OF THE STUDY: The results indicate that this immobilized laccase can be used in the removal of poisonous effluent from pulp bleaching mills.     

Optimum conditions for lipase immobilization on chitosan-coated Fe3O4 nanoparticles

Magnetic Fe₃O₄-chitosan nanoparticles are prepared by the coagulation of an aqueous solution of chitosan with Fe₃O₄ nanoparticles. The characterization of Fe₃O₄-chitosan is analyzed by FTIR, FESEM, and SQUID magnetometry. The Fe₃O₄-chitosan nanoparticles are used for the covalent immobilization of lipase from Candida rugosa using N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) as coupling agents. The response surface methodology (RSM) was employed to search the optimal immobilization conditions and understand the significance of the factors affecting the immobilized lipase activity. Based on the ridge max analysis, the optimum immobilization conditions were immobilization time 2.14 h, pH 6.37, and enzyme/support ratio 0.73 (w/w); the highest activity obtained was 20 U/g Fe₃O₄-chitosan. After twenty repeated uses, the immobilized lipase retains over 83% of its original activity. The immobilized lipase shows better operational stability, including wider thermal and pH ranges, and remains stable after 13 days of storage at 25 °C.     

Probing fundamental film parameters of immobilized enzymes--towards enhanced biosensor performance. Part I--QCM-D mass and rheological measurements

Enzyme immobilization is an ever-growing research-area for both analytical and industrial applications. Of critical importance in this area are the effects of immobilization procedures upon the functionality of the immobilized biomolecules. Both beneficial and detrimental effects can be conferred through the selection and tuning of the immobilization procedure. Quartz-crystal microbalance with dissipation (QCM-D) has been previously used to great effect in tracking alterations to thin films of biomolecules immobilized onto quartz transducers.In this study, we investigate the ability of QCM-D to track and monitor film parameters of a monolayer of laccase immobilized on a series of self-assembled monolayers (SAMs), differing in lateral density of binding residues on the SAM and height of the SAM from the quartz surface. Both mass gains and rheological parameters for these varying surfaces were measured and trends later compared to the apparent enzyme kinetics of the immobilized laccase films, assessed electroanalytically (Paper II in this two part study). For covalent attachment of proteins, both shear and viscosity were increased relative to physically adsorbed proteins. An increase in lateral density of protein-binding surface of the SAM components was shown to increase the shear/viscosity of the resultant film while an increase in distance from the electrode (through incorporation of lysine linkers) was shown to decrease the shear/viscosity while simultaneously increasing the wet mass gain of the films. Shear and viscosity may be indicative of both enzyme denaturation and increased lateral protein packing within the film structure hence it is assumed that less distortion occurs with the inclusion of linkers which allow for more optimal protein immobilization.     

漆酶在磁性壳聚糖微球上的固定及其酶学性质研究

以磁性壳聚糖微球为载体,戊二醛为交联剂,共价结合制备固定化漆酶。探讨了漆酶固定化的影响因素,并对固定化漆酶的性质进行了研究。确定漆酶固定化适宜条件为：50 mg磁性壳聚糖微球,加入10mL 0.8mg/mL 漆酶磷酸盐缓冲液（0.1mol/L,pH 7.0）,在4℃固定2h。固定化酶最适pH为3.0, 最适温度分别为10℃和55℃,均比游离酶降低5℃。在pH 3.0,温度37℃时,固定化酶对ABTS的表观米氏常数为171.1μmol/L。与游离酶相比,该固定化漆酶热稳定性明显提高,并具有良好的操作和存储稳定性。     

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。     

聚乙烯醇-明胶复合膜固定化重组大肠杆菌NAD激酶的研究

为提高烟酰胺腺嘌呤二核苷酸(NAD)激酶的稳定性,采用复合膜对NAD激酶进行固定化研究。选用聚乙烯醇(PVA)、聚乳酸(PLA)、海藻酸钠(SA)和明胶(GEL)膜材料固定化NAD激酶。通过单因素实验确定最佳固定化条件为:PVA∶GEL为4∶1,加酶量为0.6 mL,固定化时间为6h,固定化温度为35℃,此时酶活力回收率达到最高值84%。固定化酶酶学性质分析结果表明,与游离酶进行比较,固定化后NAD激酶的最适温度由50℃提高至55℃,最适pH由8.0降至7.0,NAD激酶的热稳定性和pH稳定性均得到显著提高,但固定化酶的亲和力降低。固定化NAD激酶重复利用6次后,酶活性依然可维持初始酶活性的75%以上,表明聚乙烯醇-明胶复合膜固定化酶具有良好的操作稳定性。     

Synthesis of electroconductive polyaniline using immobilized laccase

A new method for synthesis of the conductive complex between polyaniline (PANI) and poly(2-acrylamido-2-methyl-1-propanosulfonic acid) (PAMPS) was proposed; in this method, the immobilized laccase from the basidiomycete Trametes hirsuta is used as a biocatalyst for aniline oxidative polymerization. The conditions for laccase immobilization on CM cellulose by bifunctional Woodward’s reagent were optimized. The catalytic properties of immobilized and native laccases were compared. The immobilized laccase appeared an efficient catalyst for the oxidative radical polymerization of aniline on polysulfonic acid matrix at 4°C. It was demonstrated that the immobilized enzyme could be repeatedly used for enzymatic synthesis of this polymer. Several spectral characteristics of the PANI/PAMPS complexes synthesized at various pH values were studied. The conductance of PANI specimens produced using immobilized laccase as a catalyst was 13 mS/cm.     

Immobilization of Laccase by Alginate–Chitosan Microcapsules and its Use in Dye Decolorization

Laccase is a ligninolytic enzyme that is widespread in white-rot fungi. Alginate–chitosan microcapsules prepared by an emulsification–internal gelation technique were used to immobilize laccase. Parameters of the immobilization process were optimized. Under the optimal immobilization conditions (2% sodium alginate, 2% CaCl₂, 0.3% chitosan and 1:8 ratio by volume of enzyme to alginate), the loading efficiency and immobilized yield of immobilized laccase were 88.12% and 46.93%, respectively. Laccase stability was increased after immobilization. Both the free and immobilized laccase alone showed a very low decolorization efficiency when Alizarin Red was selected for dye decolorization test. When 0.1 mM 2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was added into the decolorization system, the decolorization efficiency increased significantly. Immobilized laccase retained 35.73% activity after three reaction cycles. The result demonstrated that immobilized laccase has potential application in dyestuff treatment.     

Immobilization of alpha(1)-acid glycoprotein for chromatographic studies of drug-protein binding

A new method for preparing immobilized alpha1-acid glycoprotein (AGP) for use in drug-protein binding studies was developed and optimized. In this approach, periodate was used under mild conditions to oxidize the carbohydrate chains in AGP for attachment to a hydrazide-activated support. The final conditions chosen for this oxidation involved the reaction of 5.0 mg/mL AGP at 4 degrees C and pH 7.0 with 5-20 mM periodic acid for 10 min. These conditions helped maximize the immobilization of AGP without significantly affecting its activity. This method was evaluated by using it to attach AGP to silica for use in high-performance affinity chromatography and self-competition zonal elution studies. In work with R- and S-propranolol, only one type of binding site was observed for both enantiomers on the immobilized AGP, in agreement with previous studies using soluble AGP. The association equilibrium constants measured for the immobilized AGP with R- and S-propranolol at pH 7.4 and 37 degrees C were 2.7 x 10(6) and 4.2 x 10(6) M(-1), respectively, with linear van't Hoff plots being obtained between 5 and 37 degrees C. Work performed with other drugs also gave good agreement between the behavior seen for immobilized AGP and that for soluble AGP. The same immobilization method described in this work could be used to attach AGP to other materials, such as those used for surface plasmon resonance or alternative biosensors.     

Immobilized laccase of Cerrena unicolor for elimination of endocrine disruptor micropollutants

The white-rot fungus Cerrena unicolor C-139 produced 450?000 U l⁻¹ of laccase when cultivated in submerged (50 ml) fermentation of wheat bran. Laccase (benzenediol: oxygen oxidoreductase, EC 1.10.3.2.), from C. unicolor C-139 was immobilized covalently on control porosity carrier silica beads. The activity of the immobilized laccase was approximately 15.8 units per gram of silica beads. The pH optimum was between 2.5 and 3.0 for free and immobilized laccase. The immobilization of enzyme appeared to be the main factor for retention of laccase activity at high temperature of 80 °C. The apparent K_m value (100 μmol) of immobilized laccase from C. unicolor C-139 was 6.7 times higher than free laccase (15 μmol) using 2,2-azino-bis-[3-ethylthiazoline-6-sulfonate] (ABTS) as the substrate. Immobilized laccase was able to eliminate 80 % of Bisphenol A, 40 % of Nonylphenol, and 60 % of Triclosan from solutions containing 50 μmol of each micropollutant separately. The experiments were run three times consecutively with the same immobilized laccase without loss of enzyme activity.     

Study on Remediation-improvement of 2,4-Dichlorophenol Contaminated Soil by Organic Fertilizer Immobilized Laccase

ABSTRACT

In this paper, laccase is immobilized by the cross-linking method, using organic fertilizer as a carrier and glutaraldehyde as a cross-linking agent. Here, the optimal conditions of laccase immobilization were explored and the optimal operating conditions and stabilities of free laccase and immobilized laccase were also studied. Then, free laccase and immobilized laccase were applied to the soil remediation. Meanwhile, the effect of soil improvement treated with immobilized laccase was studied through ecological evaluation. The results showed that the optimal conditions for laccase immobilization were: the volume fraction of glutaraldehyde was 5%, the amount of enzyme added was 15 mL, and the immobilization time was 6 h. Under the same conditions, thermal stability and acid-base stability of immobilized laccase were better than free laccase. Under the optimal conditions, using laccase to treat 2,4-dichlorophenol in the soil, it was found that the free laccase group degraded 44.4% within 5 days, while the immobilized laccase group degraded 58.6%. Although both the degradation trends and route are the same, the degradation effect of the latter is obviously better. Ecological evaluation showed that organic fertilizer carrier had an impact on soil physical and chemical properties and soil enzymes, playing a positive role in soil ecological security and improving the soil.     

α-环状糊精葡萄糖基转移酶的固定化及其性质研究

研究了一种α-环糊精葡萄糖基转移酶的固定化和固定化酶的性质。通过对戊二醛浓度、酶量和交联时间各单因素的考察,确定了最佳的固定化条件。与游离酶相比,以DEAE纤维素为载体的固定化酶最适pH向酸性偏移,最适温度不变,pH稳定性和热稳定性都有所提高。在40℃、150r/min下反应3h,转化率可以达到32%。固定化酶可以连续使用4次以上。固定化酶在4℃、5mmol/L CaCl2溶液里保存18d,还剩余80%以上的活力。     

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%.     

Bacterial cellulose as carrier for immobilization of laccase: Optimization and characterization

Bacterial cellulose (BC) has attracted attention as a new functional material due to its excellent mechanical strength, tridimensional nanostructure, high purity, and increased water absorption, compared to plant cellulose. In this work, commercial laccase was immobilized on BC and the influence of enzyme concentration, contact time, and pH was optimized toward the recovery activity of immobilized laccase. This optimization was carried out using a 3³ experimental design and response surface methodology. Enzyme concentration played a critical role in laccase immobilization. Under optimized conditions (0.15 μL L^?1 of enzyme concentration, 4.8 h of contact time, pH 5.4), the predicted and experimental response were equal to 47.88 and 49.30%, respectively. The thermal stability of the immobilized laccase was found to increase notably at 60 and 70°C presenting stabilization factor equal to 1.79 and 2.11, respectively. The immobilized laccase showed high operational stability, since it retained 86% of its initial activity after seven consecutive biocatalytic cycles of reaction with 2,2′‐azinobis‐(3‐ethylbenzothiazoline‐6‐sulfonic acid). Kinetic studies showed that the values of Michaelis–Menten constant and maximum reaction rate decreased upon immobilization (9.9‐ and 1.6‐fold, respectively). Globally, the use of immobilized laccase on BC offers an interesting tool for industrial biocatalytic applications.     

γ-Fe_2O_3-凹土超顺磁性纳米复合材料固载猪胰脂肪酶

以凹凸棒石黏土为原料,制备γ-Fe2O3-凹土超顺磁性纳米复合材料(γ-Fe2O3-ATP)作为猪胰脂肪酶(PPL)固定化的载体,利用透射电子显微镜(TEM)、N2吸附脱附等温图(BET)、振动试样磁强计(VSM)等对材料进行表征,同时对固定化条件和固定化酶的相关性质进行了研究。结果表明:制备的γ-Fe2O3-ATP是介孔材料,比表面积为102.63 m2/g,平均孔径为10.862 nm,饱和磁化强度为8.915 emu/g,其作为载体能实现固定化酶与反应介质简单、快速分离回收和重复利用。在固定化时间为4 h及pH 6.0时制备的固定化酶效果最佳;经过6 h高温保存后固定化酶可保留初始酶活的52%,而游离酶仅保留初始酶活的19%,同时固定化酶在重复使用5次后酶活仍保留初始酶活的60%。     

Laccase immobilization on enzymatically functionalized polyamide 6,6 fibres

Polyamide matrices, such as membranes, gels and non-wovens, have been applied as supports for enzyme immobilization, although in literature the enzyme immobilization on woven nylon matrices is rarely reported. In this work, a protocol for a Trametes hirsuta laccase immobilization using woven polyamide 6,6 (nylon) was developed. A 2⁴ full factorial design was used to study the influence of pH, spacer (1,6-hexanediamine), enzyme and crosslinker concentration on the efficiency of immobilization. The factors enzyme dosage and spacer seem to have played a critical role in the immobilization of laccase onto nylon support. Under optimized working conditions (29 U mL⁻¹ of laccase, 10% of glutaraldehyde, pH = 5.5, with the presence of the spacer), the half-life time attained was about 78 h (18% higher than that of free enzyme), the protein retention was 30% and the immobilization yield was 2%. The immobilized laccase has potential for application in the continuous decolourization of textile effluents, where it can be applied into a membrane reactor.     

A hydroquinone biosensor using modified core-shell magnetic nanoparticles supported on carbon paste electrode

A hydroquinone biosensor was developed and used to determine hydroquinone concentration in compost extracts based on the immobilization of laccase on the surface of modified magnetic core-shell (Fe(3)O(4)-SiO2) nanoparticles. Laccase was covalently immobilized on the magnetic nanoparticles by glutaraldehyde, which was modified with amino groups on its surface. The obtained magnetic bio-nanoparticles were attached to the surface of carbon paste electrode with the aid of a permanent magnet to determine hydroquinone. A good microenvironment for retaining the bioactivity of laccase was provided by the immobilization matrix. The linear range for hydroquinone determination was 1 x 10(-7) to 1.375 x 10(-4)M, with a detection limit of 1.5 x 10(-8)M. The current reached 95% of the steady-state current within about 60s. Hydroquinone concentration in compost extracts was determined by laccase biosensor and HPLC, the results of the two methods were approximately the same.     

Degradation of chlorophenols catalyzed by laccase

The degradations of 2,4-dichlorophenol (2,4-DCP), 4-chlorophenol (4-CP) and 2-chlorophenol (2-CP) catalyzed by laccase were carried out. The optimal condition regarding degradation efficiency was also discussed, which included reaction time, pH value, temperature, concentration series of chlorophenols and laccase. Results showed that the capability of laccase was the best, while to oxidize 2,4-DCP among the above-mentioned chlorophenols. Within 10 h, the removal efficiency of 2,4-DCP, 2-CP and 4-CP could reach 94%, 75% and 69%, respectively. The optimal pH for laccase to degrade chlorophenols was around 5.5. The increase of laccase concentration or temperature might result in the degradation promotion. The trends of degradation percentage were various among these three chlorophenols with the concentration increase of chlorophenols. Degradation of 2,4-DCP is a first-order reaction and the reaction activation energy is about 44.8 kJ mol⁻¹. When laccase was immobilized on chitosan, crosslinked with glutaraldehyde, the activity of immobilized laccase was lower than that of free laccase, but the stability improved significantly. The removal efficiency of immobilized laccase to 2,4-DCP still remained over 65% after six cycles of operation.