Enzyme immobilization by adsorption: a review

Among many inorganic carriers used for immobilization of enzymes by adsorption, silicas are apparently those carriers, which have drawn most attention (Erhardt and Jordening 2007; Magner 2013; Hartmann and Kostrov 2013). Silicas of different dispersive-morphological parameters and porous structures have been proposed. A representative silica used for the enzyme immobilization on a large scale is mesoporous silica SBA-15 (Santa Barbara Amorphous) with hexagonal array of pores (Salis et al. 2010; Thorn et al. 2011). It is characterized by small pores, from 5 to about 30 nm in diameter and a hexagonal array of pores (Grudzień et al., 2006; 2007; Hartmann and Kostrov 2013). Large volume of mesopores, close to 1.0 cm³/g and micropores of about 0.8 cm³/g, and also a very well developed surface area from 500 to 1400 m²/g [Hartmann and Kostrov 2013] make this silica an excellent support for the enzyme immobilization. Another mesoporous silica MSU-H (Yu and Fang 2013) has the specific surface area reaching 750 m²/g, pore radius from 7 to 10 nm and pore volume from 0.9 to 1.0 cm³/g. By contrast, in mesoporous silica MCM-41 (Mobil Composition of Matter), with hexagonally ordered mesopores (Choma et al. 2004; Weber et al. 2010), the pore size is from 2 to 8 nm, which is controlled by adjusting the synthesis conditions and/or by applying surfactants with different chain lengths (pore sizes 2–5 nm) or expanders (pore sizes up to 8 nm) in their preparation. The pore volume in this silica is close to 1.0 cm³/g and its surface area exceeds 1,200 m²/g (Magner 2013), which are features that classify this material as a carrier for the enzyme immobilization. FDU-12 (Fudan University Material) (Hartono et al. 2010) is another mesoporous silica material with face-centered cubic structures of spherical mesopores and surface area of about 700 m²/g, pore size from 10 to 15 nm and pore volume from 0.6 to 0.7 cm³/g. This silica and other silica matrices (Falahti et al. 2012) differing in pore size and structure, are also excellent carriers for enzyme immobilization. Likewise, highly ordered mesoporous silicas with 2D and 3D structures and mesopores from 2 to about 30 nm, obtained by a surfactant and block copolymer templating, can be readily applied. The material of 3D structure is a better adsorbent and permits immobilization of a greater amount of the enzyme. The small particle of mesoporous silica (Chang et al. 2011) was reported to have a surface area of 820 m²/g and a pore diameter varying from 2 to 5 nm, while the diameter of particles was close to 150 nm. This small particle mesoporous silica was compared with the large particle mesoporous silica (Chang et al. 2011), which surface area was near 260 m²/g, pore diameter varied from 20 to 40 nm and particle size reached 600 nm. It was found that the larger surface area and smaller particle diameter favoured an immobilization of a greater amount of the enzymes. Cubic Ia3d mesoporous silica nanoparticles (Falahati et al. 2011) have the surface area of over 820 m²/g, pore diameters of about 7 nm and pore volume greater than 1.5 cm³/g. To increase the surface area of the carrier available to the enzyme, the folded sheet mesoporous silica was proposed (Nara et al. 2010). Such a configuration permits an immobilization of the greater amounts of the enzymes at only insubstantial growth of cost of the carrier production. Silicas of smaller surface areas obtained mainly in the processes of hydrolysis and condensation of tetraalkoxysilanes (Grabicka and Jaroniec 2010; Fornera and Bauer 2012; Zheng et al. 2012) were also used for the enzyme immobilization.

Other silica carriers widely used for the enzyme adsorption are silica gels (Bhattacharyya et al. 2010; Lee et al. 2010). They have very well developed porous structures and surface areas, as well as high mechanical strength and thermal stability. The size of silica gel particles varies from 70 to 150 μm depending on the type and the pore size reaches 250 nm.

Other types of silicas used for the enzyme immobilization are vesicular silica and fumed silica. Vesicular silica (Zhou et al. 2011; Wu et al. 2012) has pores of a diameter ranging from 15 to 20 nm, a pore volume from 0.6 to 1.4 cm³/g and surface area reaching 360 m²/g, while fumed silica (Kramer et al. 2010) has particles of a diameter from 7 to 50 nm and a surface area of 255 m²/g. These silicas have well-developed surface areas, small particles and high mechanical strength, which make them attractive alternatives to the other silicas described.

In order to enhance the affinity of the enzymes to silicas, the modifications of the matrices with polymers were proposed. The modifications consist in mixing silicas with polymers or coating them with polymers (Kwon et al. 2012). One group of polymers are polyamidoamine dendrimers (Wang et al. 2013). These are highly branched complex compounds, which due to the presence of amino groups in their structure, facilitate the development of the enzyme-carrier bonds, thereby giving rise to a more effective immobilization. For instance, it was observed that with an increasing content of the dendrimer on the silica surface, the amount of immobilized enzyme increased from 32 mg/g to almost 87 mg/g after a full modification of the silica. Polyamidoamine dendrimers were reported to enhance the affinity to cellulases, a group of enzymes catalysing the decomposition of cellulose by cleaving β-1,4-glycoside bonds. Importantly, the enzymes immobilized on polyamidoamine dendrimers modified-silicas retained over 80 % of their activity after three full catalytic cycles. A similar effect was reported for a mesoporous silica-polypyrrole composite. The effect was ascribed to the presence of hydroxyl groups in polypyrrole. Another group of polymers applied for silica modifications are aptamers (Xiao et al. 2012). These are short oligonucleotide chains (DNA or RNA fragments) able to form specific bonds with the carrier and the biocatalyst.

The noble metal applied as a carrier for the enzyme immobilization is gold. The preparations made on the basis of gold are used mainly in the electrodes mounted in biosensors (Delvaux and Demoustier-Champagne 2003), but they can also be employed in biodegradation of cellulose (Cho et al. 2012). Gold is hardly soluble but easily malleable so its form can be well managed. For the enzyme adsorption, it is used in the form of nanoparticles, gold-doped magnetic silica nanoparticles (Cho et al. 2012), and nanotubes (Delvaux and Demoustier-Champagne 2003).

Another inorganic carrier employed for the enzyme immobilization is a titania sol–gel film (Yu and Ju 2002). Titanium dioxide is a white solid of high melting point and good adsorption parameters.

Zirconia, a white crystalline solid with a high melting point and high chemical resistance, is also an attractive for the enzyme immobilization, where it is used in the form of nanoparticles (Chen et al. 2008), layered γ-zirconium phosphate (Geng et al. 2003) and as pure zirconium (Reshmi et al. 2007).

In addition to the above-mentioned materials, also alumina gel (Gupta and Prabhu 1995) and aluminium (Reshmi et al. 2006) were tested as the enzyme carriers. Aluminium is a common, malleable and plastic metal. Its derivatives are mesoporous aluminosilicates (Wanjari et al. 2012), well characterized (Jaroniec and Fulvio 2013), which are a class of compounds made of aluminium, silicon and oxygen. They can be of either natural (zeolites) or synthetic origin.

Some enzymes were also reported to be immobilized on cordierite and mullite (de Lathouder et al. 2008). The former is a rare mineral belonging to the group of silicates, whereas the latter, a mineral related to aluminosilicates in structure and composition and it was used in the immobilization in the form of acicular mullite.

Other minerals reported as the enzyme carriers are halloysite (Zhai et al. 2010) and mica (Zaidan et al. 2012). Mica is a multi-element mineral of a complex chemical composition that includes mainly aluminium, silicon, calcium, sodium and potassium, and in smaller amounts, lithium, magnesium, iron and manganese. The benefits offered by mica are its high thermal and chemical resistance.

Hydroxyapatite is another mineral used as a carrier for the immobilization of enzymes by adsorption (Fargues et al. 1998; Marzadori et al. 1998; Bronshyteyn and Steinberg 2002). Built of calcium, phosphorus, oxygen and hydrogen, hydroxyapatite is easily available; it occurs in nature and can also be chemically synthesized. Being a component of bones, hydroxyapatite shows high biocompatibility. It also displays high resistance to a wide range of reaction conditions. Its important advantage is the ability to bind practically all enzymes, where it is typically used as a powdered solid or as ceramic hydroxyapatite (Liu et al. 2013).

Also bentonite was reported to be an enzyme carrier with high protein adsorption capacity (Erhardt and Jordening 2007). Bentonite does not dissolve in water but readily swells, which is why bentonite-supported enzymes can be used in water environments.

Useful as a carrier in the enzyme immobilization also appeared to be a mesoporous activated carbon (Kennedy et al. 2007) of different pore sizes, such as MAC 200 and MAC 4000.

Of particular interest among organic carriers for the enzyme adsorption is chitosan (Krajewska 2004; Nasratun et al. 2010; Bayramoglu et al. 2012). Chitosan is a polyaminosaccharide obtained from chitin by deacetylation. Chitosan is a nontoxic, biocompatible and gel-forming cationic compound that can readily be prepared in different geometrical configurations, such as membranes, beads, nanoparticles, fibers, hollow fibers or sponges (Krajewska 2005). It can also be applied in the microcrystalline form (Castro et al. 2007). The special advantage of chitosan is that when dissolved in acidic solutions, it bears multiple positive charges on –NH₃ ⁺ groups along its linear chains. This feature allows it to readily develop electrostatic interactions with molecules containing negatively charged groups (Alatorre-Meda et al. 2009; Krajewska et al. 2011, 2013a, b). Another adavantage of chitosan is that it can easily be chemically modified, which is possible due to the presence of modifiable functional groups (–NH₂ and –OH) on chitosan chains (Honarkar and Barikani 2009).

A common organic compound used as an enzyme carrier is calcium alginate [Gupta and Prabhu 1995]. Alginate is an anionic polysaccharide that offers attractive gel-forming, concentrating and stabilizing properties. Commercial varieties of alginate are extracted from seaweeds, including the kelp Macrocystis pyrifera, Ascophyllum nodosum, and various types of algae from Phaeophyceae family. In addition to its pure form, it can also be used admixtured, e.g. with gelatin and transglutaminase (Kawaguti et al. 2011). It easily forms spherical particles with a well-developed surface area that endow it with good adsorption properties.

Alternatively used, organic carrier is cellulose. This is a polysaccharide of natural origin, made of glucose molecules. On the industrial scale, cellulose is obtained from wood. It is frequently used in the form of colourless cellulose acetate. It is a thermoplastic but hardly combustible polymer, insoluble in water. Different structures made of cellulose acetate are utilized, e.g. cellulose acetate membranes (Gulec 2013), cellulose acetate disc membranes (Labus et al. 2012) or ultrathin film of cellulose admixtured with acetate propionate and acetate butyrate (Kosaka et al. 2007).

Agarose gel (Pesic et al. 2012), a polysaccharide polymer, typically applied for separation of nucleic acids, is also used for enzyme immobilization, which is due to its morphological structure and beneficial adsorption properties.

In addition to natural polymers, synthetic polymers form a large and varied group of the enzyme carriers (Kumar and Kanwar 2011; Brem et al. 2011). Effectively, any polymerization can be designed to prepare a polymer with the customized properties. These properties can also be adapted by preparing the polymer composites. The synthetic polymers most commonly used as enzyme carriers include: poly(vinyl alcohol) (PVA) (Mendes et al. 2012) (commercial product LentiKats) (Kubac et al. 2006); cross-linked poly(vinyl alcohol) (Ozturk and Kilinc 2010); poly(N-methylolacrylamide) (Santos et al. 2007); polypropylene (commercial products Accurel EP100 (Torres et al. 2008) and Accurel MP1000 (Hita et al. 2009); polystyrene in the form of foam (Akdogan and Pazarlioglu 2011), in which immobilization is facilitated by a large number of pores; and poly(acrylic acid-co-acrylamide)/hydrotalcite nanocomposite hydrogels (Zhang et al. 2009). An interesting enzyme carrier is the biodegradable and thermo-shrinkable hydroxybutyrate. It is used in the form of poly(hydroxybutyrate) nanoparticles (Deepak et al. 2009) and small or large poly(hydroxybutyrate) beads (Mendes et al. 2011). Another carrier, poly(o-toluidine) built of particles of o-toluidine isomer has active –NH₂ groups (Ashly and Mohanan 2010). Furthermore, poly(acrylonitrile) (PAN), a polymer widely used in the production of synthetic fibres, as an enzyme carrier is used in the form of electrospun fibres (Sakai et al. 2010). PAN mats are about 25 μm thick and they are made to have a radius of about 400 nm. The material is very simple and its production is inexpensive. The stiff and elastic PAN carriers show high porosity and ability to interact with other materials, including enzymes and can be used in the various types of the reactors.

The carriers for the enzyme adsorption, if prepared in the form of membranes, e.g. as porous hollow fibre membranes (Kawakita et al. 2002), cotton flannel cloth (Karimpil et al. 2012) and nylon membranes (Xu et al. 2010), are special as they serve both as an enzyme support and at the same time as a separation phase, which, for instance, can separate the reagents of different molar masses.

An interesting option seems to be the carriers containing magnetic particles in their structure (Zhang et al. 2008; Zhu et al. 2009; Tran et al. 2012). Such magnetic matrices provide a good control of the process, as upon application of the magnetic field, the immobilized enzyme can be isolated and the catalyzed reaction terminated. Examples of such magnetic carriers include: a mixture of silicas with iron(II) and iron(III) oxides (Zhu et al. 2007); chitosan microspheres (Xie and Wang 2012); magnetic poly(acrylamide-allylglycydyl ether) cryogels (Tuzmen et al. 2012); and hydrophobic magnetic particles (Liu et al. 2012a, b).

Commercially available, ion-exchange resins, such as Lewatit (Wu et al. 2009), Amberlite IR-120H (Spinelli et al. 2012) or Amberlite XAD-2000 and Amberlite XAD-7 (Akdogan and Pazarlioglu 2011), typically used in the form of gels, are characterized with a highly developed porous structures and the presence of multiple active groups. These characteristics allow them to act as good enzyme supports.

Beside the materials described above, there are also substances, which although less frequently utilized in enzyme immobilizations, feature good adsorption properties, examples being artificial oil bodies (Hung et al. 2008) and olive pomace powder (Yucel 2012). An interesting approach to the immobilization of enzymes is also the use of reverse micelles (Thudi et al. 2012), in which the hydrophobic part is directed outside the micelle to allow an enzyme attachment.

An example of commercially available enzyme carriers is Stremaline DEAE (Erhardt and Jordening 2007), which is a composite made of agarose with a quartz core and diethylaminoethyl ligands on the surface. Another example are the epoxy-activated polymer supports, such as Eupergit (Katchalski-Katzir and Kraemer 2000; Erhardt and Jordening 2007) commercialized by Rhon Haas and Sepabeads commercialized by Resindion (Barbosa et al. 2013). Both are available in the form of macroporous beads. Eupergit supports are copolymers, of which a main component is poly(methacryl amide), while Sepabeads are polystyrenic adsorbents. Commercially available are also Celite (Kumar and Kanwar 2011) and Celite 545 silica carriers (Brem et al. 2011). Their main component is diatomaceous earth, which is the sedimentary rock formed as a result of diatoms exoskeletons deterioration. In the naturally occurring form it is admixtured with crystobalite, quartz and alumina. Its particle size varies from a few micrometers to a millimetre, but in the commercial product it has particles of diameters from 10 to 200 μm. Also worthy of note is Sepharose, a crosslinked, beaded-form of agarose, a polysaccharide polymer material extracted from seaweed. The great advantage of Sepharose is that its surface can be chemically modified in order to better adapt it to the functional groups of the protein (Benassi et al. 2013). Chitosan is another biopolymer manufactured for the enzyme immobilization and marketed under the brand name Chitopearl (Fuji Spinning, Tokyo, Japan) (Krajewska 2004). Different Chitopearl beads are produced and they can differ in the type and length of side ligands to be rightly chosen for a particular immobilization. The commercial products utilized in enzyme adsorption also include a group of polyvinyl supports available under the name Lentikats (Kubac et al. 2006), as well as the polymer matrices Accurel EP100 (Torres et al. 2008) and Accurel MP1000 (Hita et al. 2009).

The foregoing presentation of materials considered and studied for immobilizing enzymes by adsorption, shows that their variety is very rich. It includes organic and inorganic, natural and synthetic materials, that may be configured as beads of different sizes, membranes, fibers, hollow fibers, capsules, sponges in order to best match the conditions of a specific biotransformation in a given bioreactor. Importantly, it also shows that there are no universal carriers for all enzymes and their applications. Effectively, the choice of a specific material is determined by many factors and for each enzyme and each process this should be made individually, as it may happen that a drawback of one material in one process can be its advantageous feature in another one. The following general comment on the enzyme carrier materials can, however, be proposed. Silicas are perhaps the most common enzyme carriers. Their features, advantageous for this application result chiefly from the well-developed surface area, high availability and low cost. High thermal stability and chemical resistance shown by silica materials are also characteristic for minerals, such as mica or hydroxyapatite. The carriers based on metals, such as titanium, aluminium or zirconium, also show high mechanical strength; however, they show higher affinity to some groups of enzymes, which restricts their application. The magnetic organic and inorganic carriers, which use permits a good control of the enzymatic process, have become very popular but their widespread use is limited by their high cost. For the same reason, the use of carriers based on gold is limited. The interest in materials of natural origin, such as chitin, chitosan or cellulose, stems from their high biocompatibility and availability, but their application is limited by their selective affinity to certain enzymes, but foremost by their lower durability in the process conditions as compared to inorganic materials. By contrast, synthetic polymer matrices are widely used for enzyme adsorption, as they can be tailored to suite the specific enzyme and the conditions of a specific process. Moreover, their production is relatively facile and rapid, and what is more, they show high thermal and chemical resistance.