固定化酶扩散-反应问题的近似解

固定化酶在生物技术领域具有重要的理论和实际意义。由于固定化酶内部存在扩散传质阻力,因此固定化酶系统的化学反应速率不同于游离酶的反应速率。固定化酶反应动力学一般是基质浓度的非线性函数。通过求解固定化酶扩散 反应微分方程,可以得到固定化酶有效因子,而有效因子是固定化酶反应系统设计和模拟的重要参数,也是评定固定化酶系统性能优劣的重要因素之一。有效因子的计算通常采用将扩散 反应微分方程离散化的方法,例如正交配置法求解[1,2]。本文基于大参数的假设,在寻求固定化酶扩散 反应问题近似解的基础上,导出计算固定化酶有效…     

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.     

酶固定化技术用载体材料的研究进展

酶的固定化是生物技术中最为活跃的研究领域之一.作为固定化酶技术的重要组成部分,载体的结构及性能在很大程度上直接影响着所得固定化酶的催化活性及操作稳定性.综合性能优良的载体材料的设计与制备是固定化酶技术领域的一个非常重要的研究内容.通过对传统材料的改性和新型载体材料的研究开发,必将促进固定化酶在各个领域的广泛应用.本文综述了近年来国内外有关固定化酶载体材料的研究现状和发展趋势.     

介孔材料及其改性材料中酶的固定化研究进展

介孔材料具有高的比表面积、高的孔体积、均一可调的孔径、有序的孔道结构以及易于表面功能化等优点,可广泛用于酶的固定化.介孔材料中酶的固定化方法主要包括物理吸附、物理包埋和化学吸附.综述了介孔材料中不同固定化酶方法的优缺点、酶的固定化影响因素及固定化酶的应用,并对固定化酶的发展前景进行了展望.     

固定化多酶策略及易分离的载体材料研究进展

多酶级联反应是现代工业过程中重要的生物技术。然而,酶的分离和回收是一项繁琐而费力的工作,因此酶的固定化是实际应用中的关键问题。固定化多酶可以通过底物通道提高酶的催化活性,而易分离的载体材料有利于酶的稳定性和易于回收再利用。本文综述了近年来固定化多酶策略及易分离的载体材料相关研究,内容包括不同固定策略的多酶复合体,阐述了适合固定化酶的易于分离的载体材料,特别是磁性纳米颗粒和膜状材料无需离心即可从本体溶液中分离。总结了固定化多酶在食品生产和生物传感器领域的实际应用,最后对固定化多酶催化反应的发展前景和趋势进行了展望。     

碳纳米管固定化酶

在固定化酶技术中,载体材料的选择至关重要,碳纳米管作为一种新型高效的酶固定化载体,具有较大的比表面积、有序的纳米孔道结构、良好的力学/电学/热学性能、突出的化学稳定性、生物相容性和可控的表面官能化修饰等优良特性,应用日益广泛。本文重点介绍了水解酶、氧化还原酶等具有重要工业应用价值的酶在碳纳米管上的固定化研究现状,探讨了载体的表面修饰和固定化方式对固定化酶的酶学性质的影响,并对碳纳米管固定化酶的发展前景进行了展望。     

固定化脲酶的制备及酶学性质研究

固定化酶是束缚在一定空间内仍能进行其特有反应的生物催化剂,近年来对于固定化酶的研究非常活跃[1-2].目前酶的固定化方法主要有吸附法,交联法,包埋法以及化学共价法,这些方法各有特点[3-5],应用领域多为食品工业、制药、医学治疗等方面.     

纳米花型酶-无机杂化固定化酶研究进展

酶是一种绿色高效的生物催化剂,被广泛地应用于工业生产中,为了更好的提升游离酶的性能,酶固定化技术应运而生。然而,与游离酶相比,固定化酶活性下降以及传质受限一直是酶固定化技术亟待解决的关键问题。作为一种新型酶固定化技术,纳米花型酶-无机杂化固定化酶因具有高比表面积、高酶活性和高催化效率,且制备简单,绿色无污染受到广泛关注。本文综述了近年来纳米花型酶-无机杂化固定化酶的研究进展,根据纳米花型酶-无机杂化固定化酶的形成特点,将其分为单酶纳米花、双酶纳米花和负载型纳米花。阐述了纳米花型酶-无机杂化固定化酶的制备过程和形成机理并对纳米花型酶-无机杂化固定化酶在食品工业和检测领域的应用进展做出总结。最后,对纳米花型酶-无机杂化固定化酶的发展前景做出展望。     

壳聚糖固定L-天门冬酰胺酶的研究

研究了以壳聚糖为载体，戊二醛为交联剂，固定化Ｌ－天门冬酰胺酶的最适条件，并对固定化天门冬酰胺酶的理化性质进行了初步探讨。分别从不同固定化程序、缓冲液及保护剂等方面进行了固定化天门冬酰胺酶的条件优化；固定化酶的活力回收可达２０％左右。固定化酶的最适范围变宽，由游离酶的最适ｐＨ＝４～６变为ｐＨ＝６～１０。连续使用６次后仍可基本维持固定化酶的活力。固定化酶对胃蛋白酶的水解性也较游离酶大大提高。     

酶生物传感器中酶的固定化技术

综述了近年来国内外酶生物传感器的进展,介绍了制作酶生物传感器的关键技术——酶的固定化。固定化方法主要有吸附法、包埋法、共价键合法和交联法。固定化材料分为无机材料、有机聚合物材料、凝胶以及生物材料等。探讨了固定化方法和固定化材料对酶的固定化及酶生物传感器性能的影响,并结合自己的工作展望了酶生物传感器的发展方向和趋势。     

Expanding the substrate repertoire of a DNA polymerase by directed evolution

Nucleic acid polymerases are the most important reagents in biotechnology. Unfortunately, their high substrate specificity severely limits their applications. Polymerases with tailored substrate repertoires would significantly expand their potential and allow enzymatic synthesis of unnatural polymers for in vivo and in vitro applications. For example, the ability to synthesize 2'-O-methyl-modified polymers would provide access to materials possessing properties that make them attractive for biotechnology and therapeutic applications, but unfortunately, no known polymerases are capable of efficiently accepting these modified substrates. To evolve such enzymes, we have developed an activity-based selection method which isolates polymerase mutants with the desired property from libraries of the enzyme displayed on phage. In this report, mutants that could efficiently synthesize an unnatural polymer from 2'-O-methyl ribonucleoside triphosphates were immobilized and isolated by means of their activity-dependent modification of a DNA oligonucleotide primer attached to the same phage particle. In each case, directed evolution resulted in relocating a critical side chain to a different position in the polypeptide, thus re-engineering the overall active site while preserving critical protein-DNA interactions. Remarkably, one evolved polymerase is shown to incorporate the modified substrates with an efficiency and fidelity equivalent to that of the wild-type enzyme with natural substrates.     

Immobilized bacterial luciferase and its applications

This review discusses the properties of the bioluminescent bacterial system as well as the methods for immobilization of bacterial luciferases and for their co-immobilization with other enzymes. The analytical systems using immobilized bacterial luciferases and their applications in analytical biochemistry and biotechnology have been described.     

Flow‐through immobilized enzyme reactors based on monoliths: II. Kinetics study and application

In the last decade, the application of monolithic materials has rapidly expanded to the realization of flow‐through bioconversion processes. Up to these days, different classes of enzymes such as hydrolases, lyases, and oxidoreductases have been immobilized on organic, inorganic, or hybrid monolithic materials to prepare the effective flow‐through enzymes reactors for application in proteomics, biotechnology, pharmaceutics, organic synthesis, and biosensoring. Current review describes the results of kinetic study and specialties of flow‐through immobilized enzyme reactors based on the existing monolithic materials.     

Thiol-mediated anchoring of ligands to self-assembled monolayers for studies of biospecific interactions

We report a method to immobilize thiol-containing ligands onto self-assembled monolayers (SAMs) of alkanethiolates presenting chloracetylated hexa(ethylene glycol) groups. The chloroacetyl groups react with thiols under mild basic conditions, enabling the stable immobilization of biologically active ligands in a well-defined orientation. These SAMs on gold are well suited for studies of biospecific interactions of immobilized ligands with proteins and cells. As a demonstration, we functionalized these SAMs with thiol-containing derivatives of biotin and benzene sulfonamide and observed the specific binding of neutravidin and carbonic anhydrase, respectively. We also used this method to generate mixed SAMs presenting the Arg-Gly-Asp (RGD) peptide sequence and demonstrated the integrin-mediated adhesion of fibroblast cells to these SAMs. This approach would allow the immobilization of proteins and other sensitive biomolecules and ligands for a wide variety of applications in biotechnology.     

Where is the sodium in self-assembled monolayers of single-stranded DNA?

Monolayers of single-stranded DNA (ssDNA) immobilized on surfaces form the basis of a number of important biotechnology applications, including DNA microarrays and biosensors. The organization of ssDNA as layer on a solid substrate allows one to investigate various properties of the DNA in a controlled manner and to use DNA for analytical applications as well as for exploring futuristic schemes for molecular electronics. It is commonly assumed that the adsorbed DNA layer contains some structural water and the cations. Here we show, based on XPS studies, that when monolayers of ssDNA are formed from sodium phosphate buffer and washed thoroughly, no Na+ signal is detected. A finite concentration of ions is observed when the DNA is made from a solution of Mg2+ ions, but it is still only a fifth of what it would be if all the phosphate ions were fully neutralized by the metal cations.     

Immobilization in biotechnology and biorecognition: from macro- to nanoscale systems

Biological molecules such as enzymes, cells, antibodies, lectins, peptide aptamers, and cellular components in an immobilized form are extensively used in biotechnology, in biorecognition and in many medicinal applications. This review provides a comprehensive summary of the developments in new immobilization materials, techniques, and their practical applications previously developed by the authors. A detailed overview of several immobilization materials and technologies is given here, including bead cellulose, encapsulation in ionotropic gels and polyelectrolyte complexes, and various immobilization protocols applied onto surfaces. In addition, the review summarises the screening and design of an immobilization protocol, practical applications of immobilized biocatalysts in the industrial production of metabolites, monitoring, and control of fermentation processes, preparation of electrochemical/optical biosensors and biofuel cells.     

Atomic force microscopy for the characterization of immobilized enzyme molecules on biosensor surfaces

The development of biosensors has been one of the key areas in biotechnology and biomedical studies. Often it is difficult to investigate the immobilized biomolecules on the surfaces for biosensor optimization. Atomic force microscopy (AFM) should provide an ideal means for the visualization of biosensor surface and for the investigation of biomolecule activities. Therefore, AFM has been employed to study the surface topography of immobilized glutamate dehydrogenase (GDH) on two-dimensional glutamate biosensor surfaces. Correlation between the surface topography and the activity of the biosensor was investigated. Surface analysis has revealed that the enzymatic activity of the immobilized GDH molecules on the biosensor surface is linked to surface roughness, as measured by the peak-to-valley distance. Fractal dimension of the immobilization sensor surface was found to be a good parameter for judging the quality of the immobilized biosensors. As enzyme immobilization time increases, the biosensor has its maximum activity with around 18 h of immobilization in 10(-6) M GDH solution. Various biosensors prepared under different experimental conditions have been studied by AFM. This technique is shown to be an effective tool to characterize biosensor surfaces.