Advances in enzyme immobilisation
TL;DR: Improvements in current strategies for carrier-based immobilisation have been developed using hetero-functionalised supports that enhance the binding efficacy and stability through multipoint attachment, and promise to enhance the roles of immobilisation enzymes in industry, while opening the door for novel applications.
Abstract: Improvements in current strategies for carrier-based immobilisation have been developed using hetero-functionalised supports that enhance the binding efficacy and stability through multipoint attachment. New commercial resins (Sepabeads) exhibit improved protein binding capacity. Novel methods of enzyme self-immobilisation have been developed (CLEC, CLEA, Spherezyme), as well as carrier materials (Dendrispheres), encapsulation (PEI Microspheres), and entrapment. Apart from retention, recovery and stabilisation, other advantages to enzyme immobilisation have emerged, such as enhanced enzyme activity, modification of substrate selectivity and enantioselectivity, and multi-enzyme reactions. These advances promise to enhance the roles of immobilisation enzymes in industry, while opening the door for novel applications.
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TL;DR: An overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented and emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).
Abstract: In this tutorial review, an overview of the why, what and how of enzyme immobilisation for use in biocatalysis is presented. The importance of biocatalysis in the context of green and sustainable chemicals manufacture is discussed and the necessity for immobilisation of enzymes as a key enabling technology for practical and commercial viability is emphasised. The underlying reasons for immobilisation are the need to improve the stability and recyclability of the biocatalyst compared to the free enzyme. The lower risk of product contamination with enzyme residues and low or no allergenicity are further advantages of immobilised enzymes. Methods for immobilisation are divided into three categories: adsorption on a carrier (support), encapsulation in a carrier, and cross-linking (carrier-free). General considerations regarding immobilisation, regardless of the method used, are immobilisation yield, immobilisation efficiency, activity recovery, enzyme loading (wt% in the biocatalyst) and the physical properties, e.g. particle size and density, hydrophobicity and mechanical robustness of the immobilisate, i.e. the immobilised enzyme as a whole (enzyme + support). The choice of immobilisate is also strongly dependent on the reactor configuration used, e.g. stirred tank, fixed bed, fluidised bed, and the mode of downstream processing. Emphasis is placed on relatively recent developments, such as the use of novel supports such as mesoporous silicas, hydrogels, and smart polymers, and cross-linked enzyme aggregates (CLEAs).
2,013 citations
TL;DR: In this tutorial review, some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization are listed.
Abstract: Immobilization of enzymes may produce alterations in their observed activity, specificity or selectivity. Although in many cases an impoverishment of the enzyme properties is observed upon immobilization (caused by the distortion of the enzyme due to the interaction with the support) in some instances such properties may be enhanced by this immobilization. These alterations in enzyme properties are sometimes associated with changes in the enzyme structure. Occasionally, these variations will be positive. For example, they may be related to the stabilization of a hyperactivated form of the enzyme, like in the case of lipases immobilized on hydrophobic supports via interfacial activation. In some other instances, these improvements will be just a consequence of random modifications in the enzyme properties that in some reactions will be positive while in others may be negative. For this reason, the preparation of a library of biocatalysts as broad as possible may be a key turning point to find an immobilized biocatalyst with improved properties when compared to the free enzyme. Immobilized enzymes will be dispersed on the support surface and aggregation will no longer be possible, while the free enzyme may suffer aggregation, which greatly decreases enzyme activity. Moreover, enzyme rigidification may lead to preservation of the enzyme properties under drastic conditions in which the enzyme tends to become distorted thus decreasing its activity. Furthermore, immobilization of enzymes on a support, mainly on a porous support, may in many cases also have a positive impact on the observed enzyme behavior, not really related to structural changes. For example, the promotion of diffusional problems (e.g., pH gradients, substrate or product gradients), partition (towards or away from the enzyme environment, for substrate or products), or the blocking of some areas (e.g., reducing inhibitions) may greatly improve enzyme performance. Thus, in this tutorial review, we will try to list and explain some of the main reasons that may produce an improvement in enzyme activity, specificity or selectivity, either real or apparent, due to immobilization.
1,487 citations
TL;DR: Glutaraldehyde, an apparently old fashioned reactive, remains the most widely used and with broadest application possibilities among the compounds used for the design of biocatalyst.
Abstract: Glutaraldehyde is one of the most widely used reagents in the design of biocatalysts. It is a powerful crosslinker, able to react with itself, with the advantages that this may bring forth. In this review, we intend to give a general vision of its potential and the precautions that must be taken when using this effective reagent. First, the chemistry of the glutaraldehyde/amino reaction will be commented upon. This reaction is still not fully clarified, but it seems to be based on the formation of 6-membered heterocycles formed by 5 C and one O. Then, we will discuss the production of intra- and inter-molecular enzyme crosslinks (increasing enzyme rigidity or preventing subunit dissociation in multimeric enzymes). Special emphasis will be placed on the preparation of cross-linked enzyme aggregates (CLEAs), mainly in enzymes that have low density of surface reactive groups and, therefore, may be problematic to obtain a final solid catalyst. Next, we will comment on the uses of glutaraldehyde in enzymes previously immobilized on supports. First, the treatment of enzymes immobilized on supports that cannot react with glutaraldehyde (only inter and intramolecular cross-linkings will be possible) to prevent enzyme leakage and obtain some enzyme stabilization via cross-linking. Second, the cross-linking of enzymes adsorbed on aminated supports, where together with other reactions enzyme/support crosslinking is also possible; the enzyme is incorporated into the support. Finally, we will present the use of aminated supports preactivated with glutaraldehyde. Optimal glutaraldehyde modifications will be discussed in each specific case (one or two glutaraldehyde molecules for amino group in the support and/or the protein). Using preactivated supports, the heterofunctional nature of the supports will be highlighted, with the drawbacks and advantages that the heterofunctionality may have. Particular attention will be paid to the control of the first event that causes the immobilization depending on the experimental conditions to alter the enzyme orientation regarding the support surface. Thus, glutaraldehyde, an apparently old fashioned reactive, remains the most widely used and with broadest application possibilities among the compounds used for the design of biocatalyst.
639 citations
TL;DR: In this paper, a comparative analysis of the literature reports on the recent trends in the enzyme immobilization by adsorption is presented, where both carriers, carrier modifiers and procedures developed for effective adaption of the enzymes are discussed.
Abstract: Endowed with unparalleled high catalytic activity and selectivity, enzymes offer enormous potential as catalysts in practical applications. These applications, however, are seriously hampered by enzymes’ low thermal and chemical stabilities. One way to improve these stabilities is the enzyme immobilization. Among various tested methods of this process that make use of different enzyme-carrier interactions, immobilization by adsorption on solid carriers has appeared most common. According to these findings, in this review we present a comparative analysis of the literature reports on the recent trends in the immobilization of the enzymes by adsorption. This thorough study was prepared in order to provide a deeper understanding of the process. Both carriers, carrier modifiers and procedures developed for effective adsorption of the enzymes are discussed. The review may thus be helpful in choosing the right adsorption scheme for a given enzyme to achieve the improvement of its stability and activity for a specific application.
633 citations
Cites background from "Advances in enzyme immobilisation"
...However, the enormous catalytic potential offered by the enzymes for innumerable transformations, has stimulated intense studies aimed at the improvement of their properties (Mateo et al. 2007; Brady and Jordon 2009; FernandezLafuente 2009; Garcia-Galan et al. 2011; Cowan and Fernandez-Lafuente 2011; Rodrigues et al. 2013)....
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...…potential offered by the enzymes for innumerable transformations, has stimulated intense studies aimed at the improvement of their properties (Mateo et al. 2007; Brady and Jordon 2009; FernandezLafuente 2009; Garcia-Galan et al. 2011; Cowan and Fernandez-Lafuente 2011; Rodrigues et al. 2013)....
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21 Nov 2012
TL;DR: The underlying causes of membrane biofouling are highlighted and a review on recent developments of potential monitoring and control methods in water and wastewater treatment is provided with the aim of identifying the remaining issues and challenges in this area.
Abstract: Biofouling is a critical issue in membrane water and wastewater treatment as it greatly compromises the efficiency of the treatment processes. It is difficult to control, and significant economic resources have been dedicated to the development of effective biofouling monitoring and control strategies. This paper highlights the underlying causes of membrane biofouling and provides a review on recent developments of potential monitoring and control methods in water and wastewater treatment with the aim of identifying the remaining issues and challenges in this area.
630 citations
Cites background from "Advances in enzyme immobilisation"
...as: (a) the activity of enzymes is pH-dependent and they are sensitive to temperature and salt concentration; (b) high production costs; and (c) soluble enzymes are difficult to recover from an aqueous medium [243]....
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References
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TL;DR: In all cases, enzyme engineering via immobilization techniques is perfectly compatible with other chemical or biological approaches to improve enzyme functions and the final success depend on the availability of a wide battery of immobilization protocols.
3,016 citations
TL;DR: Different methods for the immobilization of enzymes are critically reviewed, with emphasis on relatively recent developments, such as the use of novel supports, e.g., mesoporous silicas, hydrogels, and smart polymers, novel entrapment methods and cross-linked enzyme aggregates (CLEAs).
Abstract: Immobilization is often the key to optimizing the operational performance of an enzyme in industrial processes, particularly for use in non-aqueous media. Different methods for the immobilization of enzymes are critically reviewed. The methods are divided into three main categories, viz. (i) binding to a prefabricated support (carrier), (ii) entrapment in organic or inorganic polymer matrices, and (iii) cross-linking of enzyme molecules. Emphasis is placed on relatively recent developments, such as the use of novel supports, e.g., mesoporous silicas, hydrogels, and smart polymers, novel entrapment methods and cross-linked enzyme aggregates (CLEAs).
1,857 citations
TL;DR: An analysis of 134 industrial biotransformations reveals that hydrolases and redox biocatalysts are the most prominent categories and the implications of this for future research and development onBiocatalysis are discussed.
692 citations
"Advances in enzyme immobilisation" refers background in this paper
...In spite of the long history and obvious advantages of enzyme immobilisation (Katchalski-Katzir and Kraemer 2000), Straathof et al. (2002) estimated that only 20% of biocatalytic processes involve immobilised enzymes....
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TL;DR: In this paper, the authors discuss the recent advances in using nanofibers as hosts for enzyme immobilization by two different methods, surface attachment and encapsulation, and highlight their distinct characteristics.
Abstract: Enzyme immobilization has attracted continuous attention in the fields of fine chemistry, biomedicine, and biosensor. The performance of immobilized enzyme largely depends on the structure of supports. Nanostructured supports are believed to be able to retain the catalytic activity as well as ensure the immobilization efficiency of enzyme to a high extent. Electrospinning provides a simple and versatile method to fabricate nanofibrous supports. Compared with other nanostructured supports (e.g. mesoporous silica, nanoparticles), nanofibrous supports show many advantages for their high porosity and interconnectivity. This review mainly discusses the recent advances in using nanofibers as hosts for enzyme immobilization by two different methods, surface attachment and encapsulation. Surface attachment refers to physical adsorption or covalent attachment of enzymes on pristine or modified nanofibrous supports, and encapsulation means electrospinning a mixture of enzyme and polymer. We make a detailed comparison between these two immobilization approaches and highlight their distinct characteristics. The prospective applications of enzyme immobilized electrospun nanofibers in the development of biosensors, biofuel cells and biocatalysts are also discussed.
479 citations
TL;DR: An automatic reactor simulating, at laboratory scale, the performance of an industrial stirred tank reactor (STR) is described, and its utilization for evaluating theperformance of immobilized enzymes is shown.
Abstract: Eupergit® C is a carrier consisting of macroporous beads for immobilizing enzymes of industrial potential for the production of fine chemicals and pharmaceuticals. Various enzymes immobilized on Eupergit® C are reviewed in comparison with other carrier materials in terms of the operational stability of the respective biocatalysts at substrate concentrations realistic for industrial production. Other aspects of relevance in that field, such as the demand for purity of enzyme to be immobilized or type of reactor optimal for a given application, are also discussed. An automatic reactor simulating, at laboratory scale, the performance of an industrial stirred tank reactor (STR) is described, and its utilization for evaluating the performance of immobilized enzymes is shown.
423 citations
"Advances in enzyme immobilisation" refers background in this paper
...…average diameter) activated with epoxides and made of a co-polymer of N,N0-methylene-bis-(methacrylamide), glycidyl methacrylate, allyl glycidyl ether and methacrylamide, has long been used for enzyme covalent immobilisation, including commercial applications (Katchalski-Katzir and Kraemer 2000)....
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...The commercial support Eupergit (Evonik, previously Degussa), a macroporus sphere (170 lm average diameter) activated with epoxides and made of a co-polymer of N,N0-methylene-bis-(methacrylamide), glycidyl methacrylate, allyl glycidyl ether and methacrylamide, has long been used for enzyme covalent immobilisation, including commercial applications (Katchalski-Katzir and Kraemer 2000)....
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...In spite of the long history and obvious advantages of enzyme immobilisation (Katchalski-Katzir and Kraemer 2000), Straathof et al. (2002) estimated that only 20% of biocatalytic processes involve immobilised enzymes....
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...of enzyme immobilisation (Katchalski-Katzir and Kraemer 2000), Straathof et al....
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