F.
Kazenwadel
,
H.
Wagner
,
B. E.
Rapp
and
M.
Franzreb
*
Karlsruhe Institute of Technology, Institute of Functional Interfaces, Hermann-von-Helmholtz-Platz 1, 76344 Eggenstein-Leopoldshafen, Germany. E-mail: Matthias.Franzreb@kit.edu
First published on 6th November 2015
Enzyme immobilization is a versatile tool in biotransformation processes to enhance enzyme activity and to secure an easy separation of catalysts and products and the reusability of enzymes. A simple and commonly used method for crosslinking enzymes to a solid support is the zero-length crosslinking agent 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). This work shows the optimization of the EDC-crosslinking protocol for two enzymes, glucose oxidase (GOx) and horseradish peroxidase (HRP), to functionalized magnetic microparticles. For GOx the optimization of the immobilization parameters pH-value and the enzyme to particle ratio results in activity yields of up to 36%, which is in the usual range for undirected enzyme immobilisations. In contrast, for HRP the activity yield does not exceed 6% even after optimization of the protocols. The main reasons for this unusually low activity yield are the presence of multiple HRP isoforms in the enzyme solution used for immobilisation and the observed tendency of HRP to be inactive even in the case of simple physisorption to the particle surface.
Besides this variety of potential crosslinkers, different supports can be applied when proteins are to be immobilized. To obtain optimal results carriers should provide a large specific surface for biocatalyst attachment while being easily separable from the reaction solution. Magnetic carriers combine both requirements as nano- or micro-particles provide large specific surfaces and can be separated rapidly and reliably by applying an external magnetic field.20 Magnetic particles can be purchased with a wide variety of different chemical functionalities and can be easily further modified.
Table 1 shows an overview of publications reporting the use of chemical crosslinkers to immobilize enyzmes to magnetic carriers. The immobilization method is specified, naming the crosslinking method and the pH-value used in the coupling process. Furthermore, the isoelectric point and molecular size of the target-protein are listed and yields in binding and activity upon immobilization compared to free enzymes, if published, are cited. Publications range from the early 1970s to the current research state, pointing out that chemical crosslinking has a long history in the immobilization of enzymes to magnetic carriers. Most enzymes are technical enzymes that are needed for the conversion of substrates used in industrial processes, such as invertase or lipase. Other enzymes are immobilized to serve as sensoric tools, as for example cholesterol esterase. Crosslinking agents mainly contain carbodiimides, but also glutaraldehyde or epoxy groups are used for the crosslinking of amino groups. In addition in some cases stable non-covalent binding can be found. pH-values during the immobilization process are for this discussion expected to be optimized for the respective enzyme and range from 4.0 to 8.0. The pI-values of the enzymes used also vary from 3.8 (invertase from yeast) to 10.8 (bovine trypsin). It is often recommended to adjust the pH-value during the coupling process close to the isoelectric point of the enzyme in order to avoid superficial loading that may lead to a repulsion of the enzyme from the carriers. The pH-value is adjusted near the pI (±0.5–1) in 4 of the cited publications, and the rest differs for more than 1.5 pH magnitudes. Looking at Table 1 it is hard to find general trends. However, it becomes obvious that if low enzyme amounts per g of magnetic particles are offered, the resulting binding yields are high and vice versa. Most articles report initial conditions of less than 50 mg enzyme per g of particles and binding yields of 70–100%, demonstrating the effectiveness of using crosslinking agents for the immobilization of enzymes. Choosing an optimum, it has to be considered that while increasing the specific activity of the immobilisates by increasing the enzyme amount bound, the activity of the immobilized enzyme might decrease because of steric hindrance.21 Activity yields, saying how much of the initially offered activity of free enzymes finally is detectable on the immobilisates, are much less predictable. If published, they range between less than 10% to more than 100%. The same variety holds true, even if we restrict our analysis to the cases in which the same particles as in our study were used (magnetic polyvinylalcohol particles of the company Perkin-Elmer chemagen). Assuming that all protocols are optimized, these findings show that not all enzymes are identically well suited for immobilization.
Literature | Carrier | Protein | Crosslinking agent | pH | pI | Enzyme offered | Mass | Binding yield | Activity yield |
---|---|---|---|---|---|---|---|---|---|
van Leemputten & Horisberger (1974)8 | Aminoalkylsilylated magnetite (Fe3O4) | Trypsin | Glutaraldehyde | 8.0 | 10.8 | 50 mg enzyme per g particles | 23.3 kDa | 72% | 46%, 8% |
Invertase | 3.8 | 40 mg enzyme per g particles | 270 kDa | 11% | 8.7% (+1% sucrose) | ||||
Bahar & Celebi (1999)24 | Magnetic poly(styrene) particles | Glucoamylase | Aldehyde-groups | 4.0 | 4.2 | 8 mg enzyme per g particles | 72 kDa | 70% | 70% |
Liao & Chen (2001)25 | Fe3O4 magnetic nanoparticles (10.6 nm) | Yeast alcohol dehydrogenase | Carbodiimide | 6.0 | 5.4–5.8 | 0.05:1 (w/w) | 141–151 kDa | 100% | 62% |
Akgöl et al. (2001)26 | Magnetic PVA microspheres | Invertase | 1,1′-Carbonyldiimidazole | 7.0 | 3.8 | N/A | 270 kDa | 7.18 mg g per particles | 74% |
Zheng et al. (2003)21 | Magnetic poly(VAc–DVB) microspheres | Candida cylindracea lipase | Adsorptive | 7.0 | 4.5 | N/A | 43 kDa | 8–35 mg per g particles | 6750 IU per g carrier |
Wang & Lee (2003)27 | Fe3O4 magnetic nanoparticles | Trypsin | Carbodiimide | — | 10.8 | 17 mg enzyme per g particles | 23.3 kDa | 86% | N/A |
Avidin | Cyanamid | 10.5 | 66 kDa | 100% | N/A | ||||
Bozhinova et al. (2004)28 | Magnetic PVA microparticles | E.coli penicillin amidase | Epoxygroups glutaraldehyde | 7.5 | 4.3–7.0 | 30 mg enzyme per g particles | 70 kDa | 10–93% | 50–100% |
Kouassi et al. (2005)29 | Magnetic nanoparticles | Cholesterol oxidase | Carbodiimide | 7.4 | 5.1–5.4 | 7–10 mg enzyme per g particles | 34 kDa | 98–100% | >100% |
Bruno et al. (2005)30 | Magnetic POS–PVA particles | Mucor miehei lipase | Glutaraldehyde | 7.0 | 3.8 | N/A | 32 kDa | N/A | 65% |
N. Schultz (2007)31 | Magnetic PVA microparticles | Candida antarctica lipase A (CALA) | Carbodiimide | 7.0 | 7.5 | 16.7 mg enzyme per g particles | 45 kDa | 30% | 8% |
Huang et al. (2008)7 | Fe3O4 magnetic particles (12.7 nm) | Candida rugosa lipase | Carbodiimide | 6.0 | 4.5 | <33 mg enzyme per g particle | 43 kDa | 100% | 141% |
Magario et al. (2008)32 | Magnetic PVA microparticles | Naringinase | Carbodiimide | 7.0 | ∼5 | 3.7 mg enzyme per g particles | 90 kDa | 82% | 36% |
Ricco et al. (2014)33 | Magnetic nanoparticles | Almond beta-glucosidase | Carbodiimide | 4.6 | 7.3 | 400–1000 mg enzyme per g particle | 110 kDa | 18–24% | N/A |
Morhardt et al. (2014)34 | Magnetic PVA microparticles | Chymotrypsine | SMCC | 7.2 | 8.75 | 2.9–95.5 mg enzyme per g particles | 25 kDa | 75–100% | 9–45% |
Adsorptive | 5.5 | ||||||||
Carbodiimide | 5.3 | 75–100% | |||||||
Kazenwadel et al. (2015) | Magnetic PVA microparticles | Glucose oxidase | Carbodiimide | 4.0 | 4.2 | 5–15 mg enzyme per g particles | 160 kDa | 67% | 34% |
Horseradish peroxidase | |||||||||
4.0 | 3.0–9.0 | 44 kDa | 100% | 6.5% |
Table 1 also shows that 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) is one of the most commonly used crosslinking molecules. As it only activates carboxy-groups and mediates the linkage with superficial primary amino groups without the introduction of any spacer molecules, it can be classed among the so-called zero-length crosslinking agents. A neutral pH value around 7.0 during the immobilization process is required according to the standard protocols. The reaction is performed in two steps (see Fig. 1): first, the carboxy-groups of the carrier are activated by adding EDC. An active acyl-isourea intermediate product is formed. Second, the enzyme is added and a peptide bond is formed between the carboxy-groups on the surface and the superficial lysine amino side chains of the enzyme.16
In this article, the optimization of enzyme crosslinking to magnetic particles using EDC as a zero-length crosslinking agent is shown. By optimizing the protocol concerning coupling pH-value and enzyme loading, the activity of the used model enzymes glucose oxidase (GOx, Aspergillus niger, see ref. 22 for detailed information) and peroxidase (HRP, Armoracia rusticana, see ref. 23 for detailed information) could be enhanced significantly. Nevertheless, the achieved activity yields remained low, especially in the case of HRP. Possible reasons for this will be discussed and the reversible physical adsorption/desorption of the enzyme on the particle surface is identified as one critical process.
Control experiments for the detection of enzyme inactivation during the immobilization process were performed by omitting the addition of magnetic particles and by replacing the EDC-activated carboxy-terminated magnetic particles by native M-PVA-C22 particles. Adsorption was measured by detecting the protein concentration in supernatants after coupling. The specific activity of the immobilisates [U per mg particles] was calculated by dividing the activity of the particle suspension [U ml−1] by the particle concentration [mg ml−1]. Enzyme activity [U per mg enzyme] was determined by dividing the specific activity of the immobilisates [U per mg particles] by the enzyme to particle ratio of the particles [mg enzyme per mg particle]. The binding yield [%] was determined by dividing the bound enzyme mass by the applied enzyme mass. At the end, the yield in activity [%] was determined by dividing the total activity of the immobilized enzyme by the total activity of the free enzyme initially offered for immobilisation.
The pH-value during the enzyme attachment was varied and resulting activity of the particle suspension was measured. For GOx it could be clearly shown that a decrease of the pH-value from 7.1 to 4.0 led to a significant enhancement of the specific activity of the immobilisates (Fig. 2A). It also resulted in an enhancement of the activity of control particles to which enzymes were physically adsorbed. However, particles with chemically bound enzymes (+EDC) showed more than 3.2-fold activity compared to the adsorption controls (−EDC).
In order to find a reason for this behavior, the amount of bound enzymes was determined by probing the protein concentration in the supernatants of coupling and washing steps and the yield of enzymes that bound to the particles was calculated (Fig. 2B). At a pH value of 7.0 only about 23% of the added enzyme bound to the particles, while at a pH value of 4.0 no enzyme in the supernatant could be detected after the coupling process. Interestingly, the binding yield does not increase steadily when probing pH-values between 7 and 4 during immobilization, but shows a clear minimum with binding yields of approximately 14% (pH 6.0) and 8% (pH 5.3). The difference in the GOx binding yield comparing chemically crosslinked (+EDC) and physically adsorbed samples (−EDC) was not significant. Together with the finding that enzyme activity is 3.2-fold lower for adsorbed samples, this indicates that enzymes were stabilized during the immobilization process and activity was preserved using covalent binding. This effect can also be accentuated by calculating the activity of the immobilized GOx [U per mg enzyme] for adsorbed and covalently bound samples (Fig. 2C). While there is practically no or only a little activity detectable for the pH-values 5.3, 6 and 7, the activity of crosslinked GOx at pH 4.0 is 4-fold higher compared to physically adsorbed enzymes, highlighting the importance of a covalent bond for the activity of glucose oxidase. For the immobilization of 10 mg GOx to functionalized PVA-coated magnetic particles, a total yield in the activity of approximately 24% could be achieved for covalently bound GOx and 5.5% for adsorbed GOx for pH 4, while when starting the trials using the published protocol (pH 7.0) hardly any activity could be detected (Fig. 2D).
The second model enzyme, horseradish peroxidase, showed in many aspects a comparable behavior (Fig. 3). At a pH value of 7.0, a specific activity of the immobilisates of approximately 0.09 U per mg particles could be detected for covalent binding (+EDC). After decreasing the pH-value to 4.0, the specific activity could be increased significantly to 1.9 U per mg particles. The binding yield for pH 7 was only 2.5%, but it could be enhanced to 80%, when the pH-value in the coupling step was regulated to 4.0. An adjustment of the pH-value to 6.0 led to a binding yield of 18%, an adjustment to pH 5.3 to 58%. Adsorptive binding (−EDC) occurred for all pH-values (approximately 30–50% compared to covalent binding). However, the activity of physically bound enzymes was not as high as for covalent bound HRP, underlining the importance of a chemical bond for enzyme stabilization while immobilization also in this case. Fig. 3D shows the activity of immobilized enzyme. Although enzyme immobilized at pH 5.3 shows the highest activity related to immobilized mass, yield in activity is highest for pH 4.0, as more enzymes can be bound to the particle surface. The yield of immobilized activity is less than 4.5% for 10 mg HRP immobilized covalently on functionalized magnetic particles at a pH value of 4.0. For physically adsorbed enzymes it is approximately 1.5%, which is less than half compared to the crosslinking approach.
These results prove that for both enzymes the recommended immobilization conditions of pH 7 are not ideal and that the yields in immobilized mass and activity could be significantly enhanced by adjusting the pH-value in the coupling process. As it can be derived from the binding yield trend, the pH-value influences enzyme binding efficiency and thus the specific activity of enzyme immobilisates. This may be due to charging phenomena at the surface of the enzyme that depend on the isoelectric point (pI) and lead to different physical adsorption on the surface at different ambient pH-values. In the literature it is often recommended to adjust the coupling pH-value near the isoelectric point of the enzyme, in order to avoid superficial protein loading and thus repulsion from charged surfaces. This recommendation holds true for glucose oxidase, which showed a binding maximum at pH 4.0, while the isoelectric point of the enzyme is reported to be 4.2 (Sigma Aldrich, Material Data Sheet). However, for horseradish peroxidase, there is not such an explicit result. This is because the HRP used for this work is extracted from horseradish roots and contains at least 7 different isoforms, whose pI-values vary from 3.0–9.0 (Sigma Aldrich, Material Data Sheet). In view of this variety it becomes obvious that no real optimum can be found, which would result in high binding and activity yields of all HRP isoforms offered for immobilization.
Initial enzyme to particle mass ratio [mg enzyme per g particles] | Specific activity [U per mg particles] | Activity yield [%] | Binding yield [%] |
---|---|---|---|
5 | 0.08 | 14.0 | 100.0 |
10 | 0.16 | 17.2 | 100.0 |
15 | 0.49 | 34.1 | 67.0 |
20 | 0.50 | 33.1 | 51.8 |
30 | 0.53 | 25.3 | 32.1 |
For horseradish peroxidase (Table 3), a maximum in specific activity (2.6 U per mg particles) could be proven for 20 mg HRP per g particles. After further increasing the loading, the activity yield dropped to 1.87 U per mg particles for 30 mg HRP per g particles. This effect might be due to the enzyme density that may be too high at 30 mg HRP per g particles to ensure good substrate accessibility and flexibility of the enzyme to move during the conversion process. When comparing the yield in activity compared to free enzymes a maximum of 6.5% could be found for the lowest loading of 5 mg HRP per g particles. By increasing the initial enzyme to particle mass ratio, the binding yield decreases to 1.5%. In order to further investigate the yield of bound enzyme mass, supernatants were investigated. It could be seen that only for the lowest enzyme to particle ratio (5 mg HRP per g particles) all enzymes bound to the surface. By further increasing the loading, the binding yield decreased (approximately 79% for 10 mg HRP per g particles, 41% for 15 mg HRP per g particles, 32% for 20 mg HRP per g particles and 21% for 30 mg HRP per g particles). Binding yields for physically adsorbed HRP were significantly lower than covalently bound enzymes in all cases. In addition, an adsorption of enzymes resulted in much lower activity values. Although a loading of 5 mg HRP per g particles resulted in the highest activity and binding yields, the overall activity was relatively small. This is why for further experiments, a loading of 10–15 mg HRP g particles was considered to be optimal, as only smaller amounts of enzymes were washed off while specific activity was maximal.
Initial enzyme to particle mass ratio [mg enzyme per g particles] | Specific activity [U per mg particles] | Activity yield [%] | Binding yield [%] |
---|---|---|---|
5 | 1.26 | 6.5 | 100.0 |
10 | 1.91 | 4.3 | 79.4 |
15 | 2.31 | 4.0 | 41.0 |
20 | 2.60 | 3.4 | 32.2 |
30 | 1.89 | 1.5 | 21.9 |
The samples which were chemically crosslinked showed much better reusability compared to the samples, in which HRP was only physically adsorbed. In the latter case, the activity was almost completely lost after the first reuse of the particles. In contrast, the activity of the chemically crosslinked samples could be preserved for at least 6 cycles at a value of about more than 30% of the original activity. The activity loss of about 70% during recycling might be due to a fraction of physically adsorbed enzymes that desorbed during the activity testing cycles. Although examples of stable adsorptive binding of enzymes to magnetic particles can be found in the literature,21 at least for HRP these findings point out the importance of a stable chemical bond in enzyme immobilization. The partially higher standard deviations of the recycling experiments are mainly caused by the small amount of enzyme immobilisates used in the experiments. As particles easily adsorb to the pipette tip during the recycling process, particle mass may decrease over the course of the cycles. However, as was shown by our group, working with larger volumes and higher particle concentrations reuse of magnetic enzyme immobilisates results in consistent values over twenty cycles.34
For HRP it could be proven that, except for pH 4.0, there was an inactivation caused by the immobilization process conditions even without contact to magnetic particles, indicating a susceptibility of enzyme activity to higher pH-values during the coupling process (Fig. 5). The percentaged inactivation is further increased if particles are added to the solution. This might be due to a reversible adsorption and desorption or short term contact with the particle surface, which might affect enzyme stability. Also in the experiments with varying pH-values and enzyme to particle ratios it could be proven that adsorptive bound HRP does not show a comparably high specific activity as the covalent bound one.
In the case of HRP it could be shown that already the coupling process itself leads to a partial inactivation. This is most probably due to the pH-enzyme to particle ratio values higher than 4.0 and the addition of magnetic particles that leads to a consistent adsorption and desorption of proteins what might affect enzyme activity.
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