The use of enzymes in industrial processes requires the improvement of their features in many instances. Enzyme immobilization, a requirement to facilitate the recovery and reuse of these ...water-soluble catalysts, is one of the tools that researchers may utilize to improve many of their properties. This review is focused on how enzyme immobilization may improve enzyme stability. Starting from the stabilization effects that an enzyme may experience by the mere fact of being inside a solid particle, we detail other possibilities to stabilize enzymes: generation of favorable enzyme environments, prevention of enzyme subunit dissociation in multimeric enzymes, generation of more stable enzyme conformations, or enzyme rigidification via multipoint covalent attachment. In this last point, we will discuss the features of an “ideal” immobilization protocol to maximize the intensity of the enzyme-support interactions. The most interesting active groups in the support (glutaraldehyde, epoxide, glyoxyl and vinyl sulfone) will be also presented, discussing their main properties and uses. Some instances in which the number of enzyme-support bonds is not directly related to a higher stabilization will be also presented. Finally, the possibility of coupling site-directed mutagenesis or chemical modification to get a more intense multipoint covalent immobilization will be discussed.
•Enzyme immobilization in a porous structure may protect the enzyme from some inactivating causes•Enzyme immobilization may freeze some stable enzyme conformation•Multi-subunit enzyme immobilization may prevent enzyme subunit dissociation•Enzyme immobilization may enhance enzyme stability by generating special environments•Enzyme multipoint covalent attachment should increase enzyme rigidity
In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains ...with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize–stabilize–purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review.
•Use of immobilized antibodies to get the one step immobilization–purification•Use of domains with specific affinity for some ligands to immobilize/purify tagged proteins•Immobilization/purification by using of domains that increase the enzyme affinity for standard supports•Glyoxyl supports and one step immobilization–purification of multimeric proteins•Medium engineering and standard supports for immobilization/purification•Tailor made heterofunctional supports for large protein immobilization/purification
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.
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.
•Lipases immobilization on hydrophobic supports via interfacial activation is described in this review•This protocol permits the one step immobilization, purification, stabilization and hyperactivation of lipases•Lipases may be released from the support under certain conditions•Intermolecular crosslinking may prevent enzyme release•Heterofunctional supports prevent enzyme release but they have some limitations•There are many ways of taking full advantage of the new heterofunctional supports
Enzyme biocatalysis plays a very relevant role in the development of many chemical industries, e.g., energy, food or fine chemistry. To achieve this goal, enzyme immobilization is a usual ...pre‐requisite as a solution to get reusable biocatalysts and thus decrease the price of this relatively expensive compound. However, a proper immobilization technique may permit far more than to get a reusable enzyme; it may be used to improve enzyme performance by improving some enzyme limitations: enzyme purity, stability (including the possibility of enzyme reactivation), activity, specificity, selectivity, or inhibitions. Among the diverse immobilization techniques, the use of pre‐existing supports to immobilize enzymes (via covalent or physical coupling) and the immobilization without supports enzyme crosslinked aggregates (CLEAs) or crystals (CLECs) are the most used or promising ones. This paper intends to give the advantages and disadvantages of the different existing immobilization strategies to solve the different aforementioned enzyme limitations. Moreover, the use of nanoparticles as immobilization supports is achieving an increasing importance, as the nanoparticles versatility increases and becomes more accessible to the researchers. We will also discuss here some of the advantages and drawbacks of these non porous supports compared to conventional porous supports. Although there are no universal optimal solutions for all cases, we will try to give some advice to select the optimal strategy for each particular enzyme and process, considering the enzyme properties, nature of the process and of the substrate. In some occasions the selection will be compulsory, for example due to the nature of the substrate. In other cases the optimal biocatalyst may depend on the company requirements (e.g., volumetric activity, enzyme stability, etc).
The increasing relevance of cascade reactions in biocatalysis has sparked a great interest for enzyme co-immobilization. Enzyme co-immobilization allows access to some kinetic advantages that in some ...instances are necessary to get the desired product, avoiding side-reactions. However, the kinetic effect is very relevant mainly at the initial reaction rates, while it may be less relevant in the whole reaction course, depending on the kinetic parameters of the involved enzymes. This review not only critically discusses the advantages but also the drawbacks of enzymes co-immobilization: immobilization on the same support and surface, under similar conditions, discarding the whole biocatalyst when one of the co-immobilized enzymes is inactivated. We will discuss when co-immobilization is almost compulsory, when the advantages of co-immobilization may not be enough to compensate their problems and when it should be fully discarded. The co-immobilization of cofactors and enzymes bears special interest, as this can open up the opportunity to the building of artificial cells and extremely complex one-pot transformations. Finally, some recent strategies to overcome some the co-immobilization problems will be presented.
•Enzyme co-immobilization raises many problems in biocatalysts design.•Enzyme co-immobilization has some kinetic advantages regarding initial rates.•Enzyme co-immobilization is, in certain cases, necessary.•Co-immobilization of enzymes and cofactors opens up the possibility of building “synthetic cells”.•Some strategies have been developed to solve the problems of enzymes co-immobilization.
Lipases are being extensively researched for the production of biodiesel as a “silver bullet” in order to avoid the drawbacks of the traditional alkaline transesterification. In this review, we ...analyzed the main factors involved in the enzymatic synthesis of biodiesel, focusing in the choice of the immobilization protocol, and the parameters involved in the choice and configuration of the reactors. An extensive discussion is presented about the advantages and disadvantages of each type of reactor and their mode of operation. The current scenario of the market for enzymatic biodiesel and some future prospects and necessary developments are also briefly presented.
The lipase from
Rhizomucor miehei (formerly
Mucor miehei) (RML) is a commercially available enzyme in both soluble and immobilized form with very high activity and good stability under diverse ...conditions (anhydrous organic solvents, supercritical fluids, etc.). Although this lipase was initially produced to be used in food industry, in this review we will focus our attention on the application of this enzyme in organic chemistry, from biodiesel production to fine chemicals (mainly in enantio or regioselective or specific processes). After showing the enzyme features, some of the most efficient methods of RML immobilization will be commented (entrapping on reverse micelles, preparation of cross-linked RML aggregates or immobilization on pre-existing solids). Finally, the main uses of the enzyme in organic chemistry will be revised. The use of RML in the production of biodiesel will be analyzed, and compared to the performance of other lipases. The synthesis of esters of carboxylic acids as flavors is other example where RML has been successfully employed. Taking advantage of the wide specificity of the enzyme, mainly a high enantiospecificity, many examples of the use of RML in the resolution of racemic mixtures of chiral carboxylic acids, alcohols or esters will be presented. Special mention requires the use of the regioselectivity of RML, mainly the chemistry of sugars. Finally, more unusual uses of RML will be presented (anomalous substrates, novel uses, etc.). In general, this enzyme seems very adequate for esterification reactions due to its high stability in anhydrous media and good esterification activity.
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