Research review paperStrategies for the one-step immobilization–purification of enzymes as industrial biocatalysts
Introduction
Enzymes are biocatalysts with outstanding prospects as catalysts in industrial processes which include high activity under very mild environmental conditions, high selectivity, and high specificity (Gröger and Hummel, 2014, Reetz, 2013, Schrittwieser and Resch, 2013, Teixeira et al., 2014, Wells and Meyer, 2014). However, enzymes have also some limitations that may hinder their industrial implementation (Schoemaker et al., 2003).
Enzymes are water-soluble molecules that need to be separated from the reaction media to be re-used. This is important for improving the economy of the process and also for facilitating the control in the reactor (Brady and Jordaan, 2009, Garcia-Galan et al., 2011, Sheldon, 2007). Furthermore, they may neither be stable enough under industrially relevant conditions (presence of organic solvents, high temperatures to avoid contamination, etc.) nor have high enough activity, selectivity or specificity towards the target industrial substrate (sometimes quite far from the physiological substrates). Moreover, they are produced in conjunction with many other similar proteins (some of them with undesired catalytic activity versus the substrates or even the products) that decrease the final volumetric activity because some surface of the support will be occupied by other proteins. The activity of minority enzymes with opposite catalytic activity may also decrease the enantio or regioselectivity or specificity of the “biocatalyst” if it includes any of these contaminant enzymes.
The latter issue is tackled using purification strategies, which in some cases may be a long and tedious process, while in other cases it includes just one chromatographic step (Clonis et al., 2000, Porath, 1992, Wilchek et al., 1984, Zeng and Ruckenstein, 1999). Nevertheless, even in the best case scenario this may have a negative economic impact in the final cost of the biocatalyst.
On the other hand, the most obvious solution to get a simpler recovery of the enzyme is its immobilization (Brady and Jordaan, 2009, Garcia-Galan et al., 2011, Sheldon, 2007). Moreover, considering that immobilization is in most cases a requirement to use the enzyme as an industrial biocatalyst, many researchers have endeavored to couple immobilization with the improvement of other enzyme properties (Garcia-Galan et al., 2011, Guzik et al., 2014, Hernandez and Fernandez-Lafuente, 2011, Hwang and Gu, 2013, Rodrigues et al., 2013, Stepankova et al., 2013, Zucca and Sanjust, 2014). Multipoint (Mateo et al., 2007c) or multisubunit (in multimeric enzymes (Fernandez-Lafuente, 2009)) immobilization may improve enzyme rigidity and thus, improve enzyme stability (Fig. 1). The rigidification of certain areas of the protein’s surface and its controlled distortion resulting from the immobilization process have been shown to tune (in some instances significantly improving) enzyme activity, selectivity or specificity (Mateo et al., 2007c, Rodrigues et al., 2013).
Moreover, in certain cases, the immobilization protocol (including support, enzyme modification and immobilization conditions) has been designed to couple the immobilization of the enzyme and its purification in just one process preferably without sacrificing other potential enzyme improvements (Garcia-Galan et al., 2011). The present review will discuss the use of techniques that permit to join, in a single step, immobilization and purification. To this goal, it is very important to know if the interaction of the enzyme molecule with just one active group of the support is enough to keep the enzyme coupled to the support under the immobilization conditions, or, on the contrary, only after several enzyme–support interactions the protein molecule remains attached to the support. These will be the key for the final adsorption selectivity, even though the final objective will be a multipoint or multisubunit attachment to improve enzyme stability (Fig. 2) (Garcia-Galan et al., 2011).
First, a rapid view of different affinity immobilization strategies using supports bearing specific receptors to domains included in the target protein structure will be presented (Binz et al., 2005, Linder and Teeri, 1997, Ong et al., 1989, Saleemuddin, 1999). In general, these immobilizations will be just via one point (the domain) with scarce effect on enzyme stability (except those effects derived from the immobilization of enzymes inside a porous support), but in certain cases they may include several enzyme subunits of multimeric enzymes, with the positive effect on enzyme stability that this may have (Bolivar and Nidetzky, 2012b, Hernandez and Fernandez-Lafuente, 2011). A special case will be the immobilization of lipases on hydrophobic supports via interfacial activation, which produces some stabilization (Palomo et al., 2002). The use of tailor-made supports to specifically immobilize proteins with certain structural particular features (large or small proteins, lipases via interfacial activation), with the final development of heterofunctional supports to achieve the specific enzyme immobilization followed by its stabilization via multipoint or multisubunit immobilization will be also an important part of this review (Barbosa et al., 2013). Finally, the coupling use of site directed mutagenesis (to introduce specific domains in the desired areas of the protein) to these heterofunctional supports to achieve the immobilization/stabilization of the proteins will be discussed (Barbosa et al., 2013).
This strategy requires having in mind that immobilization involves different steps with different objectives. The first one is a somehow rapid immobilization, via the moieties that we have introduced which are able to recognize the protein. The second one is the promotion of covalent attachments (as many as possible to improve stability) between the enzyme and the support which may be quite a slow process and proceed at different conditions.
Section snippets
Coupled immobilization/purification of proteins via antibody specific adsorption
One general strategy to couple immobilization with purification with any protein is to immobilize it on a previously immobilized anti-target protein (Saleemuddin, 1999). This strategy may use monoclonal or polyclonal antibodies, and permits an extremely selective protein adsorption; only the target protein becomes immobilized (i) if the antibody is properly immobilized (Ahmed et al., 2006, Batalla et al., 2008, Cho et al., 2007, Iwata et al., 2008, Schmid et al., 2006) and (ii) if we can
Coupled immobilization/purification of enzymes and proteins via specific domains
There are many different peptides and proteins which have a high affinity for different groups or structures, which may be added to the structure of the target protein by genetic routes, and thus, transfer this affinity property to the employed protein (Fig. 4). These peptides may be very small (just a dozen of residues or even less), like in the poly-His tags, or domains with several kD (e.g. cellulose binding domain) (Linder and Teeri, 1997, Nordon et al., 2009, Ong et al., 1989). Perhaps the
The case of lipases immobilization via interfacial activation on hydrophobic supports
In some cases, it is possible to use some specific particularities of the catalytic mechanism of an enzyme to differentiate it from others. That is the case of lipases. These enzymes are capable of acting in the surface of drops of oils (Brzozowski et al., 1991, Van Tilbeurgh et al., 1993). To reach this goal, lipases have a mechanism of action called interfacial activation (Verger, 1997). In aqueous media, they usually have the hydrophobic catalytic center blocked by a polypeptide chain,
Coupled immobilization, purification and multipoint or multisubunit immobilization of enzymes and proteins via covalent immobilization on heterofunctional supports
Now, we will present the development of tailor-made heterofunctional supports to get the specific immobilization of target proteins. Heterofunctional supports have been recently reviewed (Barbosa et al., 2013); here we will focus on the prospects to use them to perform one step immobilization–purification. Heterofunctional supports are defined as those matrices that present several functionalities on their surface, with different physical or chemical properties, able to interact with a protein (
Coupled immobilization, purification and multipoint or multisubunit immobilization of domain tagged enzymes and proteins via covalent immobilization on heterofunctional supports
In some cases, the mere attachment of the enzyme and the support achieved by using some of the tags described in Section 3 of this review is not desired by different reasons, for example by the risk of some desorption of the enzyme during operation, a necessity for improving enzyme stability, or the intention of submitting the enzyme to processes of unfolding/refolding (Bolivar et al., 2010b). In these situations, the use of heterofunctional supports bearing a few groups able to give the
Immobilization–purification based on different immobilization rates
In some cases, mainly using strategies based that require a multipoint enzyme–support interaction, the target enzyme may have a much faster immobilization rate than the other contaminant proteins. This may not be enough for having a good purification if other proteins are also very rapidly immobilized because the difficulty in stopping the immobilization, and less at industrial level where the volumes that they manage may make it almost impossible to have a strict control of the immobilization
Conclusions
The coupling of immobilization to purification of enzymes and proteins has undoubted interest. The interest goes further if the final biocatalyst has an improved stability via multisubunit or multipoint covalent attachment, or we can get enzymes with a better orientation. The better understanding of the immobilization mechanism on the different supports may open new strategies to reach this objective. For example, glyoxyl supports have shown their real impact in this area only after recognizing
Acknowledgments
This work has been supported by grant CTQ2013-41507-R from Spanish MINECO, grant no.1102-489-25428 from COLCIENCIAS and Universidad Industrial de Santander (VIE-UIS Research Program) (Colombia) and CNPq grant 403505/2013-5 (Brazil). A. Berenguer-Murcia thanks the Spanish MINECO for a Ramon y Cajal fellowship (RyC-2009-03813). The authors would like to thank Mr. Ramiro Martinez (Novozymes, Spain S.A.) for his continuous kind support to our research.
References (178)
- et al.
Specific capture of target proteins by oriented antibodies bound to tyrosinase-immobilized Protein A on a polyallylamine affinity membrane surface
J Membr Sci
(2006) - et al.
The slow-down of the CALB immobilization rate permits to control the inter and intra molecular modification produced by glutaraldehyde
Process Biochem
(2012) - et al.
Indirect immobilization of recombinant proteins to a solid phase using the albumin binding domain of streptococcal protein G and immobilized albumin
J Immunol Methods
(1998) - et al.
Stabilization of enzymes by multipoint covalent attachment to agarose-aldehyde gels. Borohydride reduction of trypsin-agarose derivatives
Enzyme Microb Technol
(1989) - et al.
The co-operative effect of physical and covalent protein adsorption on heterofunctional supports
Process Biochem
(2009) - et al.
The adsorption of multimeric enzymes on very lowly activated supports involves more enzyme subunits: stabilization of a glutamate dehydrogenase from Thermus thermophilus by immobilization on heterofunctional supports
Enzyme Microb Technol
(2009) - et al.
Purification and stabilization of a glutamate dehygrogenase from Thermus thermophilus via oriented multisubunit plus multipoint covalent immobilization
J Mol Catal B: Enzym
(2009) - et al.
Heterofunctional supports for the one-step purification, immobilization and stabilization of large multimeric enzymes: amino-glyoxyl versus amino-epoxy supports
Process Biochem
(2010) - et al.
Complete reactivation of immobilized derivatives of a trimeric glutamate dehydrogenase from Thermus thermophillus
Process Biochem
(2010) - et al.
Hydrophobic adsorption and covalent immobilization of Candida antarctica lipase B on mixed-function-grafted silica gel supports for continuous-flow biotransformations
Process Biochem
(2013)