Chem Soc Rev TUTORIAL REVIEW. Modifying enzyme activity and selectivity by immobilization. 1. Introduction

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1 TUTORIAL REVIEW View Article Online View Journal View Issue Cite this: Chem. Soc. Rev., 2013, 42, 6290 Received 29th June 2012 DOI: /c2cs35231a Modifying enzyme activity and selectivity by immobilization Rafael C. Rodrigues, a Claudia Ortiz, b Ángel Berenguer-Murcia, c Rodrigo Torres d and Roberto Fernández-Lafuente* e 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. Introduction a Biocatalysis and Enzyme Technology Lab, Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box 15090, ZC , Porto Alegre, RS, Brazil b Escuela de Bacteriología y Laboratorio Clínico, Universidad Industrial de Santander, Bucaramanga, Colombia c Instituto Universitario de Materiales, Departamento de Química Inorgánica, Universidad de Alicante, Campus de San Vicente del Raspeig, Ap , Alicante, Spain d Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia e Departamento de Biocatalisis, Instituto de Catálisis-CSIC, Campus UAM-CSIC, C/Marie Curie 2, Cantoblanco, 28049, Madrid, Spain. rfl@icp.csic.es; Fax: ; Tel: Part of a themed issue on enzyme immobilization. Enzymes are nowadays reaching high levels of implementation in areas as diverse as fine and pharmaceutical chemistry, food modification or energy production (e.g., biodiesel and bioethanol). 1 Immobilization of enzymes is a requisite for their use as industrial biocatalysts in most of these instances, since immobilization permits the simple reuse of the enzyme and simplifies the overall design and performance control of the bioreactors. 2 4 Thus, many efforts have been devoted to convert this requirement into a powerful tool to greatly improve enzyme performance. 5 For example, stabilization of monomeric enzymes via multipoint covalent attachment or generation of favorable environments surrounding the enzyme has been reported in many instances, 6 while multimeric enzymes have been stabilized by immobilizing all enzyme subunits, thus 6290 Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

2 preventing subunit dissociation. 7 In any case, immobilization is compatible with any other strategies to yield a more stable biocatalyst, such as chemical modification, 8 use of enzymes from the thermophile microorganisms, 9 or genetic manipulation. 10 Immobilization is in many instances associated with a decrease in enzyme activity or a worsening of other catalytic features. However, some reports in the literature show how immobilization of an enzyme may also improve its activity (the rate of the reaction per milligram of enzyme), specificity (discrimination between substrates) and selectivity (production of one among several possible products). 11 Immobilization, in most cases, will produce slight distortions in the enzymes structure, and this may alter the final properties of the enzyme. 12 These changes will be largely uncontrolled, but Prof. Rafael Costa Rodrigues was born in Rio Grande, RS, Brazil, in He obtained his PhD in enzymatic synthesis of biodiesel under the supervision of Prof. Ayub at the UFRGS (Brazil). He performed a part of his research in the group of Prof. Guisan (ICP-CSIC, Spain) studying new immobilization stabilization methods for lipases to apply in biodiesel production reactions. In Rafael C. Rodrigues 2010 he obtained a lectureship at the Food Science and Technology Institute, UFRGS (Brazil). His research interests are immobilization-stabilization of enzymes and reaction engineering. He has coauthored 32 papers and 1 patent, presenting an H number of 11. Dr Ángel Berenguer-Murcia obtained his Degree in Chemistry at the University of Alicante in 2000, and in 2005 he obtained his PhD. In 2006 he moved to the University of Cambridge (UK) to work under the supervision of Prof. Brian F. G. Johnson on the design of smart materials. In 2009 he moved back to the Materials Institute of the University of Alicante where he Ángel Berenguer-Murcia is a Research Fellow. His research interests include the development of membranes, nanoparticle synthesis, and the design of porous materials. building a large library of biocatalysts prepared following quite different immobilization strategies to have a large diversity of situations may permit to find solutions where the enzyme properties improve. 11 However, the improvements in enzyme performance after immobilization are not always really related to the production of a more active or selective enzyme molecule, but to some artifact which can alter the activity of the free or immobilized enzyme, or just affect the stability of the enzyme. Thus, this review will try to present and discuss the facts and artifacts that can promote improvements in enzyme activity, specificity and selectivity after immobilization. These improvements in enzyme activity may be considered in any case more the exception than the rule, as in most instances immobilized Prof. Claudia Cristina Ortiz Lopez was born in In 2004 she obtained her PhD in possible uses of the complex mechanism of interfacial activation of lipases as a useful tool to improve biocatalytical processes with immobilized enzymes. This work was supervised by Professors Guisan and Fernandez-Lafuente at ICP-CSIC (Spain). In 2004, she obtained a tenure track position Claudia Ortiz at the School of Bacteriology, where currently she works as an Associate Professor. She also directs the Research Group in Biochemistry and Microbiology at the Universidad Industrial de Santander, Colombia, from Her research interests include Industrial Microbiology, Bioprocess Technology, Biocatalysis and Biotransformations. Prof. Rodrigo Torres was born in Valparaíso, Chile, in He obtained his PhD in 2005 working under the supervision of Profs José Manuel Guisán and Roberto Fernández-Lafuente. After a stay as a visiting scholar at the Department of Microbiology of Cornell University, he moved back to the School of Chemistry of Universidad Industrial de Santander, Bucaramanga, Rodrigo Torres Colombia, where he is currently an associate professor. His research interests include enzyme immobilization, biocatalysis and biotransformation, proteomics, peptide synthesis and nanobiotechnology for the development of new applications of nanocompounds in environmental and pharmaceutical applications. He has coauthored 42 papers and 2 patents. This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

3 enzymes will exhibit a lower catalytic performance (also cause by real effects on enzyme structure or artifacts similar to those described here). In this sense, this review is quite far from other reviews on immobilization methods that may be found in the literature that usually list immobilization methods or the different uses of them Improvements in enzyme activity by immobilization 2.1 Aggregation of the soluble enzyme In some instances, the researcher compares the activity of the immobilized enzyme under conditions in which the free enzyme is insoluble, i.e. comparison of an aggregated enzyme (formed by enzyme precipitation in that medium) with an enzyme immobilized and dispersed on the surface of the support (Fig. 1). This systematically occurs when using anhydrous media, where an enzyme is not soluble, 13 but may also occur under other reaction conditions (e.g., ph near the isoelectric point, high protein concentration, etc.). Actually, this may be viewed as a comparison between two immobilized forms of the enzyme, an aggregated enzyme with severe diffusional problems versus an enzyme immobilized on a porous support with lower diffusional limitations. 14 The result may be that, in certain cases, an improved activity is observed using the immobilized enzyme compared to the aggregated enzyme. However, this improved activity should be considered an artifact and critically scrutinized. 2.2 Prevention of enzyme inhibition Inhibition may be another cause for enzyme activity alteration. Some enzymes may be inhibited by high concentrations of the substrate or by some of the reaction products, decreasing the observed activity. 15,16 Immobilization has been reported to prevent or at least diminish enzyme inhibition in certain Prof. Roberto Fernandez-Lafuente was born in He obtained his PhD under the supervision of Prof. Guisan in ICP-CSIC (Spain). After a postdoctoral period in UCL (UK) under the supervision of Prof. Cowan, he returned to ICP-CSIC, where he obtained a permanent position in Since 2008, he is a Research Professor. His research interests are the development of strategies Roberto Fernández-Lafuente for the preparation of improved biocatalysts and biosensors: enzyme purification, immobilization, stabilization and also reaction design. He has coauthored over 270 papers and 20 patents, and has supervised 15 doctoral theses, presenting a h number of 45. Fig. 1 Prevention of enzyme precipitation by immobilization. instances; therefore, in this specific case an increase in enzyme activity after immobilization may be expected (Fig. 2). 11 Caldolysin, a metal chelator-sensitive extracellular protease from Thermus aquaticus strain T351 is the first reported example of how after its immobilization, enzyme substrate inhibition may be avoided. 17 The proposed mechanism involved steric exclusion of the substrate from an inhibition site without significant interference with the active site. Lactases from different origins are inhibited by the substrates (lactose) and reaction products (glucose and galactose); it has been shown that this inhibition may be reduced by partial blocking or just by inducing a certain distortion in the inhibition site by immobilization 18 (Fig. 2). In other examples, rigidification of the enzyme structure by multipoint covalent immobilization has reduced some allosteric inhibitions (e.g., as shown in the synthesis of antibiotics catalyzed by penicillin G acylase). 19 Thus, a higher activity of a given enzyme after immobilization may be in some instances derived from a decrease in enzyme inhibition, and not from the production of a more active conformation of the enzyme. From an applied point of view, this inhibition reduction problem may have the same, or even more merit than an actual increase in activity (e.g., yields may progress until conversion reaches 100%), but the researcher needs to check on the existence of this kind of problem before assessing the real cause for the improvement in enzyme activity. 2.3 Activity determination under harsh conditions It should be considered that enzymes are quite unstable biocatalysts (i.e. their optimum operating range is considerably narrow) whose activity strongly depends on the experimental conditions. 20,21 The immobilization of an enzyme inside a porous support may have several protective effects on the enzyme structure under different situations. For example, many 6292 Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

4 View Article Online Fig. 2 Prevention of enzyme inhibition by immobilization. detergents may produce a decrease in enzyme activity by inhibition or by enzyme distortion,22 and if the enzyme is inside the pores of a support, it may be partially protected from this cause of activity loss (e.g., micelles may have more problems in penetrating inside the pores) (Fig. 3). The final results may be an apparent increase in activity, if measured in the presence of detergent. Similar effects may be produced if Fig. 3 the enzyme is subjected to strong stirring which is able to inactivate the enzyme (e.g. to disperse the substrate, introduce oxygen into the system, etc.). An enzyme inside the pores of a support will also be protected from this negative effect14 (Fig. 3). Another variable that strongly determines enzyme activity is the reaction ph. This is important considering that in many instances the support may be an ionic exchanger. These ionic Prevention of interaction of immobilized enzymes with external surfaces. This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

5 Fig. 4 Immobilization support as a solid buffer : effects on enzyme activity. exchangers may behave as a solid buffer, generating a ph inside the biocatalyst bead that may greatly differ from the ph value in the reaction medium (Fig. 4). If the immobilized enzyme is stored at a ph near its optimal value, while the activity measurement is far from this ph and it is performed in short times, the immobilized enzyme will remain in the optimal ph value even though the ph in the bulk may be far from it. Thus, the immobilized enzyme may be apparently more active at a ph value far from the optimal value. Thus, some precautions should be considered before stating that enzyme activity increases after immobilization, and that it is not a protective effect caused by the immobilization step. 2.4 Enzyme rigidification When considering enzyme performance, there are several factors that should be weighed in apart from purely chemical ones. Another point to be considered is that enzyme activity is linked to the stability of its structure. Changes in this structure tend to decrease enzyme catalytic activity. 20 A proper enzyme immobilization may produce a strong rigidification of the enzyme structure, mainly if a very intense multipoint covalent attachment is achieved. 23 In these cases, if the support matrix is rigid and the spacer arms are short, all enzyme groups involved in the enzyme immobilization process should maintain their relative positions under any circumstance, because one group cannot move regardless of the others. This multipoint covalent attachment may not be simple to achieve but using a proper support and suitable enzyme support reaction conditions, it has revealed itself as one of the most powerful strategies to stabilize enzymes. 11,14 Thus, a stabilized-immobilized enzyme should be expected to retain its structure under much more drastic conditions than the free enzyme (for example presenting a higher optimal temperature). 11 If a very high stabilization has been achieved, this optimal temperature for the immobilized form may yield a free enzyme activity almost null due to thermal enzyme distortion (Fig. 5). Thus, activity measurements of an immobilized enzyme at temperaturesabovetheoptimalforthefreeenzymemayresultin a significant increase in enzyme activity after immobilization. Considering that multipoint covalent attachment should prevent enzyme conformational changes induced by any reagent, it is expected that enzyme activity may be retained under any distorting conditions. Thus, similar improvements in enzyme activity upon immobilization may be caused by the presence of organic solvents, urea, guanidine and any other distorting agents. These will decrease the activity of the free enzyme while having a lower effect on the activity of the stabilized-immobilized enzyme. A special case lies in multimeric enzymes, formed by different subunits that may be in association dissociation equilibrium. 24 Multisubunit immobilization is able to fully prevent this phenomenon, thus avoiding this effect on enzyme activity and stability when assayed under dissociation conditions 7 (Fig. 6). To use the term artifact when the researcher finds an increased activity under these drastic conditions due to immobilization perhaps is not exact. The most accurate way to express it would be that the observed enzyme activity under these conditions increases due to immobilization-induced rigidification. The increased enzymatic activity under those conditions, however, is not caused by the generation of a more active enzyme form, but by avoiding distortion of theenzymestructure Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

6 Fig. 5 Enzyme rigidification by immobilization decreases enzyme distortion. Fig. 6 Multisubunit enzyme immobilization prevents subunit dissociation. 2.5 Effect of medium partition In some instances, immobilization may greatly alter the physicochemical properties of the enzyme surroundings, generating a much more hydrophobic or hydrophilic environment that can produce some partition of different compounds away or towards the enzyme. 14 This is in fact a strategy to stabilize enzymes versus some inactivation agents, such as oxygen, hydrogen peroxide, dissolved gases or organic solvents If the immobilized enzyme is further modified with polymers, 8 the stabilizing effect becomes impressive (Fig. 7). In an aqueous organic co-solvent system, the organic solvent may in many instances greatly reduce enzyme activity (by inhibition or enzyme distortion). 28 If the enzyme is exposed to lower organic co-solvent concentration by partition, the observed result is an increase in enzyme activity (Fig. 7). In this case, we are reducing the cause for decrease in enzyme activity. However, as in the aforementioned cases, the researcher will observe an increase in activity after immobilization, which once again will be fully unrelated to the production of a hyperactivated form of the enzyme. This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

7 Fig. 7 Effect of medium partition on properties of immobilized enzymes. 2.6 Effect of substrate or product partition As explained above, immobilization may in some instances produce a partition of different compounds. 14 If a partition of the substrates or products is achieved after immobilization, this may affect enzyme activity depending on the different possibilities of the enzymatic kinetics. And in some cases, the effect may be positive. If the used substrate concentration is below that required to saturate the enzyme, and the enzyme environment permits partitioning of the substrate towards the enzyme, an apparent increase in activity will be observed. The actual situation will be a decrease in the apparent K M,whileK cat will remain unaltered (Fig. 8). If a high concentration of substrate is used and the partition effect reduces the concentration of substrate, this can promote Fig. 8 Effect of substrate partition towards or away the enzyme environment on the enzyme properties Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

8 a positive effect on the observed activity, provided that the substrate is able to inhibit the enzyme. Once again, K cat will remain unaltered, but an increase in K M and K i will be observed (Fig. 8). A similar positive effect could be detected if the products were excluded from the enzyme environment and were able to inhibit the enzyme. These effects will be similar to that found in any biphasic system. 29 In all these cases, a complete kinetic study of the reaction will clarify the actual causes for the observed increases in activity after immobilization. As in the other cases, this operational increase in enzyme activity upon immobilization is unrelated to the production of an enzyme structure with better properties induced by its fixation to the support. 2.7 Diffusional limitations Diffusion limitations have been usually considered to be a problem that reduces enzyme activity. 14 If substrate diffusion inside the support particle is slower than its catalytic modification, enzymes in the core of the catalyst particle will not receive the same substrate concentration as the enzyme near the surface of the particle. However, in some instances these diffusional problems may turn out for the best. The decrease of substrate in the enzyme environment can only produce an improved activity if the substrate may produce a strong inhibition on the enzyme and we are using substrate concentrations high enough to produce this negative effect on enzyme activity, as it has occurred in many industrial processes. 19 This way, the decrease in substrate concentration in the enzyme environment far from producing a decrease in enzyme activity may actually increase it (Fig. 8). Internal ph gradients may be formed in enzymes immobilized on porous supports if the activity of the biocatalyst is high enough 30,31 (Fig. 9). This is usually considered a disadvantage since the particle internal ph becomes different from that of the bulk. These different ph values have been used to increase enzyme operational stability. 32 In a similar way, if the external ph value does not correspond to the optimal ph for the enzyme activity, it is possible that the situation in the presence of ph gradients may result in enhanced activity, e.g. if the ph value inside the biocatalyst particle is nearer to the optimal ph value than the one in the bulk. This may occur in the laboratory when using standard measurements protocols, and also in industry if we must work under conditions far from the optimal ones of the enzyme, some times due to substrate solubility or stability, or process thermodynamics. Another possibility where diffusion may increase enzyme activity is when two coupled enzymes are used, co-immobilized on the same porous particle, mainly when determining the final product to state the global activity 14,33 (Fig. 10). If the production of product 1 is fast enough, this compound will accumulate inside the pore and can cause the second enzyme to act under a higher substrate concentration. In these cascade reactions, the second enzyme will be frequently working at concentrations of substrate under the saturation conditions, and this increase in its substrate concentration may yield an improved activity. This has been recently exemplified using two coupled redox enzymes. 34 The activity using low cofactor concentrations was higher using the co-immobilized enzymes, not only compared to the enzymes immobilized in different particles, but also when using similar amounts of soluble enzymes. However, measuring each enzyme individually and using the adequate concentrations of their respective substrates, enzyme activity did not improve for any of the enzymes after immobilization, because the enzyme structure was unaltered. This effect was only observed when measuring the whole biocatalyst Fig. 9 Effect of ph gradients inside the particle of biocatalyst on enzyme properties. This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

9 Fig. 10 Improving enzyme activity of co-immobilized enzymes due to partition of substrates and products inside the pores of the biocatalysts. using substrate 1 and the whole cascade (the actual industrial target), where substrate availability for the second enzyme is improved. 2.8 Freezing of a more active conformation It is important to remark that some enzymes exist in different conformational states with different activities and/or stabilities. 35 Moreover, many multimeric enzymes may exist in different degrees of aggregation, having different catalytic properties. 36 Perhaps the best known case is that of lipases. 35 Lipases exist in two main forms, open and closed forms. 37,38 In aqueous medium, the equilibrium between these two forms is displaced to the closed form, where the active center is secluded from the reaction media by a polypeptide chain called lid or flap, which in many cases is almost fully inactive. On the other hand, in the presence of a hydrophobic interface, like drops of their natural substrate (oils), the lid is displaced and the active center becomes exposed to the medium, displacing the equilibrium to the open and active form. The open form of the lipases becomes adsorbed via the large hydrophobic pocket exposed (formed by the internal face of the lid and the area surrounding the active center) to the hydrophobic surface (Fig. 11). This is the so-called interfacial activation of lipases. 35 Immobilization may become a tool to fix this open form of the lipase. It has been shown that this may be easily achieved by immobilization of the enzyme at low ionic strengths on hydrophobic supports, 39 and also by cross linking 40 or lyophilization in the presence of detergents. 41 The effect of immobilization on hydrophobic supports using lipases against fully soluble substrates may be even more beneficial. Lipases have a trend to form bimolecular aggregates, interfacing the active centers of two open forms of lipases, and that enzyme conformation tends to be less active than the monomeric form, because the lipase active center is partially blocked. 42 Adsorption on hydrophobic supports (presenting large surfaces) will give a dispersed open form, cleaving these dimers (Fig. 12). Thus, in this case, the observed enzyme activity is improved by displacing the equilibrium towards the monomeric open form of the lipase without the need for adding an external hydrophobic interface. Moreover, by changing the support morphology and hydrophobicity, it has been shown that it is likely to have an optimal open form of the lipase, with an activity even higher than that of the enzyme adsorbed on drops of insoluble substrates. 14 Some other enzymes may be hyperactivated by a conformational change induced by an effector (an activator, some Fig. 11 Interfacial activation of lipases on hydrophobic supports: congealing a hyperactivated enzyme form Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

10 Fig. 12 Hyperactivation of lipases by breaking the dimers by immobilization on hydrophobic supports. specific medium). 43 If the enzyme is attached to the support via many points, or if lyophilization is performed in the presence of this effector, enzyme molecules with this hyperactivated form may be produced, which will remain hyperactivated in the absence of the effector (Fig. 13). In this case, immobilization is stabilizing a hyperactivated enzyme form induced by a molecule or reaction medium, in a way that this hyperactive form will be retained even in the absence of the effector. 2.9 Production of a new more active conformation Immobilization of enzymes produces conformational changes and/or chemical modifications when incorporated to the support. It is very likely that enzyme activity versus its natural substrate may suffer a certain decrease. However, in many instances the target substrate is quite far from the physiological one. Although we will discuss this point more extensively at a later stage, if we prepare a large library of immobilization methods, for example involving different enzyme regions in Fig. 13 Effect of effectors during enzyme immobilization on enzyme properties. This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

11 the immobilization and giving different degrees of enzyme support interaction or generating different enzyme microenvironments, it is not unlikely to find some biocatalysts with a higher specific activity versus a particular substrate. 11 This random hyperactivation produced by a particular immobilization method against a particular substrate will be based on the casual generation of a more active enzyme form, and it would be more likely to occur with enzymes having a flexible active center (e.g., lipases, multimeric enzymes), and if a sufficiently large biocatalyst library is prepared. 3. Changes in enzyme specificity or selectivity by immobilization In this section we will discus how immobilization may greatly affect enzyme specificity and/or selectivity. 11 These changes may deeply alter enzyme performance in several reactions of industrial relevance: - Resolution of racemic mixtures: 44 enzymes are in many instances used as catalysts in the dynamic resolution of racemic mixtures of substrates different from the natural ones (where a complete specificity should be expected), via hydrolysis, esterification, amination, transesterification, etc. Using these unnatural substrates, specificity may not be complete. If immobilization alters K M or K cat towards one or both enantiomers, the enantioselectivity of the enzyme and the obtained enantiomeric excess may be greatly affected. - Enantioselective modification of prochiral compounds (e.g., reduction of prochiral ketones, asymmetric hydrolysis of prochiral dicarboxylic esters or asymmetric acylation of prochiral carboxylic acids, etc.): 44 again, the use of substrates far from those natural to the enzyme may produce moderate enantioselectivity values. Immobilization may alter the preferred produced isomer, by favoring one or the other transition state. - Regioselective modifications of poly-functional compounds: 45 for instance, regioselective hydrolysis of peracylated polyhydroxy compounds, regioselective synthesis using polyhydroxy (e.g., sugars, glycerin) or poly carboxylic acids, oxidations of poly-alcohols, etc. In this case, immobilization may affect the adsorption of the substrate on the enzyme active site, confronting different groups with the catalytic residue thus modifying the selectivity of the process. The final percentage of the target molecule will also depend on the rate of the successive modifications of the other groups in the substrate. Therefore it should also be related to the specificity of the enzyme between the different possible compounds. - Kinetically controlled synthesis: this process is based on the use of an activated acyl donor 46 such as an ester or an amide (or vinyl adduct) to reach maximum transient yields that depend on the balance of three different reactions catalyzed simultaneously by the enzyme: the formation of the target product, the hydrolysis of the activated acyl donor, and the hydrolysis of the target product. Examples of this reaction are transesterifications, transformations of esters by amides, transamidations, transglycosylations, etc. The kinetically controlled synthesis of antibiotics 47 or the synthesis of biodiesel 48 may be some of the most relevant examples. Enzyme performance on kinetically controlled synthesis depends on the adsorption of the nucleophile on the active center of the enzyme, the specificity of the enzyme versus the active acyl donor and the product, the possible inhibitions of one or the other reaction, etc. 46 (Fig. 14). Obviously, all these processes depend on the enzyme and will be deeply modulated by the enzyme structure after immobilization Interesterification and acidolysis (e.g., to produce structured triglycerides): 49 the mechanism of these reactions is quite complex. Interesterification may be carried out using a blend composed of several oils of different sources, employing just one oil which presents different fatty acids or mixing one oil together with esters of the desired fatty acid. In the case of glycerides, if we want to introduce a new fatty acid into the glycerol moiety, the ester bond between the native fatty acid residue (the original substituent group) and the glycerol moiety Fig. 14 General scheme of kinetically controlled synthesis of ampicillin Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

12 Fig. 15 Fig. 16 General scheme of interesterification. General scheme of acidolysis. must first be hydrolyzed. This reaction liberates the native fatty acid and produces a lower (less substituted) glyceride containing at least one hydroxyl group. The hydrolysis step is followed by the formation of a new ester bond by reaction of the newly created hydroxyl group with the incoming replacing fatty acid (that also needs to be released from the ester) 49 (Fig. 15). The acidolysis mechanism in this reaction is similar to an interesterification. After hydrolysis of an ester bond between the native fatty acid residue (the original substituent group) and the glycerol moiety of the triglyceride, the native fatty acid is released and a glyceride containing at least one hydroxyl group is produced. The hydrolysis step is followed by the formation of a new ester bond by reaction of the newly created hydroxyl group with the incoming new free fatty acid 49 (Fig. 16). Thus, the specificity of the enzyme by the different fatty acids and triglyceride positions becomes a key point towards the final yields of the structured triglyceride. Like in the case of the activity, the enzyme properties in all these processes may be strongly modulated by immobilization via different ways. Next, we will explain some of the most relevant. 3.1 Effect of diffusion limitations At first glance, diffusional problems will always have either a null or a negative effect on the observed results in these processes. In any reaction where enzyme specificity may play an important role, the concentration of the best substrate will decrease more rapidly than that of the less suitable substrate. Thus, for example, in the resolution of racemic mixtures, substrate diffusional problems can only produce a decrease in the apparent enzyme enantiospecificity. In kinetically controlled syntheses, the consumption of the nucleophile and the accumulation of the product along the pore of the support can only produce a decrease in the maximum yields, by decreasing the saturation of the enzyme by the nucleophile and favoring the hydrolysis of the formed product. 14 In selective reactions, enzyme selectivity did not appear to be influenced by the substrate concentration, and only a decrease in the reaction rate should be observed. Thus, at first sight, low enzyme loadings and enzyme distributions along the particle pores in a way that overcome these diffusional limitations (e.g., forming a crown on the external part of the bead) should be always advantageous. 34 However, as discussed above, diffusional problems may alter not only substrate concentration, but also the ph inside the particle. 30 The reaction ph may exert a critical influence on any enzyme property, and this effect of the ph on such properties may also be altered by immobilization (as will be discussed later). Thus, while substrate diffusional problems can hardly have any positive effect on the enzyme performance in this kind of processes, ph gradients may be used as a tool to improve the results after enzyme immobilization, and can bring forth improvements in the enantiomeric excesses obtained (in the dynamic resolution of racemic mixtures and in enantioselective processes) and also in the maximum yields in kinetically controlled processes. 45,46 Thus, even without directly affecting the enzyme structure, the promotion of internal ph gradients inside the particles of porous biocatalysts may produce a significant improvement in enzyme performance upon immobilization in this kind of reaction. 3.2 Generation of micro-environments around the enzyme Enzyme properties and performance on the processes described above are strongly influenced by the concentration of both substrates and products. If the enzyme is immobilized in a very hydrophobic (e.g., supports made of divinylbenzene) 50 or hydrophilic environment (e.g., polymeric beds anchored to the support surface, formed by polyethylenimine or dextransulfate), some partition of the substrates may be expected. 8,14 A positive partition of the substrates may have some beneficial effects on the enzyme performance, e.g., the enzyme may be saturated for longer periods of time by the preferred substrate in dynamic resolutions or by the nucleophile in kinetically This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

13 controlled processes. A partition of the product that will reduce the product concentration around the enzyme may be positive in a kinetically controlled process, by reducing the hydrolysis of the product and increasing the maximum yields. 46 In some cases, inhibitions caused by the products may be also relevant for the final results (e.g., after hydrolysis of the preferred isomer, the product may be an effective inhibitor of the hydrolysis of the undesired isomer). Thus, some partition of substrates and/or products from the immobilized enzyme environment in the right direction may greatly improve enzyme performance, and this may be achieved without really affecting enzyme conformation but just altering the availability of the different compounds involved in the reaction. Enzyme properties are also governed by the experimental conditions, and the nature of the support may promote some partition on the components of the medium. This is very clear in the presence of organic solvents. If the enzyme is in a highly hydrophilic polymeric bed, 8,14 the concentration of solvent around the enzyme will be lower than that in the reaction medium. If this lower concentration of organic solvent improves enzyme performance (e.g., producing a higher selectivity or specificity), after immobilization we can detect an improvement in the enzyme performance in the reaction. 45 This will not be a consequence of changes in the enzyme structure, but will be due to changes in the reaction conditions under which the enzyme operates. In any case, the final effect will be an improvement in enzyme performance in this kind of processes. 3.3 Immobilization of mixtures of enzymes In some instances, commercial preparations of enzymes or the crude extract obtained in a laboratory contain several enzymes that are able to catalyze a similar reaction Although this is not an ideal situation, many researches have been carried out using this mixture of enzymes. In some instances, it is difficult to even detect the presence of the contaminant enzyme that may be in trace amounts but being very active versus some specific substrates (e.g., chymotrypsinogen B in preparations of porcine pancreatic lipase). 51 In others the microorganism produces a collection of isoforms (e.g., the isoforms of lipases in Candida rugosa). 54 The properties observed using these crude preparations will be the average of the whole mixture of enzymes able to catalyze the target reactions (and will depend on the exact batch). Upon immobilization, several factors may decrease the relevance in the reaction for some of the enzymes. First, not all enzymes will become immobilized on all supports, and either by chance or on purpose (e.g., when immobilization is designed to associate immobilization and purification of the target enzyme) the contaminant enzyme may not become immobilized on the support. 51 If the enzyme that did not become immobilized on the support is the one having the poorest performance in the process, we can observe an improvement in the results obtained using the immobilized preparation when compared to the free enzyme. This is produced by the purification of the enzyme during immobilization, not by an actual alteration of the enzyme properties (Fig. 17). Another possible effect derived from immobilization is that some enzymes become significantly more inactivated versus the target substrate than others. The practical effect will be similar to the one described above, if the most inactivated enzyme is the one having the worst performance, the immobilized biocatalyst will exhibit a better behavior than the collection of free Fig. 17 Effect of the selective immobilization of a determined enzyme when using mixtures of enzymes Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013

14 enzymes. In this case, this improvement will be a consequence of the selective inactivation of one of the enzymes during immobilization. Different stabilizations due to the immobilization of the different components of the crude extract which are able to perform the reaction may also produce an effect on the performance of the immobilized biocatalyst. This mainly produces different behaviors when using conditions where the free enzyme suffers conformational changes reducing its activity. If one enzyme is much more stabilized than another after immobilization, it may retain more activity under these drastic conditions. 11 If it is the one having the best performance, an improved behavior of the immobilized biocatalyst compared to the free enzymes preparation under these conditions will be observed. Now, this will be a consequence of the preferential stabilization of one enzyme during insolubilization of the enzymes. Obviously, all these positive effects derived from immobilization of an enzyme mixture will depend on the substrate used (e.g., using a substrate where only one enzyme has activity, this effect is not possible). Thus, a deep characterization of the crude preparation may be necessary to fully understand the changes in enantio- or regioselectivity or specificity, or in kinetically controlled synthesis after immobilization. 3.4 Enzyme rigidification As commented in Section 2.4, a strong rigidification of the enzyme structure via multipoint covalent attachment may help keeping this conformation when the conditions are altered. 11 If the enzyme is utilized under conditions (e.g., using solvents to solubilize the substrate) under which the free enzyme suffers some distortion that decreases its specificity or selectivity, and the immobilization permits to keep the enzyme features, the observed result after immobilization may be an improvement in the results (Fig. 5). 3.5 Changes in enzyme structure due to immobilization As it was previously mentioned in Section 2.9, immobilization of an enzyme will most probably alters its structure to some degree, due to unspecific enzyme support interactions or the interactions that cause the immobilization. 10,11,14 In many cases, immobilization produces a change in enzyme activity, but these changes may also be correlated to changes in the behavior of the enzyme in any of the aforementioned processes. If the enzyme has a rigid active center, it may be very hard to find an immobilized preparation with improved properties. However, the situation may be different if the enzyme has a flexible active center Conformational engineering of enzymes suffering structural changes during catalysis. Some enzymes suffer drastic conformational changes during the catalytic process. As stated above, lipases are perhaps the best known enzymes in this aspect. All lipases have the capability of acting at the interface of oil drops by interfacial activation, their adsorption on these drops takes place via the large hydrophobic pocket formed by the internal face of the lid and the hydrophobic areas surrounding the active center that interact with it. 37,38 The lid may be very small, like the one of the lipase B from Candida antarctica 55 not fully secluding the active center from the medium, or quite complex, like the double lid recently described for the thermoalkaline lipase from Bacillus thermocatenulatus. 56 These movements do not affect only the lid, but alter the overall structure of the lipases (Fig. 18). In fact, although the crystal structure of a lipase shows only one open form, it is not hard to imagine that depending on the conditions under which the lipase moves its lid, different conformations of the active center may be found. That will mean that lipases will have a very flexible active center that may tolerate some distortions without losing its catalytic activity. Based on this idea, it has been proposed that the immobilization of this kind of enzymes on a battery of different supports, under different immobilization conditions, involving different regions of the enzyme surface in the immobilization, giving different degrees of rigidification, establishing different interactions between the enzyme and the support, or generating different microenvironments (Fig. 19), may generate a library of biocatalysts based on a single enzyme exhibiting very Fig. 18 Structure of open and closed forms of RML. The 3D structure was obtained from the Protein Data Bank (PDB) using Pymol vs This journal is c The Royal Society of Chemistry 2013 Chem. Soc. Rev., 2013, 42,

15 different enantioselectivity, enantiospecificity, regioselectivity, and even alter the yields in kinetically controlled synthesis. 10,11,14,45 This modulation of the enzyme properties is currently uncontrolled, because the in silico techniques can point the areas involved in the immobilization step, but still are far from predicting small changes caused by the enzyme support interactions. Moreover, the changes in enzyme properties by immobilization will be produced even if we already have a suitable biocatalyst and we do not want to alter these properties. In this case a very mild immobilization (e.g., using a lowly activated aldehyde dextran as a spacer arm) 11 is preferable. The effects of the immobilization strongly depend on the substrate. For a particular substrate the best biocatalyst may be one, while for other substrates the best biocatalyst may be completely different. This is usually observed using different lipases against different substrates. 45 Furthermore, it has been established that the reaction medium conditions exert very different effects on the enzyme catalytic features when changing the immobilization protocol. 11 Thus, in many instances a change in the reaction conditions improves enzyme performance when it is immobilized on a support, while it decreases the enzyme activity that was immobilized by using another protocol. 45 This may be explained by the influence of the experimental conditions on the interaction between the enzyme and the support (e.g., if the support is not fully inert), by a different effect on the same change in the medium when the enzyme structure is different (e.g. caused by different immobilization protocols), or if some particular region of the enzyme is stabilized and cannot move. The suitability of this strategy to tune the enzyme performance depends on the size of the biocatalyst library (Fig. 19). Immobilization affects the enzyme features, but in some cases has a deleterious effect on enzyme performance and only in some others will improve it. Thus, the wider and more different the biocatalysts that are included in said library, the higher the possibility of finally finding a catalyst able to exhibit adequate properties in a particular reaction. Therefore, while in order to have a stabilized biocatalyst there are some preferred strategies, such as to give an intense multipoint covalent attachment, 11 to modulate the enzyme catalytic features almost any immobilization protocol that yields a stable enough preparation may be interesting. 14 In this sense, some immobilization protocols that may be used to immobilize enzymes via different orientation but using the same chemical groups in the support have substantial interest. One of these examples is the use of epoxideactivated supports. 57 Epoxide activated supports, despite being reactive with many different groups of proteins, react very slowly with free enzymes. The enzyme requires to be first adsorbed on the support, and then it reacts covalently with the support. It has been shown that by adding some adsorbing groups to the support surface, these groups adsorb the enzyme, and the orientation of the enzyme on the support may be fully altered, and that produces a change in the enzyme features. 57 Another example is the adsorption of enzymes on polymeric beds formed by ionic polymers coating the support surface. The ionic strength during immobilization may permit to control the penetration of the enzyme in the polymeric bed, 58 while orientation may also depend on the immobilization ph value. One of the oldest methods found for enzyme covalent immobilization is the use of glutaraldehyde chemistry. This protocol may be used to have different forms of the lipase, at least five. 59 Using high ionic strength, ionic adsorption is avoided, but CALB is adsorbed on the support by interfacial activation. Using nonionic detergents (e.g., Triton X-100), the enzyme becomes Fig. 19 A library of biocatalyst from just one enzyme: different orientations, rigidification or microenvironments Chem.Soc.Rev.,2013, 42, This journal is c The Royal Society of Chemistry 2013