One-Step Purification, Covalent Immobilization, and Additional

Krzysztof Makowski , Aneta Białkowska , Mirosława Szczęsna-Antczak , Halina Kalinowska , Józef Kur , Hubert Cieśliński , Marianna Turkiewicz...
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Biomacromolecules 2003, 4, 107-113

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One-Step Purification, Covalent Immobilization, and Additional Stabilization of a Thermophilic Poly-His-Tagged β-Galactosidase from Thermus sp. Strain T2 by using Novel Heterofunctional Chelate-Epoxy Sepabeads Benevides C. C. Pessela,† Cesar Mateo,† Alfonso V. Carrascosa,‡ Alejandro Vian,‡ Jose´ L. Garcı´a,§ German Rivas,§ Carlos Alfonso,§ Jose´ M. Guisan,*,† and Roberto Ferna´ ndez-Lafuente*,† Departamento de Biocata´ lisis, Instituto de Cata´ lisis, CSIC, Campus Universidad Auto´ noma, 28049 Madrid, Spain, Centro de Investigaciones Biolo´ gicas, CSIC, Vela´ zquez 144, 28006 Madrid, Spain, and Departamento de Microbiologı´a, Instituto de Fermentaciones Industriales, CSIC, Juan de la Cierva 3, 28006 Madrid, Spain Received July 22, 2002

Using the poly-His-tagged-β-galactosidase from Thermus sp. strain T2 overexpressed in Escherichia coli (MC1116) as a model enzyme, we have developed a strategy to purify and immobilize proteins in a single step, combining the excellent properties of epoxy groups for enzyme immobilization with the good performance of immobilized metal-chelate affinity chromatography for protein purification. The aforementioned enzyme could not be immobilized onto standard epoxy supports with good yields, and after purification and storage, it exhibited a strong trend to yield very large aggregates as shown by ultracentrifugation experiments. That preparation could not be immobilized in any support, very likely because the pores of the solid became clogged by the large aggregates. These novel epoxy-metal chelate heterofunctional supports contain a low concentration of Co2+ chelated in IDA groups and a high density of epoxy groups. This enabled the selective adsorption of poly-His-tagged enzymes, and as this adsorption step is necessary for the covalent immobilization procedure, the selective covalent immobilization of the target enzyme could take place. This strategy allowed similar maximum loadings of the target enzyme using either pure or crude preparations of the enzyme. The enzyme derivative presented a very high activity at 70 °C (over 1000 IU in the hydrolysis of lactose) and very high stability and stabilization when compared to its soluble counterpart (activity remained unaltered after several days of incubation at 50 °C). In fact, this preparation was much more stable than when the same enzyme was immobilized onto standard epoxy Sepabeads. Introduction The availability of pure enzymes is a key factor in the preparation of very active immobilized biocatalysts, since it facilitates full occupation of the surface of the support by the enzyme. Moreover, the use of pure enzymes avoids undesired side reactions catalyzed by contaminant enzymes of crude preparations. To overcome the problems derived from enzyme purification (time-consuming, high cost, enzyme inactivation, etc.), one suitable solution would be to link the purification and immobilization steps by designing a highly selective immobilization process. Immobilized metal-chelate affinity chromatography (IMAC) is a well-developed tool to purify proteins fused to polyHis-tags at industrial scale.1-7 In some cases, the adsorption of a poly-His-tagged protein on the chelate support is quite strong and could be used for enzyme immobilization.8,9 However, the reversibility of the binding process may be a * Authors to whom correspondence may be addressed: fax, +34915854760; tel +34-915854809; e-mail, [email protected], [email protected]. † Instituto de Cata ´ lisis. ‡ Instituto de Fermentaciones Industriales. § Centro de Investigaciones Biolo ´ gicas.

drawback when used to develop an industrial immobilization procedure. Also, the undesired release of metals to the reaction media may become a problem in many cases. For these reasons, IMAC is mainly used for enzyme purification and not for protein immobilization. On the other hand, one of the most suitable methods for the industrial immobilization of proteins is based on epoxy supports.10-16 Such type of supports present several advantages, viz., (i) they are very stable, allowing a long storage and a prolonged transport from manufacturers to consumers, and (ii) they permit large enzyme-support reaction periods. In addition, they are reactive with different moieties of proteins (amine, thiol, hydroxyl groups) yielding very stable protein-support bonds (secondary amine, thioether, ether). Moreover, the remaining epoxy groups may be easily blocked after enzyme immobilization with different compounds yielding an inert surface. More recently, it has been shown that epoxy supports may be used for enzyme stabilization via an intense enzyme-support multipoint covalent attachment by controlling the incubation conditions.17,18 The mechanism of enzyme immobilization in epoxy supports19-22 provides new opportunities for linking im-

10.1021/bm020086j CCC: $25.00 © 2003 American Chemical Society Published on Web 12/10/2002

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Scheme 1. Proposed Strategy for the Purification-Immobilization of Poly-His-Tagged Proteins Using Metal Chelate-Epoxy Supports (CES)a

a The control of adsorption of proteins onto metal chelate may allow the selective adsorption of poly-His-tagged proteins. Only the adsorbed proteins react with the epoxy groups; therefore only poly-His-tagged protein becomes immobilized on the CES.

mobilization to purification. It has been described that the previous adsorption of protein on the epoxy support is necessary to achieve a significant covalent immobilization of the proteins due to the extremely low reactivity of the epoxy supports with soluble proteins.19-22 In fact, immobilization of proteins on commercial epoxy supports follows a two-step mechanism: first, the enzyme is hydrophobically adsorbed on a fairly hydrophobic support (e.g., Eupergit, Sepabeads) at very high ionic strength, and then, the covalent reaction between the enzyme and the support proceeds. With this premise, the use of multifunctional epoxy supports to immobilize proteins has been recently reported.23 This second generation of epoxy supports has different moieties that are able to physically adsorb proteins via different structural features, plus a dense layer of epoxy groups that are able to covalently react with the enzyme. One of the types of multifunctional supports that may be easily produced are the metal chelate-epoxy supports (CES).24 These supports may combine the good properties of epoxy supports for enzyme immobilization-stabilization with the high possibilities of IMAC chromatography to purify poly-His-tagged proteins (Scheme 1). An example of their use may be found in the work by Mateo et al.24 The use of epoxy-chelate supports for protein purification-immobilization requires that the adsorption selectivity becomes very high, because all adsorbed protein molecules may be covalently and irreversibly immobilized on the support. Here, epoxy-chelate supports have been optimized to achieve in a single step the purification, covalent immobilization, and stabilization of poly-His-tagged β-galactosidase from Thermus sp. strain T2, overexpressed in Escherichia coli MC1116.25 β-Galactosidases are very useful in the dairy industry and have been widely used for lactosefree milk production and cheese whey. In this area, thermophilic enzymes have a great interest because of the convenience to perform the processes at high temperatures to reduce both viscosity and the risk of microbial contamination.26,27 The enzyme that has been used in this paper is a homomultimeric one, and the molecular mass of the monomer is around 67 kDa.25,28 The enzyme is very thermostable and seems to have good activity in a broad pH and temperature range. The purified enzyme may be easily obtained via IMAC techniques.29

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Materials and Methods Crude extract of poly-His-tagged β-galactosidase cloned and overexpressed in E. coli (MC1116) (Htag-BgaA) was produced and occasionally purified as previously described.25,29 Epoxy Sepabead (FP-EP E902 P05) supports were kindly donated by Resindion S.R.L (Mitsubishi Chemical Corp.). Iminodiacetic acid disodium salt monohydrate (IDA) was from Fluka (Buchs, Switzerland). All other reagents were of analytical grade. Preparation of Sepabeads-IDA-Epoxy Support. Sepabeads (FP-EP E902 P05) were incubated in 18 mL of 100 mM sodium borate/2 M iminodiacetic acid pH 8.5 at 25 °C under very gentle stirring for 2 h. This allowed introduction of ca. 2 µmol of IDA groups per milliliter of support and maintained ca. 30 µmol of epoxy groups/mL of support in the matrix.24 Then, the support was washed with an excess of distilled water and resuspended in 50 mM sodium phosphate buffer, pH 6.0, containing 1.0 M NaCl plus 5 mg/ mL of CoCl2 . Finally the support was thoroughly washed with distilled water. Immobilization Experiments. Enzyme immobilizations were performed at pH 7 and 25 °C, by suspending 1 g of support in 10 mL of enzyme solution for 24 h. When standard supports were used, 1 M phosphate was used, but when IDA-epoxy supports were used, 25 mM sodium phosphate was used. Covalent attachment was verified by incubating the derivatives under conditions in which all enzyme was desorbed from similar but epoxy-blocked supports.23,24 Standard experiments were performed using only 1 mg of protein/g of support, although to determine the loading capacity the offered amount of enzyme reached 1 g of protein/g of support. In these experiments, maximum protein concentration in the suspension was 3 mg/mL. To block residual epoxy groups, the enzyme derivatives were incubated with 3 M glycine at pH 8.5 for 24 h.18 Metal was eliminated in some cases by addition of 0.5 M mercaptoethanol. The protocol for support preparation and immobilization is represented in Scheme 2. Enzymatic Assays. Activity standard assay was performed with o-nitrophenyl-β-D-galactopyranoside (ONPG) at 25 °C. ONPG was dissolved in Novo buffer pH 6.5 (buffer designed to reproduce the composition of milk (2.7 mM sodium citrate, 7.91 mM citric acid, 2.99 mM potassium biphosphate, 10.84 mM potassium phosphate, 19.43 mM potassium hydroxide, 4.08 mM magnesium chloride, 5.1 mM calcium chloride, and 3.33 mM sodium carbonate))30 and used at a final concentration of 13.3 mM. Activity on lactose was determined by high-performance liquid chromatography analysis. These assays were carried out in 0.1 M sodium phosphate buffer, pH 7.0, at 70 °C, using 138 mM lactose as substrate. One unit of β-galactosidase activity is defined as the amount of enzyme that produces 1 µmol of o-nitrophenol (using ONPG) or glucose (lactose) per minute under the conditions described above. Protein concentration was estimated via the Bradford method,31 using bovine serum albumin as standard. SDS-PAGE Analysis. Soluble or immobilized enzyme preparations were boiled in the presence of 1% (wt/vol) SDS

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Scheme 2. Preparation of Metal Chelate-Epoxy Supports and Their Use for Immobilization of Enzymesa

a Epoxy supports are incubated in the presence of iminodiacetic acid to introduce a few IDA groups in the support. The IDA supports are incubated with cobalt salts to obtain metal chelate-epoxy supports. These supports are used to immobilize the proteins, after which they are incubated in the presence of glycine to block the remaining epoxy groups. To eliminate Co from the support, an incubation with mercaptoethanol may be enough.

and 2% (vol/vol) 2-mercaptoethanol, and the solutions or supernatants were submitted to SDS-polyacrylamide gel electrophoresis (SDS-PAGE). This treatment ensures the release of all protein subunits not covalently attached to the support.32-34 SDS-PAGE was performed with a precast 12% gradient gel (model, protean 16, Bio-Rad Laboratories, CA), following the supplier’s instructions and according to the method described by Laemmli.35 The gels were stained with Coomassie Brilliant Blue R-250. Thermal Inactivations. Samples of derivative or soluble β-galactosidase from Thermus sp. T2 were suspended in 10 mL of Novo buffer pH 6.5 (1 IU in hydrolysis of ONPG at 25 °C/mL of suspension or solution) and incubated at different temperatures. At different times aliquots were withdrawn and their residual activity was assayed at the same pH and 25 °C, with ONPG (13.3 mM). Stabilization refers to the increment in the half-life. Sedimentation Velocity. The sedimentation velocity experiments were carried out at 30 000 rpm and 20 °C in an XL-A analytical ultracentrifuge (Beckman-Coulter Inc.) equipped with UV-vis absorbance optics, an An50Ti rotor, and 12 mm double-sector centerpieces. The protein (loading concentrations ranging from 0.03 to 0.3 mg/mL) was equilibrated in 50 mM phosphate buffer, pH 7.0. Differential sedimentation coefficient distributions, c(s), were calculated

by least-squares boundary modeling of sedimentation velocity data using the program SEDFIT (Schuck, 2000; Schuck et al., 2002).36,37 Results Immobilization of Htag-BgaA on Standard Epoxy Supports. Figure 1 shows the immobilization courses of freshly purified enzyme and of purified enzyme stored for 1 day at 4 °C on conventional epoxy supports (having only epoxy groups). For this experiment we used an amount of enzyme (1 mg of protein/g of support) much lower than the maximum loading capacity of the support (60 mg of protein/g of support). The conventional support immobilized about 40% of freshly purified enzyme under the assay conditions, while yields dropped to 10% using stored purified enzyme. In both cases, the activity of the immobilized enzyme was similar to that of the free enzyme. By use of crude samples, the immobilization yields were around 40%, independent of the time of protein storage (data not shown). This low immobilization yield together with a further decrease after storing of the purified samples complicates the design of proper immobilization protocols using conventional epoxy supports. Immobilization of Different Htag-BgaA Preparations on DEAE-Sepabeads. To analyze the influence of high

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Figure 1. Immobilization course of poli-His tagged β-galactosidase from Thermus sp. strain T2 on standard epoxy support: (9) enzyme activity of the supernatant of the immobilization suspension of pure and fresh enzyme; (b) enzyme activity of the supernatant of the immobilization suspension of purified and stored enzyme; (2) activity of both immobilization suspensions. Specifications are as described in Materials and Methods.

Figure 2. Immobilization course of poli-His tagged β-galactosidase from Thermus sp. strain T2 on DEAE Sepabeads: (b) enzyme activity on the supernatant of the immobilization of pure and fresh enzyme; (9) enzyme activity on the supernatant of the immobilization of stored purified enzyme. Specifications are as described in Materials and Methods.

ionic strength in the Htag-BgaA immobilization, we performed the adsorption of the enzyme on DEAE Sepabeads at pH 7 and low ionic strength. Figure 2 shows that the freshly purified enzyme could be fully and rapidly immobilized, while the stored enzyme only allowed a marginal immobilization. This result suggests that although the high ionic strength could cause some problems in the immobilization of the Htag-BgaA on conventional Sepabeads, the storing of the pure enzyme promoted additional difficulties to the immobilization process. Analysis of the Quaternary Structure of Htag-BgaA by Ultracentrifugation. We studied the state of association of the enzyme in solution by means of sedimentation velocity (Figure 3). From the sedimentation profile it is evident that the enzyme is polydispersed in size. The main sedimenting species (ca. 55%) has an apparent sedimentation coefficient of ca. 25 S which, assuming an spherical shape of the oligomer, would correspond to a 12-mer of the protein (monomer molar mass ) 67 000). Most of the remaining protein sediments as higher molar mass species (with apparent sedimentation coefficients of ca. 36 and 48 S). Only a minor proportion (not higher than 5%) sedimented as a lower molar mass species, although not well resolved. These results strongly suggest that the pure enzyme has a marked tendency to self-associate in solution to form large oligomeric

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Figure 3. Size-distribution analysis of poli-His-β-galactosidase of Thermus sp. STRAIN T2. Sedimentation velocity experiments of the protein were done as described in Materials and Methods section. The experiment corresponding to 0.3 mg/mL of protein is shown. Measured (292 nm) absorbance distributions (circles) and best-fit distribution from the Lamm equation model c(s) (solid line) are shown. The inset shows the best-fit c(s) sedimentation coefficient distribution, allowing for systematic time invariant noise.

species. That is, the pure enzyme is far larger than the expected tetramer.38 This made much more complicated its further handling and immobilization, because these large oligomers obstruct the pores of the matrix (Scheme 3). In this way, it seems very complicated to immobilize the pure enzyme in any kind of support, and therefore preparation of a biocatalyst with the maximum allowed load of enzyme looks difficult, hampering important targets to industrial purposes where the volumetric activity of the enzyme may be a key point. One-Step Purification-Stabilization. Very recently, a new kind of heterofunctional support has been proposed to achieve the one-step immobilization-stabilization of polyHis-tagged proteins.24 This one-step technique, although advantageous, may be critical in the specific case of HtagBgaA to eliminate the tendency of the pure enzyme to aggregate. To overcome these problems, we have prepared a support having a very low density of IDA-chelated Co2+ (2 µmol/mL of gel) and leaving the rest of the reactive groups as epoxy groups to facilitate covalent immobilization (Scheme 2). Figure 4 shows the immobilization courses of fresh pure and crude Htag-BgaA on this new heterofunctional support. In both cases, immobilization rates were very similar and residual activity was virtually 100%. This support allowed an almost quantitative immobilization of the enzyme in a much more rapid way than using the standard epoxy Sepabeads. However, after storage of the pure enzyme for 24 h, immobilization yields decreased significantly. Therefore, only if we can associate the immobilization and the purification in the same step, it would be possible to prepare biocatalysts that contain mainly the target protein To detect the selectivity of immobilization on conventional and IDA-Co2+-epoxy supports, the immobilization of crude preparation of enzyme was performed and samples of supernatants were taken after the immobilization process. The immobilization on the new support seemed to be much more selective than the immobilization on the conventional

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Figure 4. Immobilization course of fresh pure and crude extract of poly-His-tagged beta-galactosidase from Thermus sp. strain T2, on IDA-Co-epoxy chelate support: (2) enzyme activity of all the immobilization suspensions (fresh purified enzyme, crude extract and stored purified enzyme); (b) immobilization of the fresh and pure enzyme; ([) immobilization of crude extract; (9) immobilization of stored purified enzyme. Experiments were performed as described in “Materials and Methods”.

one. In fact, while the standard support immobilized most of the proteins, the new support mainly immobilized a protein of 67 kDa, which corresponds to the monomer of Htag-BgaA (Figure 5). To confirm the selectivity of immobilization, we have boiled both derivatives in the presence of SDS. In this way, any multimeric enzyme immobilized on the support through only some of its subunits, should release to the supernatant the subunits that were not covalently attached to the support, while the other subunits remained attached to the support.33,34 Figure 5 shows that while many protein subunits could be detected using the standard support, only a band of molecular mass 67 kDa could be detected in the new support, strongly suggesting that the immobilization in this support is very selective for the Htag-BgaA. If this immobilization was actually very selective, we should achieve similar maximum immobilization loadings using pure or crude preparations. In fact, with the new

Figure 5. Analysis by SDS-PAGE of the selectivity of the immobilization of poly-His-tagged β-galactosidase on different Sepabeads supports: lane 1, molecular weight markers; lane 2, crude preparation of β-galactosidase; lane 3, proteins that are not immobilized on IDA-Co-Sepabeads; lane 4, proteins that are desorbed from glycine blocked IDA-Co-Sepabeads derivatives; lane 5, proteins that are not immobilized on standard Sepabeads; lane 6, proteins that are desorbed from glycine blocked standard Sepabeads derivatives. Experiments were performed as indicated in Materials and Methods section.

supports, the maximum activity that we can immobilize was similar starting from pure (65 mg of pure enzyme/g) or crude (equivalent to 90% of the results achieved using pure enzyme) preparations (Figure 6). Considering all these results together, we can conclude that we have designed a very selective immobilization protocol, which allows combination in a single step of the purification and immobilization processes, solving, in this way, the problems of handling this enzyme. This excellent result was far from that achieved with standard epoxy immobilization protocols that offer very low immobilization yields under any loading capacity conditions (only 40% using 10 mg of total protein/g of support). Adsorbing the enzyme on anionic exchanger Sepabeads,

Scheme 3. Problems Generated during Immobilization of Large Protein Aggregates

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Figure 6. Effect of purification degree in the immobilization yields at different protein loadings: dotted line, empty symbols, immobilization of crude preparations; solid line, solid symbols, immobilization of the fresh and pure enzyme; circles, immobilization on DEAE-Sepabeads; squares, immobilization on Co-epoxy-Sepabeads. Note the semilogaritmic scale. Specific activity of pure enzyme was 45 IU; crude preparation was around 2 IU (in hydrolysis of ONPG at 25 °C, Pessela et al., 2002). Other specifications are as described in Materials and Methods section.

Figure 7. Inactivation of different derivatives of β-galactosidase from Thermus sp. strain T2: ([) soluble β-galactosidase of Thermus sp. strain T2; (2) beta-galactosidase of Thermus sp. strain T2 immobilized on standard epoxy support (9) β-galactosidase of Thermus sp. strain T2 immobilized on IDA-Co-epoxy supports. Inactivation were carried out in pH 6.5 and 80 °C. Other specifications are as described in Materials and Methods.

100% immobilization yields were achieved only until the amount of total protein (that only contained 3-5% of the target enzyme) reached a value around 100 mg of protein/g of support. After this value, the immobilization yield tends to decrease, due to the coimmobilization of target and contaminant enzymes that compete with the target enzyme by the support surface. Characterization of the Enzyme Derivative. Figure 7 shows that the new derivative of this Htag-BgaA was much more stable than the soluble enzyme and even than that prepared using the conventional epoxy supports. The KM and maximum activity of the enzyme for ONPG were almost unaltered by the immobilization process, i.e., the KM value was 3.1 mM for soluble enzyme and around 5 mM for the immobilized preparation, while the Vmax was 4550 IU in both cases. The preservation of enzyme properties allowed preparation of excellent immobilized derivatives of this thermophilic enzyme, starting from crude preparations that contain 90 IU of enzyme/g of support for the hydrolysis of 5% lactose at 25 °C and more than 1000 IU of enzyme/g of support when

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Figure 8. Effect of the incubation at 50 °C and pH 6.5 of the immobilized enzyme preparation. Other specification are described in Methods.

assayed at 70 °C. This activity was almost constant at pH values between 5.5 and 6.5, while at pH 5, the enzyme activity decreased to around 500 UI/g at 70 °C. Most important, this immobilized derivative retained almost 100% of the initial activity after several weeks of incubation at 50 °C (Figure 8) Conclusions The tendency of Htag-BgaA from Thermus strain T2 to aggregate made very difficult the preparation of immobilized derivatives of the enzyme with the maximum loading, that is, with the maximum activity, and without contaminating enzymes that could alter the properties of the product at an industrial level. Thus, only fresh enzyme preparations could be immobilized on the support. We have taken advantage of the two-step mechanism of immobilization of proteins in epoxy supports to develop a new tool that permits exploitation of the benefits of the IMAC chromatography for protein purification and the advantages of the epoxy supports for enzyme immobilization (Scheme 1). Thus, the control of both steps (adsorption and immobilization) has permitted achievement of the one-step purification-immobilization-stabilization of Htag-BgaA from Thermus sp. This methodology allows preparation of an immobilizedstabilized enzyme derivative almost fully loaded with polyHis-tagged protein (more than 90% of the protein was the tagged protein) using as starting material a crude protein extract with only 3-5% of enzyme content. Bearing in mind that these results are based on general properties of a protein surface, these methodologies should be easily utilized with any other poly-His-tagged protein to achieve, in a very easy way, the purification-immobilization-stabilization of these recombinant proteins. Acknowledgment. Resindion S.R.L (Mitsubishi Chemical Corp.) is gratefully recognized for the kind supply of supports and the economical support. Authors thank the Government of Angola for a PhD fellowship for Dr. Pessela and CAM for economical support. Specially, we want to thank Mr. Pedro Sebastia¨o, Angolan ambassador in Spain, for his support to Dr. Pessela. We thank Mr. Coumo (Resindion), Mr. Daminati (Resindion), and Mr. Miyata (Resindion) for support and interesting suggestions. We also gratefully recognize the suggestions of Dr. Hidalgo and Dr. Balcao during the writing of this manuscript.

Enzyme Purification

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