Elsevier

Biotechnology Advances

Volume 33, Issue 5, September–October 2015, Pages 435-456
Biotechnology Advances

Research review paper
Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

https://doi.org/10.1016/j.biotechadv.2015.03.006Get rights and content

Highlights

  • Use of immobilized antibodies to get the one step immobilization–purification

  • Use of domains with specific affinity for some ligands to immobilize/purify tagged proteins

  • Immobilization/purification by using of domains that increase the enzyme affinity for standard supports

  • Glyoxyl supports and one step immobilization–purification of multimeric proteins

  • Medium engineering and standard supports for immobilization/purification

  • Tailor made heterofunctional supports for large protein immobilization/purification

Abstract

In this review, we detail the efforts performed to couple the purification and the immobilization of industrial enzymes in a single step. The use of antibodies, the development of specific domains with affinity for some specific supports will be revised. Moreover, we will discuss the use of domains that increase the affinity for standard matrices (ionic exchangers, silicates). We will show how the control of the immobilization conditions may convert some unspecific supports in largely specific ones. The development of tailor-made heterofunctional supports as a tool to immobilize–stabilize–purify some proteins will be discussed in deep, using low concentration of adsorbent groups and a dense layer of groups able to give an intense multipoint covalent attachment. The final coupling of mutagenesis and tailor made supports will be the last part of the review.

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)

  • C. Carrasco-López et al.

    Activation of bacterial thermo alkalophilic lipases is spurred by dramatic structural rearrangements

    J Biol Chem

    (2009)
  • J.T. Chern et al.

    Chitin-binding domain based immobilization of d-hydantoinase

    J Biotechnol

    (2005)
  • I.H. Cho et al.

    Site-directed biotinylation of antibodies for controlled immobilization on solid surfaces

    Anal Biochem

    (2007)
  • Y.D. Clonis et al.

    Biomimetic dyes as affinity chromatography tools in enzyme purification

    J Chromatogr A

    (2000)
  • S. Daunert et al.

    Calmodulin-mediated reversible immobilization of enzymes

    Colloids Surf B Biointerfaces

    (2007)
  • Z.S. Derewenda et al.

    The crystal and molecular structure of the Rhizomucor miehei triacylglyceride lipase at 1.9 Å resolution

    J Mol Biol

    (1992)
  • JCS dos Santos et al.

    Stabilizing hyperactivated lecitase structures through physical treatment with ionic polymers

    Process Biochem

    (2014)
  • JCS dos Santos et al.

    Improving the catalytic properties of immobilized Lecitase via physical coating with ionic polymers

    Enzyme Microb Technol

    (2014)
  • A. Fatima et al.

    Polyclonal antibodies mediated immobilization of a peroxidase from ammonium sulphate fractionated bitter gourd (Momordica charantia) proteins

    Biomol Eng

    (2007)
  • R. Fernandez-Lafuente

    Stabilization of multimeric enzymes: strategies to prevent subunit dissociation

    Enzyme Microb Technol

    (2009)
  • R. Fernandez-Lafuente et al.

    Immobilization of lipases by selective adsorption on hydrophobic supports

    Chem Phys Lipids

    (1998)
  • G. Fernández-Lorente et al.

    Specificity enhancement towards hydrophobic substrates by immobilization of lipases by interfacial activation on hydrophobic supports

    Enzyme Microb Technol

    (2007)
  • G. Fernandez-Lorente et al.

    Interfacially activated lipases against hydrophobic supports: Effect of the support nature on the biocatalytic properties

    Process Biochem

    (2008)
  • A. Fishman et al.

    Stabilization of horseradish peroxidase in aqueous-organic media by immobilization onto cellulose using a cellulose-binding-domain

    J Mol Catal B: Enzym

    (2002)
  • M. Fuentes et al.

    Detection and purification of two antibody-antigen complexes via selective adsorption on lowly activated anion exchangers

    J Chromatogr A

    (2004)
  • M. Fuentes et al.

    Preparation of inert magnetic nano-particles for the directed immobilization of antibodies

    Biosens Bioelectron

    (2005)
  • M. Fuentes et al.

    Solid phase proteomics: dramatic reinforcement of very weak protein–protein interactions

    J Chromatogr B Anal Technol Biomed Life Sci

    (2007)
  • T. Gräslund et al.

    Strategy for highly selective ion-exchange capture using a charge-polarizing fusion partner

    J Chromatogr A

    (2002)
  • T. Gräslund et al.

    Integrated strategy for selective expanded bed ion-exchange adsorption and site-specific protein processing using gene fusion technology

    J Biotechnol

    (2002)
  • V. Grazu et al.

    Glyoxyl agarose as a new chromatographic matrix

    Enzyme Microb Technol

    (2006)
  • V. Grazu et al.

    Tailor-made design of penicillin G acylase surface enables its site-directed immobilization and stabilization onto commercial mono-functional epoxy supports

    Process Biochem

    (2012)
  • H. Gröger et al.

    Combining the ‘two worlds’ of chemocatalysis and biocatalysis towards multi-step one-pot processes in aqueous media

    Curr Opin Chem Biol

    (2014)
  • T. Haider et al.

    Immobilization of β galactosidase from Aspergillus oryzae via immunoaffinity support

    Biochem Eng J

    (2009)
  • M. Hedhammar et al.

    Zbasic-A novel purification tag for efficient protein recovery

    J Chromatogr A

    (2007)
  • K. Hernandez et al.

    Control of protein immobilization: coupling immobilization and site-directed mutagenesis to improve biocatalyst or biosensor performance

    Enzyme Microb Technol

    (2011)
  • K. Hernandez et al.

    Simple and efficient immobilization of lipase B from Candida antarctica on porous styrene–divinylbenzene beads

    Enzyme Microb Technol

    (2011)
  • R. Iwata et al.

    Covalent immobilization of antibody fragments on well-defined polymer brushes via site-directed method

    Colloids Surf B Biointerfaces

    (2008)
  • A.A. Khan et al.

    Simultaneous purification and immobilization of mushroom tyrosinase on an immunoaffinity support

    Process Biochem

    (2005)
  • A. Kumar et al.

    Polymer displacement/shielding in protein chromatography

    J Chromatogr B Biomed Sci Appl

    (2000)
  • D.H. Kweon et al.

    Immobilization of Bacillus macerans cyclodextrin glycosyltransferase fused with poly-lysine using cation exchanger

    Enzyme Microb Technol

    (2005)
  • J. Li et al.

    Comparison of magnetic carboxymethyl chitosan nanoparticles and cation exchange resin for the efficient purification of lysine-tagged small ubiquitin-like modifier protease

    J Chromatogr B Analyt Technol Biomed Life Sci

    (2012)
  • M. Linder et al.

    The roles and function of cellulose-binding domains

    J Biotechnol

    (1997)
  • L. Lu et al.

    Synthesis of galactooligosaccharides by CBD fusion β-galactosidase immobilized on cellulose

    Bioresour Technol

    (2012)
  • C. Mateo et al.

    Affinity chromatography of polyhistidine tagged enzymes — new dextran-coated immobilized metal ion affinity chromatography matrices for prevention of undesired multipoint adsorptions

    J Chromatogr A

    (2001)
  • C. Mateo et al.

    Some special features of glyoxyl supports to immobilize proteins

    Enzyme Microb Technol

    (2005)
  • C. Mateo et al.

    Glyoxyl agarose: a fully inert and hydrophilic support for immobilization and high stabilization of proteins

    Enzyme Microb Technol

    (2006)
  • C. Mateo et al.

    Improvement of enzyme activity, stability and selectivity via immobilization techniques

    Enzyme Microb Technol

    (2007)
  • E. Abaházi et al.

    Additives enhancing the catalytic properties of lipase from Burkholderia cepacia immobilized on mixed-function-grafted mesoporous silica gel

    Molecules

    (2014)
  • O. Abian et al.

    Stabilization of penicillin G acylase from Escherichia coli: site-directed mutagenesis of the protein surface to increase multipoint covalent attachment

    Appl Environ Microbiol

    (2004)
  • M. Arroyo et al.

    Characterization of a novel immobilized biocatalyst obtained by matrix-assisted refolding of recombinant polyhydroxyoctanoate depolymerase from Pseudomonas putida KT2442 isolated from inclusion bodies

    J Ind Microbiol Biotechnol

    (2011)
  • Cited by (0)

    View full text