Enzyme Immobilization

Enzyme immobilization can be defined as the confinement of enzyme molecules onto/within a support/matrix physically or chemically or both, in such a way that it retains its full activity or most of its activity.

From: Methods in Enzymology , 2016

Nanoarmoring of Enzymes with Carbon Nanotubes and Magnetic Nanoparticles

Diego E. Sastre , ... Caterina G.C. Marques Netto , in Methods in Enzymology, 2020

Abstract

Enzyme immobilization is a widespread empiric technology to achieve more stable, active and reusable enzymes. The empiricism can be reduced by the application of rational design procedures employing bioinformatic tools, engineered-proteins and detailed analyses of existent data. In this chapter, we describe relevant approaches to rationalize the design of enzyme immobilization protocols, with special attention to the modulation of immobilization pH to regulate the operational stability of glutaraldehyde cross-linked enzymes and the coating of iron-containing supports to preserve the integrity of iron-sensitive enzymes. Other strategies, such as the use of factorial planning, optimization of specific enzyme orientation through protein engineering and the use of mathematical algorithms and in silico prediction tools are also described to reduce the classical empiricism. Finally, a public repository creation is proposed as a new promising tool to develop an improvement on future rational design procedures of enzyme immobilization.

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Engineering Fundamentals of Biotechnology

L. Cao , in Comprehensive Biotechnology (Second Edition), 2011

Abstract

Enzyme immobilization technology is one of the key modern industrial biotechnologies. Since the commercial use of first immobilized enzymes in the 1960s, enzyme immobilization technologies and theories as well as immobilization materials and chemistry have gained rapid development. Nowadays, the design of the immobilized enzymes, which suits different specific applications, has abandoned the traditional trial-and-error approach and gradually transited to the rational design, which is characterized by the fact that the enzyme immobilization technology is now used not only to realize the reuse of the costly enzymes a better control of the process, but also to improve the enzyme catalytic functions such as activity, stability, and selectivity. To some extent, the enzyme immobilization technology is becoming a complimentary technology to the genetic engineering.

Currently, it is becoming increasingly appreciated that the availability of a robust immobilized enzyme in the early stage of process development will definitively enable early insight into process development and save costs not only in process development but also in production. However, the lack of guidelines for selection of the method of immobilization and the performance to be expected of an immobilized enzyme for a specific application seriously hampers application of a rational approach to the design of such robust immobilized enzymes.

In this context, the article systematically delineates the basic principles governing the individual approaches in the design of robust enzymes, which can be classified into the following four approaches: (1) rational versus trial–error, (2) diversity versus versatility, (3) complimentary versus alternative, and (4) modification versus immobilization approach. Moreover, the article attempts to provide a rational basis for future development of immobilized enzymes.

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Synthetic Biology and Metabolic Engineering in Plants and Microbes Part A: Metabolism in Microbes

J.T. Payne , ... J.C. Lewis , in Methods in Enzymology, 2016

2.3 Improving Enzyme Stability Through Immobilization

Enzyme immobilization has been reported to increase enzyme stability without significantly compromising activity, for example, in improving the organic solvent tolerance ( Truppo, Strotman, & Hughes, 2012) and pH range (Koszelewski, Müller, Schrittwieser, Faber, & Kroutil, 2010) of transaminases. Frese and Sewald recently reported on the enzymatic halogenation of l-tryptophan on a gram scale by forming cross-linked enzyme aggregates (CLEAs) from crude RebH lysate (Frese & Sewald, 2015). The authors reported that RebH combiCLEAs had significantly increased retention of activity relative to free, purified RebH after extended storage at 4°C, and that RebH combiCLEAs also displayed significantly increased catalyst lifetime in an active biohalogenation reaction (although relative total turnover numbers were not reported). Furthermore, RebH combiCLEAs could be reused up to 10 times while still displaying significant halogenation activity. CombiCLEAs, which have been demonstrated to be used successfully with RebH, and other enzyme immobilization methods, which as of yet lack specific demonstration with FDHs, are most likely to be useful in preparative-scale halogenation reactions, rather than high-throughput screening applications. The reported procedure for the particular immobilization strategy employed would thus be substituted for steps 31–34 of the procedure described in Section 2.1.1.

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NanoArmoring of Enzymes: Rational Design of Polymer-Wrapped Enzymes

M. Rita Correro , ... Patrick Shahgaldian , in Methods in Enzymology, 2017

4.4.1 General Procedure and Experimental Set-Up

Enzyme immobilization is performed after functionalization of the silica nanoparticles' surface. First, the surface of the silica carrier is amino modified by incubating the particle suspension with 3-aminopropyl triethoxysilane (APTES). Afterward, in order to provide anchoring residues for the enzyme, the amino-modified particles are chemically modified with the homo-bifunctional crosslinker glutaraldehyde. As model enzyme, β-galactosidase from K. lactis is chosen. The enzyme shielding is performed by incubating the immobilized β-galactosidase with a mixture of tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane at 20°C (Fig. 5).

Fig. 5. Schematic representation of the strategy developed for enzyme immobilization and shielding. The silica nanoparticles suspension is sequentially incubated with (3-aminopropyl)triethoxysilane (APTES) (step 1), glutaraldehyde (step 2), enzyme (step 3), and with APTES and tetraethyl orthosilicate (step 4) for the synthesis of the protective organosilica layer. Details of the chemical reactions occurring at the surface of the silica nanoparticles during the reagents addition are showed.

The experiment is performed in glass vials, at 400   rpm under magnetic stirring at 20°C (Fig. 3). A β-galactosidase stock solution (0.63   mg mL−   1) in buffer (100   mM phosphate, 5   mM MgCl2, pH 6.5) is prepared and kept on ice prior to use.

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Nanoarmoring of Enzymes with Carbon Nanotubes and Magnetic Nanoparticles

Veli C. Ozalp , ... M. Yakup Arica , in Methods in Enzymology, 2020

5 Conclusions

Enzyme immobilization is a useful technique for improving catalysis efficiency under production conditions. The fibrous polymer grafting on magnetic particles results in some invaluable advantages due to: (i) desired reactive chemical groups can easily be obtained via SI-ATRP method, (ii) being mild immobilization method for enzymes, (iii) providing high surface area and low volume ratio, (iv) easy separation from the reaction content with an external magnet. Immobilization might change the optimum pH and wider pH activity range. Immobilization might also raise the optimum temperature and improve thermal stability of the enzyme. The stability changes results from interactions of enzyme with hydrophilic fibrous polymer, providing conformational flexibility to immobilized enzyme. Additionally, fibrous polymer brushes provide a higher density of enzymes on the nanoparticle surface, leading to higher stability of protein conformations. Kinetic parameters for free and immobilized forms of enzyme can stay unchanged. The shelf life of immobilized enzyme might be prolonged several weeks with little loss in enzyme activity after tens of times reuse of the same magnetic particles. Thus, the immobilization of enzymes provides easy recovery and reuse of the enzyme.

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Enzymes, Aptamers, and Their Use in Sensors

Piyanut Pinyou , ... Vincent Blay , in Reference Module in Biomedical Sciences, 2021

Affinity binding

Enzyme immobilization can be also done by leveraging complementary interactions between specific biomolecules ( Guisan, 2006), such as antibody-antigen, lectin-saccharidic chain, avidin-biotin, polyhistidine tag-metal ion, etc. In affinity binding, the support is typically functionalized with one affinity partner, while the enzyme displays the complementary partner (Kłos-Witkowska, 2015). Polyhistidine tags, in particular, are widely used for purification of proteins expressed in bioreactors. In some cases, these same tags can also be used for biocatalysis or sensing without additional engineering. The advantages of affinity binding are a highly selective immobilization and often a small conformational change of the enzyme, preserving its activity (Mohamad et al., 2015). In one example, silica-coated magnetic nanoparticles (SiMNPs) were functionalized with 3-(triethoxysilyl)propyl isocyanate-nitrilotriacetic acid (ICPTES-NTA), which forms a complex with Ni2   + ions with its carboxylic acid groups. Then, the Ni2   + cations in the Ni2   +-SiMNPs complex were used to immobilize Escherichia coli prolidase through a polyhistidine tag fused to the enzyme (Wang et al., 2019a,b).

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Nanoarmoring of Enzymes with Carbon Nanotubes and Magnetic Nanoparticles

Ram Sarup Singh , Kanika Chauhan , in Methods in Enzymology, 2020

3.6 Enzyme immobilization onto homo- and hetero-functionalized MWCNTs

Enzyme immobilization onto homo- or hetero-functionalized MWCNTs can be performed using the following steps:

1.

Re-suspend homo- or hetero-functionalized MWCNTs in 2–3   mL of buffer (which is to be used for protein of interest) in glass vials/tubes with caps. Carefully re-suspend the hetero-functionalized MWCNTs in the buffer without any wastage.

2.

Add appropriate amount of enzyme of interest using a volumetric pipette into the above mentioned suspension in glass vials/tubes with caps. Always use fresh tips for pipetting the enzyme using a volumetric pipette to prevent its mixing with other samples.

3.

Incubate enzyme-MWCNTs mixture for 2–3   h, under gentle rotation using a tube rotator for enzyme coupling. Always use gentle rotation during coupling of enzyme to the MWCNTs.

4.

After coupling of enzyme, recover the immobilized biocatalyst by centrifugation (5000   rpm, 10   min, 4   °C) and wash it two to three times with buffer (which is to be used for protein of interest), to remove uncoupled enzyme. Cautiously recover the developed immobilized biocatalyst after centrifugation with nominal wastage.

5.

Thereafter, determine enzyme activity and protein content in supernatant to examine immobilization efficiency of immobilized enzyme in terms of activity yield and immobilization yield.

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Stability and Stabilization of Biocatalysts

H.-G. Hicke , ... M. Ulbricht , in Progress in Biotechnology, 1998

3.2 Symmetric enzyme microilltration membranes

Enzyme immobilization was performed sequentially: First, reactive groups were activated (g-PAA: 10  g/1   N-dimethylaminopropyl N′-ethyl carbodiimide at pH   =   4.6, 4°C; g-PAEMA: 100   g/1   glutaraldehyde in water, 25°C). After rinsing followed the coupling reaction with enzyme (AG or INV; 20   g/l at pH   =   7.5, 50°C). An example of the achieved enzyme distribution inside the pores of a fimctionalized PET capillary MFM is shown in Figure 2.

Figure 2. SEM of a PET-g-PAEMA / INV membrane after Auimmunolabelling of the enzyme

Substrate hydrolysis (AG: starch or maltose; INV : sucrose; 20   g/1 at pH   =   4.6, 50°C) produced glucose measured by a glucose sensor. Representative results for enzyme binding capacities and enzyme activities without flow through the membranes are shown in Table 3.

Table 3. Overview on heterogeneously photo-functionalized MFM for covalent enzyme immobilization

Membrane Pores Function. Permeability Bound Enzyme Specific
dp DG pH   =   4.6 enzyme activity activity
(μm) (μg/cm2) J/p (l/m2hbar) m (μg/cm2) z (U/cm2) Zsp (U/mg)
PP-g-PAA 0.2 120 7200 INV: 540 3.6 6.7
PP-g-PAEMA 0.2 90 8100 INV: 950 7.0 7.4
AG: 335 8.5 25.4
PET-g-PAA 0.2 30 15000 INV: 110 0.30 2.8
PET-g-PAEMA 0.2 30 20000 INV: 95 0.37 3.8
PET-g-PAEMA 1.0 6 120000 INV: 49 0.22 4.5
PET-g-PAEMA 3.0 5 200000 INV: 32 0.04 1.2

PP … polypropylene; PET … polyethylene terephthalate; g-PAA … grafted poly(acrylic acid); g-PAEMA … 2-aminoethyl methacrylate (AEMA)

Permeabilities of both unmodified (not shown) and g-PAEMA membranes were almost identic; while the g-PAA membranes had significantly lower values. This is caused by the expansion of the g-PAA "tentacles" due to charge repulsion at a pH around and above the pKa of the graft polymer [1], Coupling of enzyme did slightly reduce the membrane permeabilities, but large perfusion rates were possible (cf. 3.3., 3.4.).

Decreasing enzyme binding capacities with increasing MFM pore size were due to decreasing specific surface area. Specific enzyme activities were always lower for g-PAA membranes. This may be due to steric hindrance of enzyme in the "graft-tentacle" layer [2], while g-PAEMA membranes can be described as "grafted-coat" structures with enzyme bound to reactive groups on the surface of a hydrophilic, flexible layer.

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Enzyme Nanoarchitectures: Enzymes Armored with Graphene

Alexandra V. Chatzikonstantinou , ... Haralambos Stamatis , in Methods in Enzymology, 2018

1.2 Biocatalytic Systems in Nanobiotechnology

Enzyme immobilization is commonly recognized as a technology to stabilize biocatalysts against chemical and environmental attacks. Especially during large-scale industrial applications, such obstacles may lead to high operational costs due to low recovery and reusability of the biocatalyst ( de Jesús Rostro-Alanis et al., 2016). Nevertheless, once immobilized, enzymes demonstrate high stability through a favorable microenvironment for them to function. Namely, immobilization can lead to improved substrate accessibility, since aggregation of the hydrophilic protein particles is avoided and the catalytic properties of immobilized enzymes can be easily modulated, thus creating robust biocatalysts for the development of commercial biocatalytic processes (Hwang & Gu, 2013; Pavlidis, Patila, Bornscheuer, Gournis, & Stamatis, 2014). Furthermore, immobilization improves enzyme performance under extreme reaction conditions, such as the use of organic solvents and elevated temperatures, allowing applications in industrial synthesis, while a facile separation of the catalyst from the reaction products is also possible, a key advantage regarding reactions with dissolved enzymes (Hwang & Gu, 2013; Küchler, Yoshimoto, Luginbühl, Mavelli, & Walde, 2016).

There are extensive reports on laccase immobilization, in an effort to improve the enzyme's performance. Studies include carrier-based and carrier-free immobilized forms of laccase (CLEAs and Combi-CLEAs). When it comes to stability enhancement, the research focuses on carrier-based biocatalytic systems, in order to create a rigid biocatalyst that functions in harsh conditions and can easily be recycled (Ba & Vinoth Kumar, 2017).

The combined research in the fields of biotechnology and nanotechnology during the last decade has led to an upsurge of innovative biological nanosystems. Nanobiocatalysis constitutes one of the most thriving research fields nowadays, as new nanobiocatalytic systems are developed, based mainly on the immobilization of enzymes on nanostructured materials with tailor-made properties (Pavlidis, Patila, Bornscheuer, et al., 2014; Pavlidis, Patila, Polydera, Gournis, & Stamatis, 2014). Different modified nanoscale supports have been studied and found to reduce diffusion limitations, thus enhancing biocatalytic efficiency, as well as increasing enzyme loading given the superior surface area per mass unit (Pavlidis, Tsoufis, Enotiadis, Gournis, & Stamatis, 2010; Tzialla et al., 2010). Consequently, the functionalized nanocarriers serve as nanoscale-processing systems that provide a suitable microenvironment for the enzyme to react in the maximal efficiency. In this direction, nanomaterials are carefully designed to enable a long-term storage and recycling integrity for the immobilized biocatalyst, as well as large surface area and low mass transfer resistance (Hwang & Gu, 2013). Overall, elaborating the possibilities for biotechnological applications of immobilized enzymes requires an optimization of both the enzyme localization and the enzyme's storage and operational stability (Küchler et al., 2016).

A focal point to take into consideration is the fact that immobilization entails the interaction of the enzyme and the nanocarrier. Considering this, the optimal conditions for each nanobiocatalytic system have to be investigated. However, enzymes immobilized on nanomaterials are usually advantageous over native enzymes, showing wider temperature range, broader working pH, increased reusability and greater thermal, storage and operational stability. For example, nanoimmobilized laccase and lipase were shown to retain up to 90% of their activity in nonconventional media or in the presence of denaturing agents (Tzialla et al., 2010, 2009), while lipase was also shown to exhibit great storage stability while working as a nanobiocatalytic system (Pavlidis et al., 2010). Besides these, nanocarriers' properties, such as high surface-to-volume ratio, chemical, electrical, and optical properties, favor enzymes' performance by increasing enzyme loading and affecting their diffusion and catalytic activity (Chen, Zeng, Xu, Lai, & Tang, 2017).

Another aspect to consider pertains to the immobilization method. In particular, enzyme immobilization onto a support is possible through adsorption, covalent binding, entrapment, or encapsulation, with each method demonstrating different merits and demerits (Barrios-Estrada et al., 2018). Physical bonding (such as hydrophobic or van der Waals interactions) is generally too weak to keep the enzyme stable under industrial conditions. However, covalent binding has been extensively studied and is considered as a highly reliable method for achieving immobilization through specific binding sites as well as preventing enzyme leaching (de Jesús Rostro-Alanis et al., 2016; Hwang & Gu, 2013).

Concerning laccase immobilization, covalent binding has been widely studied during the last decade as it has been proven that the tertiary structure of the enzyme is stabilized, offering great resistance to denaturing agents. Providing the enzyme's rigidity, an optimal support should contain short spacer arms and a high density of reactive groups on its surface so as to be activated and react with nucleophilic groups on the protein (Fernández-Fernández, Sanromán, & Moldes, 2013).

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Nanoarmoring of Enzymes with Carbon Nanotubes and Magnetic Nanoparticles

Lorico D.S. LapitanJr., Dejian Zhou , in Methods in Enzymology, 2020

1.1 Enzyme immobilization strategies on magnetic nanoparticles

Enzyme immobilization on MNP surface provides an indispensable tool for separating the catalyst from the sample matrix by use of an external magnetic field. A variety of techniques have been used to prepare enzyme-MNP conjugates, including covalent bonding, physical adsorption, and biospecific interactions, etc. ( Ansari & Husain, 2012; Bilal et al., 2018; Zhang & Zhou, 2012). Each method has its own advantages and also drawbacks. For example, physical adsorption is simple to do, but the resulting enzyme-MNP conjugates often lack of long term stability. On the other hand, covalent immobilization gives more robust conjugate, but it is often difficult to control the enzyme orientation to maximize active site access and to maximize the activity of immobilized enzymes (Bornscheuer, 2003). A common method used in covalent immobilization is using a heterobifunctional linker having amine and thiol dual reactive function groups, such as the commercial N-hydroxysuccinamide-poly(ethylene glycol)-maleimide (NHS-PEG12-maleimide) shown in Scheme 1 (Kuhn, Finch, Hallahan, & Giorgio, 2007).

Scheme 1

Scheme 1. The chemical structure of heterobifunctional linker, NHS-PEG12-Maleimide. The group drawn in red is the amine reactive N-hydroxysuccinamide ester and that shown in blue is the thiol reactive maleimide. The two functional groups are linked by a flexible, hydrophilic linker containing 12 repeat units of ethylene glycol.

The activated NHS ester can readily react with nucleophiles to release the NHS group and form a stable amide bond as shown in Scheme 1. Unfortunately, the NHS esters are not very stable in aqueous solutions and have a half-live on the order of hours under physiological pH (pH 7.0–7.5) and the rate of hydrolysis increases with the increasing pH (Kalkhof & Sinz, 2008). The maleimide moiety can readily react with the free sulfhydryl groups present in the enzyme via Michael addition (Belbekhouche, Guerrouache, & Carbonnier, 2016; Ravasco, Faustino, Trindade, & Gois, 2019). The advantage of using the NHS-maleimide cross linker is that all conjugation steps are carried in aqueous media under mild physiological conditions, reducing the chances of enzyme denaturation and activity reduction. The success of this cross-linking conjugation is highly dependent on controlling the solution pH. The coupling of NHS with amines and Maleimide conjugation with free thiols are best carried out at buffered pH 7.5–8.3 and 6.5–7.3, respectively (Kalkhof & Sinz, 2008).

An important application of using heterobifunctional linkers in bioanalytical chemistry is to conjugate biomolecules on the surface of nanomaterials. An example here is a simple MNP-based DNA sandwich biosensor developed in the Zhou group (Zhang, Pilapong, et al., 2013). It uses two pairs of MNP-linked capture-DNA and enzyme-linked signal-DNA probes whose sequence are complementary to each half of its target-DNAs. Here two different enzymes, horseradish peroxidase (HRP) and alkaline phosphatase (ALP), are used to detect the two target-DNAs, respectively. The thiolated single-stranded capture-DNA was conjugated onto an aminated silica coated MNP using the NHS-PEG12-maleimide coupling chemistry. The capture-DNA and signal-DNA-enzyme probes sandwich hybridize with a specific target-DNA, linking either an HRP or ALP to the MNP surface for enzymatic signal amplification. Using two pairs of unique capture-DNA and signal-DNA-enzyme probes, this MNP-enzyme sandwich assay can simultaneously quantitate two different target-DNAs with a detection limit down to 50   fM for each target-DNA (Zhang, Pilapong, et al., 2013).

On the other hand, the direct immobilization of enzymes on the MNP surfaces using the NHS-PEG-maleimide hetero-cross linker can be challenging. It often requires a reduction step to introduce a free thiol on the enzyme and followed by purification, several rounds of washing to remove any unbound cross-linkers, and change buffer pH to favor the specific reactions. Another issue is the limit stability of maleimide in a buffer solution: it can be hydrolyzed at pH   >   5.5 (Baldwin & Kiick, 2011; Kirchhof et al., 2015). This side reaction can lead to low conjugation efficiency and batch-to-batch variations. To avoid such complications, the use of strong bio-specific interactions such as the extremely strong biotin-avidin (streptavidin/neutravidin, NAV) interaction (with an equilibrium dissociation constant, Kd  ~   10−   15  M) is an attractive alternative strategy. Among the three avidin variants, NAV's isoelectric point (PI) is the close to the physiological pH and gives the lowest non-specific interactions. This property is highly beneficial for biosensing, allowing for significantly reduced background and hence high sensitivity. Notably, popular enzymes, such as horseradish peroxidase (HRP) and alkaline phosphatase (ALP), are also commercially available as their NAV-conjugates and are widely used in enzymatic assays. An additional advantage of using enzyme-NAV conjugate in biosensing is that the enzyme part is not directly involved in immobilization (hence its active site will not be blocked by steric hindrance), allowing enzyme to retain high activity and benefit sensitivity. Moreover, as a tetrameric protein, each (neutr-)avidin contains four biotin-binding sites, two on each side of the protein, this makes it very useful in assembling multilayer protein structures for bionanotechnology applications (Kim, Sohn, Zhou, Duke, & Kang, 2010; Rauf et al., 2006; Zhou et al., 2003).

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