酶固化技术最新研究进展

酶的本质是一种具有催化功能的蛋白质,能影响化学反应。然而,与传统的天然酶分子比较,固化酶相对更为脆弱,而传统的有机或无机催化剂其活性则比较固定。固化酶对于优化产业生产过程非常重要,近几十年来已开发出多种新型固化酶。本文在回顾酶固定化技术最新发展的同时。着重将其最新技术分别从吸附于载体,诱惑侦查及交联等三个方面进行综述。     

纤维素基固定化酶载体的研究进展

固定化酶是工业生物催化中广泛应用的一种生物催化剂形式。作为固定化酶最重要的一个组成部分,载体材料的种类、结构和性能都会对酶的固定化效果产生重要影响,是成功制备固定化酶的关键。近年来,纤维素因具有可再生、可降解和廉价等优良特性而成为一种备受关注的固定化酶载体材料。纤维素基载体可以通过吸附、交联、共价结合以及包埋等方式实现酶的固定化,从而提高酶的热稳定性、酸碱耐受性、贮藏稳定性及其重复使用性。纤维素独特的结构及其可修饰性赋予纤维素基载体许多新的功能,如果以此作为酶固定化载体时,能够使固定化效率或固定工艺有更多的变化。本文综述改性纤维素、纤维素膜、纤维素小球等载体在酶固定化领域的研究进展、催化机制及应用,为相关的研究者提供参考。     

纳米材料固定化酶的研究进展

近年来,纳米技术为酶固定化提供了多种纳米级材料,纳米材料固定化酶不仅具有高的酶负载量,而且具有良好的酶稳定性。本文基于纳米材料固定化酶,对纳米材料的种类进行了总结,分析了纳米材料对固定化酶性能的影响,并介绍了纳米级固定化方法及纳米材料固定化酶在生物转化、生物传感器、生物燃料电池等领域的应用。     

固定化酶及其应用研究进展

酶作为一种生物催化剂 ,一经发现就被人们广泛应用在酿造、食品、医药等领域。由于酶可以在常温、常压等温和的反应条件下高效地催化反应 ,一些难以进行的化学反应在酶的催化下能顺利地完成。酶的开发利用在2 0世纪得到了巨大的发展 ,但由于酶一般必需在温和的条件下才有催化作用 ,在实际运用中也就带来了很多问题 ,从而限制了酶制剂产品的使用和开发 ,固定化酶就是在这种情况下产生的。1　固定化酶简介1916年 Nelson和 Griffin最先发现了酶的固定化现象后 ,科学家就开始了固定化酶的研究工作。 196 9年日本一家制药公司第 1次将固定化的酰…     

多酶共固定化的研究进展

固定化酶技术是现代生物催化的核心技术。过去几十年里,固定化酶技术的研究主要集中在单酶固定化。近年来,多酶共固定化由于具有可增加反应的局部浓度、提高反应收率等优点而得到研究者的广泛关注。本文根据国内外研究现状并结合本实验研究从多酶非特异性共价共固定化、非特异性非共价共固定化、非共价包埋固定化以及位点特异性固定化四个方面阐述多酶固定化方法的研究进展,并分析和展望了其在工业上的应用前景。     

酶固化技术最新研究进展(英文)

酶的本质是一种具有催化功能的蛋白质,能影响化学反应。然而,与传统的天然酶分子比较,固化酶相对更为脆弱,而传统的有机或无机催化剂其活性则比较固定。固化酶对于优化产业生产过程非常重要,近几十年来已开发出多种新型固化酶。本文在回顾酶固定化技术最新发展的同时。着重将其最新技术分别从吸附于载体,诱惑侦查及交联等三个方面进行综述。     

固定化酶载体研究进展

固定化酶技术的应用提高了酶的稳定性和重复使用性,为酶在工业上的大规模运用提供了条件,其中载体是固定化酶技术的关键环节之一,已成为固定化酶技术目前研究的热点。介绍了介孔材料、纳米材料、磁性材料、天然高分子材料在固定化酶领域的的优缺点、研究现状及其应用情况,综述了载体材料固定化酶研究过程中的分析表征手段,包括形貌分析、结构分析、元素分析、比表面积和孔径分析,并提出了固定化酶载体今后的研究方向,为固定化酶载体进一步的研究和合理利用提供参考。     

多孔纳米材料固定化酶研究进展

酶是一种天然生物催化剂,有催化效率高、底物选择性强和绿色环保等优点,但酶结构不稳定且重复利用率低,制约了其产业化应用。随着技术的发展,酶的固定化可以提高酶的活性和稳定性,为生物酶的工程化应用带来了新的机遇。多孔纳米材料具有比表面积大、孔隙率高、机械和化学性能稳定等特点和优异的成本效益,是理想的固定化酶载体。本文综述了近些年来金属有机框架、共价有机框架和多孔微球等纳米材料固定化酶的研究进展和应用,重点介绍了载体固定酶的方式,并总结了每种载体的特点,最后讨论了多孔纳米材料固定化酶面临的挑战和发展趋势。     

固定化技术进展

近些年来 ,为了得到更加符合人们生产和使用要求的固定化产物 ,人们针对固定化技术的一些特点 ,分别从减少固定化过程中活性的损失 ,改善固定化产物的通透性 ,提高固定化产物的机械性能 ,提高固定化产物的稳定性等方面对固定化技术进行了研究。从以上几个方面对近些年来国内外固定化技术取得的新成果进行了介绍 ,并对其中部分技术方法进行了详细的阐述。可以预测 ,随着固定化技术的不断发展 ,此项技术将展现出良好的发展前景。     

农药残留检测关键用酶固定化研究进展

为保障消费者食用安全,迫切需要研发农产品和食品中的农药残留快速检测技术.酶抑制法检测是目前农药残留快速检测技术中的主要研究方向之一,而酶的固定化是用基于酶抑制法原理对农药残留检测研究中的重要步骤.通过物理或化学的方法高效地将酶固定于载体上,同时保持酶的催化活性是开发各类基于酶抑制法检测农药残留传感器的关键.本文将从固定...     

Recent advances in immobilization strategies for glycosidases

Glycans play important biological roles in cell‐to‐cell interactions, protection against pathogens, as well as in proper protein folding and stability, and are thus interesting targets for scientists. Although their mechanisms of action have been widely investigated and hypothesized, their biological functions are not well understood due to the lack of deglycosylation methods for large‐scale isolation of these compounds. Isolation of glycans in their native state is crucial for the investigation of their biological functions. However, current enzymatic and chemical deglycosylation techniques require harsh pretreatment and reaction conditions (high temperature and use of detergents) that hinder the isolation of native glycan structures. Indeed, the recent isolation of new endoglycosidases that are able to cleave a wider variety of linkages and efficiently hydrolyze native proteins has opened up the opportunity to elucidate the biological roles of a higher variety of glycans in their native state. As an example, our research group recently isolated a novel Endo‐β‐N‐acetylglucosaminidase from Bifidobacterium longum subsp. infantis ATCC 15697 (EndoBI‐1) that cleaves N‐N′‐diacetyl chitobiose moieties found in the N‐linked glycan (N‐glycan) core of high mannose, hybrid, and complex N‐glycans. This enzyme is also active on native proteins, which enables native glycan isolation, a key advantage when evaluating their biological activities. Efficient, stable, and economically viable enzymatic release of N‐glycans requires the selection of appropriate immobilization strategies. In this review, we discuss the state‐of‐the‐art of various immobilization techniques (physical adsorption, covalent binding, aggregation, and entrapment) for glycosidases, as well as their potential substrates and matrices. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:104–112, 2017     

Recent trends using natural polymeric nanofibers as supports for enzyme immobilization and catalysis

All the disciplines of science, especially biotechnology, have given continuous attention to the area of enzyme immobilization. However, the structural support made by material science intervention determines the performance of immobilized enzymes. Studies have proven that nanostructured supports can maintain better catalytic performance and improve immobilization efficiency. The recent trends in the application of nanofibers using natural polymers for enzyme immobilization have been addressed in this review article. A comprehensive survey about the immobilization strategies and their characteristics are highlighted. The natural polymers, e.g., chitin, chitosan, silk fibroin, gelatin, cellulose, and their blends with other synthetic polymers capable of immobilizing enzymes in their 1D nanofibrous form, are discussed. The multiple applications of enzymes immobilized on nanofibers in biocatalysis, biosensors, biofuels, antifouling, regenerative medicine, biomolecule degradation, etc.; some of these are discussed in this review article.     

应用荧光分析法检测酶的研究进展

酶是细胞新陈代谢的基础,酶的检测在生物技术、疾病诊断及药物开发等领域都具有十分重要的意义。在检测酶的诸多方法中,荧光法因其灵敏度高、检测限低等优势发展迅速。以下简述了近年来荧光法在酶检测领域的研究,根据检测方法的不同分为直接荧光检测法和间接荧光检测法,其中直接检测法又根据不同的底物标记及检测机理进行分类。以下介绍了各种方法的应用,并展望了此类方法的前景和发展趋势,为酶工程及生命科学其他领域的相关研究提供信息。     

Strategies of enzyme immobilization on nanomatrix supports and their intracellular delivery

Abstract

Enzymes are one of the foundations and regulators for all major biological activities in living bodies. Hence, enormous efforts have been made for enhancing the efficiency of enzymes under different conditions. The use of nanomaterials as novel carriers for enzyme delivery and regulating the activities of enzymes has stimulated significant interests in the field of nano-biotechnology for biomedical applications. Since, all types of nanoparticles (NPs) offer large surface to volume ratios, the use of NPs as enzyme carriers affect the structure, performance, loading efficiency, and the reaction kinetics of enzymes. Hence, the immobilization of enzymes on nanomatrices can be used as a useful approach for direct delivery of therapeutic enzymes to the targeted sites. In other words, NPs can be used as advanced enzyme delivery nanocarriers. In this paper, we present an overview of different binding of enzymes to the nanomaterials as well as different types of nanomatrix supports for immobilization of enzymes. Afterwards, the enzyme immobilization on nanomaterials as a potential system for enzyme delivery has been discussed. Finally, the challenges associated with the enzyme delivery using nano matrices and their future perspective have been discussed.

Communicated by Ramasamy H. Sarma     

Recent advances in structure-based enzyme engineering for functional reconstruction

Structural information can help engineer enzymes. Usually, specific amino acids in particular regions are targeted for functional reconstruction to enhance the catalytic performance, including activity, stereoselectivity, and thermostability. Appropriate selection of target sites is the key to structure-based design, which requires elucidation of the structure–function relationships. Here, we summarize the mutations of residues in different specific regions, including active center, access tunnels, and flexible loops, on fine-tuning the catalytic performance of enzymes, and discuss the effects of altering the local structural environment on the functions. In addition, we keep up with the recent progress of structure-based approaches for enzyme engineering, aiming to provide some guidance on how to take advantage of the structural information.     

Hydrophobic salt-modified Nafion for enzyme immobilization and stabilization

Over the last decade, there has been a wealth of application for immobilized and stabilized enzymes including biocatalysis, biosensors, and biofuel cells. In most bioelectrochemical applications, enzymes or organelles are immobilized onto an electrode surface with the use of some type of polymer matrix. This polymer scaffold should keep the enzymes stable and allow for the facile diffusion of molecules and ions in and out of the matrix. Most polymers used for this type of immobilization are based on polyamines or polyalcohols - polymers that mimic the natural environment of the enzymes that they encapsulate and stabilize the enzyme through hydrogen or ionic bonding. Another method for stabilizing enzymes involves the use of micelles, which contain hydrophobic regions that can encapsulate and stabilize enzymes. In particular, the Minteer group has developed a micellar polymer based on commercially available Nafion. Nafion itself is a micellar polymer that allows for the channel-assisted diffusion of protons and other small cations, but the micelles and channels are extremely small and the polymer is very acidic due to sulfonic acid side chains, which is unfavorable for enzyme immobilization. However, when Nafion is mixed with an excess of hydrophobic alkyl ammonium salts such as tetrabutylammonium bromide (TBAB), the quaternary ammonium cations replace the protons and become the counter ions to the sulfonate groups on the polymer side chains (Figure 1). This results in larger micelles and channels within the polymer that allow for the diffusion of large substrates and ions that are necessary for enzymatic function such as nicotinamide adenine dinucleotide (NAD). This modified Nafion polymer has been used to immobilize many different types of enzymes as well as mitochondria for use in biosensors and biofuel cells. This paper describes a novel procedure for making this micellar polymer enzyme immobilization membrane that can stabilize enzymes. The synthesis of the micellar enzyme immobilization membrane, the procedure for immobilizing enzymes within the membrane, and the assays for studying enzymatic specific activity of the immobilized enzyme are detailed below.     

Reversible covalent enzyme immobilization methods for reuse of carriers

Reversible immobilization techniques which allow for multiple use of the carrier are relevant for applications, such as enzymatic microreactors, biosensors with specific setups and for expensive carriers such as superparamagnetic particles. The activity of immobilized enzymes reduces with time, so that the introduction of fresh immobilized enzyme becomes necessary. Thus, methods for reversible immobilization and multiple carrier reuse can help to reduce purchase costs and facilitate reactor construction. In this work, we present a method that makes use of the reduction and oxidation of cystamine, a cleavable linker with disulfide bond and amine functionality. For a proof of principle, α-chymotrypsin was immobilized on polyethylene glycol with terminal epoxy groups using cystamine as a crosslinker. The enzyme was highly active and could be used in repeated cycles. After the enzymatic reaction was demonstrated, α-chymotrypsin was cleaved off the particle by reducing agents. The resulting thiols on the particle surface were oxidized to disulfides by means of cysteamine, the reduction product of cystamine. This way, an almost complete oxidation of surface thiols with cysteamine was possible, restoring amine functionalization for further reactions. Reduction and oxidation were repeated several times without a decrease in the extent of amine coupling. Finally, immobilization of α-chymotrypsin could be repeated with results comparable to first run.     

A two‐enzyme immobilization approach using carbon nanotubes/silica as support

Multiple enzyme mixtures are attractive for the production of many compounds at an industrial level. We report a practical and novel approach for coimmobilization of two enzymes. The system consists of a silica microsphere core coated with two layers of individually immobilized enzymes. The model enzymes α‐amylase (AA) and glucoamylase (GluA) were individually immobilized on carbon nanotubes (CNTs). A CNT‐GluA layer was formed by adsorbing CNT‐GluA onto silica microsphere. A sol‐gel layer with entrapped CNT‐AA was then formed outside the CNT‐GluA/silica microsphere conjugate. The coimmobilized α‐amylase and glucoamylase exhibited 95.1% of the activity of the mixture of free α‐amylase and glucoamylase. The consecutive use exhibited a good stability of the coimmobilized enzymes. The developed approach demonstrates advantages, including controlling the ratio of coimmobilized enzymes in an easy way, facilitating diffusion of small molecules in and out of the matrix, and preventing the leaching of enzymes. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:42–47, 2015     

Directed immobilization of CGTase: The effect of the enzyme orientation on the enzyme activity and its use in packed-bed reactor for continuous production of cyclodextrins

In this study, two different approaches were assessed in order to direct the immobilization of a cyclodextrin glycosyltransferase on functionalized silica support, one by amino groups using glutaraldehyde activation (Si-NH-G-CGTase) and other by disulfide bond through the Cys on the enzyme surface (Si-SH-CGTase). The efficiency of the immobilization of the enzyme by the Cys in Si-SH was four times higher than with the amino group linkage in Si-NH-G (2.86% and 11.91%, respectively). After immobilization, the optimum pH remained at 5.5 for the two derivatives and the optimum temperature was 70 °C for the free enzyme, 80 °C for Si-SH-CGTase and 90 °C for Si-NH-G-CGTase. Both preparations were used for continuous production of cyclodextrins, and Si-NH-G-CGTase presented higher total productivity, retaining 100% of its initial activity for at least 200 h, while the Si-SH-CGTase presented only 40% at the same time. The Si-SH-CGTase could be reloaded with new enzymes linked by disulfide bonds and was able to be used for more than 200 h.