Even having some disadvantages compared to inorganic matrices, like lower stability at high temperatures, organic solvents or microbial attack, the major part of supports already employed for large-scale applications are organic materials. The major argument for utilization of organic carriers for enzyme immobilization is the variety of functional organic groups which can be attached, making possible numerous options to bind the enzyme, particularly by covalent linkages. Typically, organic support materials are of polymeric nature and can be classified as natural macromolecules or synthetic polymers (Kennedy and Cabral, 1987).
Table 1.3. Organic polymeric supports used for immobilization of enzymes
Alginate dextransucrase entrapment Berensmeyer et al., 2004
Alginate β-D-galactosidase entrapment Becerra et al., 2001 K-carrageenan peroxidase entrapment Shukla et al., 2004 Chitosan β-D-galactosidase entrapment Wentworth et al., 2004
Poly(3-hydroxybutyrate-co-hydroxyvalerate)
lipase adsorption Cabrera-Padilla et al., 2012
Diethylaminoethyl (DEAE) cellulose
peroxidase ionic adsorption Kulshrestha and Husain, 2006 Chitosan β-D-galactosidase covalent binding,
glutaraldehyde
covalent binding Mateo et al., 2005 Synthetic
Poly(glycidyl
methacrylate-co-ethylene glycol dimethacrylate)
lipase adsorption Mojovic et al., 1998
Polyethylene (Accurel EP-400)
lipase adsorption Murray et al., 1997 Poly(methacrylic
acid-co-methyl methacrylate) (Eudragit L-100)
xylanase adsorption Roy et al., 2003
Poly(acrylamide), cross-linked
glucose oxidase entrapment Rubio Retama et al., 2003
Polytetrafluorethylene (Teflon)
α-chymotrypsin entrapment Afrin et al., 2000 Nylon, grafted and
covalent binding Kotha et al., 1996
Poly(ethylene glycol
β-glucosidase covalent binding Li et al., 2010 Magnetic nitrocellulose glucoamylase covalent binding,
glutaraldehyde
glucose oxidase covalent binding Varlan et al., 1996
It must be pointed out that natural polymers (alginate, carrageenan, chitosan) are rarely used in unmodified form for immobilization, mainly by entrapment techniques. The most widely applied organic supports are polysaccharides. Consequently, the main functional groups available for interaction with the enzyme will be hydroxyl groups, which need activation before performing the immobilization. Selected examples of organic support utilized for immobilization of enzymes are presented in Table 1.3.
Natural polymers
Adsorption is used mainly in association with inorganic supports, as showed before.
However, organic supports are also suitable for adsorption immobilization, since they could have adequate porosity and surface properties to allow physical binding via non-specific forces, as hydrogen bonds or Van der Waals forces.
A natural polyester, poly(3-hydroxybutyrate-co-hydroxyvalerate), produced by different microorganisms, was recently proposed as new immobilization carrier for enzymes, having several advantages as biocompatibility, biodegradability, strength, non-toxicity and eco-friendliness. Physical adsorption of lipase from Candida rugosa has been carried out on this support, with 30% immobilization efficiency. The half-lives of immobilized lipase were 77 h and 2 h at 40°C and 60°C, respectively. The operational stability was considered appropriate for possible industrial application, exhibiting 50% residual activity after 12 cycles of reuse (Cabrera-Padilla et al., 2012).
Cellulose is the most abundant natural polymer; therefore it was one the first materials used for immobilization of enzymes. The hydroxyl groups of cellulose are not enough reactive to form covalent bonds with the enzyme, but they can be subjected to different chemical modifications, to obtain modified celluloses. Several activated cellulosic supports contain cationic or anionic ion exchange groups and are used as carriers for enzyme immobilization by ionic adsorption. Aminoethyl (AE), diethylaminoethyl (DEAE), or triethylaminoethyl (TEAE)-modified cellulose can interact with the anionic functional groups of enzymes through charge-charge interactions. Additionally, adsorption procedures demonstrated to be useful for immobilization of enzymes directly from crude or partially purified fermentation medium or enzyme extracts, avoiding the high cost of enzyme purification. Kulshrestha and Husain immobilized peroxidases on diethyl aminoethyl (DEAE) cellulose, directly from the salt fractionated proteins and dialyzed
proteins of bitter gourd (Momordica charantia). The binding efficiency was 590 enzyme units per g of the matrix. Bitter gourd peroxidase immobilized on this anion exchanger was more stable than the free enzyme against denaturation induced by pH, heat, urea, proteolytic enzyme, detergents, and water-miscible organic solvents (Kulshrestha and Husain, 2006).
Entrapment is particularly suited for enzyme immobilization in both natural and synthetic support materials. As entrapments in chitosan and sol-gel type materials are subject of separate sub-sections, in this part utilization other polymeric supports will be reviewed.
Alginate, obtained from brown algae, is a natural polysaccharide intensively used for entrapment of enzymes, especially as calcium alginate beads. Dextransucrase from Leuconostoc mesenteroides was immobilized in Ca alginate beads and optimized for applications in a fluidised bed reactor with concentrated sugar solutions, in order to allow continuous formation of defined oligosaccharides. Addition of an inorganic material (silica sand) to the alginate resulted in higher densities than classical alginate beads, and higher fluidizing velocities with smaller particle diameters. Interestingly, addition of sand with appropriate particle size stabilized the alginate beads, without abrasion in stirred or fluidised bed reactors (Berensmeyer et al., 2004). Cross-linking of alginate with divalent ions (like Ca2+) and glutaraldehyde was also reported to improve the stability of the enzyme. Tanrisevan and Doğan entrapped Saccharomyces cerevisiae invertase in alginate, treating the formed Ca alginate capsules with 1% glutaraldehyde to prevent leakage of the enzyme. The immobilization resulted in 87% relative activity and 36 days operational stability without decrease in activity (Tanrisevan and Doğan, 2001).
Carrageenan, a linear sulphated polysaccharide, is a food-grade and biocompatible support material extracted from red seaweeds, with several applications for enzyme entrapment. In its natural form, is a pseudoplastic support, being able to thin under shear stress and regain the initial viscosity when the stress has been removed (Datta et al., 2012). Carrageenan, like other thermoreversible gels, melts at elevated temperature, limiting its applicability.
Gelling can be promoted by lowering the temperature, or by the addition of gelation- inducing agents. There are four methods described in the literature to accomplish the embedding of enzymes in carrageenan: gel method, droplet method, emulsion method, and dehydration method (van de Velde et al., 2002). Entrapment of horseradish peroxidase in K-carrageenan beads was carried out using the droplet method, with polyethyleneimine as hardening agent. Heat and storage stabilities were improved for entrapped horseradish
peroxidase, compared to the free enzyme. The entrapped enzyme was considered suitable for industrial application, retaining 50% of the initial activity after 4 reuse cycles (Shukla et al., 2004).
Agarose (isolated from natural agar) has not so many applications for immobilization as other algal polysaccharides, being a more expensive matrix, with poorer mechanical and chemical resistance. However, cross-linked agarose (named Sepharose) can be activated like as cellulose or other polyhydroxylic materials, to obtain a valuable immobilization support. Sepharose-4B activated with cyanogen bromide is a commercially available support with reactive substituted imidocarbonate groups. This support has been tested for mild covalent immobilization of lipase by one-point covalent attachment through the amino terminal group. The immobilization yields were between 20 and 40%. The lipases covalently immobilized on CNBr-activated Sepharose were tested for hydrolysis of fish oil, but they displayed lower activities than the same lipases immobilized by adsorption on Octyl-Sepharose (Fernández-Lorente et al., 2011).
In the last years, an important number of publications was published by Guisán and co-workers, dealing with the topic of glyoxyl-agarose as immobilization support (Palomo et al., 2004, Mateo et al., 2005; Mateo et al., 2006; Mateo et al., 2007; Pedroche et al., 2007;
Fernández-Lorente et al., 2011, Vieira et al., 2011). The main finding was to attach aldehyde groups to the support by short spacer arms, followed by covalent multipoint binding of the enzyme molecules to this activated support. The Schiff bases formed with the amino groups of enzymes were subsequently reduced by sodium borohydride, leaving the enzyme attached to the support by very stable secondary amino bonds, while the residual aldehyde groups have been converted into inert hydroxyl groups (Fig. 1.4).
Fig. 1.4. Immobilization of enzymes by multipoint covalent attachment on glyoxyl agarose supports (Palomo et al., 2004)
Immobilization of enzymes on glyoxyl supports occurs at alkaline pH. Glyoxyl agarose was successfully utilized for covalent immobilization of many enzymes, like trypsin, chymotrypsin, penicillin G acylase, lipases, thermolysin, alcalase, urokinase, etc., with activity recoveries ranging from 60 to 100%. The activation degree was dependent on the percentage of agarose used to prepare the beads. When 10% agarose has been used, the support was activated with around 220 µmol of glyoxyl groups/mL, resulting in a surface density of approx. 17 glyoxyl groups/1000 Å, and a loading capacity of 100-120 mg/mL penicillin G acylase (Mateo et al., 2006). Glyoxyl-agarose supports were prepared by activating the agarose matrix with glycidol, followed by oxidation with periodate (Guisan, 2008).
Synthetic polymers
A large number of synthetic polymers are available as carriers for immobilization of enzymes, mainly by covalent binding. The principle is to use a pre-designed insoluble scaffold with active binding functionality that is able to react with amino acids from the structure of enzymes in suitable conditions. The structure of carrier could have significant influence on the activity, stability and selectivity of the enzyme (Cao, 2005a). Since every enzyme and application has specific characteristics and requirements, numerous carriers, as well as activation and coupling methods, have been proposed. Comprehensive evaluation of synthetic polymeric materials and activation techniques used in connection with these supports can be retrieved in the most important books published in the topic of immobilization, during the past decades (Mosbach, 1976; Chibata, 1978; Kennedy and Cabral, 1985, Cao, 2005b; Buchholz et al., 2005). A few examples will be subsequently presented, to highlight some specific applications.
Although covalent binding is the most applied immobilization method on synthetic polymers, adsorption and entrapment were also studied. Mojovic et al. immobilized lipase from Candida rugosa by adsorption on a macroporous copolymer, poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate), obtaining 15.4 mg/g bound protein. The immobilized enzyme activity, measured for hydrolysis of palm oil, was 70% of that of free enzyme. The operational stability was rather low, at only 57% residual activity after five hydrolysis cycles (Mojovic et al., 1998). The same lipase was adsorbed on two different polymeric supports: non-porous polystyrene microspheres and porous low density
polyethylene powder (Accurel EP400). Among these carriers, polystyrene latex demonstrated high affinity for adsorption of lipase, but the hydrolytic activity of the immobilized enzyme was very low. By comparison, the porous polyethylene support showed acceptable immobilization efficiency only at high enzyme loading, but the rate of hydrolysis was comparable to that achieved with free lipase and the recycle potential of the enzyme was considered satisfactory, with 35% decrease of activity after five hydrolysis cycles (Murray et al., 1997). Commercial pectinase was immobilized by ionic adsorption on a weak basic anion exchange support (Dowex WRB). Better thermal stability, reusability with only 20% activity loss after nine batches, and up to 20% productivity increase have been reported for the immobilized enzyme (Demir et al., 2001).
Polyacrylamides matrices have been considered suitable for entrapment of enzymes, due to their hydrophilic character (Kennedy and Cabral, 1985). To obtain microgels, new methods of acrylamide polymerization have been developed, like polymerization in micelles, concentrated microemulsions and nanoemulsions. Rubio-Retama et al. prepared cross-linked polyacrylamide microgels for entrapment of glucose oxidase. N,N’-methylene bisacrylamide was the cross-linking agent, generating microgel particles with diameters between 0.2 and 6.5 µm and cross-links number equal or greater than 0.021 mol/g.
Physical characteristics of the formed microgels were determined, but data concerning the immobilization efficiency were not given (Rubio Retama et al., 2003). Despite the easy immobilization protocol, encapsulation in polyacrylamide matrixes was considered not appropriate for industrial application due to leaking problems and insufficient mechanical strength of the formed gel structures. Activation of the polyacrylamide structures, to make the matrices appropriate for covalent coupling of the enzymes, could resolve these problems. Pectinase was covalently immobilized onto macroporous polyacrylamide microspheres activated with glutaraldehyde, at 81.7% immobilization yield and 296 mg of enzyme loading per gram of the carrier particles. The immobilized enzyme retained more than 75% of its initial activity over 30 days and exhibited excellent operational stability, with more than 75% residual activity after 10 batch reactions (Lei and Jiang, 2011).
Polytetrafluoroethylene (Teflon) particles were also investigated as matrices for entrapment of enzymes. α-Chymotrypsin was added to a solution of Teflon in an organic solvent, followed by evaporation of the solvent to dryness, to obtain sticky particles containing the embedded enzyme. Both peptide synthetic and hydrolytic activities were enhanced next to immobilization. The polytetrafluoroethylene matrix created a
hydrophobic environment, leading to enhanced peptide synthesis in aqueous solution (Afrin et al., 2000).
Nylon (common name of copolymers of dicarboxylic acids with diamines) was also investigated as immobilization carrier. Alike polytetrafluoroethylene, nylon lacks functional groups available for direct activation and covalent binding of enzymes.
Therefore, chemical grafting of the nylon pellets with diethylene glycol dimethacrylate was necessary to create active centres on the copolymer matrix. In the next step, 1,6-hexamethylene diamine was introduced as spacer between the grafted membrane and the enzyme, while glutaraldehyde was employed as coupling agent. Penicillin G acylase was covalently immobilized on this aminoalkylated nylon support, but only 12% of the free enzyme activity was recovered, probably caused by binding part of enzyme in wrong orientation, or binding groups from the active site. Addition of phenylacetic acid to protect the active site of enzyme through the immobilization process led to improvement of activity by a factor of 2 (Mohy-Eldin et al., 2000).
Several other polymeric materials were obtained with appropriate structures to allow activation of functional groups for covalent attachment of enzymes. The active functional groups of polymers include acyl azide, acid anhydride, halogen, epoxy, isocyanate, thioisocyanate, carbonyl, aldehyde, activated esters, etc. They are usually prepared in one step by suspension polymerization in presence of one or more co-monomer and a linker, but synthesis of homo- or heteropolymers, followed by linking with a cross-linker, was also carried out (Cao, 2005b).
Polymers with epoxy groups offer the advantage of single-step binding of enzyme. Epoxy groups bear short spacer arms and can react with several nucleophilic groups present in the enzyme structure, like as amino, sulfhydryl, imidazolyl, tyrosyl, or even with carboxylic groups at a slower rate. Moreover, epoxy groups are very stable, allowing long-term incubation with the enzyme in alkaline conditions, leading to multipoint covalent attachment. However, to overcome the low intermolecular reactivity between epoxy support and proteins, a two-step procedure was proposed, involving (i) rapid physical fixation of protein on the support surface, and (ii) slower intermolecular multipoint binding of the nucleophilic groups of the protein and the epoxy groups of the carrier. It must be pointed out that commercial epoxy supports (as Eupergit C) do not need any extra functionalization, as they can adsorb the enzymes through the hydrophobic zones present on the surface of the protein (Fig. 1.5, Mateo et al., 2007b).
Fig. 1.5. Multipoint covalent attachment of nucleophilic functional groups of enzymes onto epoxy-functionalized supports (Mateo et al., 2007)
Kotha et al. synthesized reactive, macroporous and beaded glycidyl methacrylate-divinyl benzene copolymers of controlled particle size. Besides the surface properties of the polymer beads, the most important parameters influencing the binding and activity of the enzyme are the pore size and the pore size distribution, which influence the diffusion phenomena during the biocatalytic process. For immobilization of penicillin G acylase, the best result (99% binding efficiency and 39% activity recovered following immobilization) was obtained at 25 mol% cross-linking and a pore radius of 10.8 nm. The immobilized enzyme was reused in 100 reaction cycles, with only 10% loss of activity (Kotha et al., 1996).
Epoxy-activated acrylic supports are among the most studied immobilization carriers.
Eupergit C and Eupergit C 250 L (with larger pores and lower oxirane group density), which are co-polymers of methacrylamide, N,N′-methylen-bis(acrylamide) and a monomer carrying oxirane group, were comprehensively reviewed as supports for immobilization of numerous oxidoreductases, transferases, hydrolases and lyases. The remarkable stability and selectivity of the biocatalysts obtained with these support types strongly recommend them for industrial application (Boller et al., 2002).
Polyacrylic and polymethacrylic backbones are present in a large diversity of supports, with different functional groups (amino, hydroxyl, carboxyl) which can be further activated to allow the covalent binding of the enzymes. Poly(hydroxyalkyl methacrylates), bearing hydroxyl functionality, are carriers with increased hydrophilicity compared to other synthetic polymers and can be considered synthetic analogs of polysaccharides. Most often, they are obtained by copolymerization of 2-hydroxyethyl methacrylate with a cross-linking agent, such as ethylene dimethacrylate. Activation of the resulted copolymer can be achieved by chemical modification in different ways, depending on the active functional
group needed to be grafted. Using epichlorohydrin, the carrier will bear epoxy groups which can easily bind the enzymes, as it was showed previously. Smalla et al. used this support to immobilize aminoacylase, pepsin, trypsin, chymotrypsin, elastase, subtilisin, carboxypeptidase A, and other enzymes (Smalla et al., 1988). Ayhan et al. obtained poly(ethylene glycol methacrylate)/2-hydroxyethylene methacrylate) microbeads by suspension polymerization, in the range of 50-250 µm diameter. Activation of the hydroxyl groups has been accomplished by oxidation with sodium periodate to yield aldehyde groups, which were further reacted with hexamethylene diamine to introduce a spacer arm, and finally glutaraldehyde was employed to obtain the reactive aldehyde functions. Urease was immobilized on these non-porous microbeads. Although the immobilization resulted in a drastic decrease of the kinetic parameter Vm, the operational stability was excellent, the immobilized enzyme retaining 73% of the original activity after 75 days of repeated use (Ayhan et al., 2002).
Many other functionalized polymers were tested as immobilization supports for enzymes.
As a last example, polystyrene-based carriers will be presented. They can be prepared through nitration and reduction to introduce aromatic amino groups in the polymer structure. Activation of the aromatic amino groups is usually accomplished through diazotization (Fig. 1.6).
Li et al. have proposed a new immobilization technique involving polystyrene-based diazonium salt (PS-DAS), by using it as polymeric adhesive on the surface of other polymers, like as polyethylene, polypropylene, and poly(ethylene terephthalate). Using this immobilization method, presence of reactive groups or specific activation processes is no more required for the polymers. β-glucosidase immobilized using this methodology showed significantly improved thermal stability and reasonable reusability (70% residual activity after eight reaction cycles) (Li et al., 2010).
Fig. 1.6. Synthetic route to the diazonium salt of poly(4-aminostyrene) (Li et al., 2010)