Reversible Covalent Immobilization of Enzymes Via Their Thiol Groups

Francisco Batista-Viera, Karen Ovsejevi, and Carmen Manta

Summary

This enzyme immobilization approach involves the formation of disulfide (S-S) bonds with the support. Thus, enzymes bearing exposed nonessential thiol (SH) groups can be immobilized onto thiol-reactive supports provided with reactive disulfides or disulfide oxides under mild conditions. The great potential advantage of this approach is the reversibility of the bonds formed between the activated solid phase and the thiol-enzyme, because the bound protein can be released with an excess of a low-molecular-weight thiol (e.g., dithiothreitol [DTT]). This is of particular interest when the enzyme degrades much faster than the adsorbent, which can be reloaded afterwards. The possibility of reusing the polymeric support after inactivation of the enzyme may be of interest for the practical use of immobilized enzymes in large-scale processes in industry, where their use has often been hampered by the high cost of the support material. Disulfide oxides (thiolsulfinate or thiolsulfonate groups) can be introduced onto a wide variety of support materials with different degrees of porosity and with different mechanical resistances. Procedures are given for the preparation of thiol-reactive solid phases and the covalent attachment of thiol-enzymes to the support material via disulfide bonds. The possibility of reusing the polymeric support is also shown.

Key Words: Thiol-enzymes; enzyme thiolation; reversible enzyme immobilization; thiol-reactive supports; pyridyldisulfide-agarose; solid-phase disulfide oxides; thiolsulfinate-agarose; thiolsulfonate-agarose; P-galactosidase.

1. Introduction

Immobilization methods based on thiol-disulfide exchange reactions are unique since they allow the formation of a stable and reversible covalent bond of disulfide (S-S) type (1). Thus, enzymes bearing exposed nonessential thiol (SH) groups can

From: Methods in Biotechnology: Immobilization of Enzymes and Cells, Second Edition

Edited by: J. M. Guisan © Humana Press Inc., Totowa, NJ

Immobilization Methods Enzymes
Fig. 1. Reversible covalent immobilization of thiol-enzymes onto 2-pyridyldisulfide-agarose. (1) Enzyme coupling with liberation of 2-thiopyridone, (2) elution of gel-bound enzyme with an excess of a low molecular-weight thiol (e.g., DTT or P-mercaptoethanol).

be immobilized onto thiol-reactive supports under mild conditions (e.g., low-ionic-strength buffer with pH 7.0-8.0 at room temperature). However, the applicability of these methods is not restricted to those thiol enzymes. Enzymes containing masked or unreactive thiol groups, or not containing thiol groups at all, can be modified chemically or by genetic engineering techniques, in order to provide them with reactive SH groups.

The great potential advantage of this approach is the reversibility of the bonds formed between the activated solid phase and the thiol-enzyme, because the bound protein can be released by the reduction of the disulfide bonds with an excess of a low-molecular-weight (MW) thiol (e.g., P-mercaptoethanol or dithiothreitol [DTT]). This is of particular interest when the enzyme degrades much faster than the adsorbent, which can be reloaded afterwards.

This chapter focuses exclusively on enzyme immobilization onto thiol-reactive solid phases provided with reactive disulfides or disulfide oxides. In the most traditional method, 2-pyridyldisulfide-agarose (so called PyS2-gel), reacts with thiol groups in proteins, forming a gel-bound mixed disulfide with the protein, with concomitant liberation of 2-thiopyridone (see Fig. 1). The coupling reaction is driven essentially to completion because of the formation of the thione, a compound stabilized by thiol-thione tautomerism. The release of 2-thiopyridone (the leaving group) can be monitored in order to follow the advance of the reaction of the activated solid phase with thiols. However, its release contaminates the nonimmobilized material, which sometimes can be of interest. The use of 2-pyridyldisulfide as a ligand is very advantageous because it is reactive in a wide pH range.

2-Pyridyldisulfide-agarose has been used both for enzyme immobilization and for purification of thiol-proteins by covalent chromatography (1). Thus, crude Jack bean meal urease (a thiol-rich multimeric protein containing several nonessential thiol groups) has been reversibly immobilized onto PyS2-gel by thiol-disulfide exchange with concomitant purification (2). A column packed with this immobilized urease derivative could hydrolyze urea very efficiently when a solution of the substrate was passed through it. Because the gel-bound active enzyme could be eluted quantitatively after incubation with low MW thiols (see Fig. 1), the method was also useful for the purification of urease (e.g., 167-fold) by covalent chromatography.

Pancreatic hog a-amylase (containing thiol groups required for high activity but unreactive towards pyridyldisulfide-agarose) was provided with "de novo" thiol groups through a mild thiolation process, allowing its immobilization onto PyS2-gel with high yields (3). The immobilized a-amylase derivative was used in a packed-bed reactor for the continuous hydrolysis of starch; when the enzymati-cally active gel had lost its activity, it could be regenerated in situ by reductive uncoupling of the inactive protein (see Fig. 1) and attachment of a new portion of thiolated a-amylase.

A disadvantage of these gels is that at high ionic strength (e.g., 0.5 M sodium or potassium sulfate) 2-pyridyldisulfide-gels bind some proteins lacking thiol groups, especially immunoglobulins, through a noncovalent interaction, a property that has been utilized in the so-called thiophilic adsorption chromatography (4).

Thiol-reactive adsorbents based on pyridyldisulfide groups are commercially available. Thus, Amersham Biosciences (Uppsala, Sweden), provides two types of such agarose derivatives which differ both in the length of the spacer arm and the degree of substitution. Activated Thiol Sepharose 4B contains about 1 ^mol pyridyldisulfide groups per milliliter packed gel, and a glutathione (10 atoms) spacer arm. Thiopropyl Sepharose 6B contains about 20 ^mol pyridyldisulfide groups per milliliter packed gel, and a 2-hydroxypropyl (4 atoms) spacer arm. It should be borne in mind that in spite of its trade name, Thiopropyl Sepharose 6B is not a thiol-gel but a mixed reactive disulfide gel.

More recently, a new approach based on agarose-bound disulfide oxides groups (thiolsulfinates and thiolsulfonates) has been developed for the reversible immobilization of thiol-containing substances (5). These agarose-derivatives display high reactivity and selectivity towards both low- and high-MW thiols and can be used for the reversible immobilization of thiol-peptides and thiol-proteins (5-8). Enzymes containing exposed SH groups are covalently immobilized onto solidphase disulfide oxides under mild conditions. The immobilization process involves the formation of disulfide bonds between thiol groups on the enzyme and disulfide oxide structures on the supports.

Because of displacement of the electrons around the two sulfur atoms, disulfide oxides show high S reactivity. Thiol-containing molecules react with the more electrophilic of the two sulfur atoms (the unoxidized one) and become, as a result, immobilized to the solid phase by disulfide bonds (see Figs. 2 and 3). Contrary to the case with 2-pyridyldisulfide-based gels, the leaving groups (sulfenic or sulfinic acid) remain attached to the support.

All the techniques for introducing either reactive disulfides of 2-pyridyldisulfide type or disulfide oxides (thiolsulfonates or thiolsulfinates) into beaded agarose gels start with the same support derivative: thiol-agarose, which is prepared by means of a three-step thiolation process. Thus, to obtain a thiol-substituted agarose, the support is first reacted with epichlorohydrin in a strong alkaline medium to introduce oxirane moieties into the gel; the oxirane groups are then converted with sodium thiosulfate to gel-bound thiosulfate groups (Bunte salt derivative), which finally are reduced with DTT to thiol groups (see Fig. 4). The degree of substitution in the thiol-agarose can be regulated by the amount of epichlorohy-drin added as well as by the incubation conditions (e.g., incubation period, temperature) (9).

Oxidation Thiol Group

Fig. 2. Reversible covalent immobilization of thiol-enzymes onto a thiolsulfonate-support. (1) Preparation of a thiolsulfonate-support through oxidation of thiol-support. (2) Immobilization of a thiol-enzyme onto a thiolsulfonate-support; the leaving group (sulfinic acid group) remains attached to the support. (3) Release of gel-bound enzyme with an excess of a low-molecular weight thiol.

Fig. 2. Reversible covalent immobilization of thiol-enzymes onto a thiolsulfonate-support. (1) Preparation of a thiolsulfonate-support through oxidation of thiol-support. (2) Immobilization of a thiol-enzyme onto a thiolsulfonate-support; the leaving group (sulfinic acid group) remains attached to the support. (3) Release of gel-bound enzyme with an excess of a low-molecular weight thiol.

Thiol-agarose can be subsequently converted to thiolsulfonate-agarose (TS-gel) or thiolsulfinate-agarose (TSI-gel) through different oxidation procedures. Thus, oxidation of thiol-agarose with hydrogen peroxide at moderately acidic pH and room temperature for extended periods (20-30 h) converts thiol groups on the support (via disulfide and thiolsulfinate) into thiolsulfonate (disulfide dioxide) moieties (see Fig. 2) (6). The thiolsulfinate (disulfide monoxide) groups are introduced by oxidation of thiol-agarose. The method comprises two steps: first, mild oxidation of agarose-bound thiol groups to disulfide structures with potassium fer-ricyanide; second, controlled oxidation of the agarose disulfide groups so formed to thiolsulfinate groups is performed with the oxidizing agent magnesium monoperoxyphthalate (see Fig. 3) (7,8). This reagent makes possible the introduction of only one oxygen atom per immobilized aliphatic disulfide group to form a thiolsulfinate moiety. The number of thiolsulfinate groups introduced can be regulated at will by choosing a certain molar ratio between the oxidizing agent and the gel-bound disulfide structures. When the stoichiometric quantity of monoperoxyphthalate is used, maximum thiol-binding capacity is achieved; if half of this amount is used, 50% of the maximum thiol-binding capacity is obtained, and so on.

Thus, it is possible to prepare thiolsulfinate-agarose gels with different thiol-binding capacities from the same thiol-agarose batch.

The gel-bound thiolsulfinate/thiolsulfonate groups are very stable in the pH range of 3.0 to 8.0; therefore, solid phases containing these groups can be stored as suspensions at pH 5.0 at 4°C for extended periods, without a decrease in their thiol-binding capacity. Furthermore, disulfide oxide gels do not need any preservative agent since no bacterial or fungal growth was observed after storing at 4°C at pH 5.0 for 2 yr.

Enzyme Immobilization Covalent Binding

Fig. 3. Reversible covalent immobilization of thiol-enzymes onto a thiolsulfinate-sup-port. (1) Preparation of a thiolsulfinate-support through oxidation of thiol-support. (2) Immobilization of a thiol-enzyme onto a thiolsulfinate-support; the leaving group (sulfenic acid group) remains attached to the support. (3) Release of gel-bound enzyme with an excess of a low-molecular weight thiol.

Fig. 3. Reversible covalent immobilization of thiol-enzymes onto a thiolsulfinate-sup-port. (1) Preparation of a thiolsulfinate-support through oxidation of thiol-support. (2) Immobilization of a thiol-enzyme onto a thiolsulfinate-support; the leaving group (sulfenic acid group) remains attached to the support. (3) Release of gel-bound enzyme with an excess of a low-molecular weight thiol.

Oxidation Thiol Groups
Fig. 4. Synthesis of a thiol-support through a three-step procedure. (1) Epoxy-activa-tion of the solid phase. (2) Formation of thiosulfate-ester estructures (Bunte salt derivative). (3) Reduction of the Bunte salt by an excess of a low-molecular-weight thiol.

After extensive reuse of an enzyme derivative until its inactivation, the disulfide bonds can be split under reducing conditions at alkaline pH to remove the bound protein (see Figs. 2 and 3). Then, the gel can be regenerated following the activation techniques described above and reloaded with fresh enzyme. This is achieved more efficiently with a TSI-gel, resulting from the fact that the sulfenic acid groups (SOH) formed during thiol coupling can easily be converted back to SH moieties by using an excess of a mild reducing agent (see Fig. 3). Thus thiolsulfinate-agarose can, at least in theory (as is also the case with 2-pyridyldisulfide-agarose), be regenerated an unlimited number of times. In contrast, TS-gels can be regenerated only a few times, owing to the formation of gel-bound nonreducible sulfinate groups (see Fig. 2), with a concomitant decrease of about 50% of its thiol-binding capacity after each cycle (7).

Besides beaded agarose, thiolsulfonate and thiolsulfinate groups can be introduced onto a wide variety of support materials with different degrees of porosity and with different mechanical resistances (e.g., soft particles such as Sephadex and cellulose; semirigid ones such as Toyopearl® resins, Sephacryl™, Eupergit®, Superdex™, and Superose™; and rigid particles such as controlled porous glass). Thiolation of the different supports is carried out using the same procedure as reported for agarose, except for porous glass which is directly thiolated by silanization in organic solvent with 3-mercaptopropyltriethoxysilane, and for oxirane-carrying acrylic beads (Eupergit C, Sepabeads® EP), for which the first step in the thiolation procedure is omitted (8).

Table 1 shows thiol-binding capacities of different thiolsulfinate-supports prepared according to the methods described. These figures represent the total number of thiol-reactive structures, because they are determined by measuring the maximum binding of a thiol-peptide (reduced glutathione). In spite of the disparate types of support assayed, it is possible to provide all of them with thiol-reactive groups.

In some cases the degrees of activation achieved were lower than expected, as a consequence of the rigidity of some matrices that makes impossible the formation of disulfide bonds between SH groups located far from each other.

The application of TS- and TSI-gels to the immobilization of high-MW thiols was assessed using different enzymes: with exposed nonessential SH groups, with buried thiol groups, with de novo thiol groups, and with reducible disulfides. Thus, immobilization of P-galactosidase (Escherichia coli) on thiolsulfonate- and thiolsulfinate-agarose has been performed with yields higher than 80% and a high percentage of expressed activity (7,10). The maximum capacity of these thiol-reac-tive adsorbents is on the order of 10 mg protein/mL. In the case of commercial P-galactosidases, such as those from Kluyveromyces lactis and Aspergillus oryzae, a previous reduction process was shown to be essential in order to unblock their more exposed thiol groups (11). This can be done either with soluble reducing agents such as DTT or more conveniently with solid-phase reducing agents (12). When enzymes lack reactive SH groups, one alternative can be the introduction of de novo thiol groups by a thiolation process using suitable heterobifunctional reagents, as in the case of sweet potato P-amylase and pullulanase (13,14). Another possibility is site-directed mutagenesis techniques that can introduce a free cysteine at a suitable position on the protein, which can be used subsequently

Table 1

Thiol-Binding Capacities for Different Thiolsulfinate-Supports Determined by Glutathione Binding (8)

Table 1

Thiol-Binding Capacities for Different Thiolsulfinate-Supports Determined by Glutathione Binding (8)

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  • gorbulas
    What when how thiol enzymes?
    7 years ago

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