- Email: [email protected]
[3] Tresyl chloride-activated supports for enzyme immobilization
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beaded form of magnetic support materials with each other and with their development of alginite/magnetite spheres. Alginite is frequently employed as an entrapping medium for enzymes and less frequently as a matrix for covalent immobilization of enzymes and ligands. The alginite/ magnetite dispersion produced spheres that were significantly better than magnetic spheres produced by any other methods in terms of mechanical strength, high surface area and high capacity for covalent immobilization of enzymes. Potential applications of magnetized materials are numerous. Mosbach and colleagues 249 have used them in an intriguing fashion to target beads of various magnetic materials that have been injected into a rabbit's veins to an area of the animal where a magnet has been placed. Such magnetic targeting may well prove to be a valuable addition to research on antibody-drug targeting ("magic bullet") approaches to tumor killing. Comparison of Immobilization Methods No single immobilization method is best for all enzymes or all applications of any given enzyme. While this outline concentrates on covalent coupling methods, the noncovalent methods are often valuable. The choice of a given immobilization method, an appropriate matrix, or even the best enzyme to use is very much dependent upon the application. Hopefully this outline and the detailed immobilization methodology in subsequent chapters will provide some help in designing appropriate systems for many applications.
[3] T r e s y l C h l o r i d e - A c t i v a t e d S u p p o r t s for Enzyme Immobilization
By KURT NILSSON and KLAUS MOSBACH Although a number of methods for the covalent immobilization of enzymes to solid phases are known and applied, I-3 few, if any, can be considered to be the "ideal" one. As enzymes are costly and quite unstable molecules it is desirable that the method used for immobilization be 1 W. B. Jakoby and M. Wilchek, eds., this series, Vol. 34. 2 K. M o s b a c h , ed., this series, Vol. 44. 3 This v o l u m e [1,2].
METHODS IN ENZYMOLOGY, VOL. 135
Copyright c(c) 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
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IMMOBILIZATION TECHNIQUES FOR ENZYMES
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efficient and mild, giving a high yield of bound active enzyme even at neutral pH and at low temperature. For engineering purposes it is advantageous if the enzyme is stabilized by its immobilization. The method used should of course also be easy to reproduce, giving consistent couplings. Alkyl sulfonate esters, R'SOzOR", have been used in organic chemistry for over 100 years, alkylating nucleophiles of great diversity, such as halides, mercaptides, and amines. 4 We have found that sulfonate esters also are suitable for affinity ligand and enzyme immobilization to hydroxyl group carrying supports, such as agarose, cellulose, glycophase glass, and glycerylpropyl-silica.5-8 The activation and coupling of the enzyme to the support are thought to involve the following steps: Activation: Support-CH2OH + R-SO2CI ~ support-CH2OSO2-R Coupling: Support-CH2OSO2-R + H 2 N - e n z y m e - ~ support-CH2NH-enzyme + HOSO2-R Support-CH2OSO2-R + HS-enzyme--~ support-CH2-S-enzyme + HOSO2-R
(R = CHECF3 or C6H4CH3). Tosylates (R = C6H4CH3) and tresylates (R -CHECF3) were found to be highly convenient reagents for enzyme immobilization. Tresylated supports allow efficient immobilization (coupling yield 75100%) at near neutral pH and at 4°. Such activated supports are now commercially available (tresyl-activated Sepharose 4B, Pharmacia AB, Uppsala, Sweden; tresyl-activated glycerylpropyl-silica 500 A, Rockford, IL). Tosylated supports are useful for coupling at higher pH (above 9) and at room temperature (details on tosylated supports can be found in references 5, 6, and 7). In addition to the activation of agarose and glycerylpropyl-silica, that will be described here, cellulose, hydroxyethyl methacrylate, and glycophase glass have also been activated with tresyl chloride. 7 In addition, water-soluble tresyl-PEG (polyethylene glycol, MW 4000 or 6000) has been prepared. 7 This compound should be suitable for preparation of PEG-ligand derivatives used in membrane reactors, in affinity partitioning, or in affinity partitioning ligand assay. PEG activated with cyanuric chloride 9 or carbonyldiimidazole ~°has been applied for covalent modification of several enzymes so as to render them nonimmunogenic. Tresyl4 K. K. Andersen, Compr. Org. Chem. 3, 331 (1979). 5 K. Nilsson and K. Mosbach, Eur. J. Biochem. 112, 397 (1980). 6 K. Nilsson, O. Norrl6w, and K. Mosbach, Acta Chem. Scand., Ser. B B35, 19 (1981). 7 K. Nilsson and K. Mosbach, this series, Vol. 104, p. 56. s K. Nilsson and K. Mosbach, Biochem. Biophys. Res. Commun. 102, 449 (1981). 9 A. Abuchowsky, T. van Es, N. C. Palczuk, and F. F. Davis, J. Biol. Chem. 252, 3578 (1977). z0 C. O. Beauchamp, S. L. Gonias, D. P. Menapace, and S. V. Pizzo, Anal. Biochem. 131, 25 (1983).
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67
PEG is probably more reactive than the PEG activated as above, thus allowing modification under milder conditions. Recently, the sulfonate method has been used for immobilization of proteins (IgG, Con A, lactate dehydrogenase) to glycerylpropyl-coated silicon plates. H Experimental Procedures
Reaction of Supports with Tresyl Chloride (Activation) The activation is performed in a water-free solvent in order to avoid hydrolysis of the sulfonyl chloride. Pyridine is added to neutralize liberated HC1. Higher yields are obtained when using dried solvents rather than the commercially available analytical grade. Complete anhydrous conditions are, however, unnecessary and in this work molecular sieve 4 A (Merck, Darmstadt, FRG) has been used as a drying agent (30 g/liter solvent). The reaction is performed in a well-ventilated hood. The procedure for activation of agarose is the same whether it is crosslinked or not. In order to obtain a high activation level (about 1 mmol tresyl groups per g dry support) the following procedure is used7: Sepharose CL-4B (I0 g, Pharmacia AB, Uppsala, Sweden) is washed in succession with 100 ml of each of the following: 30:70 and 70:30 of acetone : water (v/v), acetone p.a. (twice), and three times with dry acetone (dried over molecular sieve 4 A (Merck, Darmstadt, FRG). The gel is then transferred to a predried beaker containing 3 ml dry acetone and 220 /zl dry pyridine (dried with molecular sieve). During magnetic stirring, 220 /xl tresyl chloride (Fluka AG, Buchs, Switzerland; also available from Fluorochem Ltd., UK) is added dropwise. After 10 min the gel is washed twice with 100 ml of each of the following: acetone p.a., 30:70, 50:50, and 70 : 30 of 5 mM HC1 : acetone (v/v) and finally with 1 mM HCI and stored at 4° until used. The amount of introduced tresyl groups is determined on freeze-dried gel by elemental analysis of sulfur. To obtain a lower activation level (0.2 mmol tresyl groups/g dry support) the procedure is the same except that 45/xl tresyl chloride and 150 ~1 pyridine are used.
Activation of Glyceryipropyl-Silica and Glycophase Glass with Tresyl Chloride Glass and silica are not amenable to direct activation by tresyl chloride. A hydrophilic layer consisting of glycerylpropyl groups is therefore N C. F. Mandenius, S. Welin, B. Danielsson, I. Lundstr6m, and K. Mosbach, Anal. Biochem. 137, 106 (1984).
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IMMOBILIZATION TECHNIQUES FOR ENZYMES
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introduced to the surface of these particles.~2 This cqating reaction also minimizes nonspecific adsorption of biomolecules. Such coated glass particles are commercially available in pore sizes ranging from 40 to 500 A (CPG/Glycophase, Pierce Chemicals, Rockford, IL). Glycerylpropylsilica, or diol-silica, is commercially available from Merck, Darmstadt, FRG, in 100 A pore size. The procedure for preparation of diol-silica is, however, simple. Glycerylpropyl-silica was prepared from LiChrospher Si 100, 300, or 1000 ,~ (Merck, Darmstadt, FRG) by treatment with 3'glycidoxypropyltrimethoxysilane according to previous publications. 13,14 Activation of glycophase glass and glycerylpropyl-silica is similar 7 and given below is a typical procedure which yields a low activation level (50 /zmol tresyl groups/g dry 300 A silica, i.e., about 10% saturation of the available diol groups).14 The dry support is washed three times with 50 ml each of dried acetone. The moist gel (about 10 g) is added to a dry beaker containing 5 ml of dry acetone and 260 /xl of dry pyridine with magnetic stirring. Tresyl chloride (70/zl) is added to the suspension. After 15 min at 0 ° the gel is washed as described for agarose. If not used directly for coupling, the gels are preferably washed with water, 50:50 water-acetone and acetone, dried, and stored in desiccator until required. Activation with 180 /zl tresyl chloride leads to the introduction of about 130 /zmol tresyl groups/g. Large pore silica (1000 /k) activated with 130/zl tresyl chloride/g dry support contains about 50/xmol tresyl groups/g (i.e., about 100% substitution). Coupling of Enzymes to Tresyl Chloride-Activated Supports The procedures for coupling of biomolecules to tresylated support are very similar to those for coupling to supports activated with CNBr. Generally, the buffer used for coupling should not contain strong nucleophiles, e.g., free amino groups that might react with the activated support and thus compete with the biomolecule during coupling. Phosphate, carbonate, and HEPES buffers can be used. Since sulfonic acid is liberated during coupling the pH will decrease if the buffer concentration is too low. Usually, 0.1-0.2 M buffer is sufficient to minimize the change in pH. It is usually sufficient to treat the support after coupling of protein with 0.2 M Tris-HC1 buffer at pH 8 for 2 hr at room temperature in order to 12 F. E. Reqnier and R. Noel, J. Chromatogr. Sci. 14, 316 (1976). 13 P.-O. Larsson, M. Glad, L. Hansson, M.-O. M~nsson, S. Ohlson, and K. Mosbach, Adv. Chromatogr. 21, 41 (1983). 14 K. Nilsson and P.-O. Larsson, Anal. Biochem. 134, 60 (1983).
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69
remove any interference from remaining tresyl grOups.8 Mercaptoethanol has also been used with biomolecules not sensitive to this compound. Below is given a standard procedure for coupling of an enzyme to tresyl-agarose) 5 Coupling of other enzymes is exemplified in references 5-8 and 16-19.
Coupling of a-Chymotrypsin to Tresyl-Agarose ot-Chymotrypsin (25 mg bovine pancreas, type II, 38 U/mg, EC 3.4.2.1.1; Sigma, St. Louis, MO) is dissolved in 5 ml 0.2 M HEPES-HCI, pH 7.8, 4°, containing 5 mM Ac-o-Trp-OMe (an inhibitor that minimizes autolysis). The solution is added to 5 g of tresyl-Sepharose CL-4B (0.97 /~mol tresyl groups/g dry wt), which has been rapidly washed with the above buffer. Coupling proceeds with gentle agitation for 12 hr at 4°. The enzyme is expected to be bound in its monomeric form under the conditions used for coupling. E°The gel is rigorously washed with the following cold solutions: 0.1 M sodium acetate containing 0.5 M sodium chloride, pH 4, 0.5 M sodium chloride, and distilled water. The remaining reactive tresyl groups are quenched by incubating the gels with 0.2 M Tris-HCl, pH 8.5, for 2 hr at room temperature. The gel is then washed in distilled water and finally with buffer for storage, 0.1 M sodium acetate, pH 4. The gel is stored at 4 °. This procedure introduced 89 mg enzyme/g dry gel, i.e., 78% yield as determined by amino acid analyses of freeze-dried sample. ~5
Coupling of Horse Liver Alcohol Dehydrogenase to Tresyl-Silica The commercial enzyme preparation (25 mg horse liver alcohol dehydrogenase, 2.7 U/mg, EC 1.1.1.1; Boehringer-Mannheim, Mannheim, FRG) is dialyzed against 0.075 M sodium phosphate, pH 9, and cleared by centrifugation. Tresyl-silica (1000 A; 10/.~m; 0.71 g dry wt) is suspended in 2 ml cold 0.4 M sodium phosphate buffer, pH 7.0, containing 2 mM NADH and 0.2 mM isobutyramide. The suspension is deaerated in vacuum and 2 ml of a ~ K. Nilsson and K. Mosbach, Biotechnol. Bioeng, 26, 1146 (1984). 16 L. B01ow and K. Mosbach, Biochem. Biophys. Res. Comrnun. 107, 458 (1982). t7 L. Hedbys, P.-O. Larsson, K. Mosbach, and S. Svensson, Biochem. Biophys. Res. Comrnun. 123, 8 (1984). ~8 M.-O. M~nsson, N. Siegbahn, and K. Mosbach, Proc. Natl. Acad. Sci. U.S.A. 80, 1487 (1983). 19 B. Kozulic et al., to be published. 20 R. Ega, H. O. Michel, R. Schloeter, and B. J. Jandorf, Arch. Biochem. Biophys. 66, 366 (1957).
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IMMOBILIZATION TECHNIQUES FOR ENZYMES
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dialyzate containing 15 mg of alcohol dehydrogenase is added. Coupling proceeds for 20 hr at pH 8.0 and at room temperature. Any remaining tresyl groups are subsequently removed by treatment with 0.2 M TrisHC1 + 1 mM dithioerythritol, pH 8.0, for 1 hr. The gel is then washed extensively with 0.5 M NaC1 + 1 mM dithioerythritol in 0.1 M sodium phosphate buffer, pH 7.5, followed by 1 mM dithioerythritol, pH 7.5, in 0.15 M sodium phosphate buffer. The horse liver alcohol dehydrogenasesilica (HLADH-silica) is stored at 4° until used. This procedure introduced 21 mg enzyme/g dry wt as determined by elemental analyses of freeze-dried sample.
Coupling of Soybean Trypsin Inhibitor (STI) to Tresyl-Silica Tresyl-silica (300 ,~; 10 /zm; 100 mg dry weight; 400 /zmol tresyl groups/g dry wt) is suspended in 1.3 ml cold coupling buffer (0.3 M sodium phosphate + 0.3 M sodium chloride, pH 8.0) containing 35 mg STI (type l-S; Sigma, St. Louis, MO). Coupling proceeds for 1 hr at 4°. After rigorous washing with coupling buffer, water, and 0.24 M sodium acetate, the gel is freeze-dried and protein determined by amino acid analysis. This procedure introduced 220 mg protein/g dry wt as determined by elemental analysis.
Coupling and Activity Yield Activation and coupling data for the immobilization of chymotrypsin to tresyl-agarose are shown in Table I. As can be seen the activation level is easily adjusted simply by changing the amount of tresyl chloride. The yield of tresyl groups introduced can be increased by increasing the time for activation. If Sepharose 4B is activated under the same conditions, TABLE I IMMOBILIZATION OF CHYMOTRYPSIN TO TRESYL-AGAROSE
Tresyl chloride added (mmol/g dry support)
Tresyl groups introduced (mmol/g dry support)
Bound chymotrypsin (mmol/g dry support)
Coupling yield (%)
Activity ° (mmol/min, g dry gel)
1.2 3.4 5.8
0.22 0.57 0.97
70 78 89
60 68 78
13.5 12.9 11.1
o Calculated from the initial linear conversion of 0.1 M Ac-Phe-OMe into the products Ac-Phe-GIy-NH2 and Ac-Phe-OH in the presence of 0.2 M GIy-NH2 in 0.2 M NaHCO3, pH 9. (Data taken from Nilsson and Mosbach z5 with permission.)
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SUPPORTS FOR ENZYME IMMOBILIZATION
71
practically the same amount of tresyl groups is introduced. Sepharose 6B and CL-6B give a slightly higher yield than the 4B series, which is probably due to the higher concentration of OH groups in the former gel types. As can be seen from Table I, the enzyme is coupled almost quantitatively (60-80% yield) to the different tresyl-agaroses at 4° and at pH 7.8. Even the less activated gel is thus very efficient. As a further example, a 90% coupling yield of protein A (70 mg/g dry support) can be achieved with a low activated gel (150/xmol tresyl/g dry wt) when coupled at pH 8.5 for 15 hr at room temperature, zl Generally, a less activated support is expected to affect the enzyme properties (activity, stability, etc.), less than a higher one (cf. below). Similar coupling yields and retained activities have been obtained with other enzymes. Thus, trypsin (10 mg/g wet carrier) was bound to tresylated agarose, cellulose, and glycerylpropyl-silica with 70, 87, and 80% yield, respectively, at 4 ° and pH 8.2. 8 The retained apparent activity relative to soluble enzyme was highest for glycerylpropyl-silica (63%), probably because the small particle size of the carrier leads to less restricted diffusion of substrate. Hexokinase was bound to tresyl-agarose with 53% yield at pH 7 and at 40.8 The retained apparent activity was in this case 25% relative to the free enzyme. These values for trypsin and hexokinase, immobilized to tresyl-agarose, are in agreement with results obtained when the enzymes are bound to CNBr-activated agarose (unpublished). No competitive inhibitor was present during the coupling of these enzymes and therefore autolysis (trypsin), active site modification, and diffusion limited kinetics might contribute to the decreased activity of these enzymes after immobilization. T4 DNA ligase, 16 fl-galactosidase, 17 horse liver alcohol dehydrogenase, ~8and endo H 19 (cf. below) have also been immobilized efficiently by tresyl-agarose using similar conditions. An advantage in the coupling step over CNBr-activated supports is that tresylated supports can be stored for several weeks in 1 mM HCI at 4° without losing more than a few percent coupling capacity (as tested by low-molecular-weight compounds, e.g., 3,-aminobutyric acid). Even at neutral pH the stability against hydrolysis is quite high. Thus, after storage at 4 ° and pH 7.5 overnight, the decrease in tresyl groups from agarose was only about 5%. Freeze-dried tresyl-silica has been stored for I year in a desiccator without losing coupling capacity. The effect of pH and reaction time 8 on coupling of a protein (STI) at 4 ° to tresyl-agarose (Sepharose 4B, 1.28 mmol tresyl groups/g dry support) is shown in Table II. The optimal pH is around 9.5 but the yield decreases 2~ M. Ramstorp, K. Nilsson, R. Mosbach, and K. Mosbach, unpublished observations.
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IMMOBILIZATION TECHNIQUES FOR ENZYMES
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TABLE II IMMOBILIZATION OF SOYBEAN TRYPSIN INHIBITOR TO TRESYL-AGAROSEa Inhibitor added (mg/g wet support)
Time for coupling (hr)
pH
Bound inhibitor (mg/g dry support)
Yield (%)
10 lO lO (20) lO lO lO
15 15 15 15 15 1.5
6.5 7.5 8.5 9.5 10.5 8.5
70 195 211 (366) 223 195 200
25 68 74 (64) 78 68 70
a
Tresyl groups (1.28 mol)/g dry weight. (Data taken from Nilsson and Mosbach 8 with permission.)
by only about 10% when coupling is instead carrier out at pH 7.5. At pH 8.5 the coupling is almost complete in 1.5 hr. Similar results are obtained with tresyl-silica 14 (Table III). As can be seen from Tables II and III a very high loading with protein on the supports is possible. In fact, when a silica with a high tresyl group content was used, virtually all of the available surface area of the glycerylpropyl-silica could be covered with STI as calculated from the dimensions of the protein molecule. TABLE III IMMOBILIZATION OF SOYBEAN TRYPSIN INHIBITOR TO TRESYL-SILICAa Bound inhibitor (mg/g dry support) after
Pore size (/~)
Tresyl chloride added (/zmol/g dry support)
Tresy| groups introduced (/xmol/g dry support)
Inhibitor added (mg/g dry support)
1 hr
20 hr
100 100 300 300 300 1000 1000
2900 2900 160 410 2450 1800 1800
450 450 50 125 400 60 60
300 900 350 350 350 50 200
n.d. n.d. 42 90 220 n.d. n.d.
250 250 103 115 279 46 54
a The amounts of pyridine used were 300 and 200 p,1/g dry glycerylpropyl-silica 100 and 1000/~, respectively. The amount of pyridine used when activating the three samples of 300/~ diol-silica was 100, 100, and 300/zl/g dry diol-silica, respectively. (Data taken from Nilsson and Larsson 14 with permission.)
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73
SUPPORTS FOR ENZYME IMMOBILIZATION TABLE IV IMMOBILIZATION OF HORSE LIVER ALCOHOL DEHYDROGENASE TO TRESYL-SILICAa
Tresyl chloride added (/~mol/g dry support)
Enzyme bound (mg/g dry support)
Relative activity of bound enzyme compared with free enzyme (%)
Retention of coenzyme binding sites (%)
Dissociation constant NADH (/~M)
100 300
12 21
100 95
100 97
0.87 0.80
" 1000A, 10 ~m. (Data taken fromNilsson and LarssonE4with permission.)
Characterization of the Intrinsic Properties of an Immobilized Enzyme Diffusional hindrances are usually considerable when assaying enzymes immobilized to commercially available agarose particles (40-120 /xm in diameter). We therefore chose small particulate glycerylpropylsilica (10/zm) with large pores (1000 A) in order to obtain a better assessment of the intrinsic properties of the immobilized enzyme.14 The obtained coupling data for horse liver alcohol dehydrogenase (HLADH) are shown in Table IV. Obviously, the yield of bound enzyme approach 100% using the higher activation level. Despite quite a high loading of enzyme virtually complete retention of the enzymatic activity is obtained. Obviously no diffusion limitations exist at the assay conditions employed, since the activity increases proportionally with the loading of enzyme on the support. Furthermore, the Km for ethanol and acetaldehyde is very similar to that of the soluble enzyme (0.4 and 0.3 raM, respectively, at pH 7.5). Coenzyme binding site determination with isobutyramide and radioactive NADH indicates virtually complete retention of these sites after immobilization (Table IV). A blank silica, tresyl-silica treated with TrisHCI, does not bind any NADH. Since the rate-limiting step for free HLADH catalysis is the dissociation of the coenzyme, the retained coenzyme binding sites and specific activity indicate that the dissociation rate constant for NADH from the immobilized enzyme is as high as from the soluble enzyme. The dissociation constant (Ko) of the immobilized enzyme-NADH complex is very similar to that reported for soluble enzyme (0.6 m M ) ) 2 The dissociation constant is determined by plotting the amount of bound radioactive NADH to the immobilized enzyme according to Scatchard (Fig. I). Furthermore, the linear Scatchard plots indicate that the vast majority of the coenzyme binding sites are homogeneous. A small popula2z H. Theorell and A. D. Winer, Arch. Biochem. Biophys. 83, 291 (1959).
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IMMOBILIZATION TECHNIQUES FOR ENZYMES
[3]
4-
-'r
3 z
_.=o 2 . -ia
z
. 1
,4 2
3
pM
[NADH]boun d FIG. 1. Binding of NADH to HLADH-silica containing 21 (0) or 12 mg enzyme/g dry silica (©). (Reproduced from Nilsson and Larsson 14 with permission.)
tion of weaker binding sites is, however, inferred from the independent coenzyme binding site determination with radioactive NADH + isobutyramide (Kd is about 10-8/zM for NADH and soluble enzyme in this ternary complex). 23 The result of this measurement is indicated by arrows in Fig. I. The pH dependence for binding of NAD, ADP-ribose, and AMP to HLADH also seems to be well conserved after immobilization. Summarizing, the intrinsic properties of HLADH, i.e., the activity, the coenzyme binding, the pH dependence, and the Km value for acetaldehyde/ethanol, seem to be well preserved after immobilization to tresyl chloride-activated 1000/~ silica. Doing these experiments it was found that the porosity of the support as well as the degree of activation influence the intrinsic properties of the immobilized enzyme.24 HLADH undergoes a large conformational change upon binding of NADH. When the enzyme was immobilized in the presence of either NADH or ADP-ribose (which does not induce any conformational change) to highly activated tresyl-silica 100/~, we obtained two preparations with different activity and different Kd. The NADH preparation showed the lowest catalytic activity and had the lowest Kd for NADH and for NAD. Kd for AMP (which is bound in a region not affected by the conformational change) was, however, the same in the two preparations. 23 H. Theorell and J. S. McKinley-McKee, Acta Chem. Scand. 15, 1811 (1961). 24 K. Nilsson, doctoral dissertation, Lund (1984).
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SUPPORTS FOR ENZYME IMMOBILIZATION
75
It could thus well be that the enzyme was "frozen" in its NADH-binding conformation after immobilization in the 100 ,~, pores (which are of similar dimensions as the enzyme molecule). The effect was not observed with low activated silica or with 1000 * silica. Examples of Applications of Immobilized Enzymes Proteases
During the last two decades there has been an increased interest in utilizing enzymes as efficient, specific catalysts for syntheses of fine chemicals. By doing this, the number of synthetic steps often can be drastically reduced and the overall product yield considerably increased compared with conventional organic synthesis. As shown in this volume, immobilization of the enzyme is advantageous as it allows easy separation and reutilization of the expensive catalyst. Chymotrypsin immobilized to tresyl-Sepharose CL-4B has been used for repeated ester and peptide synthesis in aqueous-organic solvent systems. Quantitative yields of N-acetyl-L-tyrosine ethyl ester was thus obtained when N-acetyl-L-tyrosine dissolved in cyclohexane (saturated with water) and 30% ethanol was pumped through a column with chymotrypsin-Sepharose. 25 The stability of the enzyme preparation increased with the level of tresyl chloride activation. As discussed elsewhere in this series serine and thiol proteases catalyze peptide bond formation by aminolysis of amino acid ester substrates (kinetic or nonequilibrium approach). The use of organic cosolvents (such as DMF, acetone, methanol, and butanediol) is advantageous as they increase the solubility of hydrophobic substrates and decrease the competing hydrolysis steps. Soluble proteases, however, tend to denature and aggregate in high concentrations of organic cosolvent and have therefore mainly been used in systems with 70-100% water. Chymotrypsin-agarose (cross-linked Sepharose 4B) was found to catalyze peptide bond formation efficiently even in high concentrations of organic cosolvents. ~5 As expected, the high concentrations of organic cosolvents used were found to increase the yield of peptide (up to 97% yield was obtained). Continuous use of the enzyme was possible as product precipitation did not occur. It was also found that the rate of peptide synthesis was high even in high concentrations of organic cosolvent. In fact, with 0.5 M AC-L-PheOMe and 0.5 M GIy-NH2, the initial liner rate of ester breakdown was higher in 50% DMF than in 10% DMF. Obviously, the higher K~ in 50% 25 T. Mori, K. Nilsson, P.-O. Larsson, and K. Mosbach, to be published.
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IMMOBILIZATION TECHNIQUES FOR ENZYMES
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DMF can be compensated for by the possibility to use a higher substrate concentration (which also increased the peptide yield). As judged from the product yields obtained with L- or D-alaninamide and with glycinamide as acceptor amino acids, the secondary specificity (i.e., the specificity for the acceptor substance) of chymotrypsin was well conserved in both 50% DMF and in 80% butanediol. The immobilized enzyme could be repeatedly used for several weeks when operated in 50% DMF or 90% butanediol. Again the stability of the enzyme was increased by increasing the level of tresyl chloride activation. The difference in stability increased with increased temperature, pH, and DMF concentration. Thus, after 3 weeks in 50% DMF at 20° there was twice as much activity left on the high-activated preparation than on the low-activated support, while there was a 10 times difference in activity after 1 day at 37°. The increased stability obtained on coupling to the highly activated support can be explained by the fact that this support allows more linkages to be formed with the enzyme, which decreases the tendency for unfolding of the enzyme. This is in analogy with previous reports on inactivation of immobilized enzymes by heat 25 or urea. 27 As water-miscible organic solvents are known to decrease the temperature of enzyme denaturation, enzyme stability in such solvents can be increased by lowering the temperature. This is well known from cryoenzymology where water-miscible solvents are used in high concentration at subzero temperatures, zS,z9 Immobilized chymotrypsin was shown to be rapidly inactivated in 50% DMF at 37°, but was quite stable at 4 °, 70% of the original synthetic activity remaining after 3 weeks. As an additional advantage the yield of peptide increased considerably on lowering of the temperature. Thus, the yield of Ac-Phe-GIy-NH2 was about 67% at 22 °, but 86% at - 1 0 °. The same effect was found with trypsin immobilized to tresylSepharose CL-4B and with the corresponding soluble enzymes in 10% DMF (unpublished observation).
Other Enzymes Besides proteases other enzymes immobilized to tresyl-agarose have been used in various applications (Table V). Thus, immobilized T4 DNA ligase could be repeatedly used for joining DNA fragments and remained 26 A.-C. Koch-Schmidt and K. Mosbach, Biochemistry 16, 2105 (1977). 27 D. Gabel, Eur. J. Biochem. 33, 348 (1973). 2s p. Douzou, Adv. Enzymol. 45, 157 (1977). 29 A. L. Fink and G. A. Petsko, Adv. Enzymol. 52, 177 (1981).
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77
SUPPORTS FOR ENZYME IMMOBILIZATION TABLE V VARIOUSPROTEINSAND ENZYMESIMMOBILIZEDON TRESYL CHLORIDE-ACTIVATEDAGAROSE Enzyme
Application
Reference
Soybean trypsin inhibitor Concanavalin A Protein A Chymotrypsin Trypsin T4 DNA ligase /3-Galactosidase Alcohol dehydrogenase Endoglycosidase H
Separation of proteases Purification of commercial peroxidase Separation of IgG subclasses Peptide synthesis Peptide synthesis Joining of DNA fragments Carbohydrate synthesis Site-to-site directed immobilization Deglycosylation of enzymes
8 8 21 15 Unpublished 16 17 18 19
active for over 3 months.16 The retained activity of this sensitive enzyme after immobilization was strongly dependent on the degree of activation of the support. The reproducibility of the activation even with low amounts of tresyl chloride was an important advantage in this application. The transferase activity of an immobilized glycosidase,/3-galactosidase, was used to mediate repeated carbohydrate synthesis. 17Endoglycosidase H has been immobilized at pH 6.5 with high retention of activity. The preparation was used for efficient deglycosylation of enzymes. The immobilized enzyme was repeatedly used without apparent loss of activity. ,9
Affinity Chromatography with Immobilized Enzyme In the literature one can find only a few examples of immobilized enzymes that have been used as adsorbents for low-molecular-weight biomolecules. This is probably due to the low capacity and high cost of such systems. By the introduction of the HPLC technique and of minicolumns these problems may be overcome and the analytical potential of such systems may be realized. Thus, the suitability of HLADH immobilized to tresyl-silica (10/xm, 1000 A) for affinity HPLC has been demonstrated. 14A number of adenine nucleotides and their derivatives as well as impurities in commercial nucleotide preparations can be separated on this type of column. This is exemplified in Fig. 2. The same column can also be used for the rapid screening of the dissociation constants of the substances injected on the column. Ko can be estimated from the equation Kj = (HLADH)/k', where (HLADH) is the concentration of HLADH binding sites and k' is the capacity factor of the retained substance.
78
IMMOBILIZATION TECHNIQUES FOR ENZYMES
[3]
A M P - DERIVATIVES
o,x= CH2C H; b~x=? E t.-
C~ X = H
002
to
d~ x = CH2COO-
o,I
et x = (CHz)s NH3+
,.J 0 Z <
H.
X
IZ~ 0.01 0 tip
\
0.00
O-
I
I
I
0
5
I0
OH OH
MIN.
Fro. 2. separation of AMP analogs on a HLADH-silica column (50 x 5 mm). The binding site concentration in the column was 200/zM. Sample concentrations: 40-100/zM. Flow rate: 0.5 ml/min. Eluent: 0.25 M sodium phosphate, pH 7.5, containing 1 /zM ZnSO4. (Reproduced from Nilsson and Larsson t4 with permission.)
The correlation of experimental Ko values with literature data was generally satisfactory. Attempts to estimate kinetic rate constants, i.e., the association and dissociation rate constants, for retarded material gave, however, a low correlation with literature values. Better estimates are to be expected when nonporous and smaller beads, preferably down to 1/~m diameter, become available. This type of reversed affinity HPLC (that utilizes immobilized enzyme) should have a potential for purification of complex isomeric or racemic mixtures. Furthermore, it would be a way to detect and purify new chemotherapeutic agents interacting with enzymes. In addition, subunit interactions of multimeric enzymes and enzyme-enzyme interactions should be possible to quantitate with this type of HPLC technique. In this type of application, obvious requirements of the immobilization method are that it is mild, leaving the enzyme's binding characteristics as unchanged as possible, and that the method is efficient. As shown above, the tresyl chloride-activated silica seemed to fulfill these requirements as virtually 100% of H L A D H was immobilized with retained binding characteristics.
SUPPORTS FOR ENZYME IMMOBILIZATION
65
beaded form of magnetic support materials with each other and with their development of alginite/magnetite spheres. Alginite is frequently employed as an entrapping medium for enzymes and less frequently as a matrix for covalent immobilization of enzymes and ligands. The alginite/ magnetite dispersion produced spheres that were significantly better than magnetic spheres produced by any other methods in terms of mechanical strength, high surface area and high capacity for covalent immobilization of enzymes. Potential applications of magnetized materials are numerous. Mosbach and colleagues 249 have used them in an intriguing fashion to target beads of various magnetic materials that have been injected into a rabbit's veins to an area of the animal where a magnet has been placed. Such magnetic targeting may well prove to be a valuable addition to research on antibody-drug targeting ("magic bullet") approaches to tumor killing. Comparison of Immobilization Methods No single immobilization method is best for all enzymes or all applications of any given enzyme. While this outline concentrates on covalent coupling methods, the noncovalent methods are often valuable. The choice of a given immobilization method, an appropriate matrix, or even the best enzyme to use is very much dependent upon the application. Hopefully this outline and the detailed immobilization methodology in subsequent chapters will provide some help in designing appropriate systems for many applications.
[3] T r e s y l C h l o r i d e - A c t i v a t e d S u p p o r t s for Enzyme Immobilization
By KURT NILSSON and KLAUS MOSBACH Although a number of methods for the covalent immobilization of enzymes to solid phases are known and applied, I-3 few, if any, can be considered to be the "ideal" one. As enzymes are costly and quite unstable molecules it is desirable that the method used for immobilization be 1 W. B. Jakoby and M. Wilchek, eds., this series, Vol. 34. 2 K. M o s b a c h , ed., this series, Vol. 44. 3 This v o l u m e [1,2].
METHODS IN ENZYMOLOGY, VOL. 135
Copyright c(c) 1987 by Academic Press, Inc. All rights of reproduction in any form reserved.
66
IMMOBILIZATION TECHNIQUES FOR ENZYMES
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efficient and mild, giving a high yield of bound active enzyme even at neutral pH and at low temperature. For engineering purposes it is advantageous if the enzyme is stabilized by its immobilization. The method used should of course also be easy to reproduce, giving consistent couplings. Alkyl sulfonate esters, R'SOzOR", have been used in organic chemistry for over 100 years, alkylating nucleophiles of great diversity, such as halides, mercaptides, and amines. 4 We have found that sulfonate esters also are suitable for affinity ligand and enzyme immobilization to hydroxyl group carrying supports, such as agarose, cellulose, glycophase glass, and glycerylpropyl-silica.5-8 The activation and coupling of the enzyme to the support are thought to involve the following steps: Activation: Support-CH2OH + R-SO2CI ~ support-CH2OSO2-R Coupling: Support-CH2OSO2-R + H 2 N - e n z y m e - ~ support-CH2NH-enzyme + HOSO2-R Support-CH2OSO2-R + HS-enzyme--~ support-CH2-S-enzyme + HOSO2-R
(R = CHECF3 or C6H4CH3). Tosylates (R = C6H4CH3) and tresylates (R -CHECF3) were found to be highly convenient reagents for enzyme immobilization. Tresylated supports allow efficient immobilization (coupling yield 75100%) at near neutral pH and at 4°. Such activated supports are now commercially available (tresyl-activated Sepharose 4B, Pharmacia AB, Uppsala, Sweden; tresyl-activated glycerylpropyl-silica 500 A, Rockford, IL). Tosylated supports are useful for coupling at higher pH (above 9) and at room temperature (details on tosylated supports can be found in references 5, 6, and 7). In addition to the activation of agarose and glycerylpropyl-silica, that will be described here, cellulose, hydroxyethyl methacrylate, and glycophase glass have also been activated with tresyl chloride. 7 In addition, water-soluble tresyl-PEG (polyethylene glycol, MW 4000 or 6000) has been prepared. 7 This compound should be suitable for preparation of PEG-ligand derivatives used in membrane reactors, in affinity partitioning, or in affinity partitioning ligand assay. PEG activated with cyanuric chloride 9 or carbonyldiimidazole ~°has been applied for covalent modification of several enzymes so as to render them nonimmunogenic. Tresyl4 K. K. Andersen, Compr. Org. Chem. 3, 331 (1979). 5 K. Nilsson and K. Mosbach, Eur. J. Biochem. 112, 397 (1980). 6 K. Nilsson, O. Norrl6w, and K. Mosbach, Acta Chem. Scand., Ser. B B35, 19 (1981). 7 K. Nilsson and K. Mosbach, this series, Vol. 104, p. 56. s K. Nilsson and K. Mosbach, Biochem. Biophys. Res. Commun. 102, 449 (1981). 9 A. Abuchowsky, T. van Es, N. C. Palczuk, and F. F. Davis, J. Biol. Chem. 252, 3578 (1977). z0 C. O. Beauchamp, S. L. Gonias, D. P. Menapace, and S. V. Pizzo, Anal. Biochem. 131, 25 (1983).
[3]
SUPPORTS FOR ENZYME IMMOBILIZATION
67
PEG is probably more reactive than the PEG activated as above, thus allowing modification under milder conditions. Recently, the sulfonate method has been used for immobilization of proteins (IgG, Con A, lactate dehydrogenase) to glycerylpropyl-coated silicon plates. H Experimental Procedures
Reaction of Supports with Tresyl Chloride (Activation) The activation is performed in a water-free solvent in order to avoid hydrolysis of the sulfonyl chloride. Pyridine is added to neutralize liberated HC1. Higher yields are obtained when using dried solvents rather than the commercially available analytical grade. Complete anhydrous conditions are, however, unnecessary and in this work molecular sieve 4 A (Merck, Darmstadt, FRG) has been used as a drying agent (30 g/liter solvent). The reaction is performed in a well-ventilated hood. The procedure for activation of agarose is the same whether it is crosslinked or not. In order to obtain a high activation level (about 1 mmol tresyl groups per g dry support) the following procedure is used7: Sepharose CL-4B (I0 g, Pharmacia AB, Uppsala, Sweden) is washed in succession with 100 ml of each of the following: 30:70 and 70:30 of acetone : water (v/v), acetone p.a. (twice), and three times with dry acetone (dried over molecular sieve 4 A (Merck, Darmstadt, FRG). The gel is then transferred to a predried beaker containing 3 ml dry acetone and 220 /zl dry pyridine (dried with molecular sieve). During magnetic stirring, 220 /xl tresyl chloride (Fluka AG, Buchs, Switzerland; also available from Fluorochem Ltd., UK) is added dropwise. After 10 min the gel is washed twice with 100 ml of each of the following: acetone p.a., 30:70, 50:50, and 70 : 30 of 5 mM HC1 : acetone (v/v) and finally with 1 mM HCI and stored at 4° until used. The amount of introduced tresyl groups is determined on freeze-dried gel by elemental analysis of sulfur. To obtain a lower activation level (0.2 mmol tresyl groups/g dry support) the procedure is the same except that 45/xl tresyl chloride and 150 ~1 pyridine are used.
Activation of Glyceryipropyl-Silica and Glycophase Glass with Tresyl Chloride Glass and silica are not amenable to direct activation by tresyl chloride. A hydrophilic layer consisting of glycerylpropyl groups is therefore N C. F. Mandenius, S. Welin, B. Danielsson, I. Lundstr6m, and K. Mosbach, Anal. Biochem. 137, 106 (1984).
68
IMMOBILIZATION TECHNIQUES FOR ENZYMES
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introduced to the surface of these particles.~2 This cqating reaction also minimizes nonspecific adsorption of biomolecules. Such coated glass particles are commercially available in pore sizes ranging from 40 to 500 A (CPG/Glycophase, Pierce Chemicals, Rockford, IL). Glycerylpropylsilica, or diol-silica, is commercially available from Merck, Darmstadt, FRG, in 100 A pore size. The procedure for preparation of diol-silica is, however, simple. Glycerylpropyl-silica was prepared from LiChrospher Si 100, 300, or 1000 ,~ (Merck, Darmstadt, FRG) by treatment with 3'glycidoxypropyltrimethoxysilane according to previous publications. 13,14 Activation of glycophase glass and glycerylpropyl-silica is similar 7 and given below is a typical procedure which yields a low activation level (50 /zmol tresyl groups/g dry 300 A silica, i.e., about 10% saturation of the available diol groups).14 The dry support is washed three times with 50 ml each of dried acetone. The moist gel (about 10 g) is added to a dry beaker containing 5 ml of dry acetone and 260 /xl of dry pyridine with magnetic stirring. Tresyl chloride (70/zl) is added to the suspension. After 15 min at 0 ° the gel is washed as described for agarose. If not used directly for coupling, the gels are preferably washed with water, 50:50 water-acetone and acetone, dried, and stored in desiccator until required. Activation with 180 /zl tresyl chloride leads to the introduction of about 130 /zmol tresyl groups/g. Large pore silica (1000 /k) activated with 130/zl tresyl chloride/g dry support contains about 50/xmol tresyl groups/g (i.e., about 100% substitution). Coupling of Enzymes to Tresyl Chloride-Activated Supports The procedures for coupling of biomolecules to tresylated support are very similar to those for coupling to supports activated with CNBr. Generally, the buffer used for coupling should not contain strong nucleophiles, e.g., free amino groups that might react with the activated support and thus compete with the biomolecule during coupling. Phosphate, carbonate, and HEPES buffers can be used. Since sulfonic acid is liberated during coupling the pH will decrease if the buffer concentration is too low. Usually, 0.1-0.2 M buffer is sufficient to minimize the change in pH. It is usually sufficient to treat the support after coupling of protein with 0.2 M Tris-HC1 buffer at pH 8 for 2 hr at room temperature in order to 12 F. E. Reqnier and R. Noel, J. Chromatogr. Sci. 14, 316 (1976). 13 P.-O. Larsson, M. Glad, L. Hansson, M.-O. M~nsson, S. Ohlson, and K. Mosbach, Adv. Chromatogr. 21, 41 (1983). 14 K. Nilsson and P.-O. Larsson, Anal. Biochem. 134, 60 (1983).
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SUPPORTS FOR ENZYME IMMOBILIZATION
69
remove any interference from remaining tresyl grOups.8 Mercaptoethanol has also been used with biomolecules not sensitive to this compound. Below is given a standard procedure for coupling of an enzyme to tresyl-agarose) 5 Coupling of other enzymes is exemplified in references 5-8 and 16-19.
Coupling of a-Chymotrypsin to Tresyl-Agarose ot-Chymotrypsin (25 mg bovine pancreas, type II, 38 U/mg, EC 3.4.2.1.1; Sigma, St. Louis, MO) is dissolved in 5 ml 0.2 M HEPES-HCI, pH 7.8, 4°, containing 5 mM Ac-o-Trp-OMe (an inhibitor that minimizes autolysis). The solution is added to 5 g of tresyl-Sepharose CL-4B (0.97 /~mol tresyl groups/g dry wt), which has been rapidly washed with the above buffer. Coupling proceeds with gentle agitation for 12 hr at 4°. The enzyme is expected to be bound in its monomeric form under the conditions used for coupling. E°The gel is rigorously washed with the following cold solutions: 0.1 M sodium acetate containing 0.5 M sodium chloride, pH 4, 0.5 M sodium chloride, and distilled water. The remaining reactive tresyl groups are quenched by incubating the gels with 0.2 M Tris-HCl, pH 8.5, for 2 hr at room temperature. The gel is then washed in distilled water and finally with buffer for storage, 0.1 M sodium acetate, pH 4. The gel is stored at 4 °. This procedure introduced 89 mg enzyme/g dry gel, i.e., 78% yield as determined by amino acid analyses of freeze-dried sample. ~5
Coupling of Horse Liver Alcohol Dehydrogenase to Tresyl-Silica The commercial enzyme preparation (25 mg horse liver alcohol dehydrogenase, 2.7 U/mg, EC 1.1.1.1; Boehringer-Mannheim, Mannheim, FRG) is dialyzed against 0.075 M sodium phosphate, pH 9, and cleared by centrifugation. Tresyl-silica (1000 A; 10/.~m; 0.71 g dry wt) is suspended in 2 ml cold 0.4 M sodium phosphate buffer, pH 7.0, containing 2 mM NADH and 0.2 mM isobutyramide. The suspension is deaerated in vacuum and 2 ml of a ~ K. Nilsson and K. Mosbach, Biotechnol. Bioeng, 26, 1146 (1984). 16 L. B01ow and K. Mosbach, Biochem. Biophys. Res. Comrnun. 107, 458 (1982). t7 L. Hedbys, P.-O. Larsson, K. Mosbach, and S. Svensson, Biochem. Biophys. Res. Comrnun. 123, 8 (1984). ~8 M.-O. M~nsson, N. Siegbahn, and K. Mosbach, Proc. Natl. Acad. Sci. U.S.A. 80, 1487 (1983). 19 B. Kozulic et al., to be published. 20 R. Ega, H. O. Michel, R. Schloeter, and B. J. Jandorf, Arch. Biochem. Biophys. 66, 366 (1957).
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IMMOBILIZATION TECHNIQUES FOR ENZYMES
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dialyzate containing 15 mg of alcohol dehydrogenase is added. Coupling proceeds for 20 hr at pH 8.0 and at room temperature. Any remaining tresyl groups are subsequently removed by treatment with 0.2 M TrisHC1 + 1 mM dithioerythritol, pH 8.0, for 1 hr. The gel is then washed extensively with 0.5 M NaC1 + 1 mM dithioerythritol in 0.1 M sodium phosphate buffer, pH 7.5, followed by 1 mM dithioerythritol, pH 7.5, in 0.15 M sodium phosphate buffer. The horse liver alcohol dehydrogenasesilica (HLADH-silica) is stored at 4° until used. This procedure introduced 21 mg enzyme/g dry wt as determined by elemental analyses of freeze-dried sample.
Coupling of Soybean Trypsin Inhibitor (STI) to Tresyl-Silica Tresyl-silica (300 ,~; 10 /zm; 100 mg dry weight; 400 /zmol tresyl groups/g dry wt) is suspended in 1.3 ml cold coupling buffer (0.3 M sodium phosphate + 0.3 M sodium chloride, pH 8.0) containing 35 mg STI (type l-S; Sigma, St. Louis, MO). Coupling proceeds for 1 hr at 4°. After rigorous washing with coupling buffer, water, and 0.24 M sodium acetate, the gel is freeze-dried and protein determined by amino acid analysis. This procedure introduced 220 mg protein/g dry wt as determined by elemental analysis.
Coupling and Activity Yield Activation and coupling data for the immobilization of chymotrypsin to tresyl-agarose are shown in Table I. As can be seen the activation level is easily adjusted simply by changing the amount of tresyl chloride. The yield of tresyl groups introduced can be increased by increasing the time for activation. If Sepharose 4B is activated under the same conditions, TABLE I IMMOBILIZATION OF CHYMOTRYPSIN TO TRESYL-AGAROSE
Tresyl chloride added (mmol/g dry support)
Tresyl groups introduced (mmol/g dry support)
Bound chymotrypsin (mmol/g dry support)
Coupling yield (%)
Activity ° (mmol/min, g dry gel)
1.2 3.4 5.8
0.22 0.57 0.97
70 78 89
60 68 78
13.5 12.9 11.1
o Calculated from the initial linear conversion of 0.1 M Ac-Phe-OMe into the products Ac-Phe-GIy-NH2 and Ac-Phe-OH in the presence of 0.2 M GIy-NH2 in 0.2 M NaHCO3, pH 9. (Data taken from Nilsson and Mosbach z5 with permission.)
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SUPPORTS FOR ENZYME IMMOBILIZATION
71
practically the same amount of tresyl groups is introduced. Sepharose 6B and CL-6B give a slightly higher yield than the 4B series, which is probably due to the higher concentration of OH groups in the former gel types. As can be seen from Table I, the enzyme is coupled almost quantitatively (60-80% yield) to the different tresyl-agaroses at 4° and at pH 7.8. Even the less activated gel is thus very efficient. As a further example, a 90% coupling yield of protein A (70 mg/g dry support) can be achieved with a low activated gel (150/xmol tresyl/g dry wt) when coupled at pH 8.5 for 15 hr at room temperature, zl Generally, a less activated support is expected to affect the enzyme properties (activity, stability, etc.), less than a higher one (cf. below). Similar coupling yields and retained activities have been obtained with other enzymes. Thus, trypsin (10 mg/g wet carrier) was bound to tresylated agarose, cellulose, and glycerylpropyl-silica with 70, 87, and 80% yield, respectively, at 4 ° and pH 8.2. 8 The retained apparent activity relative to soluble enzyme was highest for glycerylpropyl-silica (63%), probably because the small particle size of the carrier leads to less restricted diffusion of substrate. Hexokinase was bound to tresyl-agarose with 53% yield at pH 7 and at 40.8 The retained apparent activity was in this case 25% relative to the free enzyme. These values for trypsin and hexokinase, immobilized to tresyl-agarose, are in agreement with results obtained when the enzymes are bound to CNBr-activated agarose (unpublished). No competitive inhibitor was present during the coupling of these enzymes and therefore autolysis (trypsin), active site modification, and diffusion limited kinetics might contribute to the decreased activity of these enzymes after immobilization. T4 DNA ligase, 16 fl-galactosidase, 17 horse liver alcohol dehydrogenase, ~8and endo H 19 (cf. below) have also been immobilized efficiently by tresyl-agarose using similar conditions. An advantage in the coupling step over CNBr-activated supports is that tresylated supports can be stored for several weeks in 1 mM HCI at 4° without losing more than a few percent coupling capacity (as tested by low-molecular-weight compounds, e.g., 3,-aminobutyric acid). Even at neutral pH the stability against hydrolysis is quite high. Thus, after storage at 4 ° and pH 7.5 overnight, the decrease in tresyl groups from agarose was only about 5%. Freeze-dried tresyl-silica has been stored for I year in a desiccator without losing coupling capacity. The effect of pH and reaction time 8 on coupling of a protein (STI) at 4 ° to tresyl-agarose (Sepharose 4B, 1.28 mmol tresyl groups/g dry support) is shown in Table II. The optimal pH is around 9.5 but the yield decreases 2~ M. Ramstorp, K. Nilsson, R. Mosbach, and K. Mosbach, unpublished observations.
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IMMOBILIZATION TECHNIQUES FOR ENZYMES
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TABLE II IMMOBILIZATION OF SOYBEAN TRYPSIN INHIBITOR TO TRESYL-AGAROSEa Inhibitor added (mg/g wet support)
Time for coupling (hr)
pH
Bound inhibitor (mg/g dry support)
Yield (%)
10 lO lO (20) lO lO lO
15 15 15 15 15 1.5
6.5 7.5 8.5 9.5 10.5 8.5
70 195 211 (366) 223 195 200
25 68 74 (64) 78 68 70
a
Tresyl groups (1.28 mol)/g dry weight. (Data taken from Nilsson and Mosbach 8 with permission.)
by only about 10% when coupling is instead carrier out at pH 7.5. At pH 8.5 the coupling is almost complete in 1.5 hr. Similar results are obtained with tresyl-silica 14 (Table III). As can be seen from Tables II and III a very high loading with protein on the supports is possible. In fact, when a silica with a high tresyl group content was used, virtually all of the available surface area of the glycerylpropyl-silica could be covered with STI as calculated from the dimensions of the protein molecule. TABLE III IMMOBILIZATION OF SOYBEAN TRYPSIN INHIBITOR TO TRESYL-SILICAa Bound inhibitor (mg/g dry support) after
Pore size (/~)
Tresyl chloride added (/zmol/g dry support)
Tresy| groups introduced (/xmol/g dry support)
Inhibitor added (mg/g dry support)
1 hr
20 hr
100 100 300 300 300 1000 1000
2900 2900 160 410 2450 1800 1800
450 450 50 125 400 60 60
300 900 350 350 350 50 200
n.d. n.d. 42 90 220 n.d. n.d.
250 250 103 115 279 46 54
a The amounts of pyridine used were 300 and 200 p,1/g dry glycerylpropyl-silica 100 and 1000/~, respectively. The amount of pyridine used when activating the three samples of 300/~ diol-silica was 100, 100, and 300/zl/g dry diol-silica, respectively. (Data taken from Nilsson and Larsson 14 with permission.)
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73
SUPPORTS FOR ENZYME IMMOBILIZATION TABLE IV IMMOBILIZATION OF HORSE LIVER ALCOHOL DEHYDROGENASE TO TRESYL-SILICAa
Tresyl chloride added (/~mol/g dry support)
Enzyme bound (mg/g dry support)
Relative activity of bound enzyme compared with free enzyme (%)
Retention of coenzyme binding sites (%)
Dissociation constant NADH (/~M)
100 300
12 21
100 95
100 97
0.87 0.80
" 1000A, 10 ~m. (Data taken fromNilsson and LarssonE4with permission.)
Characterization of the Intrinsic Properties of an Immobilized Enzyme Diffusional hindrances are usually considerable when assaying enzymes immobilized to commercially available agarose particles (40-120 /xm in diameter). We therefore chose small particulate glycerylpropylsilica (10/zm) with large pores (1000 A) in order to obtain a better assessment of the intrinsic properties of the immobilized enzyme.14 The obtained coupling data for horse liver alcohol dehydrogenase (HLADH) are shown in Table IV. Obviously, the yield of bound enzyme approach 100% using the higher activation level. Despite quite a high loading of enzyme virtually complete retention of the enzymatic activity is obtained. Obviously no diffusion limitations exist at the assay conditions employed, since the activity increases proportionally with the loading of enzyme on the support. Furthermore, the Km for ethanol and acetaldehyde is very similar to that of the soluble enzyme (0.4 and 0.3 raM, respectively, at pH 7.5). Coenzyme binding site determination with isobutyramide and radioactive NADH indicates virtually complete retention of these sites after immobilization (Table IV). A blank silica, tresyl-silica treated with TrisHCI, does not bind any NADH. Since the rate-limiting step for free HLADH catalysis is the dissociation of the coenzyme, the retained coenzyme binding sites and specific activity indicate that the dissociation rate constant for NADH from the immobilized enzyme is as high as from the soluble enzyme. The dissociation constant (Ko) of the immobilized enzyme-NADH complex is very similar to that reported for soluble enzyme (0.6 m M ) ) 2 The dissociation constant is determined by plotting the amount of bound radioactive NADH to the immobilized enzyme according to Scatchard (Fig. I). Furthermore, the linear Scatchard plots indicate that the vast majority of the coenzyme binding sites are homogeneous. A small popula2z H. Theorell and A. D. Winer, Arch. Biochem. Biophys. 83, 291 (1959).
74
IMMOBILIZATION TECHNIQUES FOR ENZYMES
[3]
4-
-'r
3 z
_.=o 2 . -ia
z
. 1
,4 2
3
pM
[NADH]boun d FIG. 1. Binding of NADH to HLADH-silica containing 21 (0) or 12 mg enzyme/g dry silica (©). (Reproduced from Nilsson and Larsson 14 with permission.)
tion of weaker binding sites is, however, inferred from the independent coenzyme binding site determination with radioactive NADH + isobutyramide (Kd is about 10-8/zM for NADH and soluble enzyme in this ternary complex). 23 The result of this measurement is indicated by arrows in Fig. I. The pH dependence for binding of NAD, ADP-ribose, and AMP to HLADH also seems to be well conserved after immobilization. Summarizing, the intrinsic properties of HLADH, i.e., the activity, the coenzyme binding, the pH dependence, and the Km value for acetaldehyde/ethanol, seem to be well preserved after immobilization to tresyl chloride-activated 1000/~ silica. Doing these experiments it was found that the porosity of the support as well as the degree of activation influence the intrinsic properties of the immobilized enzyme.24 HLADH undergoes a large conformational change upon binding of NADH. When the enzyme was immobilized in the presence of either NADH or ADP-ribose (which does not induce any conformational change) to highly activated tresyl-silica 100/~, we obtained two preparations with different activity and different Kd. The NADH preparation showed the lowest catalytic activity and had the lowest Kd for NADH and for NAD. Kd for AMP (which is bound in a region not affected by the conformational change) was, however, the same in the two preparations. 23 H. Theorell and J. S. McKinley-McKee, Acta Chem. Scand. 15, 1811 (1961). 24 K. Nilsson, doctoral dissertation, Lund (1984).
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SUPPORTS FOR ENZYME IMMOBILIZATION
75
It could thus well be that the enzyme was "frozen" in its NADH-binding conformation after immobilization in the 100 ,~, pores (which are of similar dimensions as the enzyme molecule). The effect was not observed with low activated silica or with 1000 * silica. Examples of Applications of Immobilized Enzymes Proteases
During the last two decades there has been an increased interest in utilizing enzymes as efficient, specific catalysts for syntheses of fine chemicals. By doing this, the number of synthetic steps often can be drastically reduced and the overall product yield considerably increased compared with conventional organic synthesis. As shown in this volume, immobilization of the enzyme is advantageous as it allows easy separation and reutilization of the expensive catalyst. Chymotrypsin immobilized to tresyl-Sepharose CL-4B has been used for repeated ester and peptide synthesis in aqueous-organic solvent systems. Quantitative yields of N-acetyl-L-tyrosine ethyl ester was thus obtained when N-acetyl-L-tyrosine dissolved in cyclohexane (saturated with water) and 30% ethanol was pumped through a column with chymotrypsin-Sepharose. 25 The stability of the enzyme preparation increased with the level of tresyl chloride activation. As discussed elsewhere in this series serine and thiol proteases catalyze peptide bond formation by aminolysis of amino acid ester substrates (kinetic or nonequilibrium approach). The use of organic cosolvents (such as DMF, acetone, methanol, and butanediol) is advantageous as they increase the solubility of hydrophobic substrates and decrease the competing hydrolysis steps. Soluble proteases, however, tend to denature and aggregate in high concentrations of organic cosolvent and have therefore mainly been used in systems with 70-100% water. Chymotrypsin-agarose (cross-linked Sepharose 4B) was found to catalyze peptide bond formation efficiently even in high concentrations of organic cosolvents. ~5 As expected, the high concentrations of organic cosolvents used were found to increase the yield of peptide (up to 97% yield was obtained). Continuous use of the enzyme was possible as product precipitation did not occur. It was also found that the rate of peptide synthesis was high even in high concentrations of organic cosolvent. In fact, with 0.5 M AC-L-PheOMe and 0.5 M GIy-NH2, the initial liner rate of ester breakdown was higher in 50% DMF than in 10% DMF. Obviously, the higher K~ in 50% 25 T. Mori, K. Nilsson, P.-O. Larsson, and K. Mosbach, to be published.
76
IMMOBILIZATION TECHNIQUES FOR ENZYMES
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DMF can be compensated for by the possibility to use a higher substrate concentration (which also increased the peptide yield). As judged from the product yields obtained with L- or D-alaninamide and with glycinamide as acceptor amino acids, the secondary specificity (i.e., the specificity for the acceptor substance) of chymotrypsin was well conserved in both 50% DMF and in 80% butanediol. The immobilized enzyme could be repeatedly used for several weeks when operated in 50% DMF or 90% butanediol. Again the stability of the enzyme was increased by increasing the level of tresyl chloride activation. The difference in stability increased with increased temperature, pH, and DMF concentration. Thus, after 3 weeks in 50% DMF at 20° there was twice as much activity left on the high-activated preparation than on the low-activated support, while there was a 10 times difference in activity after 1 day at 37°. The increased stability obtained on coupling to the highly activated support can be explained by the fact that this support allows more linkages to be formed with the enzyme, which decreases the tendency for unfolding of the enzyme. This is in analogy with previous reports on inactivation of immobilized enzymes by heat 25 or urea. 27 As water-miscible organic solvents are known to decrease the temperature of enzyme denaturation, enzyme stability in such solvents can be increased by lowering the temperature. This is well known from cryoenzymology where water-miscible solvents are used in high concentration at subzero temperatures, zS,z9 Immobilized chymotrypsin was shown to be rapidly inactivated in 50% DMF at 37°, but was quite stable at 4 °, 70% of the original synthetic activity remaining after 3 weeks. As an additional advantage the yield of peptide increased considerably on lowering of the temperature. Thus, the yield of Ac-Phe-GIy-NH2 was about 67% at 22 °, but 86% at - 1 0 °. The same effect was found with trypsin immobilized to tresylSepharose CL-4B and with the corresponding soluble enzymes in 10% DMF (unpublished observation).
Other Enzymes Besides proteases other enzymes immobilized to tresyl-agarose have been used in various applications (Table V). Thus, immobilized T4 DNA ligase could be repeatedly used for joining DNA fragments and remained 26 A.-C. Koch-Schmidt and K. Mosbach, Biochemistry 16, 2105 (1977). 27 D. Gabel, Eur. J. Biochem. 33, 348 (1973). 2s p. Douzou, Adv. Enzymol. 45, 157 (1977). 29 A. L. Fink and G. A. Petsko, Adv. Enzymol. 52, 177 (1981).
[3]
77
SUPPORTS FOR ENZYME IMMOBILIZATION TABLE V VARIOUSPROTEINSAND ENZYMESIMMOBILIZEDON TRESYL CHLORIDE-ACTIVATEDAGAROSE Enzyme
Application
Reference
Soybean trypsin inhibitor Concanavalin A Protein A Chymotrypsin Trypsin T4 DNA ligase /3-Galactosidase Alcohol dehydrogenase Endoglycosidase H
Separation of proteases Purification of commercial peroxidase Separation of IgG subclasses Peptide synthesis Peptide synthesis Joining of DNA fragments Carbohydrate synthesis Site-to-site directed immobilization Deglycosylation of enzymes
8 8 21 15 Unpublished 16 17 18 19
active for over 3 months.16 The retained activity of this sensitive enzyme after immobilization was strongly dependent on the degree of activation of the support. The reproducibility of the activation even with low amounts of tresyl chloride was an important advantage in this application. The transferase activity of an immobilized glycosidase,/3-galactosidase, was used to mediate repeated carbohydrate synthesis. 17Endoglycosidase H has been immobilized at pH 6.5 with high retention of activity. The preparation was used for efficient deglycosylation of enzymes. The immobilized enzyme was repeatedly used without apparent loss of activity. ,9
Affinity Chromatography with Immobilized Enzyme In the literature one can find only a few examples of immobilized enzymes that have been used as adsorbents for low-molecular-weight biomolecules. This is probably due to the low capacity and high cost of such systems. By the introduction of the HPLC technique and of minicolumns these problems may be overcome and the analytical potential of such systems may be realized. Thus, the suitability of HLADH immobilized to tresyl-silica (10/xm, 1000 A) for affinity HPLC has been demonstrated. 14A number of adenine nucleotides and their derivatives as well as impurities in commercial nucleotide preparations can be separated on this type of column. This is exemplified in Fig. 2. The same column can also be used for the rapid screening of the dissociation constants of the substances injected on the column. Ko can be estimated from the equation Kj = (HLADH)/k', where (HLADH) is the concentration of HLADH binding sites and k' is the capacity factor of the retained substance.
78
IMMOBILIZATION TECHNIQUES FOR ENZYMES
[3]
A M P - DERIVATIVES
o,x= CH2C H; b~x=? E t.-
C~ X = H
002
to
d~ x = CH2COO-
o,I
et x = (CHz)s NH3+
,.J 0 Z <
H.
X
IZ~ 0.01 0 tip
\
0.00
O-
I
I
I
0
5
I0
OH OH
MIN.
Fro. 2. separation of AMP analogs on a HLADH-silica column (50 x 5 mm). The binding site concentration in the column was 200/zM. Sample concentrations: 40-100/zM. Flow rate: 0.5 ml/min. Eluent: 0.25 M sodium phosphate, pH 7.5, containing 1 /zM ZnSO4. (Reproduced from Nilsson and Larsson t4 with permission.)
The correlation of experimental Ko values with literature data was generally satisfactory. Attempts to estimate kinetic rate constants, i.e., the association and dissociation rate constants, for retarded material gave, however, a low correlation with literature values. Better estimates are to be expected when nonporous and smaller beads, preferably down to 1/~m diameter, become available. This type of reversed affinity HPLC (that utilizes immobilized enzyme) should have a potential for purification of complex isomeric or racemic mixtures. Furthermore, it would be a way to detect and purify new chemotherapeutic agents interacting with enzymes. In addition, subunit interactions of multimeric enzymes and enzyme-enzyme interactions should be possible to quantitate with this type of HPLC technique. In this type of application, obvious requirements of the immobilization method are that it is mild, leaving the enzyme's binding characteristics as unchanged as possible, and that the method is efficient. As shown above, the tresyl chloride-activated silica seemed to fulfill these requirements as virtually 100% of H L A D H was immobilized with retained binding characteristics.