Immobilization of laccase from Myceliophthora thermophila on functionalized silica nanoparticles: Optimization and application in lindane degradation

This work is focused on immobilization of laccase from Myceliophthora thermophila expressed in Aspergillus oryzae(Novozym 51003? laccase) on amino modified fumed nano-silica(AFNS) and the possible use in bioremediation. Hereby, for the first time, factors affecting the immobilization of Novozym 51003? laccase on AFNS were investigated for defining the immobilization mechanism and optimizing the utilization of AFNS as support for laccase immobilization. The highest specific activity(13.1 IU·mg~(-1) proteins) was achieved at offered 160 mg per g of AFNS and for the same offered protein concentration the highest activity immobilization yield, reaching68.3% after the equilibrium time, at optimum pH 5.0, was obtained. Laccase immobilization occurs by adsorption as monolayer enzyme binding in 40 min, following pseudo-first-order kinetics. The possible use of obtained immobilized preparation was investigated in degradation of pesticide lindane. Within 24 h, lindane concentration was reduced to 56.8% of initial concentration and after seven repeated reuses it retained 70% of the original activity.     

离子液体修饰超顺磁性纳米颗粒用于青霉素G酰化酶固定化(英文)

Functionalized ionic liquids containing ethyoxyl groups were synthesized and immobilized on magnetic silica nanoparticles(MSNP) prepared by two steps,i.e.,Fe3O4 synthesis and silica shell growth on the surface.This magnetic nanoparticle supported ionic liquid(MNP-IL) were applied in the immobilization of penicillin G acylase(PGA).The MSNPs and MNP-ILs were characterized by the means of Fourier transform infrared spectroscopy(FTIR),scanning electron microscopy(SEM),transmission electron microscopy(TEM),and vibrating sample magnetometer(VSM).The results showed that the average size of magnetic Fe3O4 nanoparticles and MSNPs were ~10 and ~90 nm,respectively.The saturation magnetizations of magnetic Fe3O4 nanoparticles and MNP-ILs were 63.7 and 26.9 A?m2?kg?1,respectively.The MNP-IL was successfully applied in the immobilization of PGA.The maximum amount of loaded enzyme was about 209 mg?g?1(based on carrier),and the highest enzyme activity of immobilized PGA(based on ImPGA) was 261 U?g?1.Both the amount of loaded enzyme and the activity of ImPGA are at the same level of or higher than that in previous reports.After 10 consecutive operations,ImPGA still main-tained 62% of its initial activity,indicating the good recovery property of ImPGA activity.The ionic liquid modified magnetic particles integrate the magnetic properties of Fe3O4 and the structure-tunable properties of ionic liquids,and have extensive potential uses in protein immobilization and magnetic bioseparation.This work may open up a novel strategy to immobilize proteins by ionic liquids.     

Immobilization of organophosphorus hydrolase enzyme by covalent attachment on modified cellulose microfibers using different chemical activation strategies: Characterization and stability studies

The plant cellulose powder was activated by two different methods using 1,4-butanediol diglycidyl ether(BTDE)and 1,1′-Carbonyldiimidazole(CDI) as the chemical coupling agents.Organophosphorus hydrolase(OPH) from Flavobacterium ATCC 27551 was immobilized on any of activated support through covalent bonding.The optimal conditions of affecting parameters on enzyme immobilization in both methods were found, and it was demonstrated that the highest activity yields of immobilized OPH onto epoxy and CDI treated cellulose were 68.32%and 73.51%, respectively.The surface treatment of cellulose via covalent coupling with BTDE and CDI agents was proved by FTIR analysis.The kinetic constants of the free and immobilized enzymes were determined, and it was showed that both immobilization techniques moderately increased the Kmvalue of the free OPH.The improvements in storage and thermal stability were investigated and depicted that the half-life of immobilized OPH over the surface of epoxy modified cellulose had a better growth compared to the free and immobilized enzymes onto CDI treated support.Also, the pH stability of the immobilized preparations was enhanced relative to the free counterpart and revealed that all enzyme samples would have the same optimum pH value for stability at 9.0.Additionally, the immobilized OPH onto epoxy and CDI activated cellulose retained about 59% and 68% of their initial activity after ten turns of batch operation, respectively.The results demonstrated the high performance of OPH enzyme in immobilized state onto an inexpensive support with the potential of industrial applications.     

二氧化硅表面银诱导的木瓜蛋白酶的高效固定化和催化

Complexation and interaction between silver and amino group were applied to induce an efficient immobilization of papain on silica spheres.Tbe silver nanoparticles were deposited on the silica spheres before p apainwas coupled to the silica spheres. The silica spheres with silver nanoparticles were characterized by high resolution transmission electron microscopy （HR-TEM）, Fournier transform infrared spectroscopy （FT-IR）, and UV-Vis scanning spectrometer. FT-IR spectrum was also used to characterize the immobilized and free papain. Effect of some factors on the activities of the immobilized papain was investigated. It was observed that the coupled yield and relative activity of the papain on Ag/SiO2 were 1.17 and 1.86 times of those on the bare SiO2, respectively. At an optimum concentration of silver, theobserved activity of the immobilized papain was 2.1 timesof that on the bare.silica.In addition, the maximum specific activity of papain immobilized on Ag/SiO2 was 819.9 U·mg·^-1, which is slightly lower than that of the free papain, 906.2 U·mg^-1 . Stability of the immobilized papain was also examined. The resuits indicate that the silver nanoparticles successfully induce a fine immobilization of papain.     

乳状液膜包酶制备6-APA的研究

Production of 6-aminopenicillanic acid (6-APA) by hydrolysis using penicillin acylase (PA) was studied as a model of an enzymatic emulsion liquid membrane (ELM) process. The loss of PA activity was examined for various membrane compositions (organic solvent, surfactant, carrier). The effects of some experimental variables on the stability of emulsion were investigated. It was found that the choice of organic solvent greatly affected tilestability of the emulsion. Increasing the concentration of the carrier in the membrane phase increases the transfer rate of substrate and products but also has a destabilizing effect on the emulsion. The recovery of 6-APA obtained by a di-carrier system (N263-N1923) was much higher than those when either of the di-carriers was used separately.The whole process was controlled both by the enzymatic reaction rate and by the transfer rate of the substrate and the products, however, the ratio of them could be changed by varying the composition of the system. For an optimum condition, it was obtained that the recovery ratio of 6-APA was over 80% and the conversion of benzyl penicillin (PG) was up to 90% in the external phase after 30 minutes. Meanwhile, the breakage percentage of the emulsion was less than 2%.     

蜡状芽抱杆菌壬基苯酚降解酶的分离纯化及其酶学性质(英文)

An extracellular NP-degrading enzyme secreted by Bacillus cereus.Frankland was purified to homogeneity by a combination of ammonium sulfate precipitation,Phenyl-Sepharose hydrophobic-interaction chromatography and DEAE anion-exchange chromatography.On SDS(sodium dodecyl sulfate)-polyacrylamide gel electrophoresis analysis,the purified enzyme showed a relative molecular mass of 58.3 kDa.The depolymerzation of subunits was accompanied with the loss of NP-degrading enzyme activity,and removing denaturing factors by dialysis could restore the dimer structure and enzymatic activity.The enzyme had an isoelectric point of 5.5 and an optimal temperature of 60℃,and was the most active at pH 6.0.The enzymatic activity was stable at pH 4-8 and inhibited by Cu2+.TenN-terminal amino acids were determined to be ASVNSIKIGY,demonstrating that the purified enzyme was a novel one.The hydrolysis pattern of the purified enzyme indicated that the NP-degrading enzyme was an endo NP-degrading enzyme.The extraordinary thermo-stability provided the enzyme with a good prospect to be used as a new tool for clean-production process for textile industry.     

Membrane Microfiltration Fermentation of Glucose Oxidase with Cell Recycling

Membrane microfiltration fermentation(MMF) with cell recycling was successfully ap-plied to the production of glucose oxidase(GOD).A plate microfiltration module was found suitablefor such purpose.By feeding whole medium in MMF,the productivity of GOD was much higherthan that by feeding glucose alone.With increasing dilution rate the enzyme productivity increasedand average enzyme activity decreased.The enzyme productivity of MMF under D=0.12h~(-1)and0.2Oh~(-1) were 3871 and 3945 U.h~(-1)respectively,which was about 3 times as that of batch fermen-tation(BF)and the average enzyme activity was still as high as 37 U.mL~(-1)under D=0.12h~(-1).The relative efficiency of MMF applied to low yield strain was higher than that applied to high yieldstrain.     

Attachment of Lipase on Amino Functionalized Titania Submicrospheres via Covalent Binding

A facile and effective method for immobilized lipase was presented. The titania submicrospheres were synthesized via a modified sol-gel method followed by amino functionalization through the chelation between dopamine and titania. Lipase was covalently attached on the functionalized titania surface using glutaraldehyde as the cross linking agent. The loading ratio and relative activity of the immobilized lipase were 230 mg/g titania submicrospheres and 65%, respectively. The kinetic parameters including the Michaelis constant (K_m) and the maximum reaction rate (V_max) changed slightly after immobilization. Compared to free lipase, the immobilized lipase showed favorable pH stability, thermostability, recycling stability and storage stability. The immobilized lipase retained 90% activity after incubation at 50 ℃ for 2 h, while the free lipase retained only 60% activity. The immobilized lipase retained more than 80% activity after 8 batches.     

Immobilization of urease in metal–organic frameworks via biomimetic mineralization and its application in urea degradation

Enzyme immobilization has been accepted as an efficient technique for improving the stability and recyclability of enzymes. Herein, biomimetic mineralization strategy was employed to achieve the immobilization of urease in a type of metal–organic frameworks(zeolite imidazolate framework-8, ZIF-8), and the immobilized enzyme urease@ZIF-8 was systematically evaluated for its structure, activity, stability and recyclability, using the hydrolysis of urea as a model. The entrapment of urease was found to be realized in a synchronous manner with the formation of ZIF-8 crystal. The loading of urease in ZIF-8 was measured to be ca. 10.6% through the bicinchoninic acid(BCA) protein assay. The encapsulated urease could efficiently maintain its native conformation, which endowed the immobilized urease with excellent activity and stability, even in harsh conditions(e.g., in the presence of trypsin, acidic or alkali conditions, or at high temperature). Further, urease@ZIF-8 exhibited good recyclability during the degradation of urea, in which it could keep 58.86% of initial activity after being used for 5 cycles. Thus, biomimetic mineralization could be potentially utilized as a promising method to prepare immobilized ureases with superior activity, stability and recyclability, thereby facilitating the construction of efficient catalysts for industrial biocatalysis and biosensing.     

用于环戊烯氧化合成戊二醛反应的钨基催化剂的表征

Tungsten-containing hexagonal mesoporous silica （W-HMS） supported tungsten oxide catalysts （WOx/W-HMS） was prepared for the selective oxidation of cyclopentene with aqueous hydrogen peroxide to glutaraldehyde. X-ray diffraction （XRD） results indicated that the crystal form of the active phase （tungsten oxide） of the WOx/W-HMS catalysts was dependent on the W loading and calcination temperature. X-ray photoelectron spec- troscopy （XPS） analysis revealed that the dispersed tungsten oxides on the surface of W-HMS support consisted of a mixture of W（V） and W（VI）. It was found that a high content of amorphous W species in （5＋） oxidation state resuited in the high catalytic activity. When the W loading was up to 12% （by mass） or the catalyst precursor was treated at temperature of 623 K, the catalytic activity decreased due to the presence of WO3 crystallites and the oxidation of W（V） to W（VI） on the catalyst surface. Furthermore, NH3-temperature-programmed-desorption （NH3-TPD） analysis showed that the effects of W loading and calcination temperature on the acidity of the catalysts were related to the catalytic activity. A high selectivity of 80.2% for glutaraldehyde with a complete conversion of cyclopentene was obtained over 8%WOx/W-HMS catalyst calcined at 573 K after 14 h of reaction.     

介孔纳米孔道中单维交联酶策略固定青霉素酰化酶

为解决酶固定化过程中稳定性较差的问题,将单维交联酶策略引入青霉素酰化酶的固定化中,通过在介孔泡沫硅载体(MCFs)表面初步吸附作用将酶固定,再用对本醌对酶分子进行交联,从而制得单维交联酶聚体。实验结果显示:单维交联酶聚体(CLEA)的稳定性有明显提高,最适温度从游离酶的45℃上升到55℃,催化温度的适用范围更宽,在50℃下热处理25 h仍保持74.4%活力,在测试操作稳定性方面,连续操作15次,其酶活保持在61.9%。     

Improvement of the catalytic performance of immobilized penicillin acylase through assembly of macromolecular reagents in nanopore to create a crowding environment

Macromolecular reagents were co-assembled with penicillin acylase (PA) and immobilized in mesocellular siliceous foams (MCFs) to resemble living cells. Types and concentrations of macromolecules were studied. The catalytic characteristic and stability of PA preparations were also investigated. PA assembled with dextran 10 k in MCFs showed maximum specific activity, 1.32-fold of that of the solely immobilized PA. The optimum pH of dextran and BSA derivatives shifted to neutrality, and the optimum temperature increased by 10 °C. Also, Km of BSA derivative of PA declined 56.7% compared to solely immobilized PA, while the Kcat/Km of PA assembled with BSA was enhanced to 147%. After incubation at 50 °C for 6 h, residual activity of PA assembled with BSA exhibited 53.0%. The ficoll derivative showed 82.8% of its initial activity at 4 °C after 8-week storage. The results indicated that macromolecular reagents assembled with PA in MCFs could dramatically improve the catalytic performance and stability of immobilized enzyme.     

青霉素酰化酶固定化载体材料研究进展

就青霉素酰化酶固定化载体材料的研究和应用进行了论述。重点介绍了功能基化高分子聚合物、含环氧基高分子聚合物、离子型层状水滑石和介孔分子筛等新型载体的结构特性和对青霉素酰化酶的固定化作用,并对近年来固定化生物酶载体材料的发展动向和趋势加以评述和展望。     

修饰固定化青霉素酰化酶在非水相中的催化

为提高酶的催化水解活力和稳定性,将青霉素酰化酶组装于介孔泡沫二氧化硅(MCFs)中,并应用于水/有机混合体系催化水解。分别考察了有机介质种类和体积分数、葡聚糖(Dex10k)修饰对固定化酶活力的影响,研究了不同条件下固定化酶的稳定性。实验结果显示:体积分数20%石油醚中,添加Dex10k的介孔泡沫硅固定化酶比活力达209.5U/mg,是缓冲液中MCFs固定化酶活力的196.2%。20%石油醚中,经25次连续操作,固定化酶保持初始活力的71.5%。结果表明:石油醚等烷烃形成的水/有机体系是适合青霉素酰化酶催化的二相体系,且添加Dex10k能提高固定化酶在二相体系中的催化活力及稳定性。     

亲水性环氧聚合物磁性微球的制备及其固定化青霉素酰化酶

通过设计反相悬浮聚合体系,制备了聚甲基丙烯酸缩水甘油酯（GMA）-甲基丙烯酸羟乙酯（HEMA）- N,N′-亚甲基双丙烯酰胺（MBAA）亲水性磁性聚合物GHM微球。球中的Fe₃O₄微晶呈倒立尖晶石结构,在微球表面存在着大量环氧基和亲水性的羟基及酰胺等基团,这些功能性基团为青霉素酰化酶（PGA）的固定化提供了适宜的微环境;同时,GHM微球具有的大孔结构和较高的比表面积,使其制备的固定化酶的载酶量高,这些有利因素使得固定化酶PGA/GHM在37℃下水解青霉素G钾合成6-氨基青霉烷酸的表观活性达748 IU·g^-1。 PGA/GHM经15次重复使用,其催化活性未出现明显的衰减,在使用中,固定化酶在磁场的作用下能够快速沉降与产物分离。     

小分子封闭提高固定化青霉素酰化酶的稳定性

为提高青霉素酰化酶的催化性能和热稳定性,在酶组装过程中添加小分子试剂对介孔泡沫硅载体表面过量的活化位点进行封闭。详细考察了小分子添加质量分数和种类对青霉素酰化酶负载率、催化活力及热稳定性的影响。实验结果得到:经精氨酸封闭的固定化酶活力提高至1.92倍;甘氨酸封闭的固定化酶5 h的50℃热稳定性提高至2.9倍,甘氨酸和谷氨酸封闭的固定化酶50℃热处理25 h仍保持87.9%和82.2%的残余活力;甘氨酸和谷氨酸封闭的固定化酶最适催化pH值向中性偏移且对pH值的耐受性增强。结果表明,在青霉素酰化酶共价组装过程中添加合适的小分子封闭能显著提高酶的催化性能和热稳定性。     

An integrated downstream processing strategy for the recovery and partial purification of penicillin acylase from crude media

An integrated process strategy for the recovery of penicillin acylase was developed, based on precipitation of non‐enzymatic proteins directly from Escherichia coli homogenates or crude extracts using Rolquat (quaternary ammonium salt) and adsorption of the enzyme on Amp‐Seph (3.8 µmole ampicillin cm^?3) under pseudo‐affinity conditions. The effect of pH, concentrations of ammonium sulfate and Rolquat, and also concentrations of protein and cell debris on the precipitation of non‐enzymatic proteins from homogenates and crude extracts of penicillin acylase were analysed. The method of addition of Rolquat to homogenates and crude extracts significantly influenced the size of the precipitated particles. Improved results on the specific activity of penicillin acylase were obtained for 22% and 1% (w/v) of ammonium sulfate and Rolquat, respectively, added sequentially to enzyme solutions and at room temperature. Under these experimental conditions, the specific activity of penicillin acylase in homogenates and crude extracts was enhanced 2.5–3.0‐fold. Finally, the integrated process strategy was implemented first by precipitation of non‐enzymatic proteins and recovery of penicillin acylase directly from the enzyme solution treated with Rolquat using an adsorption/filtration system with an overall yield of 86%. This system allows simultaneously the filtration of cell debris and fine precipitated particles, in situ recovery of penicillin acylase by its adsorption on Amp‐Seph, and selective desorption of the enzyme with a specific activity of 11 IU (mg prot)^?1 and a desorption yield of 95%. © 2002 Society of Chemical Industry     

Ag/P(St-MMA)纳米复合高分子微球固定化青霉素酰化酶的研究

通过溶剂热法和无皂乳液聚合相结合,制备了P(St-MMA)高分子纳米微球.并以吸附沉积的方式在其表面沉积了Ag金属纳米粒子,最后将青霉素酰化酶共价连接在微球表面.初步研究了微球直径、银的质量分数等因素对固定化酶活力的影响.结果显示随着微球直径减小,固定化酶的偶联率和活力逐渐增加;银纳米粒子最多将固定化酶的偶联率和活力分别提高了42%和72%,固定化酶的最大表观活力(以干重记)达到了1 869 u/g,明显高于其它高分子载体固定化青霉素酰化酶的活力;实验证明银纳米粒子在青霉素水解过程中没有催化活力,但能大大提高青霉素酰化酶的催化活力.     

Immobilization of GL-7-ACA acylase for the production of 7-ACA

Glutaryl-7-aminocephalosporanic acid(GL-7-ACA) acylase is an important enzyme for the production of 7-ACA (7-aminocephalosporanic acid). For an efficient immobilization of GL-7-ACA acylase, various carriers were tested. A high-porous hydrophilic carrier (FPHA) among various carriers tested was found to be the best for the immobilization of GL-7-ACA acylase. In order to develop an effective immobilization method of GL-7-ACA acylase, the parameters that affect the immobilization of GL-7-ACA acylase were also investigated under different conditions of buffer solution and different concentrations of glutaraldehyde. The highest value of GL-7-ACA acylase activity (70 Unit/g-matrix) was obtained when immobilized with 1% glutaraldehyde in a 0.1 M Tris buffer (pH 8.0). Also, in order to enhance the activity of the immobilized GL-7-ACA acylase, unreacted aldehyde groups were quenched by reaction with a low molecular weight agent such as L-lysine after immobilization. The highest activity of immobilized GL-7-ACA acylase was obtained at 0.1% of L-lysine. The immobilized GL-7-ACA acylase was tested for long-term stability and it was found that the activity was retained at about 62% of the initial value after 72 times of reuse at 25 ‡C.     

青霉素酰化酶的分离纯化和固定化研究新进展

青霉素酰化酶又称为青霉素酰胺酶或青霉素氨基水解酶,主要从大肠埃希菌胞内酶和巨大芽孢杆菌胞外酶获得,该酶已大规模应用于工业生产β-内酰胺类抗生素的关键中间体和半合成β-内酰胺类抗生素。主要介绍了青霉素酰化酶固定化技术的进展,讨论了不同固定化技术的特点,并展望了固定化青霉素酰化酶的发展前景。