Biocatalysis 2015; 1: 166–177

Review Open Access

Susana Velasco-Lozano*, Fernando López-Gallego, Juan C. Mateos-Díaz, Ernesto Favela-Torres Cross-linked enzyme aggregates (CLEA) in enzyme improvement – a review

DOI 10.1515/boca-2015-0012 industries [1-3]. However, to be profitable at the industrial Received September 17, 2015; Accepted November 25, 2015 level, industrial enzymes must allow easy handling Abstract: Structural and functional catalytic and operation procedures, stability and reuse. For this characteristics of cross-linked enzyme aggregates purpose, enzyme immobilization has emerged as a tool (CLEA) are reviewed. Firstly, advantages of enzyme to produce robust industrial biocatalysts; and it has been immobilization and existing types of immobilization employed over the last forty years for the successful are described. Then, a wide description of the factors utilization of enzymes in industrial processes [4]. that modify CLEA activity, selectivity and stability is Immobilization is defined as the confinement of presented. Nowadays CLEA offers an economic, simple enzymes in a defined space, retaining their catalytic and easy tool to reuse biocatalysts, improving their activity and allowing their repeated and continuous catalytic properties and stability. This immobilization use [5]. Furthermore, immobilization of enzymes must methodology has been widely and satisfactorily tested meet other criteria to obtain robust biocatalysts. Any with a great variety of enzymes and has demonstrated immobilized enzyme should comprise non-catalytic its potential as a future tool to optimize biocatalytic requirements (shape, size, thickness, length, etc.) processes. designed to aid separation and reuse; as well as, catalytic requirements (activity, selectivity, stability, pH and Keywords: cross-linking, enzyme, immobilization, temperature optimum, etc.) designed to convert the target stabilization compounds [6]. The immobilization of enzymes affects their conformation, rigidity and aggregation state, modifying their reactivity [7]. Thus, enzyme immobilization has 1 Introduction been also utilized to modulate enzyme selectivity [8-10]. The immobilization of enzymes offers the following Enzymes play an important role in relevant advantages: 1) The possibility of reuse, 2) The increase in biotechnological processes in energy, food and chemical stability, such as resistance to high temperatures, extreme pHs, high substrate concentration, polar solvents, mechanical shear, among others, 3) Increased volumetric *Corresponding author: Susana Velasco-Lozano, Heterogeneous activity, and 4) Increase and modulation of selectivity, Biocatalysis group, CIC Biomagune, Parque Tecnológico de San Sebastián, Edificio Empresarial “C”, Paseo Miramón 182, 20009, such as regio-, acyl-, chemo- and enantioselectivity [11-14]. Donostia-San Sebastián Guipúzcoa, Spain, E-mail: svelasco@ Methods with physical or chemical interactions are cicbiomagune.es used for enzyme immobilization. When physical methods Susana Velasco-Lozano, Ernesto Favela-Torres, Departamento de are used, the enzyme does not undergo structural changes Biotecnología, Universidad Autónoma Metropolitana-Iztapalapa. Av. as it is physically attached or trapped by the support San Rafael Atlixco #186, Col. Vicentina 09340, D.F. México Fernando López-Gallego, Heterogeneous Biocatalysis group, CIC structure. The main advantage being that the immobilized Biomagune, Parque Tecnológico de San Sebastián, Edificio Empre- enzyme, trapped or encapsulated, nearly maintains its sarial “C”, Paseo Miramón 182, 20009, Donostia-San Sebastián native state; allowing its reuse and, in most cases, an Guipúzcoa, Spain improvement in the enzyme stability [15-17]. However, the Fernando López-Gallego, Ikerbasque, Basque Foundation for Sci- main disadvantage is that the union between the support ence, 48011, Bilbao, Spain and the enzyme is very weak; causing enzyme leakage Juan C. Mateos-Díaz, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, A.C., Unidad de Biotec- while the biocatalyst is operating in aqueous medium. nología Industrial, Guadalajara, México Adsorption, entrapment, and encapsulation are the

© 2016 Susana Velasco-Lozano et al., published by De Gruyter Open. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License. Cross-linked enzyme aggregates (CLEA) in enzyme improvement – a review 167 more representative types of physical immobilization. In successfully used through the last fifteen years over a contrast, chemical immobilization may alter the protein wide range of enzymes, and has proved to be an easy, fast, structure because enzymes are attached on the surface efficient and suitable approach in enzyme optimization; and/or inside the support by chemical bonds which may therefore, in this mini-review we summarize a battery of modify its shape, rigidity and aggregation state [18]. examples where enzymes properties has been improved Covalent bonding to substrates and self-immobilization by this technology. are within these types of immobilization. Immobilization of enzymes by covalent attachment to a support is a method in which proteins are irreversibly 2 Carrier-free immobilization types, linked. To achieve immobilization, the reactive amino advantages and disadvantages. groups of amino acids such as lysine, typically present in the surface of proteins, are mainly used. With these amino The carrier-free immobilized enzymes do not need acids, the covalent attachment of enzymes to a reactive an extra inactive mass, the support. They are usually group of the support leads to a highly stable covalent produced by direct cross-linking of dissolved enzymes bond. Epoxides are commonly used as reactive groups (CLE), crystallized enzymes (CLEC), and aggregated on supports to react with the amino groups of lysine enzymes (CLEA). within the enzyme [19]. Covalent attachment usually The first report of such structures was described by provides the strongest bond between the enzyme and Quiocho and Richards [25], who found that insoluble the support compared to other types of immobilization, proteins linkage could be obtained by using bifunctional being able to minimize the leakage of enzyme from the cross-linking chemical groups, such as glutarladehyde. support [18]. Besides allowing its reuse, the majority of These linked proteins known as cross-linked enzymes supports provide the enzyme with a protective barrier (CLE) retained their catalytic activity. When the link is [14]. Nonetheless, in many cases, the supports used have based on purified enzymes, linked proteins are known as high prices raising the cost of production of the thereby cross-linked enzyme crystals (CLEC) [26]. However, at the immobilized biocatalysts [20]; on the other hand, carrier- time the work of Quiocho and Richards was published, the free immobilization or self-immobilization is possible efforts in immobilization research were focused towards since it achieves linking of two enzyme molecules through immobilization by adsorption, entrapment and chemical bifunctional cross- linking agents or simply cross-linkers bonding to supports, due to the low activity and stability without the use of a support. The bifunctional compound retention that CLE exhibited. These structures began to be most commonly used for this purpose is glutaraldehyde studied more intensively in the 1990’s [11,27]. (GA) [21], which is a small dialdehyde molecule. Some The CLE are formed with proteins in solution that are advantages of self-immobilization are greater volumetric linked together by a cross-linking agent having sizes from activity per biocatalyst mass, increased specific activity, 1 to 100 μm. Compared to the native enzymes, this type of easier production, reduced production costs, higher biocatalyst improves their thermal stability, but requires purity, less interferences and/or contaminations by the the care of various factors such as pH, the amount of cross- support and lower mass transfer limitations [6,22]. Carrier- linking agent, ionic strength and temperature. However, free immobilized enzymes are advantageous catalysts in CLE present disadvantages such as low native activity processes requiring high yields and productivities, where retention (usually less than 50%), poor reproducibility, enzymes cannot be stabilized through the conventional and low mechanical stability [6]. These inconveniences solid supports [23]. have been partially overcome by entrapping CLE in a gel Although there are many immobilization strategies, matrix or membranes [28-30]. none of them overcome all enzymes’ limitations in all Along with CLE, studies were directed to the production kinds of processes. The selection of specific strategies of CLEC, that are suitable for biotransformations in non- of immobilization will depend on many factors such aqueous media, due to its high stability under harsh as limitations inherent to the enzyme and the process conditions [25,31]. CLEC are crystalline proteins, bonded in which it will be used [24]. Thus, the selection of the together with a cross-linking agent. They achieved best enzyme immobilization technique will depend on improved mechanical strength and high stability against its application demands as well as the balance between temperature, pH, organic solvents and proteolysis cost-production and the potential uses of the immobilized [22,32,33]. For instance, lipase-CLEC have been developed biocatalyst on its direct application. In this context, cross- in order to improve drug delivery under natural low-pH linked enzyme aggregates (CLEA) technology has been gastric conditions [34]. The catalytic activity obtained 168 S. Velasco-Lozano, et al. with CLEC can be improved by controlling the size of the enzymes is clearly advantageous since it is possible to enzyme crystals. It was found that minimizing the crystal feed reactors with a higher amount of enzyme, which size increases the catalytic activity retained. However, compensates the loss of activity during reuse, without it requires laborious steps of crystallization of proteins, prolonging the reaction time [6,42]. In contrast, the which increases the production´s cost [35]. enzymes immobilized on supports occupy more than Compared to crystallization, a less expensive method a 10−20% volume of the reactor, which causes the to aggregate the enzyme molecules and further cross- prolongation of reaction time in order to achieve the same linking is precipitating the proteins by adding salts, conversion levels [43]. organic solvents or non-ionic polymers [20,36]. This idea The selectivity of carrier-free immobilized enzymes as was first introduced with penicillin G amidase in the CLEC and CLEA strongly depends on the conformation of Sheldon`s laboratories [37] and subsequently marketed the crystals or the enzyme aggregates [37]. Crystallization by CLEA Technologies (Holland). Cross-linked enzyme or aggregation conditions to obtain CLEC or CLEA can aggregates (CLEA) are obtained from non-purified alter the selectivity and/or activity of biocatalysts [21]. precipitated enzymes forming solid particles that remain However, although CLEA eliminates the need for together through non-covalently binding. These CLEA crystallization of the enzyme of interest, there remains remain permanently insoluble after covalent binding with an inherent disadvantage; since a precise protocol of a cross-linking agent such as glutaraldehyde (Fig. 1) [27,37]. aggregation and cross-linking must be established for CLEC and CLEA exhibit 10 to 1000 times greater activity each enzyme, implying that significant efforts must be (U g-1 of biocatalyst) than their corresponding biocatalysts made to optimize preparation conditions [44]. attached to a support [22,38-41]. Consequently, the use CLEA stability and activity are similar to those of CLEC of carrier-free immobilized enzymes could be beneficial [6,37]. The catalytic properties of CLEA depend on the for processes requiring high productivity and yield, long precipitating agent. Different precipitating agents induce stability and for labile enzymes that cannot be efficiently distinct conformations of enzyme aggregates, in such a stabilized by traditional methods of immobilization on way that the selectivity of CLEA strongly depends on the supports. Besides, the use of carrier-free immobilized conformation of the enzyme aggregates [37].

Figure 1. Simple process for the preparation of CLEA in an organic solvent. Reprinted from reference [70] with the permission of Springer (license number 3687170268753). Cross-linked enzyme aggregates (CLEA) in enzyme improvement – a review 169

Similar to carrier-immobilized enzymes, CLEA CLEA might form aggregates of larger size (> 1 µm), present a better stability than the free enzyme under called “clusters”, presenting mass transfer limitation. severe conditions of reaction such as pH, organic solvents, Future studies are aimed at the formation of CLEA with temperature, and higher resistance to substrates and fewer tendencies to form “clusters”. It has recently been inhibitory products or denaturants [20,45,46]; attributes reported that increasing the stirring speed and time that turn them into promising insoluble biocatalysis for avoids the formation of “clusters” as they decrease the industrial applications. size of CLEA without inactivating them, achieving an An important property of CLEA for their large scale increase in mass transfer and allowing 100% recovery application is the particle size, which has a direct effect on of the original activity of the free enzyme. With this mass transfer limitations and filterability [47]. Typically, purpose, the reciprocating cell disruptor (FastPrep®) has the particle size of a CLEA varies between 0.1−200 µm. been used. In comparison with the use of a vortex, use However, during its application in industrial processes, of FastPrep® allows decreasing the size of CLEA in just where recovery operations such as filtration and 30−60 s of treatment, thus recovering 100% of activity. centrifugation are required, increase in size particle will In contrast, the use of a vortex requires stirring for promote mass transfer limitations with the consequent 90 minutes to recover 80% of the initial activity [48,51,52]. reduction in its overall activity [48]. Among the factors Another effort to increase CLEA’s mass transfer proved determining the CLEA size, the amount of enzyme and that porous CLEA (p-CLEA) can be obtained by adding the concentration of the cross-linker play a major role starch as a pore-forming agent, which at the end of the [21]. Both parameters can modify the final result of the process was removed with α-amylase [53]. This will allow biocatalyst, as reported for CLEA of lipase from Candida the use of macromolecular substrates. rugosa, whose optimum activity in the hydrolysis of lauric Despite the advantages of carrier-free immobilized acid was obtained with 40−50 µm particles [49]. enzymes, there are certain inconveniences to solve since According to their morphology, CLEA can be classified no one knows exactly how to control the size of enzyme as: Type I. These CLEA measure around 1 µm in diameter aggregates, how to increase their flexibility, as well as the and are formed from low glycosylated enzymes with large exact methodologies to modulate the stability, selectivity hydrophobic surfaces such as CAL-B, which contains and activity [6]. Unfortunately, the design of immobilized 8 × 108 enzyme molecules per CLEA and forms aggregates enzymes for its optimal performance is still largely empirical of a spherical shape (Fig. 2A); and Type II. Which measure [27] and deep characterization studies are missing. less than 0.1 µm in diameter and are formed from enzymes with large hydrophilic surfaces and high glycosylation levels, such as lipases from Candida rugosa and 3 Cross-linking chemical nature oxynitrilase from Prunus amygdalus. These type of CLEA and most employed cross-linkers. present around 1 × 104 enzyme molecules per CLEA, and are aggregates of slightly irregular shapes and are smaller Glutaraldehyde (GA) is the cross-linking agent most than those in type 1 (Fig. 2B) [50]. frequently used for the preparation of CLEA, since it

Figure 2. CLEA morphology. A) Type 1, CLEA of CAL-B, amplification X3500; and B) Type 2, CLEA of CRL, amplification X25000. Reprinted from reference [50] with the permission of John Wiley and Sons (license number 3687171223427). 170 S. Velasco-Lozano, et al. achieves high conversion efficiency, besides being low On the other hand, the concentration of cross-linking cost and having high market availability [47]. However, agent regulates the degree of cross-linking and therefore with some enzymes, such as nitrilases, retention of modifies the final structure of the CLEA, a result impacting enzymatic activity is very low or almost null when their activity and stability. Wilson et al. [63] found using this dialdehyde as cross-linking agent [27]. Mateo that excess in the degree of cross-linking sites reduces et al. tested a large polyaldehyde molecule (dextran- productivity, stability and performance of an immobilized polyaldehyde) [54]. CLEA formed with this cross-linker enzyme of CLEA of penicillin acylase for the production presented higher activity (10−90 times) in comparison of cephalexin. Majumder et al., [64] demonstrated that to the one commonly formed with GA. Furthermore, activity, stability and enantioselectivity of CLEA of lipase CLEA of nitrilases cross-linked with GA totally lose their from Pseudomonas cepacia, can be significantly modified activity; such effect is attributed to the small size of the by using different GA concentrations. Their findings show agent (0.1 kDa) that manages to go inside the protein and that there is an increase in the thermostability of CLEA destroy its tertiary structure, resulting in the complete at a higher degree of cross-linking; however, activity loss of activity. The latter effect was not observed with and enantioselectivity are compromised, because of the dextran polyaldehyde, which is a long chain polymer the higher degree of enzyme rigidity. They also proved (100−200 kDa). However, despite being less denaturing, that the morphology of the CLEA depends on the cross- the dextran polyaldehyde (DP) is not always superior to linking degree, which increases by incrementing the GA. Valdés et al., [55] compared the activity, size, stability concentration thereof, in turn increasing the degree of and reusability of CLEA of lipase PS from Burkholderia rigidity of the molecule resulting in lower catalytic activity. cepacia cross-linked with GA and DP, finding that the In addition, cross-linking time and temperature are also small dialdehyde allows obtaining smaller, properly implicated in the final activity of CLEA, since both higher cross-linked, active, stable and reusable biocatalysts than times [65,66] and temperatures [60,67] drive higher cross- those obtained with the larger dextran polyaldehyde. linking degrees thus altering this property. pH also plays Few studies have explored other cross-linking agents an important role in the step of cross-linking since GA different from GA. Among them, the work of Mateo et can be in monomeric or polymeric forms depending upon al., [54] used for the first time the dextran polyaldehyde. the conditions of pH of the medium. The final products Latter, Miletic et al., [56] prepared CLEA of the lipase of reaction obtained in the step of cross-linking under from CAL-B using diepoxides of different chain length alkaline and acidic conditions are different [68] since GA finding a strong dependence of the thermostability of the tends to polymerize at high pH values, which does not enzyme with the degree of cross-linking and chain size of occur at pH 7 [55,63]. Also, Yu et al., [49] found that the the diepoxide used. Recently, Wang et al., [57] reported size of the particle of CLEA of CRL is influenced by pH, the use of p-benzoquinone as a cross-linking agent of the having a larger size in alkaline medium. recombinant lipase of Geobacillus sp., which is five times more thermostable at 50°C for 90 minutes than CLEA prepared with GA. Similar results were obtained with 4 Improvement of enzymatic L-lysine as the cross-linker for the preparation of higher catalytic features by CLEA’s biocompatible peroxydase and urease-CLEA [58]. However, all these cross-linker agents are based on the formation of technology. covalent bindings between protein-amino groups and the Several factors are responsible for the final catalytic reactive group of the cross-linking agents. More recently, behavior of CLEA. Some works have dedicated their efforts Talekar et al., [59] showed that pectin is a more suitable to describe and understand such factors and their effects cross-linker of glucoamilase than GA, attaining more active on CLEA’s technology preparation [21,47,69]. Herein, we and stable pectin-CLEA than GA-CLEA. summarize different strategies to improve of catalytic Another available alternative is the cross-linking of properties of enzymes via their immobilization as CLEA. carboxyl-activated protein amino groups with chitosan as cross-linker for lacasse-CLEA [60] and polyethyleneimines 4.1 Enhancement of CLEA’s activity for lipase-CLEA [61]. The carboxyl-activated cross-linking of proteins has the advantage that carboxyl groups are more Activity of CLEA depends on several factors such as abundant than amino groups. Thereby, a higher chemical precipitant agent, additive, cross-linker, cross-linking time, modification degree is attained with the consequent enzyme concentration, temperature, pH and agitation, increase in protein rigidity and thermal stability [61,62]. Cross-linked enzyme aggregates (CLEA) in enzyme improvement – a review 171 among others. Precipitation agents and precipitation from CLEA using an appropriate organic solvent leaving conditions play a crucial role in the preparation of CLEA. the immobilized enzyme in a fixed favorable catalytic They can induce a more active conformation of the conformation [27]. enzyme [70]. However, one cannot generalize the use of CLEA preparation from proteins with low content one precipitating agent for all enzymes since this agent of lysine becomes difficult because lysine residues play can drive opposite results with different enzymes. For a main role in the cross-linking step when GA is used. that, a variety of precipitating agents with each enzyme This problem can be solved by precipitating proteins should be evaluated, taking into account that the optimal with polymers containing primary amino groups, such precipitant may not be the one that will ultimately produce as polylysine [76] and polyethyleneimine [74,77]; the the optimal CLEA [47]. latter forms a positively charged microenvironment A recent study demonstrated that during CLEA that prevents the contact between the organic solvents preparation, the period of time between the precipitation with the protein providing it with greater stability in the of enzymes and the cross-linking step influences a lot presence thereof [78,79]. Another alternative is the use the structural organization of the resulting enzyme of bovine serum albumin (BSA) as a protein feeder that preparation. As an example of these different properties, serves as source of protein and amino groups to carry CLEA of penicillin acylase (PA), fresh (which were cross- out the cross-linking of the enzyme of interest [64,80]. linked immediately after precipitation) and mature (which It has been reported that CLEA prepared in the presence were cross-linked after spending seven days at 4°C), of BSA retain higher catalytic native activity than those precipitating both with polyethyleneglycol (PEG6000) prepared without BSA. Addition of BSA to obtain CLEA and cross-linking with GA were evaluated [71]. It was of lipase from Pseudomonas cepacia results in obtaining observed that mature CLEA presented larger sizes than 100% of its native activity, while the same immobilized the fresh CLEA; besides, kinetic studies showed that the enzyme with GA in the absence of BSA retained only mature CLEA were more effective in both synthesis and 0.4% [80]. The addition of BSA in the preparation of hydrolysis reactions. Therefore, they demonstrated that CLEA improves the performance of cross-linking, the size the size of the aggregates could regulate the extent of and flexibility [52], and stability of the CLEA [61,65,80]. covalent modifications of the PA and thus influence the Moreover, the addition of BSA to the CLEA preparation catalytic properties of their respective CLEA. Likewise, it prevents proteolysis, increasing the biocatalysts stability has been found that the size of CLEA is also affected by the [81]. type and concentration of precipitating salts employed Bioimpression with specific substrates during CLEA [49,51]. Organic solvents used as precipitant agents may preparation allows maintaining native enzyme activities be denaturing for enzymes. The damage caused by such favoring the cross-linking of more active configuration. agents is primarily because solvents remove water bound Imprinting substrate treatments have been applied to the protein diminishing its flexibility. To solve this for phenylalanine ammonia lyase (PAL) with trans- problem, Wang et al. [72] studied the preparationof CLEA cinnamic acids, which were more active and stable in in assisted precipitation with sugars such as glucose, comparison with the free enzyme during the synthesis trehalose and sucrose. Resulted CLEA were stabilized of L-phenylalanine [82]. Moreover, substrate imprinting towards several denaturant solvents. might be an alternative for the protection of active residues On the other hand, addition of additives during during the cross-linking step as it was observed for lipase the precipitation step has been evaluated. Use of Triton CLEA imprinted with trioctanoin, which were 2-fold more X-100, sodium dodecyl sulphate and crown ethers has active in the presence than in the absence of the employed demonstrated their influence on the activity of CLEA triglyceride [61]. [50,70,73]. Addition of Triton X-100 increases the activity of those lipases which tend to form dimers such as that 4.2 Enhancement of CLEA’s stability of Thermomyces lanuginosus (TLL), whereas the Candida antarctica B (CAL-B) is hardly influenced by the presence Frequently, the low native enzyme stability under large- of Triton X-100 [74]. scale reaction conditions is a main drawback hampering The activation of lipases due to additives such as their fast incorporation to industrial bioprocesses. Hence, surfactants and crown ethers is generally attributed to stabilization of native enzymes at several harsh production the freezing and more active conformation of the enzyme conditions must be implemented. Temperature and pH during the preparation step [75]. Since additives are not are operational parameters with detrimental effects on covalently bound to the enzyme, they can be easily rinsed enzyme activity at the industrial level. Enzyme stability 172 S. Velasco-Lozano, et al. towards denaturing temperatures is related to the degree charged polymers as PEI during CLEA preparation has of rigidity of the molecule [83]. Namely, the greater number protective effects against solvent denaturation since of inter and intra molecular covalent bonds, the higher the it hampers the contact of the solvent and the enzyme thermal stability. Therefore, the covalent immobilization [74,79,90]. of enzymes is highly correlated with the stabilization As well, industrial biocatalysts must entail high of enzymes towards temperature. Several examples of storage stability. To this aim, CLEA have demonstrated thermostabilization are described by immobilization of high storage-stabilization of enzymes. For instance, different enzymes as CLEA [46,60,62,84]. During CLEA the amyloglucosidase-CLEA maintained 98% of its preparation, the effect of cross-linking time, cross- initial activity after 60 days at 4°C, whereas the free linker concentration and cross-linker type are directly enzyme lost more than 50% of its initial activity after implicated in the thermostabilization effect of the final 5 days at the same storage conditions [91]. Moreover, CLEA. Thermostability might also depend on the type of protease-CLEA, which are applied in sea paints, have precipitant agent used for CLEA obtention. Thermostability been well stabilized under artificial seawater exhibiting of penicillin G acylase CLEA precipitated by tert-buthanol a hyperactivation of 900% after 28 days of storage at was higher than that obtained with DME and PEG [85]. On such oxidizing conditions [92]. Other good examples the other hand, the preparation of CLEA in the presence of high storage stability of CLEA are also addressed in of PEI (as an additive or cross-linker) has demonstrated literature [93,94]. beneficial effects increasing thermal stability of lipase CLEA [61,86]. 4.3 Enhancement of CLEA’s selectivity Another weakness of native enzymes is the negative effect of high substrate or product concentrations. In Enzyme selectivity is one of the attractive catalytic regards, CLEA’s approach has been used as an effective features that make enzymes into powerful tools in the tool to reduce substrate or product inhibition. Cu et al., preparation of fine valuable compounds. Hence, the [82] demonstrated that imprinted CLEA of phenylalanine modification and modulation of a desired selectivity ammonia lyase imprinted with trans-cinnamic acids is the main goal of the scientific community. Recently, significantly reduces substrate inhibition. Sheldon has summarized several enantioselective Besides temperature and substrate/product asymmetric synthetic resolutions catalyzed by inhibition, acid or alkaline conditions are important CLEA of lipases, proteases, amidases, esterases, issues in enzyme stabilization. Enzymes are more active nitrilases, peroxidases and nitrile hydratase [89]. The at certain pH intervals, showing maximum activity at improvement in enzyme selectivity is related to several a specific pH value. However, several hydrolytic and factors. Particularly, Majumder [64] demonstrated synthetic reactions entail acidic or alkaline conditions that GA concentration affects differently the activity, that frequently denature enzymes, thus hampering their stability and enantioselectivity of CLEA of lipase from direct application. CLEA immobilization has also been Pseudomonas cepacia. successfully used to enhance the stability of enzymes at The selectivity of the biocatalyst is also affected a broader pH range, for instance, the CLEA of peroxidase by the use of additives during the preparation of from Brassica rapa present higher activity at acidic CLEA. Wilson and coworkers [74] evaluated the conditions as compared to the free enzyme [87]. On enantioselectivity of CLEA of lipase from Alcaligens sp. the other hand, enhancement of stability of glutamate in presence of polyethyleneimine and dextran sulphate decarboxylase at alkaline values allows the production as precipitating agents; addition of Triton X-100 modify of γ-aminobutyric acid which is characterized by the pH the activity and enantioselectivity in the hydrolysis of increase during the reaction [88]. A similar effect was glycidyl butyrate. Furthermore, the addition of BSA observed for the CLEA of phenylalanine lyase during during lipase-CLEA preparation also showed a marked the synthesis of L-phenylalanine [82]. effect on the enzyme enantioselectivity during the Since Klibanov demonstrated that hydrolytic hydrolytic resolution of S-mandelic acid [61]. Moreover, enzymes catalyze a large number of synthetic reactions it has been recently reported that enantioselectivity in organic solvents, scientists began the intensive and activity of magnetic-CLEA of Yarrowia lipolitica research of stable biocatalysts in the presence of lipase were highly influenced by an alternating denaturing solvents. In this field, CLEA technology has magnetic field during the resolution of S-2-octanol [95]. also confirmed its competence in providing solvent- This latter effect was mainly attributed to the behavior resistant enzymatic biocatalysts [42,89]. The addition of of magnetic-CLEA as microscopic stirrers. Cross-linked enzyme aggregates (CLEA) in enzyme improvement – a review 173

5 Combi-CLEA in cascade-reactions. [105] prepared chymotrypsin and Mucor javanicus lipase CLEA that were trapped inside the pores of a mesoporous Nowadays, cascade reactions represent an advantageous mesocellular silica matrix with pores hierarchically approach for the preparation of multistep reaction ordered (HMMS). This material has cavities of 37 nm where processes which may be conducted in a single step instead the CLEA are retained after having been cross-linked with of separate operations [96]. Immobilized multi-enzymatic GA and cannot get out because each of these small holes is systems offer several advantages including avoidance of joined by channels of 13 nm in diameter through which the the accumulation of unstable intermediates, reduction of CLEA cannot pass through and are retained in the pores. secondary undesirable reactions, achievement of multi- With this type of entrapment, it was possible to increase cascade reaction processes and the regeneration in situ the stability of CLEA up to 2 weeks stirring at 200 rpm, of cofactors [24]. Similar strategies are used to produce which prevented lixiviation of CLEA of the support and combi-CLEA, which contain more than one type of the inhibition of chymotrypsin autolysis. enzyme. Combi-CLEA have been successfully employed Likewise, penicillin acylase CLEA were encapsulated for cascade reactions as the resolution of S-mandelic acid inside a hydrogel of rigid polymer matrix of polyvinylalcohol [97,98] and in processes with multipurpose reactions. (LentiKats®) which turned out quite stable in organic Examples of that are combi-CLEA of pectinase, xylanase, solvents, while maintaining 95% of its activity during and cellulose which perform the three activities at the same 50 days in mechanical stirring at 20°C [104]. time [99] and combi-CLEA of several feruloil esterases A different alternative is the stabilization through for the production of alkyl ferulates [100]. Besides the the cross-linking of CLEA with amino-functionalized combi-CLEA approach has also been applied for the magnetic nanoparticles (magnetic-CLEA) [106] or by biodegradation of acetaminophen in wastewaters by the direct cross-linking of enzymes on magnetic particles combined action of laccase and tyrosinase based CLEA, (CLEMPA) [107]. Such structures have driven biocatalysts which exhibited high stability under harsh conditions of with higher operational stability, easier separation and temperature and chemical denaturation [101]. reutilization, besides CLEA clumping is avoided [69]. Some multistep reactions frequently imply the Further, CLEA have also been cross-linked to magnetic formation of labile intermediates that must be protected beads as magnetic-coated mesoporous silica maintaining in order to prevent their degradation. Combi-CLEA their initial activity after shaking during 35 days and the composed of (S)-hydroxynitrile lyase and a nitrilase were biocatalyst could be also recycled 35 times [108]. excellent examples of such application. This combi- Immobilization technology in the form of CLEA has CLEA were applied in the two step cascade synthesis of proven to be successfully applicable to many and varied (S)-2-hydroxycarboxylic acid amides, that needs the enzymes, becoming a new option for the tools catalogue prevention of the racemization of the (S)-2-hydroxynitrile of industrial biocatalysis. In this area, some types of intermediate by the protection of the nitrile-converting reactor configurations have been proposed such as those enzyme [102]. The latter bioconversion has been also of membranes in which the immobilized aggregates achieved with whole cells as catalysts; however the combi- remain retained and substrates and products are pumped CLEA strategy presents higher stability in the required through the reactor, [109,110] the ones of microchannels in solvent (diisopropylether) and is an easier recycling which a CLEA membrane is formed around the surface of biocatalyst than the entire cells [103]. a microchannel [111] and those of a perfusion basket [112]. Also, CLEA have been employed in continuous reactors 6 Coupling CLEA with solid [113]. matrixes and nano-particles. 7 Conclusions, future challenges Carrier-free immobilized enzymes have the disadvantage and trends. that they are not yet fully scalable to industrial levels since they exhibit low mechanical stability against shear Within the vast range of immobilization techniques, stress and harsh conditions of stirring [21,69,104]. cross-linked enzyme aggregates (CLEA) offer attractive In order to improve the mechanical stability of CLEA, advantages such as simplicity of preparation, low several research groups have worked on the encapsulation, production cost and fast optimization. CLEA technology coating and entrapment of these in polymers and matrices has proven to be applicable to a wide range of enzymes that allow them to increase their stability. Moon et al. turning it into a useful tool for the catalogue of biocatalysis 174 S. Velasco-Lozano, et al.

[15] Matsumoto M., Ohashi K. Effect of immobilization on thermo- processes. Nevertheless, there will be numerous studies stability of lipase from Candida rugosa. Biochem. Eng. J., aimed to improve CLEA’s stability, performance in 2003,14,75-77. industrial processes and finding new cross-linking agents. [16] Raviyan P., Tang J., Rasco B. A. Thermal stability of α-amylase Likewise, for rational modulation of the selectivity, activity from Aspergillus oryzae entrapped in polyacrylamide gel. J. and stability of enzymes, future efforts will focus the Agric. Food Chem., 2003,51,5462-5466. understanding of the phenomena governing the behavior [17] Mazurenko I., Ghach W., Kohring G.-W., Despas C., Walcarius A., Etienne M. Immobilization of membrane-bounded and performance of enzymes in the immobilized form. (S)-mandelate dehydrogenase in sol–gel matrix for electroen- zymatic synthesis. Bioelectrochemistry, 2015,104,65-70. [18] Cao L. Covalent Enzyme Immobilization. In: L. Cao editor. References Carrier-bound immobilized enzymes, Principles, Applications and Design. The Netherlands: WILEY-VCH Verlag GmbH & Co, [1] Andualema B., Gessesse A. Microbial lipases and 2006. p. 169-293. their industrial applications: Review. Biotechnology, [19] Mateo C., Grazú V., Pessela B. C. C., Montes T., Palomo J. M., 2012,11,100-118. Torres R., López-Gallego F., Fernández-Lafuente R., Guisán [2] Bornscheuer U. T. Enzymes in lipid modification: Past J. M. Advances in the design of new epoxy supports for achievements and current trends. Eur. J. Lipid Sci. Technol., enzyme immobilization-stabilization. Biochem. Soc. Trans., 2014,116,1322-1331. 2007,35,1593-1601. [3] Illanes A. Applications of microbial enzymes in organic [20] Sheldon R. A. Cross-linked enzyme aggregates as industrial synthesis. Biotechnology of Microbial Enzymes, 2012. p. biocatalysts. Org. Process Res. Dev., 2011,15,213-223. 141-174. [21] Talekar S., Joshi A., Joshi G., Kamat P., Haripurkar R., [4] Singh R. K., Tiwari M. K., Singh R., Lee J. K. From protein Kambale S. Parameters in preparation and characterization engineering to immobilization: Promising strategies for of cross linked enzyme aggregates (CLEAs). RSC Advances, the upgrade of industrial enzymes. International Journal of 2013,3,12485-12511. Molecular Sciences, 2013,14,1232-1277. [22] Sheldon R. A. Cross-linked enzyme aggregates (CLEA®s): [5] Brena B., Batista-Viera F. Immobilization of enzymes: A Stable and recyclable biocatalysts. Biochem. Soc. Trans., literature survey. In: J. M. Guisán editor. Immobilization of 2007,35,1583-1587. enzymes and cells. Totowa NJ: Humana Press, 2006. [23] Illanes A., Wilson L., Aguirre C. Synthesis of cephalexin [6] Cao L., van Langen L., Sheldon R. A. Immobilised enzymes: in aqueous medium with carrier-bound and carrier-free Carrier-bound or carrier-free? Curr. Opin. Biotechnol., penicillin acylase biocatalysts. Appl. Biochem. Biotechnol., 2003,14,387-394. 2009,157,98-110. [7] Palomo J. M., Fernandez-Lorente G., Mateo C., Ortiz C., [24] Garcia-Galan C., Berenguer-Murcia A., Fernandez-Lafuente R., Fernandez-Lafuente R., Guisan J. M. Modulation of the enantio- Rodrigues R. C. Potential of different enzyme immobilization selectivity of lipases via controlled immobilization and medium strategies to improve enzyme performance. Adv. Synth. Catal., engineering: Hydrolytic resolution of mandelic acid esters. 2011,353,2885-2904. Enzyme Microb. Technol., 2002,31,775-783. [25] Quiocho F. A., Richards F. M. The enzymic behavior of [8] Palomo J. M. Modulation of enzymes selectivity via immobi- carboxypeptidase-A in the solid state. Biochemistry (Mosc). lization. Current Organic Synthesis, 2009,6,1-14. 1966,5,4062-4076. [9] Mateo C., Palomo J. M., Fernandez-Lorente G., Guisan J. [26] Quiocho F. A., Richards F. M. Intermolecular cross linking of M., Fernandez-Lafuente R. Improvement of enzyme activity, a protein in the crystalline state Proceedings of the National stability and selectivity via immobilization techniques. Enzyme Academy of Sciences of the United States of, 1964,52,833-839. Microb. Technol., 2007,40,1451-1463. [27] Sheldon R. A. Enzyme immobilization: The quest for optimum [10] Rodrigues R. C., Ortiz C., Berenguer-Murcia A., Torres performance. Adv. Synth. Catal., 2007,349,1289-1307. R., Fernández-Lafuente R. Modifying enzyme activity [28] Thomas D., Bourdillon C., Broun G., Kernevez J. P. Kinetic and selectivity by immobilization. Chem. Soc. Rev., behavior of enzymes in artificial membranes. Inhibition and 2013,42,6290-6307. reversibility effects. Biochemistry (Mosc). 1974,13,2995-3000. [11] Cao L. Carrier-bound Immobilized Enzymes: Principles, [29] Broun G., Selegny E., Avrameas S., Thomas D. Enzymatically Application and Design. 2006. p. 1-563. active membranes: Some properties of cellophane membranes [12] Hanefeld U., Gardossi L., Magner E. Understanding enzyme supporting cross-linked enzymes. BBA - Enzymology, immobilisation. Chem. Soc. Rev., 2009,38,453-468. 1969,185,260-262. [13] Palomo J. M., Segura R. L., Fernandez-Lorente G., Fernandez- [30] Khare S. K., Vaidya S., Gupta M. N. Entrapment of proteins Lafuente R., Guisán J. M. Glutaraldehyde modification of by aggregation within sephadex beads. Appl. Biochem. lipases adsorbed on aminated supports: A simple way to Biotechnol., 1991,27,205-216. improve their behaviour as enantioselective biocatalyst. [31] St. Clair N. L., Navia M. A. Cross-linked enzyme crystals as Enzyme Microb. Technol., 2007,40,704-707. robust biocatalysts. J. Am. Chem. Soc., 1992,114,7314-7316. [14] Brady D., Jordaan J. Advances in enzyme immobilisation. [32] Gogoi P., Hazarika S., Dutta N. N., Rao P. G. Kinetics and Biotechnol. Lett., 2009,31,1639-1650. mechanism on laccase catalyzed synthesis of poly(allylamine)– catechin conjugate. Chem. Eng. J., 2010,163,86-92. Cross-linked enzyme aggregates (CLEA) in enzyme improvement – a review 175

[33] Roy J. J., Abraham T. E. Preparation and characterization of [50] Schoevaart R., Wolbers M. W., Golubovic M., Ottens M., cross-linked enzyme crystals of laccase. J. Mol. Catal. B: Kieboom A. P. G., Van Rantwijk F., Van Der Wielen L. A. M., Enzym., 2006,38,31-36. Sheldon R. A. Preparation, optimization, and structures, of [34] Hetrick E. M., Sperry D. C., Nguyen H. K., Strege M. A. Characte- cross-linked enzyme aggregates (CLEAs). Biotechnol. Bioeng., rization of a novel cross-linked lipase: Impact of cross-linking 2004,87,754-762. on solubility and release from drug product. Mol. Pharm., [51] Aytar B. S., Bakir U. Preparation of cross-linked tyrosinase 2014,11,1189-1200. aggregates. Process Biochem., 2008,43,125-131. [35] Brady D., Steenkamp L., Skein E., Chaplin J. A., Reddy S. [52] García-García M. I., Sola-Carvajal A., Sánchez-Carrón Optimisation of the enantioselective biocatalytic hydrolysis G., García-Carmona F., Sánchez-Ferrer Á. New stabilized of naproxen ethyl ester using ChiroCLEC-CR. Enzyme Microb. FastPrep-CLEAs for sialic acid synthesis. Bioresour. Technol., Technol., 2004,34,283-291. 2011,102,6186-6191. [36] Sheldon R. A., Van Pelt S. Enzyme immobilisation in [53] Wang M., Jia C., Qi W., Yu Q., Peng X., Su R., He Z. Porous-CLEAs biocatalysis: Why, what and how. Chem. Soc. Rev., of papain: Application to enzymatic hydrolysis of macromo- 2013,42,6223-6235. lecules. Bioresour. Technol., 2011,102,3541-3545. [37] Cao L., Van Rantwijk F., Sheldon R. A. Cross-linked enzyme [54] Mateo C., Palomo J. M., Van Langen L. M., Van Rantwijk F., aggregates: A simple and effective method for the immobi- Sheldon R. A. A New, Mild Cross-Linking Methodology to lization of penicillin acylase. Org. Lett., 2000,2,1361-1364. Prepare Cross-Linked Enzyme Aggregates. Biotechnol. Bioeng., [38] Tischer W., Kasche V. Immobilized enzymes: Crystals or 2004,86,273-276. carriers? Trends Biotechnol., 1999,17,326-335. [55] Valdés E. C., Soto L. W., Arcaya G. A. Influence of the pH [39] Toral A. R., de los Ríos A. P., Hernández F. J., Janssen M. H. of glutaraldehyde and the use of dextran aldehyde on the A., Schoevaart R., van Rantwijk F., Sheldon R. A. Cross-linked preparation of cross-linked enzyme aggregates (CLEAs) of Candida antarctica lipase B is active in denaturing ionic lipase from Burkholderia cepacia. Electron. J. Biotechnol. vol. liquids. Enzyme Microb. Technol., 2007,40,1095-1099. 14; 2011. [40] Alagöz D., Tükel S. S., Yildirim D. Enantioselective Synthesis [56] Miletić N., Loos K. Over-Stabilization of Chemically Modified of Various Cyanohydrins Using Covalently Immobilized and Cross-Linked Candida antarctica Lipase B Using Various Preparations of Hydroxynitrile Lyase from Prunus dulcis. Appl. Epoxides and Diepoxides. Aust. J. Chem., 2009,62,799-805. Biochem. Biotechnol., 2015. [57] Wang A., Zhang F., Chen F., Wang M., Li H., Zeng Z., Xie T., [41] Zhou Z., Piepenbreier F., Marthala V. R. R., Karbacher K., Chen Z. A facile technique to prepare cross-linked enzyme Hartmann M. Immobilization of lipase in cage-type mesoporous aggregates using p-benzoquinone as cross-linking agent. organosilicas via covalent bonding and crosslinking. Catal. Korean J. Chem. Eng., 2011,28,1090-1095. Today, 2015,243,173-183. [58] Ayhan H., Ayhan F., Gülsu A. Highly biocompatible enzyme [42] Sheldon R. A., van Pelt S., Kanbak-Aksu S., Rasmussen J. A., aggregates crosslinked by L-lysine. Turkish Journal of Janssen M. H. A. Cross-linked enzyme aggregates (CLEAs) in Biochemistry, 2012,37,14-20. organic synthesis. Aldrichimica Acta, 2013,46,81-93. [59] Talekar S., Nadar S., Joshi A., Joshi G. Pectin cross-linked [43] Illanes A., Wilson L., Caballero E., Fernández-Lafuente R., enzyme aggregates (pectin-CLEAs) of glucoamylase. RSC Guisán J. M. Crosslinked penicillin acylase aggregates for Advances, 2014,4,59444-59453. synthesis of β-lactam antibiotics in organic medium. Appl. [60] Arsenault A., Cabana H., Jones J. P. Laccase-based CLEAs: Biochem. Biotechnol., 2006,133,189-202. Chitosan as a novel cross-linking agent. Enzyme Research, [44] Roessl U., Nahálka J., Nidetzky B. Carrier-free immobilized 2011,2011,art. no. 376015. enzymes for biocatalysis. Biotechnol. Lett., 2010,32,341-350. [61] Velasco-Lozano S., López-Gallego F., Vázquez-Duhalt R., [45] Roberge C., Amos D., Pollard D., Devine P. Preparation and Mateos-Díaz J. C., Guisán J. M., Favela-Torres E. Carrier-free application of cross-linked aggregates of chloroperoxidase immobilization of lipase from Candida rugosa with with enhanced hydrogen peroxide tolerance. J. Mol. Catal. B: polyethyleneimines by carboxyl-activated cross-linking. Enzym., 2009,56,41-45. Biomacromolecules, 2014,15,1896-1903. [46] Kartal F., Janssen M. H. A., Hollmann F., Sheldon R. A., Kilinc [62] Galvis M., Barbosa O., Ruiz M., Cruz J., Ortiz C., Torres R., A. Improved esterification activity of Candida rugosa lipase Fernandez-Lafuente R. Chemical amination of lipase B from in organic solvent by immobilization as Cross-linked enzyme Candida antarctica is an efficient solution for the preparation aggregates (CLEAs). J. Mol. Catal. B: Enzym., 2011,71,85-89. of crosslinked enzyme aggregates. Process Biochem., [47] Sheldon R. A. Characteristic features and biotechnological 2012,47,2373-2378. applications of cross-linked enzyme aggregates (CLEAs). Appl. [63] Wilson L., Illanes A., Soler L., Henríquez M. J. Effect of the Microbiol. Biotechnol., 2011,92,467-477. degree of cross-linking on the properties of different CLEAs of [48] Montoro-García S., Gil-Ortiz F., Navarro-Fernández J., penicillin acylase. Process Biochem., 2009,44,322-326. Rubio V., García-Carmona F., Sánchez-Ferrer A. Improved [64] Majumder A. B., Mondal K., Singh T. P., Gupta M. N. Designing cross-linked enzyme aggregates for the production of desacetyl cross-linked lipase aggregates for optimum performance as β-lactam antibiotics intermediates. Bioresour. Technol., biocatalysts. Biocatal. Biotransform., 2008,26,235-242. 2010,101,331-336. [65] Cruz J., Barbosa O., Rodrigues R. C., Fernandez-Lafuente R., [49] Yu H. W., Chen H., Wang X., Yang Y. Y., Ching C. B. Cross-linked Torres R., Ortiz C. Optimized preparation of CALB-CLEAs by enzyme aggregates (CLEAs) with controlled particles: response surface methodology: The necessity to employ Application to Candida rugosa lipase. J. Mol. Catal. B: Enzym., a feeder to have an effective crosslinking. J. Mol. Catal. B: 2006,43,124-127. Enzym., 2012,80,7-14. 176 S. Velasco-Lozano, et al.

[66] Kim M. H., Park S., Kim Y. H., Won K., Lee S. H. Immobilization [81] Cabana H., Jones J. P., Agathos S. N. Preparation and of formate dehydrogenase from Candida boidinii through characterization of cross-linked laccase aggregates and cross-linked enzyme aggregates. J. Mol. Catal. B: Enzym., their application to the elimination of endocrine disrupting 2013,97,209-214. chemicals. J. Biotechnol., 2007,132,23-31. [67] Yang X., Zheng P., Ni Y., Sun Z. Highly efficient biosynthesis of [82] Cui J. D., Liu R. L., Li L. L. Imprinted cross-linked enzyme sucrose-6-acetate with cross-linked aggregates of Lipozyme TL aggregate (iCLEA) of phenylalanine ammonia lyase: A new 100 L. J. Biotechnol., 2012,161,27-33. stable biocatalyst. Lecture Notes in Electrical Engineering vol. [68] Wine Y., Cohen-Hadar N., Freeman A., Frolow F. Elucidation 332; 2015. p. 223-231. of the mechanism and end products of glutaraldehyde [83] Bommarius A. S., Paye M. F. Stabilizing biocatalysts. Chem. crosslinking reaction by X-ray structure analysis. Biotechnol. Soc. Rev., 2013,42,6534-6565. Bioeng., 2007,98,711-718. [84] Dong T., Zhao L., Huang Y., Tan X. Preparation of cross-linked [69] Cui J. D., Jia S. R. Optimization protocols and improved aggregates of aminoacylase from Aspergillus melleus by using strategies of cross-linked enzyme aggregates technology: bovine serum albumin as an inert additive. Bioresour. Technol., Current development and future challenges. Crit. Rev. 2010,101,6569-6571. Biotechnol., 2015,35,15-28. [85] Kopp W., Da Costa T. P., Pereira S. C., Jafelicci Jr M., Giordano [70] López-Serrano P., Cao L., Van Rantwijk F., Sheldon R. A. R. C., Marques R. F. C., Araújo-Moreira F. M., Giordano R. L. Cross-linked enzyme aggregates with enhanced activity: C. Easily handling penicillin G acylase magnetic cross-linked Application to lipases. Biotechnol. Lett., 2002,24,1379-1383. enzymes aggregates: Catalytic and morphological studies. [71] Pchelintsev N. A., Youshko M. I., Švedas V. K. Quantitative Process Biochem., 2014,49,38-46. characteristic of the catalytic properties and microstructure of [86] Yan J., Gui X., Wang G., Yan Y. Improving stability and activity cross-linked enzyme aggregates of penicillin acylase. J. Mol. of cross-linked enzyme aggregates based on polyethylenimine Catal. B: Enzym., 2009,56,202-207. in hydrolysis of fish oil for enrichment of polyunsaturated fatty [72] Wang M., Qi W., Jia C., Ren Y., Su R., He Z. Enhancement of acids. Appl. Biochem. Biotechnol., 2012,166,925-932. activity of cross-linked enzyme aggregates by a sugar-assisted [87] Tandjaoui N., Tassist A., Abouseoud M., Couvert A., Amrane precipitation strategy: Technical development and molecular A. Preparation and characterization of cross-linked enzyme mechanism. J. Biotechnol., 2011,156,30– 38. aggregates (CLEAs) of Brassica rapa peroxidase. Biocatalysis [73] Gupta P., Dutt K., Misra S., Raghuwanshi S., Saxena R. K. and Agricultural Biotechnology, 2015,4,208-213. Characterization of cross-linked immobilized lipase from [88] Dinh T. H., Jang N. Y., McDonald K. A., Won K. Cross-linked thermophilic mould Thermomyces lanuginosa using glutar- aggregation of glutamate decarboxylase to extend its activity aldehyde. Bioresour. Technol., 2009,100,4074-4076. range toward alkaline pH. J. Chem. Technol. Biotechnol., 2015. [74] Wilson L., Fernández-Lorente G., Fernández-Lafuente R., Illanes [89] Sheldon R. A. 9.15 Industrial Applications of Asymmetric A., Guisán J. M., Palomo J. M. CLEAs of lipases and poly-ionic Synthesis using Cross-Linked Enzyme Aggregates. Compre- polymers: A simple way of preparing stable biocatalysts hensive Chirality vol. 9, 2012. p. 353-366. with improved properties. Enzyme Microb. Technol., [90] Vaidya B. K., Kuwar S. S., Golegaonkar S. B., Nene S. 2006,39,750-755. N. Preparation of cross-linked enzyme aggregates of [75] Theil F. Enhancement of selectivity and reactivity of lipases by l-aminoacylase via co-aggregation with polyethyleneimine. J. additives. Tetrahedron, 2000,56,2905-2919. Mol. Catal. B: Enzym., 2012,74,184-191. [76] Yamaguchi H., Miyazaki M., Asanomi Y., Maeda H. Poly-lysine [91] Gupta K., Jana A. K., Kumar S., Jana M. M. Solid state supported cross-linked enzyme aggregates with efficient fermentation with recovery of Amyloglucosidase from extract enzymatic activity and high operational stability. Catal. Sci. by direct immobilization in cross linked enzyme aggregate for Technol., 2011,1,1256-1261. starch hydrolysis. Biocatalysis and Agricultural Biotechnology, [77] López-Gallego F., Betancor L., Hidalgo A., Alonso N., Fernández- 2015. Lafuente R., Guisán J. M. Co-aggregation of enzymes and [92] Skovgaard J., Bak C. A., Snabe T., Sutherland D. S., Laursen polyethyleneimine: A simple method to prepare stable and B. S., Kragh K. M., Besenbacher F., Poulsen C. H., Shipovskov immobilized derivatives of glutaryl acylase. Biomacromo- S. Implementation of cross-linked enzyme aggregates of lecules, 2005,6,1839-1842. proteases for marine paint applications. J. Mater. Chem., [78] Montes T., Grazú V., Manso I., Galán B., López-Gallego 2010,20,7626-7629. F., González R., Hermoso J. A., García J. L., Guisán J. M., [93] Martínez Y. N., Cavello I., Cavalitto S., Illanes A., Castro G. R. Fernández-Lafuente R. Improved stabilization of genetically Studies on PVA pectin cryogels containing crosslinked enzyme modified penicillin G acylase in the presence of organic aggregates of keratinase. Colloids Surf. B. Biointerfaces, cosolvents by co-immobilization of the enzyme with polyethy- 2014,117,284-289. leneimine. Adv. Synth. Catal., 2007,349,459-464. [94] Park J.-M., Kim M., Park H.-S., Jang A., Min J., Kim Y.-H. Immobi- [79] Pan J., Kong X. D., Li C. X., Ye Q., Xu J. H., Imanaka T. lization of lysozyme-CLEA onto electrospun chitosan nanofiber Crosslinking of enzyme coaggregate with polyethyleneimine: A for effective antibacterial applications. Int. J. Biol. Macromol., simple and promising method for preparing stable biocatalyst 2013,54,37-43. of Serratia marcescens lipase. J. Mol. Catal. B: Enzym., [95] Liu Y., Guo C., Liu C. Z. Enhancing the resolution of 2011,68,256-261. (R,S)-2-octanol catalyzed by magnetic cross-linked lipase [80] Shah S., Sharma A., Gupta M. N. Preparation of cross-linked aggregates using an alternating magnetic field. Chem. Eng. J., enzyme aggregates by using bovine serum albumin as a proteic 2015,280,36-40. feeder. Anal. Biochem., 2006,351,207-213. Cross-linked enzyme aggregates (CLEA) in enzyme improvement – a review 177

[96] Muschiol J., Peters C., Oberleitner N., Mihovilovic M. D., a novel biocatalyst in organic media. Biotechnol. Bioeng., Bornscheuer U. T., Rudroff F. Cascade catalysis-strategies and 2004,86,558-562. challenges en route to preparative synthetic biology. Chem. [105] Moon I. K., Kim J., Lee J., Jia H., Hyon B. N., Jong K. Y., Ja H. Commun., 2015,51,5798-5811. K., Dohnalkova A., Grate J. W., Wang P., et al. Crosslinked [97] Chmura A., Rustler S., Paravidino M., van Rantwijk F., Stolz enzyme aggregates in hierarchically-ordered mesoporous A., Sheldon R. A. The combi-CLEA approach: enzymatic silica: A simple and effective method for enzyme stabilization. cascade synthesis of enantiomerically pure (S)-mandelic acid. Biotechnol. Bioeng., 2007,96,210-218. Tetrahedron: Asymmetry, 2013,24,1225-1232. [106] Talekar S., Ghodake V., Ghotage T., Rathod P., Deshmukh P., [98] Mateo C., Chmura A., Rustler S., Van Rantwijk F., Stolz A., Nadar S., Mulla M., Ladole M. Novel magnetic cross-linked Sheldon R. A. Synthesis of enantiomerically pure (S)-mandelic enzyme aggregates (magnetic CLEAs) of alpha amylase. acid using an oxynitrilase-nitrilase bienzymatic cascade: Bioresour. Technol., 2012,123,542-547. A nitrilase surprisingly shows nitrile hydratase activity. [107] Tudorache M., Nae A., Coman S., Parvulescu V. I. Strategy Tetrahedron-Asymmetr, 2006,17,320-323. of cross-linked enzyme aggregates onto magnetic particles [99] Dalal S., Sharma A., Gupta M. N. A multipurpose immobilized adapted to the green design of biocatalytic synthesis of biocatalyst with pectinase, xylanase and cellulase activities. glycerol carbonate. RSC Advances, 2013,3,4052-4058. Chemistry Central Journal, 2007,1. [108] Lee J., Na H. B., Kim B. C., Lee J. H., Lee B., Kwak J. H., Hwang [100] Vafiadi C., Topakas E., Christakopoulos P. Preparation of Y., Park J. G., Gu M. B., Kim J., et al. Magnetically-separable multipurpose cross-linked enzyme aggregates and their and highly-stable enzyme system based on crosslinked application to production of alkyl ferulates. J. Mol. Catal. B: enzyme aggregates shipped in magnetite-coated mesoporous Enzym., 2008,54,35–41. silica. J. Mater. Chem., 2009,19,7864-7870. [101] Ba S., Haroune L., Cruz-Morató C., Jacquet C., Touahar I. E., [109] Hilal N., Nigmatullin R., Alpatova A. Immobilization of Bellenger J. P., Legault C. Y., Jones J. P., Cabana H. Synthesis cross-linked lipase aggregates within microporous polymeric and characterization of combined cross-linked laccase and membranes. J. Membr. Sci., 2004,238,131-141. tyrosinase aggregates transforming acetaminophen as a [110] Sorgedrager M. J., Verdoes D., Van Der Meer H., Sheldon R. A. model phenolic compound in wastewaters. Sci. Total Environ., Chim Oggi, 2008,26,23-25. 2014,487,748-755. [111] Miyazaki M., Maeda H. Microchannel enzyme reactors [102] Van Pelt S., Van Rantwijk F., Sheldon R. A. Synthesis of and their applications for processing. Trends Biotechnol., aliphatic (S)-α-hydroxycarboxylic amides using a one-pot 2006,24,463-470. bienzymatic cascade of immobilised oxynitrilase and nitrile [112] Cabana H., Jones J. P., Agathos S. N. Utilization of cross-linked hydratase. Adv. Synth. Catal., 2009,351,397-404. laccase aggregates in a perfusion basket reactor for the [103] Van Rantwijk F., Stolz A. Enzymatic cascade synthesis of continuous elimination of endocrine-disrupting chemicals. (S)-2-hydroxycarboxylic amides and acids: Cascade reactions Biotechnol. Bioeng., 2009,102,1582-1592. employing a hydroxynitrile lyase, nitrile-converting enzymes [113] Wilson L., Illanes A., Romero O., Vergara J., Mateo C. Carrier- and an amidase. J. Mol. Catal. B: Enzym., 2015,114,25-30. bound and carrier-free penicillin acylase biocatalysts for [104] Wilson L., Illanes A., Pessela B. C. C., Abian O., Fernández- the thermodynamically controlled synthesis of β-lactam Lafuente R., Guisán J. M. Encapsulation of crosslinked compounds in organic medium. Enzyme Microb. Technol., penicillin G acylase aggregates in lentikats: Evaluation of 2008,43,442-447.