大尺寸SiO2大孔材料固定化漆酶

以表面固定Cu2+的改性大尺寸SiO2大孔材料作为载体,考察了时间、pH和给酶量对漆酶固定化效果的影响,并对固定化漆酶的活性和稳定性进行了研究。结果表明:5 h时吸附达到平衡,pH为4.5、漆酶与载体比例为5 mg·g-1时固定化效果最好,酶活回收率可达到100.4%;固定化漆酶的最适pH和最适温度较游离漆酶的均有升高且范围变宽,固定化后,漆酶的pH稳定性和热稳定性都得到显著提高;固定化漆酶的K m值略高于游离漆酶的;固定化漆酶具有良好的操作稳定性,与底物反应反复操作10批次后剩余酶活为72.7%。     

介孔SiO2/Fe3O4中空磁性微球的漆酶固定化

以介孔SiO2/Fe3O4磁性中空微球作为载体,采用物理吸附法对漆酶进行固定化,考察了时间、温度和pH值对漆酶固定化效果的影响,并对固定漆酶的活性及稳定性进行了研究.结果表明,介孔SiO2/Fe3O4磁性中空微球吸附漆酶分子后,介孔材料的比表面积与孔体积均减小.在3 h时复合微球对漆酶的吸附达到平衡,复合微球中介孔SiO2对漆酶的有效固定量为689 mg/g,大大高于纯介孔材料MCM-41的漆酶固定量(319 mg/g).在pH=3～6的条件下,复合微球中固定漆酶仍保持70%以上的相对酶活.当温度不高于60℃时,固定漆酶的相对酶活仍保持65%以上.固定漆酶的pH稳定性和热稳定性都明显优于游离漆酶,固定漆酶的米氏常数为1.05 mmol/L,与游离漆酶相比,固定漆酶与底物的亲和力有所降低.当2,4-二氯苯酚的浓度为10 mg/L时,固定漆酶对其去除率在6 h时达到81.6%,表现出很好的催化活性.     

漆酶在纳米多孔金上的固定化及其酶学性质研究

利用纳米材料为载体对酶等生物大分子进行固定化近年来引起人们的浓厚兴趣. 以Au/Ag合金为原料, 通过控制浓硝酸的腐蚀时间再辅以退火处理得到了不同孔径的纳米多孔金(NPG), 利用扫描电镜(SEM)和N2气体吸附仪对孔性质进行了表征. 以NPG为载体, 用α-硫辛酸和N-乙基-N’-(3-二甲基氨基丙基)碳酰二亚胺/N-羟基琥珀酰亚胺(EDC/NHS)对金表面进行活化, 通过化学共价偶联的方法对产自Trametes versicolor的漆酶进行了固定化. 比较了孔径大小对酶固定化量及比活力的影响. 发现小孔径更有利于对该漆酶的固定化. 与游离酶相比, 固定化酶的最适pH没有改变, 但最适温度却从原来的40 ℃升到了60 ℃. 固定化后, 漆酶的pH和热稳定性都明显提高了. 重复使用8次仍能保持初始活力的65%, 且在4 ℃下保存1个月几乎观察不到酶活力的下降. 此外, 失活的固定化酶经浓硝酸处理后, NPG载体可重复利用. 本结果初步显示出了NPG在生物技术领域中的应用潜力.     

Immobilization of laccase by Cu2+ chelate affinity interaction on surface modified magnetic silica particles and its use for the removal of pentachlorophenol

Magnetic Cu²⁺-chelated silica particles using polyacrylamide as a metal-chelating ligand was developed and used for the immobilization of laccase by coordination.The effect of pH and temperature on the enzymatic property of immobilized laccase and its catalytic capacity for pentachlorophenol（PCP） degradation were evaluated systemically.Compared with free laccase,the immobilized laccase showed improved acid adaptabihty and thermal stability.The immobilized laccase prepared in this work exhibited a good catalytic capacity for PCP removal from aqueous solutions.     

漆树酶固定化及其性质研究

本文介绍了用过渡金属水合氧化物固定化漆树漆酶的方法。考察了整合法固定漆树酶的最适条件。对固定化前后漆酶的性质进行了比较,并分析了造成固定化酶与游离酶性质差异的原因。     

Immobilized Laccase on Activated Poly(Vinyl Alcohol) Microspheres For Enzyme Thermistor Application

Poly(vinyl alcohol) (PVA) microspheres were prepared by inverse suspension crosslinked method, with glutaraldehyde as a crosslinking agent. PVA microspheres activated with aldehyde groups were employed for Trametes versicolor laccase immobilization. Fourier transform infrared spectroscopy and X-ray photoelectron spectroscopy were used to characterize the activated PVA microspheres and PVA microspheres with immobilized laccase (Lac/PVA microspheres), which show that laccase was successfully immobilized on the PVA microspheres. The optimum pH and temperature coupling conditions for the immobilized laccase were determined to be 3.3 and 30 °C, respectively. Residual activity was also investigated by soaking the immobilized laccase in organic solvents at different concentrations, proving it chemically stable. Immobilized laccase exhibited good storage stability at 4 °C. The enzyme biosensor showed good performance in 2,2-azinobis(3-ethylthiazoline-6-sulfonate) and bisphenol A, with concentration ranges of 2 to 8 mM and 0.05 to 0.25 mM, respectively. Therefore, PVA microspheres may have high potential as support for enzyme thermistor applications.     

Laccase immobilized on mesoporous SiO<Subscript>2</Subscript> and its use for degradation of chlorophenol pesticides

In this paper, mesoporous silica with large specific surface area was used to immobilize laccase by the glutaraldehyde cross-linking method, and after screening and optimization experiments, the best enzyme immobilization process conditions were found (25°C, pH 5.4, 4% glutaraldehyde and 0.2 g/L laccase, treatment time 6 h). After that, the removal and degradation ratio of 2,4-dichlorophenol (abbreviated as DCP) under different conditions were also studied. After the degradation process was performed for 6 h at 30°C, pH 5.4, and DCP initial concentration of 50 mg/L in the presence of 0.1 g of immobilized laccase, the removal ratio and the degradation ratio were 42.28 and 15.93%, respectively. Compared with free laccase, the reusability of immobilized laccase is significantly improved.     

Screening thermotolerant white-rot fungi for decolorization of wastewaters

To select a thermotolerant fungal strain for decolorization of wastewaters, ligninolytic enzyme production (lignin peroxidase, manganese peroxidase [MnP], and laccase), decolorization, and removal of total phenol and chemical oxygen demand (COD) were detected. Thirty-eight fungal strains were studied for enzyme production at 35 and 43°C on modified Kirk agar medium including 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and MnCl₂. Thirteen strains grew on manganese-containing agar and provided green color on ABTS-containing agar plates under culture at 43°C. Decolorization of wastewater from alcohol distillery (WAD) by these strains was compared under static culture at 43°C, and Pycnoporus coccineus FPF 97091303 showed the highest potential. Thereafter, immobilized mycelia were compared with free mycelia for WAD decolorization under culture conditions of 43°C and 100 rpm. The immobilized mycelia on polyurethane foam enhanced the ligninolytic enzyme production as well as total phenol and color removal. At about the same COD removal, MnP and laccase produced by immobilized mycelia were 2 and 19 times higher than by free mycelia; the simultaneous total phenol and color removal were 3.1 and 1.5 times higher than the latter. Moreover, decolorization of synthesis dye wastewater was carried out at 43°C and 100 rpm. More than 80% of 300 mg/L of reactive blue-5 was decolorized by the immobilized mycelia within 1 to 2 d for four cycles.     

Effects of fungal laccase immobilization procedures for the development of a biosensor for phenol compounds

Fungal laccase was immobilized on carbon-fiber electrodes using classical methods: physical adsorption, glutaraldehyde, carbodiimide and carbodiimide/glutaraldehyde. The highest biosensor response was obtained using carbodiimide/glutaraldehyde for coupling laccase to carboxyl groups on the carbon fibers. In this method, different percentages of glutaraldehyde had important effects on the sensitivity of the biosensor, the best percentage of glutaraldehyde being 10% (m/v). The behavior of the obtained biosensor was investigated in terms of sensitivity, operational range, pH and applied potential. The developed biosensor showed an optimum response at pH 5.0 and at an applied potential of -100 mV. The immobilized laccase retained a good activity for over 2 months.     

新型有机-无机纳米复合粒子的制备及其固定化漆酶研究

制备了四氨基酞菁钴(CoTAPc)-Fe3O4纳米复合粒子, 用红外光谱(IR)、X射线衍射(XRD)、X射线能谱(EDS)、场发射扫描电镜(FEG-SEM)及振动样品磁强计等对其进行了表征. 结果表明, 形成了CoTAPc包覆在Fe3O4纳米粒子表面的纳米复合粒子. 粒子呈现不规则球形, 平均粒径为70 nm, 矫顽力为316.4 A/m, 接近超顺磁性. 以此纳米复合粒子作为载体, 通过交联法固定漆酶, 固定化酶最适反应温度为45 ℃, 最适pH为3; 固定化酶比游离酶具有更好的热稳定性、贮存稳定性及操作稳定性, 且易于分离.     

CuTAPc-Fe3O4纳米复合粒子及其漆酶固定化研究

漆酶的固定化研究对基于漆酶催化的光纤生物传感器具有十分重要的意义，制备了四氨基酞菁铜(CuTAPc)-Fe3O4纳米复合粒子，并用红外(IR)、场发射扫描电镜(FEG—SEM)、X射线衍射(XRD)、能谱、粒径仪等对其进行了表征．结果表明形成了以CuTAPc包覆在Fe3O4纳米粒子表面的纳米复合粒子，粒子呈现不规则球形，且分布均匀，粒子平均粒径在50nm左右，用此纳米复合粒子通过戊二醛交联法固定了漆酶，固定后的酶比游离酶具有更好的贮存稳定性及操作稳定性，这为研制高性能的光纤生物传感器打下了较好的基础。     

高分子聚合物-多壁碳纳米管复合物固定漆酶及其在玻碳电极上的直接电子迁移

采用不同结构的高分子聚合物与纯化的多壁碳纳米管(MWCNTs)共混的方法,制备得到聚合物非共价功能化多壁碳管复合物,测定了这些载体对漆酶(lac)的担载量、固定漆酶的比活力及稳定性.以固定漆酶的复合物修饰玻碳(GC)电极后,采用循环伏安法研究这些电极在无氧磷酸盐缓冲液(PBS)中的直接电化学行为及催化氧还原活力,粗略地测定了固定漆酶与电极间电子转移的速率常数.实验结果表明,当聚合物中含亲漆酶基团或能与漆酶活性中心发生相互作用的官能团时利于直接电子转移,而且复合物固定漆酶保持了游离漆酶的天然构象.这些电极中,lac/NIPAM-co-BPCP-M WCNTs/GC(NIPAM-co-BPCP:N-烯丙基-1-苯甲酰基-3-苯基-4,5-2H-4-甲酰胺基吡唑-co-N-异丙基丙烯酰胺)在无氧PBS中发生直接电子转移的式电位(605mV)更接近漆酶活性中心的式电位(580mV),具有较快的异相电子转移速率(0.726s-1),较高的漆酶担载量(103.5mg/g)和固定漆酶比活力(1.68U/mg),较高的催化氧还原能力(氧还原起始电位820mV,在650mV时的催化峰电流为85.5μA)以及良好的重复使用性和长期使用性.     

Laccase–polyazetidine prepolymer–MWCNT integrated system: Biochemical properties and application to analytical determinations in real samples

Bioelectrochemical properties of Trametes versicolor Laccase (TvL) and Trametes hirsuta Laccase (ThL) immobilized by using polyazetidine prepolymer (PAP) onto multi-walled carbon nanotubes (MWCNTs) screen printed electrode (SPE) surface, have been studied with several redox mediators by cyclic voltammetry (CV). The efficient entrapment of laccase in the PAP layer was confirmed by determination of both kinetic parameters (maximum current and Michaelis–Menten apparent constant) and analytical performances by chronoamperometry. The Laccase-modified MWCNTs electrode provides an effective biosensor for determination of polyphenols and catecholamines in real matrices; performances of the considered biosensors for real samples analysis are also compared and discussed.     

云芝漆酶在N,N'-亚甲基双丙烯酰胺（BIS）交联聚甲基丙烯酸基元上的固定及其修饰玻碳电极电化学行为

以N,N′-亚甲基双丙烯酰胺（BIS）交联聚甲基丙烯酸作为固定漆酶的载体,以共价偶联法固定云芝漆酶并测定了固定基元的酶固定量和固定漆酶的比活力。 还研究了固定漆酶热稳定性、重复使用性以及固定漆酶催化2,6-二甲氧基苯酚（DMP）氧化的酶动力学参数。 实验结果表明,这种交联聚合物基元通过共价偶联法固定漆酶的量和固定漆酶的比活力分别可达26.37 mg/g和1.202 U/mg;在交联聚合物基元上固定的漆酶在50 ℃下放置2 h后仍然保持初始活力的83％,重复使用10次后仍保持初始活力的80％以上;交联聚合物固定漆酶催化DMP氧化的表观速率常数k_cat可达1090 min^-1,以固定漆酶的BIS交联聚甲基丙烯酸功能化碳纳米管修饰的玻碳电极在pH=4.4磷酸盐缓冲液中氧还原发生在+724 mV(vs.SCE)。     

CuTAPc-Fe3O4纳米复合粒子及其漆酶固定化研究

漆酶的固定化研究对基于漆酶催化的光纤生物传感器具有十分重要的意义. 制备了四氨基酞菁铜(CuTAPc)-Fe₃O₄纳米复合粒子, 并用红外(IR)、场发射扫描电镜(FEG-SEM)、X射线衍射(XRD)、能谱、粒径仪等对其进行了表征. 结果表明形成了以CuTAPc包覆在Fe₃O₄纳米粒子表面的纳米复合粒子, 粒子呈现不规则球形, 且分布均匀, 粒子平均粒径在50 nm左右. 用此纳米复合粒子通过戊二醛交联法固定了漆酶, 固定后的酶比游离酶具有更好的贮存稳定性及操作稳定性. 这为研制高性能的光纤生物传感器打下了较好的基础.     

漆酶在纳米金溶胶/多重壁碳纳米管复合载体上固定方法的比较及粒子尺寸效应

以纳米金溶胶（NGS）和多重壁碳纳米管（MWCNTs）的共混物(NGS/MWCNTs)作为固定漆酶的载体,研究了3种固定漆酶方法在酶固定量、比活力上的差异。 研究了不同的固定方法对固定酶热稳定性和重复使用性及纳米金溶胶颗粒粒径对酶固定量和固定酶动力学参数的影响。 实验结果表明,NGS/MWCNTs具有良好的固定漆酶能力和高固酶比活力,NGS/MWCNTs（NGS粒径37 nm）通过简单物理吸附法固定漆酶的量和固酶的比活力最高,分别可达33.80 mg/g和9.433 U/mg。 在NGS-MWCNTs上采用化学键合方法固定的漆酶在70 ℃放置2 h后仍然保持初始活力的75％,重复使用20次后仍保持初始活力的70％。 纳米金溶胶粒子越小（24 nm）,底物和固定漆酶间亲和力越好（KM=0.027 mmol/L）,表观速率常数越大。     

壳聚糖-g-N-羧甲基-2-硫代-4,5-2H咪唑啉酮-多壁碳纳米管复合物固定漆酶基化学传感器的性能

通过壳聚糖-g-N-羧甲基-2-硫代-4,5-2H咪唑啉酮(CTS-g-N-CSIDZ)非共价功能化多壁碳纳米管(MWCNTs)的方式制备固定漆酶载体,该复合物载体主要通过物理吸附和漆酶活性中心与载体上配体之间的配位作用来固定漆酶,较大程度地保持了游离漆酶活性位原始构象.将固定了漆酶的复合物附着在裸玻碳电极上便构筑了复合物固定漆酶修饰玻碳电极.在以分光光度法测定了这种复合物载体对漆酶的担载量、固定漆酶比活力、稳定性、重复使用性及其催化2,6-二甲氧基苯酚(DMP)氧化动力学参数的基础上,还对基于此种复合物固定漆酶修饰玻碳电极作为化学传感器(以DMP作为底物)的性能进行了研究.结果表明,该复合物具有较高的固酶担载量(81.7 mg/g)和固定漆酶比活力(1.33 U/mg);而作为电化学传感器的复合物固定漆酶修饰玻碳电极对底物DMP具有较高的亲和力(对DMP的米氏常数KM是0.0918 mmol/L),较高的灵敏度( 3680 mA· L/mol),较低的检测限(3.3×10-4 mmol/L),较高的响应选择性,良好的重现性、重复使用性和长期稳定性.这种漆酶基电极有望用作电流型特定结构的酚类传感器.     

Oriented immobilization of a fully active monolayer of histidine-tagged recombinant laccase on modified gold electrodes

The formation of a dense monolayer of histidine-tagged recombinant laccase on gold electrodes by using a short thiol-NTA linker is described, as well as a kinetic analysis of the process by cyclic voltammetry. From a detailed analysis of the catalytic reduction of dioxygen by laccase in the presence of a one-electron redox mediator it can be concluded that the immobilized enzyme remains as active as in homogeneous solution.     

环氧基修饰周期性介孔有机氧化硅对漆酶的固定化作用

以三嵌段共聚物P123为模板剂, 在酸性条件下通过1,2-三乙氧基硅基乙烷(BTESE)和3-含氧缩水甘油基-丙基-三甲氧基硅烷(GPTMS)共水解缩聚合成环氧基修饰的周期性介孔氧化硅(PMOs), 以修饰后的PMOs为载体对漆酶进行固定化, 研究了环氧基修饰对固定化酶稳定性的影响. 通过XRD、TEM、固态NMR和N₂吸附等手段表征材料的修饰效果、孔结构以及漆酶的固定化. 结果表明, 修饰后的材料保持良好的孔结构, 环氧基的修饰有利于提高固定化酶的活力, 基于环氧基修饰PMOs的固定化酶具有较高稳定性.     

Laccase-Catalyzed Oxidative Polymerization of Phenolic Compounds

Enzymatic polymerization of phenolic compounds (catechol, resorcinol, and hydroquinone) was carried out using laccase. The mechanism of polymerization and the structures of the polymers were evaluated in terms of UV–Vis and Fourier transform infrared spectroscopy. The molecular weights of the produced polyphenols were determined with GPC. The results showed that the phenolic monomers firstly turned into quinone intermediates by laccase catalysis. Through further oxidation, the intermediates formed covalent bonds. Finally, catechol units were linked together with ether bonds, and both resorcinol and hydroquinone units were linked together with C-C bonds. The number-average molecular weights of the polyphenols ranged from 1,000 to 1,400 Da (corresponding to the degree of polymerization that varied from 10 to 12) with a lower polydispersity value of about 1.10, showing selective polymerization of phenolic compounds catalyzed by laccase.