MnO_2纳米粒子固载纤维素酶用于高效水解农业废弃物制备生物乙醇（英文）

纤维素酶是一种有效的纤维质类物质水解催化剂,工业应用时可通过固定化纤维素酶来降低其成本.本文将烟曲霉原变种JCF产生的纤维素酶固定在Mn O2纳米颗粒上.MnO2可提高纤维素酶的活性,并充当一个更好的载体.采用扫描电镜表征了所制MnO2纳米粒子及其负载纤维素酶的表面性质,以傅里叶变换红外光谱分析了固定在MnO2纳米粒子上纤维素酶的官能团性质.纤维素酶在MnO2纳米粒子上最大的固定化效率为75%.考察了固定化纤维素酶的活性、操作pH值、温度、热稳定性和重复使用性等性质.结果表明,所制固定化酶的稳定性比游离酶更高.固定于MnO2纳米粒子上的纤维素酶可用于纤维质类物质的水解反应,且能在较宽的温度和pH值范围内使用.表征结果证实了该催化剂具有非常高的催化纤维素类物质水解的活性.     

磁性纳米颗粒固定化细菌纤维素酶的活性和稳定性

在氨水溶液中进行Fe+2和Fe+3离子共沉淀并水热处理后制得磁性纳米颗粒Fe3O4,通过戊二醛活化将纤维素酶固定于其上。采用基于响应面法的Box-Behnken法(BBD)优化了制备条件,如磁性纳米颗粒浓度、戊二醛浓度、酶浓度和交联时间。 BBD分析结果表明,用实验数据可合理调节二次模型。利用生成的基于统计数据的等高线评价了响应面的变化,以理解纳米颗粒和酶活性之间的关系。运用扫描电镜、X射线衍射和红外光谱表征了纳米颗粒上酶的尺寸、结构、形貌和结合情况。采用诸如pH值、温度、重复使用性和存储能力分析了固定化纤维素酶的活性和稳定性。发现固定后的纤维素酶表现出更好的稳定性和活性。     

磁性纳米颗粒固定化细菌纤维素酶的活性和稳定性（英文）

在氨水溶液中进行Fe~(+2)和Fe~(+3)离子共沉淀并水热处理后制得磁性纳米颗粒Fe_3O_4,通过戊二醛活化将纤维素酶固定于其上.采用基于响应面法的Box-Behnken法(BBD)优化了制备条件,如磁性纳米颗粒浓度、戊二醛浓度、酶浓度和交联时间.BBD分析结果表明,用实验数据可合理调节二次模型.利用生成的基于统计数据的等高线评价了响应面的变化,以理解纳米颗粒和酶活性之间的关系.运用扫描电镜、X射线衍射和红外光谱表征了纳米颗粒上酶的尺寸、结构、形貌和结合情况.采用诸如pH值、温度、重复使用性和存储能力分析了固定化纤维素酶的活性和稳定性.发现固定后的纤维素酶表现出更好的稳定性和活性.     

基于聚乙烯醇/Fe2O3纳米颗粒的纤维素酶固定化

以聚乙烯醇/Fe2O3磁性纳米颗粒为纤维素酶固定化载体, 通过反复冻融的方法成功地实现了纤维素酶固定化. 采用透射电镜、红外光谱仪、振动样品磁强度计对固定化酶复合体进行了表征, 结果显示, 固定化酶复合体为大小约1 μm的微凝胶团, 内含10 nm左右的Fe2O3纳米颗粒. 研究影响固定化因素后发现, 当pH为6, 固定化时间为11 h, 纤维素酶/PVA质量比为4, PVA/Fe质量比为50时, 固定化纤维素酶效果最好. 通过该方法固定后酶活回收率达42%, 酶水解效率显著提高, 经过5次反应后的固定化酶相对酶活力保留50%以上. 因此, 基于聚乙烯醇/Fe2O3纳米颗粒的纤维素酶固定有利于酶的循环使用并显著提高酶的使用效率, 是一种有效固定化纤维素酶的新方法.     

疏水性聚丙烯酸/聚乙烯醇/葡萄糖淀粉酶复合纳米纤维膜的制备及酶学性质

利用混合静电纺丝将葡萄糖淀粉酶(GA)固定于聚丙烯酸(PAA)/聚乙烯醇(PVA)纳米纤维膜上,并通过鉴定固定化GA的酶学特征检验PAA/PVA可否成为一种优良的酶固定化载体。 对其理化性质和酶学特征进行鉴定,经红外光谱(FT-IR)和扫描电子显微镜(SEM)表征发现,GA可成功包埋于PAA/PVA纳米纤维膜内部;对包裹固定的GA进行酶学性质鉴定,发现固定化GA的最适反应温度为68 ℃,比游离GA提高了9 ℃;固定化GA的适用pH值范围明显变宽;热稳定性和存贮稳定性显著增强且可以重复使用。PAA/PVA纳米纤维膜是一种优良的酶固定化载体,可以通过混合静电纺丝包埋法简便地将蛋白质分子固定于其内部,具有一定的应用前景。     

多孔硅球固定化木瓜蛋白酶的制备和性质

用载体交联法制备了多孔硅球固定化木瓜蛋白酶。考察了固定化时间、温度、pH值、给酶量和成二醛浓度对固定化木瓜蛋白酶活力的影响。研究了固定化木瓜蛋白酶的性质，并同溶液酶进行了比较。着重考察了固定化木瓜蛋白酶的热稳定性。所制得的固定化木瓜蛋白酶最适温育温度达到80℃，对底物酪蛋白的水解活力随温度的升高而增加，在90℃达到最高值；在70℃温育12小时后酶活力仍能保持高水平。     

超顺磁性氧化石墨烯复合材料固定辣根过氧化物酶催化去除氯酚

采用超声辅助共沉淀法成功地将磁性Fe3O4纳米颗粒沉积在氧化石墨烯表面,利用透射电镜、磁滞回归曲线和X射线光电子能谱对材料进行了表征。将该材料作为载体固定辣根过氧化物酶,考察了固定化酶催化2-氯酚、4-氯酚和2,4-二氯酚降解反应,研究了溶液pH值、反应温度、反应时间、H2O2和氯酚浓度以及固定化酶用量对酚类物质去除率的影响。基于取代基数量和位置不同,去除率排序为2-氯酚<4-氯酚<2,4-二氯酚。另外,采用GC-MS研究了降解过程中的氧化产物。固定化酶的生化性质研究表明,固定化酶比游离酶具有更好的储存稳定性、pH稳定性和热稳定性。经过4次循环利用,固定化酶仍保留66%的活性,说明磁性纳米材料可以分离回收并重复利用,在污水处理领域具有应用前景。     

可磁力回收的Fe3O4纳米颗粒负载纤维素酶生物催化剂用于玉米芯降解

面对日益枯竭的化石能源和资源危机,科研工作者加速了对生物资源回收利用的研究.其中,作为生物资源主要成分的纤维素被证实是一种可以重新利用的原料,甚至可以作为工业产品潜在的前驱体.因此,回收利用富含纤维素的农作物副产品显得尤为重要.目前,多数纤维素资源并没有得到充分利用,例如玉米芯,全世界只有大约0.5％被利用.为了高效利用玉米芯资源,人们尝试各种分解方法将其主要成分纤维素和半纤维素转化成葡萄糖、木糖、糠醛以及酒精等.其中,最有效的策略是利用纤维素酶来分解玉米芯中的纤维素.然而,纤维素酶在实际应用过程中缺乏长久稳定性,将纤维素酶从反应体系中回收并重复利用非常困难.将纤维素酶负载到固体载体上是提高传统生物酶稳定性和可回收性的有效方法.固载纤维素酶在批生产处理和连续生产中比自由酶更具优势,可使生物酶催化剂从反应体系中分离出来变得容易和可操控.可以作为纤维素酶载体的物质有很多,例如浮石、静电纺丝的PAN纤维、纳米纤维膜、甲基丙烯酸甲酯共聚物和石墨烯等.一般来讲,任何含有表面功能基团从而提供了可以和纤维素酶形成强物理、化学作用的载体都可以采用.纳米尺寸的载体具有特殊性,一方面纳米颗粒提供了较大的比表面积从而可以拥有可观的负载能力,另一方面纳米颗粒可以轻易解决大颗粒载体应用中产生的反应底物和催化剂之间的扩散受阻问题.目前,纳米磁性颗粒已广泛用于负载蛋白质、多肽和生物酶.另外,用纳米磁性粒子作载体可方便地借助外加磁场实现生物酶催化剂的选择性分离回收,避免了传统载体所需的过滤或离心等单元操作,从而降低了生产成本,使生物酶催化技术实现连续化操作并用于规模化工业生产.本文通过水热法制备了颗粒均匀的纳米Fe3O4磁性颗粒,然后用3-氨丙基三乙氧基硅烷(KH550)化学修饰,再用戊二醛作交联剂将纤维素酶通过键合作用负载到修饰后的磁性载体上,从而高效制备了一种可磁力回收的生物酶催化剂.采用透射电镜和X射线衍射表征了磁性纳米粒子、修饰后的磁性纳米粒子以及制备的生物酶催化剂的粒径、外观形貌和品格结构,用红外光谱验证了磁性纳米颗粒上固载化纤维素酶的存在,用热重分析了固载化酶和自由酶的热稳定性,计算了制备的生物酶催化剂负载量和磁性粒子含量.对影响负载酶活性的多种因素进行了考察,合适的负载温度和pH值分别为40℃和6.0,戊二醛最佳添加浓度为2.0％,适宜的固载时间为4h.在最优负载条件下得到的固载化生物酶的活性可以保持自由酶活性的99.1％.经过15次重复使用后,固定化酶活性仍能保持91.1％.将制备的生物酶催化剂用于玉米芯分解制葡萄糖反应,预处理后的玉米芯最大分解率可达61.94％.     

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.     

Schiff碱改性法制备Ag/TiO2纳米粒子催化剂及其催化性能

通过引入水杨醛衍生的Schiff 碱功能单体与钛酸正丁酯发生水解共缩聚反应,制备了二氧化钛负载的银纳米粒子催化剂。采用红外光谱分析仪(FTIR)、广角X射线衍射仪(Wide Angle XRD)、高分辨透射电子显微镜(HRTEM)和紫外-可见分光光度计(UV-Vis) 等技术手段对制得的样品进行了表征,并考察了样品催化CO氧化反应的活性。结果表明,通过改变Schiff碱功能单体的用量和样品焙烧温度可以调控Ag纳米粒子的粒径和TiO₂载体的结构,从而可以调控Ag/TiO₂纳米催化剂的活性。当Schiff碱功能单体用量为20%,焙烧温度为873 K时,Ag/TiO₂纳米催化剂活性最好,CO转化率达到99.9%。     

Facile route to enzyme immobilization: core-shell nanoenzyme particles consisting of well-defined poly(methyl methacrylate) cores and cellulase shells

A one-step method for preparing cellulase-immobilized nanoparticles that consist of well-defined poly(methyl methacrylate) (PMMA) cores and cellulase shells has been developed. The core-shell nanoparticles are synthesized from a direct graft copolymerization of methyl methacrylate (MMA) from cellulase in an aqueous medium. Particle formation strongly depends on the surface nature of the cellulase (e.g., pH of reaction media) and MMA to cellulase weight ratio. Under optimized conditions, high MMA conversions (>90%) were achieved, and the PMMA-cellulase nanoparticles produced were very stable with narrow size distributions ( Dv/Dn < 1.20). Particle sizes in the range between 80 and 124 nm (volume average diameter) could be tailored by a variation of cellulase concentration. Transmission electron microscopy micrographs revealed that the nanoparticle had a well-defined PMMA core which was evenly coated with cellulase shell. Study of cellulase activity of the PMMA-cellulase nanoparticles indicated that even though activity of immobilized cellulase on the nanoparticles was 41% less than that of the native cellulase after the polymerization, the immobilized cellulase showed improved properties such as broader working pH range and better thermal stability. Other important advantages of this approach include that the PMMA-cellulase nanoparticles could be produced in high concentrations (up to 18% w/w solids content) and the nanoparticles have thick and evenly distributed enzyme shells. Thus, this method may provide a new commercially viable route to the immobilization of thermally stable enzyme to form nanoenzyme particles.     

聚甲基丙烯酸-聚乙烯吡啶复合物固定纤维素酶的研究

<正> 作为植物细胞壁主要成份的纤维素,占植物总重的一半,是地球上最丰富的有机物,是对人类有用而且潜力很大的自然资源。将这些廉价易得的纤维素利用纤维素酶转变成葡萄糖,不仅在食品工业上有现实意义,而且对未来化学工业的发展有着不可估量的影响。但由于酶在水溶液中不稳定,反应后难以分离和回收,只能使用一次,成本较高,使其应用受到一定的限制,固定酶的发展正好弥补了上述不足。     

Immobilization of cellulase using acrylamide grafted acrylonitrile copolymer membranes

Immobilization of cellulase onto acrylamide grafted acrylonitrile copolymer (PAN) membranes by means of glutaraldehyde has been studied. The bound cellulase was verified by X-ray photoelectron spectroscopy. The activities of free cellulase and immobilized cellulase are determined by measuring the amount of glucose made from carboxymethyl cellulase in the given conditions. Results show that immobilization conditions had some effects on the activity of immobilized cellulase. The immobilized cellulase had a higher K_m than free cellulase (0.02 mg/ml) did. The immobilized cellulase had better stability with respect to pH or temperature than free cellulase.     

纳米增强型毛细管酶柱用于葡萄糖液滴生物传感器的研究

葡萄糖的检测在临床医学以及食品工业等领域中十分重要.以往的检测方法主要包括化学发光法_{[1]、吸光光度法[2]、电化学法[3]和荧光法[4]等.固定化酶柱的制作是发展葡萄糖传感器的关键技术之一.传统的固定化方法主要是将具有生物活性的酶通过物理吸附、共价键合和交联的方法固定于载体基质上或包埋于有机聚合物的基质中.近期研究[5,6]表明,采用溶胶凝胶(Sol-gel)法将蛋白质和酶等生物活性物质包埋于无机陶瓷或玻璃材料内,保持生物组分的活性,且SiO2作为基质材料具有较好的坚固性、抗磨性、化学惰性以及高的光稳定性和透过性,但目前该法多用于电化学型生物传感器[7,8].本文利用纳米颗粒的比表面积大和吸附能力强等特点,将酶吸附在SiO2纳米颗粒表面,用易成膜的聚乙烯醇缩丁醛(PVB)作辅助基质在毛细管上固定酶,并采用分立式酶柱,克服了以往混合型酶柱普遍存在的酶促效率不高和使用寿命较短的局限性.所制得的酶柱具有表面反应活性高、表面活性中心多和催化效率高等特点.结合自行设计的液滴光化学传感装置[9,10],建立了一种高效、快速、微量的葡萄糖实时检测方法.}     

Immobilization of cellulase using polyurethane foam

Cellulase was covalently immobilized using a hydrophilic polyurethane foam (Hypol®FHP 2002). Compared to the free enzyme, immobilized cellulase showed a dramatic decrease (7.5-fold) in the Michaelis constant for carboxymethylcellulose. The immobilized enzyme also had a broader and more basic pH optimum (pH 5.5–6.0), a greater stability under heat-denaturing or liquid nitrogen-freezing conditions, and was relatively more efficient in utilizing insoluble cellulose substrates. High molecular weight compounds (Blue Dextran) could move throughout the foam matrix, indicating permeability to insoluble celluloses; activity could be further improved 2.4-fold after powdering, foams under liquid nitrogen. The improved kinetic and stability features of the immobilized cellulase combined with advantageous properties of the polyurethane foam (resistance to enzymatic degradation, plasticity of shape and size) suggest that this mechanism of cellulase immobilization has high potential for application in the industrial degradation of celluloses.     

Magnetite Nanoparticles Immobilized Pectinase: Preparation,Characterization and Application for the Fruit Juices Clarification

In this work, pectinase was immobilized on the surface of silica‐coated magnetite nanoparticles via covalent attachment. The magnetite‐immobilized enzyme was characterized by Fourier transform infrared spectroscopy, X‐ray powder diffraction, scanning electron microscopy and vibrating sample magnetometery techniques. Response Surface Methodology using Minitab Software was applied for statistical designing of operating conditions in order to immobilize pectinase on magnetic nanoparticles. The optimal conditions were obtained at 30 °C and pH 5.5 with 42.97 μl pectinase for 2 h. The immobilization yield was 50.6% at optimized conditions. Compared to the free pectinase, the immobilized pectinase was found to exhibit enhanced enzyme activity, better tolerance to the variation of pH and temperature, and improved storage stability. Both free and immobilized samples reduced the viscosity of apple juice from 1.12 to 0.88 and 0.92 mm²s^?1, respectively, after 30 min at their optimum temperature. Furthermore, the immobilized enzyme could be reused six consecutive cycles and the efficiency loss in viscosity reduction was found to be only 8.16%.     

Direct introduction of amine groups into cellulosic paper for covalent immobilization of tyrosinase: support characterization and enzyme properties

Tyrosinase is used to eliminate phenolic compounds from wastewater. Therefore, its immobilization is important to enhance catalytic efficiency. Papery materials are of particular interest for use as support for enzyme immobilization since the porous microstructure of fiber networks in papers can provide a suitable reaction environment, especially in flow-type catalytic reactions. However, immobilization of protein onto papery structure needs chemical modifications in severe conditions. To overcome this challenge, a cellulosic paper was directly amine-functionalized in moderate conditions and used for tyrosinase immobilization. The support was pretreated with HCl (0.5 N) solution and then sequentially immersed in ethylenediamine (EDA), glutaraldehyde solution (2% v/v) and the crude enzyme. In comparison with the untreated one, the immobilized enzyme on the EDA-treated support offered a 3.7-fold increase in activity. The FTIR spectra as well as EDX analysis proved the presence of amine groups in the cellulosic paper and also covalent immobilization of tyrosinase on the modified support. When considering the effect of pH on the activity at 25 °C, a maximum relative activity of 134% at pH 6 was revealed. Similarly, evaluating the effect of temperature on the activity at pH 7 displayed a maximum relative activity of 152% at 35 °C. The immobilized enzyme was suitable for use for more than four cycles to degrade a phenolic compound at severe pH and temperature conditions. Additionally, the immobilized enzyme was active after treatment of the surface at different pHs and temperatures for 105 min. The chemically modified cellulosic paper can be used as a support for enzyme immobilization.