Enzyme and Microbial Technology 38 (2006) 960–966
Glyoxyl agarose as a new chromatographic matrix Valeria Grazu, Lorena Betancor, Tamara Montes, Fernando Lopez-Gallego, Jose M. Guisan ∗ , Roberto Fernandez-Lafuente ∗ Departamento de Biocat´alisis, Instituto of Cat´alisis (CSIC), Campus UAM Cantoblanco, 28049 Madrid, Spain Received 13 April 2005; received in revised form 17 August 2005; accepted 26 August 2005
Abstract At alkaline pHs, glyoxyl agarose is able to immobilize most of the proteins contained in a crude extract. However, due to its special immobilization features, at pH 7.0 only proteins that contain at least two exposed low pK amino groups in the same plane were immobilized (-galactosidase from Escherichia coli, catalase from bovine liver, and IgG from rabbit). However, with many other proteins, even multimeric ones, immobilization could not be achieved (e.g.: glucose oxidase from Aspergillus niger and Penicillium vitale; catalase from Micococcus sp., A. niger and bovine liver; alcohol oxidase from Pichia pastoris, Hansenula sp. and Candida boidinii, -galactosidase from Thermus sp., etc.). Elution of the attached proteins under mild conditions was not simple, if the number of protein-support bonds was very high, only boiling in SDS allowed the elution of the proteins. However, using glyoxyl agarose 4BCL with only 20 mol of aldheyde groups/g of support, proteins could be fully eluted by competition with amino compounds (e.g., Tris buffer). In this first approach, we have tried to take advantage of this specific immobilization at pH 7.0 to purify multimeric proteins, using a -galactosidase from E. coli as a model. The enzyme could be eluted from the support using Tris–HCl buffer as eluting agent, with a high yield (80%) and a high purification factor (32). © 2005 Elsevier Inc. All rights reserved. Keywords: Glyoxyl agarose; Reversible covalent immobilization; Selective immobilization; Purification of multimeric proteins; Immobilization via multipoint interaction
1. Introduction Most of the reactive groups used for immobilizing proteins (glutaraldehyde, cyanogen bromide, etc.) are able to yield very stable enzyme-support bonds under mild immobilization conditions (e.g., neutral pH values) [1]. However, glyoxyl-agarose immobilizes proteins via reversible Schiff’s bases and, for this reason, incorporation of the enzyme to the matrix requires, at least, the formation of two simultaneous bonds [2]. Due to this restriction glyoxyl-agarose has always been used to immobilize proteins at alkaline pH values. The driving force for the immobilization is not the reactivity of a single residue but the density of reactive groups on the protein surface; proteins with different density of exposed lysines would have different rates of immobilization. In fact this has been already used for the one step purification and immobilization of enzymes having very rich lysine areas, like glutamate racemase [2]. Moreover, the enzyme
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Corresponding authors. Tel.: +34 91 585 48 09; fax: +34 91 585 47 60. E-mail addresses:
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immobilization will be directed through the area/s having the highest concentration of Lys. This self-direction of the proteins could explain why this support has been successfully used to stabilize many proteins via multipoint covalent attachment [2–11]. Here, we intend to use the reversibility of the amine aldehyde bond together to the conditions required for immobilization to convert glyoxyl agarose in a chromatographic matrix. To this goal, it is necessary to study the proteins that can be immobilized on it under different conditions. Finally, we will study how it is possible to desorb the immobilized proteins. 2. Materials Agarose (4% cross-linked agarose beads, 4BCL) was purchased from BTG-LKB (Uppsala, Sweden). Crude extract of -galactosidase from Escherichia coli was kindly donated by Dr. Jos´e Luis Garcia (CIB, CSIC, Madrid). ␣- and -galactosidases from Thermus sp.-strain T2 (pBGT1) was over-expressed in E. coli and produced as published elsewhere [12,13]. Glycidol (2,3-epoxy propanol), sodium periodate, sodium borohydride, p- and o-nitrophenyl--d-galactopyranoside
V. Grazu et al. / Enzyme and Microbial Technology 38 (2006) 960–966
(pNPG, oNPG), 6-nitro-3-(phenylacetamido) benzoic acid (NIPAB), glucose oxidase (Aspergillus niger and Penicillium vitale), catalase (Micrococcus sp., A. niger and bovine liver), alcohol oxidase (Pichia pastoris, Hansenula sp. and Candida boidinii), -galactosidase from E. coli, immonoglobulin G (IgG) from rabbit, trysin and chymotrypsin from bovine pancreas, 2,2 -Azino-bis(3-etilbenz-tiazoline-6-sulfonic acid) (ABTS), horseradish peroxidase (HRP), and ethylendiamine (EDA) were purchased from Sigma (St. Louis, MO, USA). -Galactosidase form Kluyveromyces lactis was obtained from Gist-Brocades (Cedex, France). Penicillin G acylase (PGA) form E. coli was generously donated by Antibi´oticos S.A. (Le´on, Spain). All others chemicals were of reagent grade. 3. Methods All results represent averages of at least three experiments. In all cases, experimental error was lower than 5%.
3.1. Preparation of glyoxyl-agarose The preparation of glyoxyl-agarose (4BCL) activated with 20 or 40 mol of aldheyde groups/g of support was carried out as described in [3], using the corresponding amount of sodium peridoate after full activation with glycidol of the support.
3.2. Protein immobilization One gram of glyoxyl-agarose was added to 4 mL of 1 mg/mL protein solution (glucose oxidase from A. niger or P. vitale, catalase from Micococcus sp., A. niger or bovine liver, ␣-galactosidase from Thermus sp., -galactosidase from E. coli, Thermus sp., Aspergillus oryzae or K. lactis, alcohol oxidase from P. pastoris, Hansenula sp. or C. boidinii and IgG from rabbit) prepared in 0.1 M sodium phosphate buffer pH 7.0. The mixture was gently stirred at 25 ◦ C. Periodically, samples of the supernatant were withdrawn and protein concentration [14] and/or enzyme activity was determined.
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The percentage of desorbed enzyme was followed via enzyme activity of the supernatant compared to the activity of the suspension (which remained constant throughout the experiment). The percentage of desorbed protein was determined by the Bradford method [14]. The degree of purity of the eluted fraction was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) [15].
3.5. Determination of enzyme activity One unit of enzyme (IU) was defined as the amount of enzyme able to produce 1 mol of product per minute under the specified conditions. 3.5.1. α-Galactosidase activity Activity was followed spectrophotometrically by following the increase in absorbance at 405 nm caused by the hydrolysis of pNPG at 25 ◦ C. Aliquots of 25–200 L of enzymatic solution or derivative suspension was added to a cubette with 2 mL of 13.3 mM pNPG dissolved in 50 mM sodium phosphate buffer pH 7.0 [16]. 3.5.2. β-Galactosidase activity Activity was determined spectrophotometrically by following the increase in absorbance at 405 nm caused by the hydrolysis of oNPG at 25 ◦ C [17]. A sample of enzymatic solution or derivative suspension (25–200 L) was added to a cuvette with 2 mL of 10 mM oNPG dissolved in activity buffer. These buffers were different for the different -galactosidases from: (i) E. coli: 25 mM sodium phosphate buffer pH 7.0 with 2 mM of MgCl2 ; (ii) K. lactis: 25 mM sodium phosphate buffer pH 7.0 with 2 mM of MgCl2 and 0.1 M KCl; (iii) Thermus sp.: 25 mM sodium phosphate buffer pH 7.0; (iv) A. oryzae: 25 mM sodium phosphate buffer pH 5.0. 3.5.3. Penicillin G acylase activity PGA activity was measured at room temperature by following the increase in absorbance at 405 nm which accompanies the hydrolysis of the synthetic substrate NIPAB in 25 mM sodium phosphate pH 7.0 [18]. 3.5.4. Catalase activity Activity was determined spectrophotometrically by monitoring the decomposition of H2 O2 , measuring the change in absorbance at 240 nm [19]. Aliquots of 2.9 mL of 35 mM H2 O2 solution in 50 mM sodium phosphate buffer pH 7.0 were incubated with 0.2 mL of enzyme solution. All the measurements were carried out at 25 ◦ C.
3.3. Protein elution One gram of support with each adsorbed protein was suspended in 4 mL of different elution solutions (1–3 M Tris–HCl buffer pH 7, 3 M EDA pH 7, and 3 M ethanolamine pH 7). The mixture was gently stirred at 25 ◦ C. Samples of the supernatant were withdrawn at different time intervals and analyzed for protein concentration and/or enzyme activity. The percentage of desorbed protein was followed via protein determination and/or enzyme activity of the supernatant compared to the adsorbed protein and/or activity on the support.
3.4. E. coli β-galactosidase purification 3.4.1. Enzyme immobilization Sixteen-milliliter aliquots of crude extract of -galactosidase from E. coli (the cells were sonicated in 50 mM sodium phosphate pH 7.0) were incubated with 4 g of glyoxyl-agarose 4BCL containing 20 mol of aldheyde groups/g of support. The mixture was gently stirred at 25 ◦ C. Periodically, samples of the supernatants were withdrawn and analyzed for enzyme activity. The gel with the immobilized protein was washed with 50 mM sodium phosphate pH 7.0. 3.4.2. Enzyme elution Four grams of glyoxyl-agarose with adsorbed proteins were suspended in 16 mL 1 M Tris–HCl pH 7.0. The mixture was gently stirred at 25 ◦ C for 16 h.
3.5.5. Glucose oxidase activity Glucose oxidase activity was determined spectrophotometrically by the increase in absorbance at 414 nm resulting from the oxidation of ABTS by a coupled peroxidase-catalyzed reaction [20]. The reaction mixture consisted of 1 mL 100 mM sodium phosphate buffer pH 6.0, 0.5 mL 1 M glucose, 0.1 mL of 1 mg/mL ABTS in distilled water, and 0.1 mL of 2 mg/mL HRP in sodium phosphate buffer, pH 6.0. 3.5.6. Alcohol oxidase activity The activity of alcohol oxidase was measured following the increase in absorbance at 414 nm resulting from the oxidation of ABTS by a coupled peroxidase-catalyzed reaction [20]. The reaction was initiated by adding 0.5 mL of alcohol oxidase solution to a mixture containing 0.1 mL of a HRP solution (2 mg/mL) 0.1 mL of ABTS solution (1 mg/mL) in distilled water, 0.1 mL of ethanol and 1.2 mL of 100 mM sodium phosphate buffer pH 6.0.
3.6. SDS-PAGE analysis SDS-PAGE experiments were performed as described by Laemmli [15] in a SE 250-Mighty small II electrophoretic unit (Hoefer Co.) using (9 cm × 6 cm) separation gels of 12% polyacrylamide and 5% polyacrylamide stacking gels. Gels were stained with Coomasie brilliant blue (R250). Low-molecular weight marker kits from Pharmacia were used (MW = 14,000–94,000).
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Table 1 Influence of pH on protein immobilization yields on glyoxyl-agarose containing 40 mol of aldehyde groups/g of support
Table 2 Immobilization yields of different proteins at pH 7.0 on glyoxyl-agarose containing 40 mol of aldehyde groups/g of support
pH
Yield of protein immobilization (%)
Protein
Source
7.0 8.0 9.0 10.0
3 5 40 97
Subunit composition
Yield of immobilization
Alcohol oxidase Alcohol oxidase Alcohol oxidase Catalase Catalase Catalase ␣-Galactosidase -Galactosidase -Galactosidase -Galactosidase -Galactosidase Glucose oxidase Glucose oxidase IgG PGA Trypsin Chymotripsin
C. boidinii Hansenula sp. P. pastoris A. niger Bovine liver Micrococcus sp. Thermus sp. A. oryzae E. coli K. lactis Thermus sp. A. Niger P. vitale Rabbit E. coli Bovine pamcreas Bovine pancreas
Octameric [21] Octameric [22] Octameric [23] Tetrameric [23] Terameric [24] Tetrameric [25] Hexameric [26] Monomeric [27] Tetrameric [28] Dimeric [29] Tetrameric [13] Dimeric [30] Dimeric [30] Tetrameric [31] Dimeric [18] Monomeric [32] Trimeric [33]
–a – – – 52 – – – 92 – – – – 46 – 100 100
Crude extract from E. coli was utilized in these experiments. Immobilization yields reported were achieved after 2 h of incubation at 25 ◦ C.
4. Results and discussion 4.1. Immobilization of proteins on glyoxyl-agarose Crude preparations of proteins were offered to 4BCL glyoxyl agarose activated with 40 mol of aldehyde groups/g support at different pH values. While at pH 10 most of the proteins (97%) were immobilized on the support after 2 h incubation, immobilization yield decreased at lower pH values (Table 1). At pH 7, immobilization yield was under 5% of the total protein content. A SDS-PAGE of the supports was performed to confirm that proteins were really immobilized on the matrix and could be eluted (Fig. 1). The almost full immobilization of all proteins at alkaline pH value was confirmed. Interestingly, it was found that only certain proteins were selectively immobilized on glyoxyl-agarose at pH 7. In this first evaluation of glyoxyl agarose supports for protein purification, we decided to pay attention to these proteins. Several proteins were incubated at pH 7.0 with glyoxylagarose containing 40 mol of aldehyde groups/g of support. Table 2 shows that many proteins were not significantly immobilized on the support: -galactosidase from A. oryzae, PGA from E. coli, glucose oxidase from A. niger, P. vitale, catalase from Micococcus sp. and A. niger, alcohol oxidase from P. pas-
Immobilization yields achieved after 2 h of incubation at 25 ◦ C. a Non-significant immobilization after 24 h of incubation.
toris, Hansenula sp. and C. boidinii, ␣- and -galactosidase from Thermus sp. Only E. coli -galactosidase, rabbit IgG, bovine liver catalase, bovine pancreas trypsin and bovine pancreas chymotrypsin, could be immobilized on this support under these conditions. The structure of the enzymes was analyzed (Table 2) [13,21–33] The structure of bovine liver catalase [24], E. coli -galactosidase [28], and rabbit IgG [31] shows that all of them have at least two exposed amino terminal groups in the same plane of interaction with the support (Fig. 2). Bovine pancreas trypsin and chymotrypsin presented several low pK amino groups by proteolysis [32–33]. Thus, it is seems that only proteins having these features may be adsorbed on this support under these conditions. In fact they should be the only ones able to simultaneously form two imines between the support and the enzyme. Therefore, at a neutral pH value, this immobilization methodology seems to be highly selective towards proteins that have at least two exposed low pK amino terminal groups in the same plane of interaction with the support. This result suggests that proteins presenting this structure could be purified by selective immobilization using this new chromatographic matrix. 4.2. Protein elution
Fig. 1. SDS-PAGE analysis of the different pattern of immobilization of proteins of a crude extract of E. coli on glyoxyl-agarose at different pH values, 1 g of glyoxyl-agarose containing 40 mol of aldehyde groups/g of support was incubated with 10 mL of crude extract of E. coli at different pH values. Lane 1: molecular weight markers; Lane 2: crude extract from E. coli; Lane 3: proteins in the supernatant after immobilization at pH 7; Lane 4: proteins released from the previous glyoxyl-agarose derivatives by boiling it in the presence of SDS. Lane 5 crude extract; Lane 6: proteins in the supernatant after immobilization at pH 10; Lane 7: proteins released from the previous glyoxyl-agarose derivatives by boiling it in the presence of SDS.
The formation of at least two point enzyme-support attachments is enough to incorporate the enzyme to the glyoxyl-support. However, the Schiff’s bases resulting from the amine-aldehyde reaction are reversible links and therefore, the immobilized proteins should be eluted by addition of compounds having primary low pK amino groups that are able to compete for the aldehyde groups of the support (e.g.: ethanolamine, ethilendiamine, tris(hidroxymethil) aminomethane buffer, etc.). It was not possible to elute proteins when the immobilization onto glyoxyl-supports was carried out at pH 10. This result
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Fig. 2. 3D structure of -galactosidase from E. coli (A), catalase from bovine liver (B), IgG from rabbit (C) and PGA from E. coli (D). Amino terminal groups are represented in blue. These 3D structures were obtained from the Protein Data Bank (PDB) using Grasp version 1.2-1994. The corresponding ID was: 1DP0 for -galactosidase from E. coli, 4BLC for catalase from bovine liver, 1IGY for IgG from rabbit, and 1AI4 for PGA from E. coli.
suggests that most of the protein molecules are attached to the support by multiple points (in fact, this support has been designed to generate multipoint covalent attachment) and the simultaneous cleavage of all the bonds is a very difficult task. Thus, the use of this matrix to adsorb most of the proteins and the further selective desorption of the proteins as a function of the number and strength of attachment needs further study. Thus, we focused our attention on the proteins immobilized at pH 7. It was a surprise to find that it was quite difficult to fully desorb all the immobilized proteins. It was observed that no more than 50% of the protein was desorbed from the support using different aminated compounds (even at concentration as high as 3 M). As an example, Fig. 3 shows the elution of -galactosidase with the different amino compounds utilized. These results suggested that also at neutral pH values, after immobilization of the protein, the high apparent concentration of Lys and aldehydes could yield a certain multipoint attachment that made protein desorption difficult. This may be similar to the use of heterofuctional epoxy-supports, where the first immobilization of the enzyme to the support via physical adsorption permits the rapid covalent immobilization [34–37] However, as shown before, full desorption was achieved by boiling the support with the attached proteins in the presence of SDS and mercaptoethanol (Fig. 1). Therefore, in this situation, from a chromatographic point of view, the attachment of the protein to the support becomes “irreversible” as elution is only possible under very drastic conditions. To reduce the degree of multipoint covalent attachment, immobilization was performed on different conditions that have
been described to reduce the support-enzyme reactivity [38]: decrease of temperature to 4 ◦ C, presence of amino compounds (able to compete with the enzyme) or borate (able to reduce aldehyde reactivity [4]). However, this only permitted to marginally improve the elution of the enzyme, keeping unaltered the immobilization yields. From all these variables, the best results were obtained when immobilization was performed at a low temperature (4 ◦ C) and in 0.3 M Tris–buffer pH 7: the maximum yield of elution obtained was 75% of attached -galactosidase (Fig. 4). However, the percentage of protein that could be released from
Fig. 3. Elution of -galactosidase from E. coli from glyoxyl-agarose containing 40 mol of aldehyde groups/g of support, Enzyme derivatives were incubated for 18 h with 3 M of different amino compounds: Tris–HCl, ethanolamine, and ethilendiamine. Elution yield (% EU) was defined as: (eluted mg of protein or IU/g of wet derivative)/(bounded mg of protein or IU/g of wet derivative).
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Fig. 4. Effect of incubation time on the rate of immobilization and elution of -galactosidase from E. coli, The solid line curve shows the percentage of offered enzyme that is immobilized onto glyoxyl-agarose containing 40 mol of aldehyde groups/g of support. The immobilization was performed at 4 ◦ C in 0.3 M Tris–HCl pH 7.0. The dotted line curve represents the percentage of enzyme elution of the support. That is expressed as: (eluted IU/g of wet support)/(immobilized IU/g wet support) × 100. The elution treatment consisted in the incubation of an aliquot of enzyme insoluble derivative with 3 M ethilendiamine at pH 7.0 for 18 h at 25 ◦ C.
the support decreased with time (time has been described as one of the main variables controlling the protein support multiinteraction). This problem was solved modifying a last variable: the decrease in the amount of aldehyde groups from 40 to 20 mol of glyoxyl groups/g of support. As it can be observed in Fig. 5, it was possible to elute 100% of the -galactosidase attached to the support, for example using 1 M Tris at pH 7 and 4 ◦ C. Of course, the use of an even lower concentration of aldehyde groups may permit to have desorption under even milder conditions. This reduction in the amount of the aldehyde groups does not reduce the capacity of the support to adsorb proteins, still there are a superficial density of aldehydes of around ˚ 2 , although reduces the adsorption 8–9 glyoxyl groups/1000 A rate. Thus, 20 mg of pure betagalactosidase could be immobilized on 1 mL of this support after 24 h.
Fig. 5. Elution of -galactosidase from E. coli from glyoxyl-agarose containing 20 mol of aldehyde groups/g of support. Enzyme derivatives were incubated for 18 h with 1 M of different amino compounds: Tris–HCl, ethanolamine, and ethilendiamine. Elution yield (% EU) was defined as: (eluted mg of protein or IU/g of wet derivative)/(bounded mg of protein or IU/g of wet derivative).
Fig. 6. Immobilization course of -galactosidase from E. coli on glyoxylagarose containing 20 mol of aldehyde groups/g of support, Immobilization was performed at pH 7.0 and 25 ◦ C in 50 mM sodium phosphate. () Enzyme activity of the immobilization suspension; () enzyme activity on the supernatant of the immobilization suspension. The activity of soluble enzyme incubated under similar conditions is represented by (䊉).
Future works will try to improve the enzyme desorption of immobilized proteins at alkaline pH values. 4.3. Purification of β-galactosidase from E. coli by adsorption at pH 7 on middle activated glyoxyl agarose supports After testing the selective immobilization of proteins having very special features and their full recovery after elution, this methodology was used for purification of -galactosidase from a crude extract of E. coli. The preparation was incubated at pH 7.0 with 20 mol/g glyoxyl-agarose. Fig. 6 shows that the adsorption of -galactosidase was very fast (nearly 80% of the activity was adsorbed after 2 h of incubation). However, after 16 h of incubation, over 90% of the total protein still remained in the supernatant. Fig. 7 shows that this immobilization protocol was quite selective for -galactosidase, that appears contaminated only by some other protein bands (obviously, it may be expected that in a crude extract there might be some multimeric proteins having several terminal amino groups on one plane). After the elution of the enzyme from the support with 1 M of Tris–HCl pH 7.0, a purification factor of around 30 was found (Table 3).
Fig. 7. Analysis by SDS-PAGE of selectivity of immobilization of galactosidase from E. coli at pH 7.0 on glyoxyl-agarose containing 20 mol of aldehyde groups/g of support. Lane 1: molecular weight markers; Lane 2: crude extract from E. coli; Lane 3: proteins in the supernatant after immobilization; Lane 4: proteins released from glyoxyl-agarose derivatives by boiling in the presence of SDS.
V. Grazu et al. / Enzyme and Microbial Technology 38 (2006) 960–966
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Table 3 Purification of -galactosidase from E. coli Fraction
Volume (mL)
[Protein] (mg/mL)
Activity (U/mL)
Specific activity (U/mg)
Purification factor
Yield (%)
Crude extract Eluted fraction
24 24
7.8 0.2
20 16
2.5 80
1 32
100 80
The eluted fraction is the supernatant after treatment of the enzyme immobilized on 20 mol glyoxyl groups/g of 4BCL agarose with 1M Tris–HCl pH 7.0 for 18 h at 25 ◦ C. The elution yield was 100%.
5. Conclusions [8]
Glyoxyl agarose, a support designed to stabilize proteins via multipoint covalent attachment and that has been used to perform simultaneous immobilization and purification of some proteins [2], may be also used as a chromatographic matrix based on the reversibility of each individual bond. Immobilization of proteins on supports activated with glyoxyl groups implies the simultaneous establishment of, at least, two attachments between the protein and the support because of the low energy of a single bond. [2] At pH 7.0, this peculiarity in the immobilization process restricts the successful immobilization of proteins to those which have at least two exposed low pK amino groups at the same plane of interaction with the support. This high selectivity together with the fact that it is possible to fully elute the protein from the support, incubating with Tris–HCl, makes the use of this technique feasible for protein purification. However, it is necessary to control the degree of protein support interaction to permit the full protein elution, as it has been reported for other chromatographic methodologies (e.g., IMAC Ionic exchanger) [39,40].
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Acknowledgements
[15]
This project has been funded by the EC (Project MATINOES G5RD-CT-2002-00752). Authors thank the predoctoral fellowships for Lopez-Gallego and Montes. The authors would like ´ to thank MSc. Angel Berenguer (Departamento de Qu´ımica Inorg´anica, Universidad de Alicante) for his kind help and suggestions during the writing of this paper.
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