Stabilized water soluble enzymes and a method for their preparation

FIELD OF INVENTION

The invention concerns a stabilized enzyme product and a method for its preparation. The stabilized enzyme product may 5 be in form of an aqueous solution or a lyophilized dry product. A stabilized enzyme product according to the invention may optionally be further stabilized by immobilization.

LIST OF REFERENCES

1. Klibanov, A.M., Adv. Appl. Microb. 1983, 29, 1-28.

2. Martinek, K. and Berezin, I. V., J. Solid Phase Biochem.

1978,2,343-385.

3. Butler, L. G., Enzyme Microb. Technol.,, 1979, 1, 253 259.

4. Martinek, K., Klibanov, A.M., Goldmacher, V.S. and

Berezin, I.V., Biochim, Biophys, Acta 1977, 485, 1-12.

5. Klibanov, A.M., Anal. Biochem. 1979, 93, 1-25.

6. Schmid, R.D. Adv. Biochem. Eng. 1979, 12, 41-118.

83451-2

7. Back, J.R., Oakenfull, D. and Smith, M.B.,

Biochemistry 1979, 18, 5191-5196. ]

8. Tonchilin, V.P. and Martinek, K., Enzyme Microb, Technol. 1979, 1, 7482־.

9. Greco, G. and Gianfreda, L., Biotechnol. Bioeng. 1981, 23, 2199-2210.

10. Wykes, J.R., Dunill, P. and Lilly, M.D., Biochim. Biophys. Acta 1971, 250, 522-529.

11. Marshall, J.J., Trends in Biochem. Sci. 1978, 3, 79-83.

12. Lenders, J.P. and Crichton, R.R., Biotech. Bioeng. 1984, 26, 1343-1351.

13. Hixon, H.F., Biotech. Bioeng. 1973, 15, 1011-1016.

14. von Specht, B.U., Seinfeld, H. and Brendel, W., HoppeSeyler's Z. Physiol. Chem, 1973, 354(s), 1659-1660.

15. Paz, M.A., Blumenfeld, 0.0., Rojkind, M., Henson, E.,

Furfine, C., and Gallop, P.M., Arch. Biochem Biophys., 1965, 109, 548-559.

16. Gary, W.L., Johnson, R.N. and Kho, B.T., J. Chromatog, 1978, 156, 285-291.

17. Freeman, A., Methods. Enzymol. 1987, 135, 216-222.

18. Collinson, E., Dainton, F.S. and McNaughton, G.S., ------------------

Trans. Faraday Soc. 1957, 53, 489498־,

19. Freeman, A. and Aharonowitz, Y. , Biotech. Bioeng.,

.2759־2747 ,23 ,1981

20. Reuveny, S., Mizrahi, A., Kotler, M. and Freeman, A., Biotech. Bioeng. 1983, 25, 469-480.

83451-2

21. Blank-Koblenc, T., Tor, R and Freeman, A., Biotechnol, Appl. Biochem., 1988, 10, 32-41.

22. Nichols, C.S. and Cromartie, T.H., J. Chem. Educ. , 1979, 56. 832-834.

23. Samuni, A., Anal. Biochem., 1975, 63, 17-26.

24. Malikkides, C.O. and Weiland, R.H., Biotech. Bioeng.

1982, 24, 1911-1914.

25. Freeman, A., Blank, T. and Haimovich, B., Annal. N.Y. Acad. 1983, 413, 557-559.

26. Goldstein, L. and Manecke, G., Appl. Biochem. Bioeng.,

1976, 1, 23-126.

27. Dror, Y. , Cohen, 0. and Freeman, A., Enz. Microb. Technol., 1988, 10, 273-279.

83451-2

BACKGROUND OF THE INVENTION AND PRIOR ART

One of the intrinsic problems often hindering further / development of analytical and synthetic applications of enzymes is the inherent instability of many enzymes. This limitation is crucial in particular in the case of applications involving prolonged exposure of the enzyme to elevated temperatures or to the presence of organic solvents (refs. 1, 2, 3).

Loss of enzymic activities may result from several factors imposing their effect via different mechanisms, of which the structural unfolding of the enzyme is the most abundant (refs. 2,4).

Enzyme stabilization by means of restrictions imposed on the enzyme, limiting its large conformational changes, was in the past attempted by either of two main approaches: rigidification of protein structure by multi-point attachment to water insoluble polymeric supports (refs. 1, 2, 4, 5), or by the addition of various solutes (refs. 6, 7, 8,9).

Several reports in literature describe coupling of water soluble polymers to enzymes, aiming towards the stabilization of enzymes in soluble form (for review see ref. 1). Thus, water soluble enzyme-polymer conjugates, prepared by enzyme coupling to polysaccharides (refs. 1, 10, 11, 12), polyvinylalchohol (ref. 13), polyvinylpyrrolidone (ref. 14) and polymethacrylic acid (ref. 4) were described.

Effective stabilization of soluble enzymes via this approach strongly depends on obtaining an intensive multipoint

2־83451 polymer - enzyme interaction, in a complementary way (ref. 1,4). The effectiveness of such enzyme - polymer conjugates will also depend on the chemical nature of the polymeric moiety, creating a hydrophylic or charged microenvironment in the immediate surrounding of the enzyme. The most effective conjugate of this kind seems to be the chymotrypsin-polymethacrylic acid conjugate, prepared by the copolymerization of methacrylic acid and acryloyl chloride treated enzyme (ref. 4). The resulting poly - anionic enzyme derivative exhibited stability comparable to that of the gel entrapped enzyme analogue, prepared by copolymerization of methacrylamide or acrylamide, with bisacrylamide and acryloyl chloride treated enzyme.

SUMMARY OF THE INVENTION

In accordance with the invention there is provided a stabilized, biologically active water soluble enzyme product, characterized by enzyme molecules having a bi-layer protective structure comprising a polyaldehyde base coat linked to free amino groups of the enzyme, and cross-linked therewith an outer polymer coat, the polymers constituting said outer coat being of a kind which in the unlinked state comprises free amino and/or acyl hydrazide groups.

The invention further provides a method of making a stabilized, biologically active enzyme product of the kind specified, characterized in that in a first step a native enzyme product is reacted in aqueous solution with an excess of a water soluble polyaldehyde to produce an intermediary enzyme product in

83451-=2 which the enzyme molecules are coated with said polyaldehyde base coat, and in a second step said intemediary enzyme product is reacted in aqueous solution with a polymeric reagent bearing free amino and/or free acyl hydrazide groups.

The bi-layer protective structure provided in accordance with the invention will occasionally be referred to hereinaftaer as cage״ and its production as encagement. It provides several advantages over the prior act, such as:

- The cage concept is of a general nature and enables also the stabilization of enzymes bearing low numbers of lysil residues. This is so because the polyaldehyde used in the first step amplifies the total number of crosslinking bridges formed and consequently, as distinct from the prior art (ref. 4) the stabilization degree will not depend solely on the lysil content of the enzyme (ref. 4).

- The encagement method is based on the use of water soluble polymeric reagents, and is thus virtually free of toxic and inhibitory effects which occur when low molecular weight reagents have to be used such as, for example, acryloyl chloride, and acrylamide.

The cage concept allows for control of the microenvironment of the encaged enzyme by suitably modifying or selecting the chemical nature of the polymeric reagent used in the formation of the outer coat. For example, hydrophylicity or hydrophobicity may be introduced into the said polymeric reagent /3 as may be required by the nature of the enzyme or its intended use.

Due to the combined effect of the two constituent cage coats, the stabilization of the enzyme is more effective as compared to single coat enzyme-polymer conj ugates.

The nature of the reactants in said first and second steps is not critical. Thus, in the first step any water soluble polyaldehyde may be used that is capable of reacting with free amino groups forming part of lysil residues of the enzyme, a typical example being polyglutaraldehyde. Likewise, the selection of the water soluble polymeric reagent used in the second step is not critical, provided it bears free amino and/or acyl hydrazide groups, since it is these groups that cross-link with carbonyl groups of the polyaldehyde base coat. Typical examples of polymeric reactants used in the second stage are polyacrylamides, N-alkylated polyacrylamides, acylhydrazide derivatives of polyacrylamides, polyvinyl, hydrazides, polyvinylamides, polylysine, various proteins and the like.

If desired, a stabilized enzyme product with encaged enzyme molecules according to the invention may be further stabilized by, immobilization. As the outer coat of the stabilized enzyme according to the invention is coated with polyacrylamide-amine or acyl hydrazide derivatives, its immobilization, either by chemical binding or crosslinking, is readily available. The latter method is particularly suitable seeing that the stabilized enzyme solution obtained at the end of

51-2 834 ־«־ the second step containes a large excess of unbound polymer which may be employed to form a matrix by appropriate crosslinking. Immobilization of enzymes by means of gel matrices is known per se and was successfully employed for the immobilization of 5 enzymes in gels made of polyacrylamide-hydrazide crosslinked by glyoxal (refs. 21, 25).

It is believed that the stabilization of encaged enzymes according to the invention results from a combination of two effects. For one there occurs physical suppression of the unfolding process by the crosslinked polymeric cage built around the enzyme. Secondly there occurs a chemical suppression of conformational changes due to the microenvironmental effect of the bound hydrophylic polymeric moiety.

A stabilyzed enzyme product according to the invention • 15 may be stored in aqueous solution or be lyophilized into dry powder and stored as such. It has surprisingly been found in accordance with the invention that lyophilization of the encaged enzyme product according to the invention further increases its stability. Furthermore, immobilization of 20 lyophilized encaged enzyme in glyoxal crosslinked polyacrylamide-hydrazide gel results in maximal amplification of this stabilization approach.

The stabilized and optionally immobilized enzyme products according to this invention may be used in a variety of 25 enzyme catalyzed chemical processes, whether carried out batchwise or continuously.

DESCRIPTION OF THE DRAWINGS

I

For better understanding the invention will now be described with reference to the annexed drawings, in which:

Fig. 1 is a diagram showing the stabilization of pig liver esterase by encagement in accordance with the invention expressed by resistance to combined temperature cosolvent denaturation effects by three different solvents, in comparison to the behaviour of native carboxylesterase under similar conditions;

Fig. 2 is a diagram showing the thermal stability of a lyophilized encaged pig liver esterase entrapped in a polyacrylamide-hydrazide gel in accordance with the invention, in comparison to the behaviour of native pig liver esterase under similar conditions; and

Fig. 3 is a schematic illustration of the formation a bi-layer protective cage in accordance with the invention.

DESCRIPTION OF THE INVENTION

The stabilization of encaged enzymes according to the invention in aqueous solution and in the presence of water miscible organic solvents was investigated. Stabilization against denaturation by organic co-solvents is of great practical value as such cosolvents may be employed for the enhancement of substrate and product sollubilities and the reversal of natural reaction routes (refs. 3,21). The stabilization effect was estimated by measuring inactivation-rate constant (Kina) values for native and ״encaged enzymes (lyophilized) incubated at 55°C in the presence of 3.5M cosolvent (about 20X v/v; (ref. 21)). Cosolvents, representing the group exhibiting only mild inteference with the retention of enzyme stability in their presence (e.g. ehylene glycol, DMSO (ref. 21)), as well as cosolvents exhibiting strong denaturating effect (e.g. ethanol) were employed and the stabilization according to the invention was found to be effective with solvents of both groups. In Fig.

the empty circles stand for a stabilized encaged carboxylesterase according to the invention and the blackened circles stand for the native enzyme, and it is clearly seen that the rate of inactivation of the former is much lower than of the latter. This enhanced stability against denaturation by ethylene glycol, DMSO and ethanol allows for a significant increase in the content of the cosolvent present, without impairing the enzymic activity. Stabilized enzyme products according to the invention can thus be used to advantage as catalysts in the performance of reactions in which co-solvents are required from the outset or form in the course of the reaction.

Fig. 2 shows that a lyophilized encaged and immobilized enzyme product according to this invention (empty circles) retained its activity over sixteen days virtually without any change, while that of a native enzyme (blackened circles) dropped sharply during day 1 and from then on approached asymptotically zero activity. In these tests the enzymes were

׳6 incubated at 55°C in O.O5M Tris buffer (pH 8) and the activities were periodically tested.

Fig. 3 illustrates schematically the formation of a bilayer encagement in accordance with the invention. As shown, in a first step an enzyme molecule is reacted with a polyaldehyde to yield an enzyme with a base coat. This product is then reacted with a Polymer 2 being an amino and/or acyl hydrazide groups bearing water soluble polymer, to yield an enzyme within a bi-layered cage in accordance with the invention.

The invention will now be further described in the following examples to which it is not limited. All temperature indications are in centigrades.

Example

Stabilization of glucose oxidase (E.C.1.1.3.4) by bilayer

1. Preparation of polymeric reagents:

(a) Water soluble polymeric glutardialdehyde:

Into a 100 ml round bottomed flask, thermostated at 50°C, 20 ml of IM K₂CO₃ (pH = 10) were added, followed by 20 ml of 25° (w/v) glutardialdehyde (Merck, Cat. No. 4239). The polymerization reaction was allowed to proceed at 50°C for 2 h. The reaction mixture was then cooled to room temperature, the pH brought to 7 by the addition of concentrated HCI and precipitates removed by centrifugation (7000 rpm for 10 min.). The remaining solution was then added into a ten fold volume of acetone to remove K2CO3 and salts by precipitation. The solution was then separated, the acetone evaporated and the remaining solution lyophilized and stored at -20״C (yield: 3.3 g; 66$).

The water soluble polymeric glutaraldehyde thus obtained contained 0.35 mmole aldehyde groups per g (dry weight) determined according to J.S. Thompson and G.D. Shockman (1968) Analytical Biochemistry 22, 260-268) and its MW estimated from gel filtration (Biogel p-6) against protein standards to be 1000.

(b) Preparation of Polyacrylamides;

b-1) Polyacrylamide of MW=8000:

Into a one liter round bottomed flask, equipped with a magnetic stirrer, 680 ml of water were added. The temperature t was brought to 4 C by means of an ice bath and. under nitrogen flushing, 4 g (0.056 mole) of acrylamide monomer were added. Following complete monomer dissolution, 4.6 ml (0.034 mole) of Ν,Ν,Ν,Ν-tetramethylethylenediamine were added, followed immediately by 20 ml of 0.8777 M (4g in 20 ml) ammoniumpersulfate. The polymerization reaction (over ice, under nitrogen atmosphere) was allowed to proceed for one hour.

The polymer solution was then added dropwide into 3.5 1 ice cold methanol, the precipitate separated by filtration, stored overnight at 4°C under methanol, separated and dried on a rotavapour for 30 min at 40°C. The polymer was finally dried by dessication in vacuo over P2O5. The dry polymer was stored in a tightly closed vessel at room temperature (yield 3.2 g (80$)).

-מb-2) Alkylamine - derivative :

Into a 0.25 1 round bottomed flask equifjCd with a magnetic stirrer 100 ml ethyleneglycol were added and the temperature adjusted to 50°C (oil bath). One g of polyacrylamide was added and dissolution was allowed to proceed at 50° overnight. The temperature was then adjusted to 100°C and 13 ml (0.13 mole) 1,4 diaminobutane were added. The aminolysis reaction was allowed to proceed at 100°C for 3 hrs. The solution was then cooled over ice and mixed with 100 ml of ice cold 2N HC1. The pH of this solution was adjusted to 6.3 and the solution dialyzed (four times: against 5 litres of 0.02M phosphate, pH 6.3; 0.02M phosphate, pH 6.3; 0.02M phosphate, pH 6.3; and finally water). The polymer was finally recovered by lyophilization (yield: essentially quantitative, amine content 2 meq/g dry polymer (12.5% conversion, determined titrimetrically according to J.F. Inman and H.M. Dintzis, (1969) Biochemistry 8, 40744082־) and stored at -20°C.

2. Bilayer encagement of glucose oxidase

2-a) Into a 20 ml beaker equipped with a magnetic stirrer and thermostated at 4’C, 9.8 ml of 10 mg/ml solution of polymeric glutaraldehyde in 0.2 M phosphate pH 8.2 were added, followed by 0.2 ml of 50 mg/ml glucose oxidase solution in 0.2 M phosphate pH 6.0. The coupling reaction was allowed to proceed for 3 hrs at 4°C. The non-bound polymer was removed by dialysis (3 times against 0.2M phosphate buffer pH 8).

־142-b) Into a 20 ml beaker equipped with a magnetic stirer and thermostated at 4°C, 16 ml of aminobutyl derivative of polyacrylamide solution (0.25 mg/ml in 0.2M phosphate, pH 8.0) were added, followed by 4 ml of polyglutaraldehyde coated glucose oxidase solution (obtained as described above). The reaction was allowed to proceed for 3 hrs at 4°C and the pH adjusted to 6.0 by dialysis against 0.2M phosphate pH 6.0.

The stabilized enzyme was stored either as solution at 4°C or as a lyophilized powder.

3. Stabilization of glucose oxidase to thermal inactivation:

The stabilization of glucose oxidase to thermal denaturation was demonstrated by measuring the rate of inactivation of the native or stabilized enzyme at a fixed elevated temperature (55°C). The rate constant defined as:

Ki״a ־ - In Mt)/E(°) t where: E(t) = enzymic activity measured at time t

E(o) ^{= tf} ” ^{״ ft M w}o” (for description of this method see ref. 24).

The data are presented in Table 1.

Table Effect of bi-layer encagement on the thermal stability of glucose oxidase

Kina Relative Residual activity after

Enzyme (h,55^־C) Kina (%) 5 h at 55°C (%)

------- -----------------------------------------------------------

<td colspan="2"> native (contol)</td><td> 0.19</td><td> 100</td><td> 43</td>

<td></td><td> polyglutaraldehydetreated</td><td> 0.066</td><td> 35</td><td> 60</td>

<td> 10</td><td> bi-layer (”encaged)</td><td> 0.032</td><td> 17</td><td> 80</td>

4. Stabilization of glucose oxidase to inactivation resulting from the presence of organic cosolvents

The stabilization of glucose oxidase towards the denaturing effect of water miscible organic solvents was demonstrated by measuring the rate of inactivation (Kina) for native and stabilized enzyme at a fixed elevated temperature (55°C) in presence of 3.5 Μ (λ/20$) of cosolvent. The data are presented in table 2.

Table

2: Effect of bi-layer encagement on the tolerance of glucose oxidase to organic sovents

<td> Co-solvent (all at 3.5M)</td><td> Kina(h' Native enzyme (a)</td><td>¹⁰¢) Encaged enzyme (b)</td><td> Relative (b/a,%)</td>

<td> none</td><td> 0.19</td><td> 0.055</td><td> 29</td>

<td> ethyleneglycol</td><td> 0.16</td><td> 0.074</td><td> 46</td>

<td> DMS0</td><td> 0.19</td><td> 0.075</td><td> 40</td>

<td> DMF</td><td> 0.40</td><td> 0.22</td><td> 55</td>

<td> ethanol</td><td> 1.020</td><td> 0.40</td><td> 39</td>

<td> formamide</td><td> 1.74</td><td> 0.50</td><td> 29</td>

Example

Stabilization of pig liver esterase (E.C.3.1.1.1.) by bi-layer

1. preparation of polymeric reagents

Polymeric polyglutaraldehyde and polyacrylamide were prepared as described in Example number 1.

An acylhydrazide-derivative of polyacrylamide was prepared as follows: Into a 100 ml round bottomed flask equipped with a magnetic stirrer and maintained at 50°C, 38 ml of water and 1.5g of polyacrylamide were added. Following complete dissolution 12 ml (0.23 mole) hydrazine hydrate were added and the hydrazinolysis reaction allowed to proceed for 4 hrs. The acylhydrazide derivative thus obtained was separated by precipitation induced by the dropwise addition of the aqueous reaction solution into 250 ml of methanol, recovered by centrifugation, redissolved and reprecipitated as above, stored overnight at 4°C under methanol, separated and dried on a rotavapour (30 min at 40°C) and finally dessicated in vacuo over P^O^. The dry polymer (acyl-hydrazide content: 1.5 mmole/gm, determined according to ref. 19, yield: essentially quantitative) was stored at -20°C.

2. Bilayer encagement of pig-liver esterase

2-a) Into a 50 ml beaker equipped with a magnetic stirrer and thermostated at 419.7 ,0^ס ml of 4 mg/ml polyglutaraldehyde solution in 0.05 M phosphate pH 8.0 were added followed by 0.3 ml of 11 mg/ml enzyme stock solution (Sigma, Cat. No. E-3128). The coupling reaction was allowed to proceed at 4°C for 3 hrs and excess of polyglutaraldehyde was removed by dialysis (three times against 0.05 m phosphate, pH 8.0).

2-b) Into a 100 ml beaker, equipped with a magnetic stirrer and thermostated at 4°C, 41 ml of 5 mg/ml acylhydrazide derivataive of polyacrylamide in 0.05M phosphate, pH 8, were added, followed by 19 ml of polyglutaraldehyde enzyme solution (obtained as described above). The reaction was allowed to proceed at 4°C for 3 hrs and finally dialysed against 0.05 m Tris buffer pH 8. The stabilized enzyme was stored as solution at 4°C or as lyophilized powder.

<img file="IL83451A_D0001.tif" />

3. Stabilization of pig liver esterase to thermal inactivation

The stabilization of pig liver esterase to thermal 1 denaturation was demonstrated by measuring Kina (h ,55’C) as described in Example 1. The data are presented in Table 3.

Table 3: Effect of bilayer encagement on the thermal stability of pig liver esterase.

<td> Enzyme</td><td> Kina (Η^_1,55״Ο</td><td> Relative Kina ($)</td><td> Residual activity after 5 h at 55’C (X)</td>

<td> native (control)</td><td> 0.140</td><td> 100</td><td> 47</td>

<td> polyglutaraldehydetreated</td><td> 0.047</td><td> 34</td><td> 50</td>

<td> bi-layer (encaged)</td><td> 0.032</td><td> 23</td><td> 82</td>

4. Stabilization of pig liver esterase to the presence of water miscible solvents Stabilization to the denaturing effect of water miscible cosolvents present in 3.5 M (»20%) concentration was demonstrated as described in Example 1. The data are presented in Table 4 and they relate to lyophilized encaged enzyme preparations.

Table 4: Effect of bilayer encasement on the tolerance of pig liver esterase to organic solvents.

Kina(h,55°C)

Co-solvent Native enzyme Encaged enzyme (all at 3.5 M) (lyophilized)

<td> none</td><td> 0.14</td><td> 0.00</td>

<td> ethyleneglycol</td><td> 0.24</td><td> 0.00</td>

<td> DMSO</td><td> 0.24</td><td> 0.01</td>

<td> propyleneglycol</td><td> 0.50</td><td> 0.00</td>

<td> ethanol</td><td> 2.95</td><td> 0.093</td>

Example

Stabilization of /?-lactamase (E.C.3.5.2,6) bilayer encagement

1. Preparation of polymeric reagents: polyglutaraldeyde was prepared as described in Example 1. Polyacrylamide, partially substituted with acylhydrazide groups was prepared as described in Example 2.

2. The two stage procedure was carried out essentially as described in Example 2 for pig liver esterase.

3. Stabilization of /?-lactamase to thermal denaturation by bilayer encagement according to the invention was demonstrated by measuring Kina (h”/ 55°C) as described in Example 1. The data are shown in Table 5.

This entire page is postdated July 13, 1988 “8^345172

Table 5: Effect of bi-layer engagement on the thermal stability of /?-lactamase

<td></td><td> Enzyme</td><td> Kina (^¹,55⁰¢)</td><td> Relative Kina (%)</td><td> Residual activity after 5 h at 55°C (%)</td>

<td> 5</td><td> native (control)</td><td> 0.52</td><td> 100</td><td> 10</td>

<td></td><td> polyglutaraldehyde treated</td><td> 0.10</td><td> 19</td><td> 28</td>

<td></td><td> bi-layer (encaged)</td><td> 0.03</td><td> 6</td><td> 70</td>

Example 4

Stabilization of caged enzymes by lyophilization

Lyophilization of caged enzymes resulted in significant further improvement of enzyme stability. This effect is in contrast to the common effect of lyophilization on native 15 enzymes; in many cases their stability declines, in others it remains unchanged.

The effect of lyophilization on caged enzyme stability is demonstrated in Table 6 for pig liver esterase and /?-lactamase.

This entire page is postdated July 13,1988 ' 83451/2

־21-

<td></td><td></td><td> Table 6: Effect</td><td> of lyophilization</td><td> on the thermal stabilitv</td>

<td rowspan="2"> • 5</td><td></td><td colspan="3"> of caged and native enzymes</td>

<td> t Enzyme</td><td></td><td> Kina(h'^ Before lyophilization</td><td> ,55°C) Following lyophilization</td>

<td></td><td> Pig-liver</td><td> esterase-native</td><td> 0.12</td><td> 0.14</td>

<td></td><td></td><td> -caged</td><td> 0.032</td><td> 0.00</td>

<td></td><td colspan="2"> β lactamase - native</td><td> 0.52</td><td> 2.59</td>

<td> 10</td><td> h tr</td><td> - caged</td><td> 0.03</td><td> 0.00</td>

Example Immobilization and stabilization of lyophilized caged enzyme Immobilization procedure

Into a 1.5 g solution of polyacrylhydrazide (MW

100,000) in 8.5 ml of distilled water, 0.5 ml of IM phosphate buffer (pH 8) was added and thoroughly mixed. A buffered solution of a lyophilized encaged pig liver esterase prepared as in Example 2 (150 mg in 1 ml of 0.05M phosphate, pH 8, 30 EU) was then added, thoroughly mixed by magnetic stirring and the solution thus obtained injected (injector outlet 2 mm) into an ice-cold IX glyoxal solution. The gel noodles thus obtained were allowed to harden for one hour and then fragmented into fragments of about 2 mm by pressing through a syringe (outlet diameter 2mm). The gel was then incubated at 4 °C for 20h and 25 further fragmented into particles of about 0.25 mm by means of a blade homogenizer (Sorval Omnimixer, 7000 rpm, 1 min). The gel /2 particles were washed with 11 of ice cold 0.05M Tris buffer (pH

8), resuspended in 50 ml of the same buffer and stored at 4°C p°^‘^ed._q. until used.

July 13, 191 a

The thermal stability - at 55°C - of this preparate is shown in Fig. 2.