Overview on immobilization of enzymes on synthetic polymeric nanofibers fabricated by electrospinning

The arrangement and type of support has a significant impact on the efficiency of immobilized enzymes. 1-dimensional fibrous materials can be one of the most desirable supports for enzyme immobilization. This is due to their high surface area to volume ratio, internal porosity, ease of handling, and high mechanical stability, all of which allow a higher enzyme loading, release and finally lead to better catalytic efficiency. Fortunately, the enzymes can reside inside individual nanofibers to remain encapsulated and retain their three-dimensional structure. These properties can protect the enzyme's tolerance against harsh conditions such as pH variations and high temperature, and this can probably enhance the enzyme's stability. This review article will discuss the immobilization of enzymes on synthetic polymers, which are fabricated into nanofibers by electrospinning. This technique is rapidly gaining popularity as one of the most practical ways to fibricate polymer, metal oxide, and composite micro or nanofibers. As a result, there is interest in using nanofibers to immobilize enzymes. Furthermore, present research on electrospun nanofibers for enzyme immobilization is primarily limited to the lab scale and industrial scale is still challanging. The primary future research objectives of this paper is to investigate the use of electrospun nanofibers for enzyme immobilization, which includes increasing yield to transfer biological products into commercial applications.     

Modifying bio-catalytic properties of enzymes for efficient biocatalysis: a review from immobilization strategies viewpoint

Enzyme-based catalysis has become one of the most important disciplines in organic synthesis and plays a noteworthy role in the establishment of many chemical industries, e.g. fine chemicals, food or energy, textiles, agricultural, cosmeceutical, medicinal and pharmaceutical industries. However, pristine enzymes fail to demonstrate requisite functionalities for an industrial setting where extremely specific and stable catalysts are required. Immobilization enhances the catalytic stability and activity of enzymes and trims the overall cost burden of the enzyme. Therefore, it widely endeavours for proficient, sustainable, and environmentally responsive catalytic processes. Amongst several immobilization strategies, e.g. (1) supports-assisted, i.e. physical or covalent coupling and (2) supports-free techniques, i.e. cross-linked enzyme crystals (CLECs) or aggregates are the most promising ones and widely pursued for enzyme immobilization purposes. This perspective review focuses on up-to-date developments in the area of enzyme immobilization and presents their potentialities to upgrade and/or modify enzyme properties. Both types of immobilization strategies, i.e. supports-assisted and supports-free techniques are discussed with particular reference to CLECs or aggregates and protein-coated microcrystals. Also, several useful traits achieved after immobilization are also discussed in the second half of the review.     

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     

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.     

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.     

Enzyme stabilization by nano/microsized hybrid materials

Immobilization is a key technology for successful realization of enzyme‐based industrial processes, particularly for production of green and sustainable energy or chemicals from biomass‐derived catalytic conversion. Different methods to immobilize enzymes are critically reviewed. In principle, enzymes are immobilized via three major routes (i) binding to a support, (ii) encapsulation or entrapment, or (iii) cross‐linking (carrier free). As a result, immobilizing enzymes on certain supports can enhance storage and operational stability. In addition, recent breakthroughs in nano and hybrid technology have made various materials more affordable hosts for enzyme immobilization. This review discusses different approaches to improve enzyme stability in various materials such as nanoparticles, nanofibers, mesoporous materials, sol–gel silica, and alginate‐based microspheres. The advantages of stabilized enzyme systems are from its simple separation and ease recovery for reuse, while maintaining activity and selectivity. This review also considers the latest studies conducted on different enzymes immobilized on various support materials with immense potential for biosensor, antibiotic production, food industry, biodiesel production, and bioremediation, because stabilized enzyme systems are expected to be environmental friendly, inexpensive, and easy to use for enzyme‐based industrial applications.     

Laccase immobilization and insolubilization: from fundamentals to applications for the elimination of emerging contaminants in wastewater treatment

Over the last few decades many attempts have been made to use biocatalysts for the biotransformation of emerging contaminants in environmental matrices. Laccase, a multicopper oxidoreductase enzyme, has shown great potential in oxidizing a large number of phenolic and non-phenolic emerging contaminants. However, laccases and more broadly enzymes in their free form are biocatalysts whose applications in solution have many drawbacks rendering them currently unsuitable for large scale use. To circumvent these limitations, the enzyme can be immobilized onto carriers or entrapped within capsules; these two immobilization techniques have the disadvantage of generating a large mass of non-catalytic product. Insolubilization of the free enzymes as cross-linked enzymes (CLEAs) is found to yield a greater volume ratio of biocatalyst while improving the characteristics of the biocatalyst. Ultimately, novel techniques of enzymes insolubilization and stabilization are feasible with the combination of cross-linked enzyme aggregates (combi-CLEAs) and enzyme polymer engineered structures (EPESs) for the elimination of emerging micropollutants in wastewater. In this review, fundamental features of laccases are provided in order to elucidate their catalytic mechanism, followed by different chemical aspects of the immobilization and insolubilization techniques applicable to laccases. Finally, kinetic and reactor design effects for enzymes in relation with the potential applications of laccases as combi-CLEAs and EPESs for the biotransformation of micropollutants in wastewater treatment are discussed.     

Development of a thiol‐ene based screening platform for enzyme immobilization demonstrated using horseradish peroxidase

Efficient immobilization of enzymes on support surfaces requires an exact match between the surface chemistry and the specific enzyme. A successful match would normally be identified through time consuming screening of conventional resins in multiple experiments testing individual immobilization strategies. In this study we present a versatile strategy that largely expands the number of possible surface functionalities for enzyme immobilization in a single, generic platform. The combination of many individual surface chemistries and thus immobilization methods in one modular system permits faster and more efficient screening, which we believe will result in a higher chance of discovery of optimal surface/enzyme interactions. The proposed system consists of a thiol‐functional microplate prepared through fast photochemical curing of an off‐stoichiometric thiol‐ene (OSTE) mixture. Surface functionalization by thiol‐ene chemistry (TEC) resulted in the formation of a functional monolayer in each well, whereas, polymer surface grafts were introduced through surface chain transfer free radical polymerization (SCT‐FRP). Enzyme immobilization on the modified surfaces was evaluated by using a rhodamine labeled horseradish peroxidase (Rho‐HRP) as a model enzyme, and the amount of immobilized enzyme was qualitatively assessed by fluorescence intensity (FI) measurements. Subsequently, Rho‐HRP activity was measured directly on the surface. The broad range of utilized surface chemistries permits direct correlation of enzymatic activity to the surface functionality and improves the determination of promising enzyme‐surface candidates. The results underline the high potential of this system as a screening platform for synergistic immobilization of enzymes onto thiol‐ene polymer surfaces. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1267–1277, 2017     

甲壳素酶学研究现状

本文介绍了甲壳素在生物合成和分解代谢过程中所涉及的相关酶,如甲壳素合成酶、甲壳素水解酶和其它相关酶,讨论了它们在分离纯化、结构鉴定、作用机制与模型、酶的固定化、基因工程以及应用等方面的研究现状和进展,对甲壳素的研究开发以及相关领域具有理论和实际意义 。     

Enzyme engineering: Reshaping the biocatalytic functions

Enzyme engineering is a powerful tool to fine-tune the enzymes. It is a technique by which the stability, activity, and specificity of the enzymes can be altered. The characteristic properties of an enzyme can be amended by immobilization and protein engineering. Among them, protein engineering is the most promising, as in addition to amending the stability and activity, it is the only way to modulate the specificity and stereoselectivity of enzymes. The current review sheds light on protein engineering and the approaches applied for it on the basis of the degree of knowledge of structure and function of enzymes. Enzymes, which have been engineered are also discussed in detail and categorized on the basis of their respective applications. This will give a better insight into the revolutionary changes brought by protein engineering of enzymes in various industrial and environmental processes.     

Enzyme immobilization: an update

Compared to free enzymes in solution, immobilized enzymes are more robust and more resistant to environmental changes. More importantly, the heterogeneity of the immo-bilized enzyme systems allows an easy recovery of both enzymes and products, multiple re-use of enzymes, continuous operation of enzymatic processes, rapid termination of reactions, and greater variety of bioreactor designs. This paper is a review of the recent literatures on enzyme immobilization by various techniques, the need for immobilization and different applications in industry, covering the last two decades. The most recent papers, patents, and reviews on immobilization strategies and application are reviewed.     

Immobilization of lipases on hydrophobic supports: immobilization mechanism,advantages, problems,and solutions

Lipases are the most widely used enzymes in biocatalysis, and the most utilized method for enzyme immobilization is using hydrophobic supports at low ionic strength. This method allows the one step immobilization, purification, stabilization, and hyperactivation of lipases, and that is the main cause of their popularity. This review focuses on these lipase immobilization supports. First, the advantages of these supports for lipase immobilization will be presented and the likeliest immobilization mechanism (interfacial activation on the support surface) will be revised. Then, its main shortcoming will be discussed: enzyme desorption under certain conditions (such as high temperature, presence of cosolvents or detergent molecules). Methods to overcome this problem include physical or chemical crosslinking of the immobilized enzyme molecules or using heterofunctional supports. Thus, supports containing hydrophobic acyl chain plus epoxy, glutaraldehyde, ionic, vinylsulfone or glyoxyl groups have been designed. This prevents enzyme desorption and improved enzyme stability, but it may have some limitations, that will be discussed and some additional solutions will be proposed (e.g., chemical amination of the enzyme to have a full covalent enzyme-support reaction). These immobilized lipases may be subject to unfolding and refolding strategies to reactivate inactivated enzymes. Finally, these biocatalysts have been used in new strategies for enzyme coimmobilization, where the most stable enzyme could be reutilized after desorption of the least stable one after its inactivation.     

多级孔金属-有机框架固定化酶的研究进展

金属-有机框架(metal-organic frameworks, MOFs)作为酶固定化的优良载体,为生物催化反应提供优越的物理和化学保护。近年来,多级孔金属-有机框架(hierarchical porous metal-organic frameworks, HP-MOFs)由于其独特的结构优势,在固定化酶方面显示出更大的潜力。到目前为止,已经开发了各类具有原生多级孔或缺陷多级孔的HP-MOFs用于酶的固定化研究,并且使得固定化酶在催化活性、稳定性和重复利用性等方面得到了显著增强。本文系统总结了HP-MOFs用于固定化酶的各种策略,介绍了HP-MOFs固定化酶(enzyme@HP-MOFs)在催化合成、生物传感、生物医药等领域的最新应用进展。最后,讨论并展望了HP-MOFs固定化酶这一领域所面临的挑战和机遇。     

Enhancing the functional properties of thermophilic enzymes by chemical modification and immobilization

The immobilization of proteins (mostly typically enzymes) onto solid supports is mature technology and has been used successfully to enhance biocatalytic processes in a wide range of industrial applications. However, continued developments in immobilization technology have led to more sophisticated and specialized applications of the process. A combination of targeted chemistries, for both the support and the protein, sometimes in combination with additional chemical and/or genetic engineering, has led to the development of methods for the modification of protein functional properties, for enhancing protein stability and for the recovery of specific proteins from complex mixtures. In particular, the development of effective methods for immobilizing large multi-subunit proteins with multiple covalent linkages (multi-point immobilization) has been effective in stabilizing proteins where subunit dissociation is the initial step in enzyme inactivation. In some instances, multiple benefits are achievable in a single process.Here we comprehensively review the literature pertaining to immobilization and chemical modification of different enzyme classes from thermophiles, with emphasis on the chemistries involved and their implications for modification of the enzyme functional properties. We also highlight the potential for synergies in the combined use of immobilization and other chemical modifications.     

功能化磁性纳米粒子在固定化酶研究中的应用

酶是高效的生物催化剂,在生物技术领域有广泛的应用。然而,不可再生催化的高成本和酶的有效成分分离回收,是实现大规模工业化应用需要解决的关键问题。磁性纳米粒子(magnetic nanoparticles,MNPs)具有优异的磁回收性质。通过设计和制备功能化MNPs作为固定化酶的多功能载体,是解决这一问题的有效途径之一,可为酶的工业化大规模应用提供条件。近年来,功能化磁性纳米粒子在酶的固定化领域基于载体性质、固定化方法和应用有广泛研究。文中重点介绍了近年来各种功能化磁性纳米载体,特别是Fe3O4纳米粒子,在固定化酶中的应用。根据功能化试剂的差异分类,实例讨论了不同功能化修饰的磁性纳米载体对酶的固定化,包括硅烷修饰的磁性纳米载体、有机聚合物修饰的磁性纳米载体、介孔材料修饰的磁性纳米载体以及金属-有机骨架材料(metal-organic framework,MOF)修饰的磁性纳米载体。同时,结合可持续工业催化的发展要求,对磁性复合载体固定化酶的发展前景进行了展望。     

Cross-linked enzyme aggregates (CLEAs): A novel and versatile method for enzyme immobilization (a review)

The key to obtaining optimum performance of an enzyme is often a question of devising an effective method for its immobilization. This review describes a novel, versatile and effective methodology for enzyme immobilization, namely, as cross-linked enzyme aggregates (CLEAs). The method is exquisitely simple - involving precipitation of the enzyme from aqueous buffer followed by cross-linking of the resulting physical aggregates of enzyme molecules - and amenable to rapid optimization. It is applicable to a wide variety of enzymes, including cofactor-dependent oxidoreductases and lyases, and affords stable, recyclable catalysts with high retention of activity, sometimes higher than that of the free enzyme it was derived from. The enzyme does not need to be of high purity. Indeed, the methodology is essentially a combination of purification and immobilization in one step. The technique is also applicable to the preparation of combi-CLEAs, containing two or more enzymes, for use in one-pot, multi-step syntheses. For example, an oxynitrilase/nitrilase combi-CLEA was used for the one-pot synthesis of (S)-mandelic acid from benzaldehyde, in high yield and enantiomeric purity.     

Cross-linked enzyme aggregates (CLEAs): A novel and versatile method for enzyme immobilization (a review)

The key to obtaining optimum performance of an enzyme is often a question of devising an effective method for its immobilization. This review describes a novel, versatile and effective methodology for enzyme immobilization, namely, as cross-linked enzyme aggregates (CLEAs). The method is exquisitely simple – involving precipitation of the enzyme from aqueous buffer followed by cross-linking of the resulting physical aggregates of enzyme molecules – and amenable to rapid optimization. It is applicable to a wide variety of enzymes, including cofactor-dependent oxidoreductases and lyases, and affords stable, recyclable catalysts with high retention of activity, sometimes higher than that of the free enzyme it was derived from. The enzyme does not need to be of high purity. Indeed, the methodology is essentially a combination of purification and immobilization in one step. The technique is also applicable to the preparation of combi-CLEAs, containing two or more enzymes, for use in one-pot, multi-step syntheses. For example, an oxynitrilase/nitrilase combi-CLEA was used for the one-pot synthesis of (S)-mandelic acid from benzaldehyde, in high yield and enantiomeric purity.     

Integrating enzyme immobilization and protein engineering: An alternative path for the development of novel and improved industrial biocatalysts

Enzyme immobilization often achieves reusable biocatalysts with improved operational stability and solvent resistance. However, these modifications are generally associated with a decrease in activity or detrimental modifications in catalytic properties. On the other hand, protein engineering aims to generate enzymes with increased performance at specific conditions by means of genetic manipulation, directed evolution and rational design. However, the achieved biocatalysts are generally generated as soluble enzymes, ?thus not reusable- and their performance under real operational conditions is uncertain.Combined protein engineering and enzyme immobilization approaches have been employed as parallel or consecutive strategies for improving an enzyme of interest. Recent reports show efforts on simultaneously improving both enzymatic and immobilization components through genetic modification of enzymes and optimizing binding chemistry for site-specific and oriented immobilization. Nonetheless, enzyme engineering and immobilization are usually performed as separate workflows to achieve improved biocatalysts.In this review, we summarize and discuss recent research aiming to integrate enzyme immobilization and protein engineering and propose strategies to further converge protein engineering and enzyme immobilization efforts into a novel “immobilized biocatalyst engineering” research field. We believe that through the integration of both enzyme engineering and enzyme immobilization strategies, novel biocatalysts can be obtained, not only as the sum of independently improved intrinsic and operational properties of enzymes, but ultimately tailored specifically for increased performance as immobilized biocatalysts, potentially paving the way for a qualitative jump in the development of efficient, stable biocatalysts with greater real-world potential in challenging bioprocess applications.     

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。     

Cooperativity of covalent attachment and ion exchange on alcalase immobilization using glutaraldehyde chemistry: Enzyme stabilization and improved proteolytic activity

Alcalase was scarcely immobilized on monoaminoethyl-N-aminoethyl (MANAE)-agarose beads at different pH values (<20% at pH 7). The enzyme did not immobilize on MANAE-agarose activated with glutaraldehyde at high ionic strength, suggesting a low reactivity of the enzyme with the support functionalized in this manner. However, the immobilization is relatively rapid when using low ionic strength and glutaraldehyde activated support. Using these conditions, the enzyme was immobilized at pH 5, 7, and 9, and in all cases, the activity vs. Boc-Ala-ONp decreased to around 50%. However, the activity vs. casein greatly depends on the immobilization pH, while at pH 5 it is also 50%, at pH 7 it is around 200%, and at pH 9 it is around 140%. All immobilized enzymes were significantly stabilized compared to the free enzyme when inactivated at pH 5, 7, or 9. The highest stability was always observed when the enzyme was immobilized at pH 9, and the worst stability occurred when the enzyme was immobilized at pH 5, in agreement with the reactivity of the amino groups of the enzyme. Stabilization was lower for the three preparations when the inactivation was performed at pH 5. Thus, this is a practical example on how the cooperative effect of ion exchange and covalent immobilization may be used to immobilize an enzyme when only one independent cause of immobilization is unable to immobilize the enzyme, while adjusting the immobilization pH leads to very different properties of the final immobilized enzyme preparation. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 35: e2768, 2019.