IL34291A - Method for improving the stability of enzymes - Google Patents

Description

Method for Improving the stability, of enzymes GRAY INDUSTRIES, INC.

Oi-32581 U.S. 817,181 The invention relates to a method of improving the stability of enzymes and to so-stabilized products.

Enzymes are generally unstable on standing in liquid aqueous medium whether in their natural menstruum (after removal from the living cell source) or after isolation from their natural menstruum and resuspension or re-dissolution (reconstitution) in a prepared aqueous medium. Thus, commercial enzymes, that is enzymes prepared and marketed for industrial, including medical, use, are normally dried, although in some instances certain enzyme preparations can be marketed as concentrated syrups „ Some deterioration of the enzymes can occur by the time complete drying or concentration can be achieved. For ultimate use^ the dehydrated or concentrated enzymes are often reconstituted by suspension or solution in an aqueous medium. This re-constitution affords an opportunity for error particularly where the reconstituted material must have a definite activity value per unit weight or volume. In addition, the reconstituted enzyme is subject to deterioration; the rate and extent depending upon various factors, such as temperature, concentration, time and the nature of the reconstituting medium, such as pHo For example, in automated electronic diagnostic procedures for diagnostic study of body fluids, like blood, blood plasma, blood serum and urinea a control blood serum is v required which should be as close as possible to fresh, natural and normal blood serum. Blood serum, of course, contains many enzymes. In the past, it has been necessary to lyophilize blood serum marketed for this purpose. The technician then had to reconstitute the lyophlllzed material with water for use in his procedures. This presented limitations and disadvantages. Lyophilization itself can upset the delicate balance of constituents of the serum.

Reconstltutlon of the lyophlllzed materials often resulted in serious errors. Often a plurality of different lyophlllzed blood serum products had to be prepared from which the technician had to select one or more depending upon the particular diagnosis he was to perform. Moreover, the reconstituted material had limited shelf life, so that a fresh package of lyophlllzed material normally had to be reconstituted and used daily.

The foregoing exemplifies problems encountered through Instability of enzyme preparations, and has been dis-- cussed at length since blood serum represents a particularly delicate and unstable enzyme-containing material to which the present invention is particularly applicable. However, as will appear hereinafter, the present invention is applicable to enzymes in general, whether in their natural menstruum (after removal from their natural living cell source or environment) or reconstituted.

In accordance with the present invention there is provided a method of improving the stability of an enzyme which comprises cooling the enzyme in aqueous medium to below 50°P., and then* in quick succession, (a) subjecting the enzyme in a treating zone, while held in a closed, micro- 'V wave-permeable container within said treating zone, to microwave energy through a moving atmosphere of coolant gas, for a period short of inactivation of the enzyme a said moving atmosphere being in direct contact with microwave-permeable walls of said container but out of direct contact with said enzyme and being at a temperature below about 60°F. upon its admission to said treating zone; (b) discontinuing exposure of said enzyme to said microwave energy at the end of said period and (c) cooling said enzyme. Preferably, the circula-tion of coolant gas is continued, after discontinuance of exposure of the enzyme to the microwave energy, for a period to provide at least a portion of the stated cooling, Preferably also, the enzyme material is cooled to below 50°F., and at least the latter portion of this cooling may be achieved by other means, as by contacting a container of said enzyme material with a cold liquid, like cold water. It is also preferred that the container of enzyme-material be rotated during the exposure to the microwave energy at a rate to provide at least one complete rotation of 36Ο0 during said exposure, and preferably a plurality of 36Ο0 rotations, to insure presentation of all major walls of the container to the microwave energy during the stated exposure.

The invention also provides a blood serum which possesses improved stability against deterioration as com-pared to similar blood serum not so treated.

It has been found that the foregoing treatment is capable of markedly enhancing the stability of the treated enzymes against deterioration, that is, against permanent loss of activity. It is known that enzymes can become de-activated, even permanently, by more drastic thermal treat- ments, including exposure to microwave energy. The treatment of the present invention, through the effects of pre-coollng and rapid post-cooling, in combination with the effects of the coolant gas during exposure to microwave radiation and the limited extent of the latter, prevents deactivation of the enzyme while at the same time enhancing the stability of the enzyme This is illustrated in the examples set forth hereinafter, and especially so in the example dealing with blood serum.

Stabilization according to the present invention is accomplished rapidly—in a matter of seconds—as well as simply and economically. Conditions can be standardized for any particular enzyme or mixture of enzymes to provide reproducible results from time to time. Thus, each lot or batch can be assayed by techniques specific for such enzyme.

As far as is presently known, any enzyme should be susceptible to improvement in stability according to the present invention. Enzymes are proteins, including metalo-proteins and conjugated proteins, and are produced by living cells. Although there are various classifications of enzymes, one accepted classification is according to Webb, Biochemical Engineering, D. Van Nostrand Co., Inc., Princeton, N.J,,, 1964 (see also Encyclopedia of Chemical Technologya Kirk-Othmar, Second Ed., Vol. 8), as follows: (1) hydrolyzing enzymes -proteases and peptidases, such as pepsin, rennin, and the like; carbohydrases, such as amylases; esterases, such as lipases, phosphatases, and the like; urease; deaminase; etc.; (2) transferring enzymes - dehydrogenases, like lactic dehydrogenase; oxidases; transaminases, like glutamic trans-aminases; kinases, like creatine phosphokinase; (3) addition and subtraction enzymes - like aconitase, enolase, carboxylase and aldolase; (4) isomerases - like alanine racemase; (5) synthetases - like glutamine synthetase; and (6) nucleases - like deoxyribonuclease . As stated, the present invention is particularly applicable to the treatment of enzymes in blood serum which include phosphatases, transaminases, dehydrogenases and phosphokinase . Any one or all of these may be isolated from blood serum and treated separately, after reconstitutlon, if necessary, or blood serum as such may be treated. Blood serum, which is the clear liquid remaining after removing cellular elements (red and white cells and platelets) and the coagulating mechanism (fibrinogen) from whole blood, is one of the particularly preferred materials treated in accordance with the present invention.

The enzyme treated in accordance with the present invention will be in an aqueous medium—that is, it will not be dry. It may be in its natural menstruum with or without concentration or it may be reconstituted by suspension or dissolution in a prepared aqueous medium after isolation from or concentration in its natural menstruum. The amount of aqueous medium associated with the enzyme is not critical, so' long as sufficient is present to wet the enzyme, and may well be dictated by the ultimate use for the product or handling considerations. For example, it is often most con-venient to work with a liquid suspension or solution.

As is well known, microwave energy is the electromagnetic wave energy of the wave length falling in the microwave region of the electromagnetic spectrum. The United States of America Federal Communications Commission has presently set aside, for microwave processing, bands of microwave energy within the range of between about 400 and about 20,000 megacycles per second, with a wave length ranging from about 13 inches for the lower frequencies to about 0.7 inches Tor the highest frequencies; specifically frequencies of about 890-940 with a wave length of about 13 inches; frequencies of about 2400-2500 with a wave length of about 4-5 inches 8.nd frequencies of 17,850-18,000 with a wave length of about 0.7 inch. However, the presently preferred microwave energy for use according to the present invention is an intermediate range having a frequency of from about 1000 to about $000, and more particularly from about 2000 to about 3000, megacycles per second. Microwave energy is generated from a suitable high frequency source, such as a magnetron.

One feature of the present invention is the precool-ing of the enzyme material. Thus, the material at the time it Is first exposed to the microwave energy should be well . ' o below room temperature, that is below about 50 P. While it may actually be frozen, since it will thaw upon exposure to the "microwave energy, there is no need for this and, for ease of handling, it is preferably at a temperature above freezing. A temperature in the range of from about 35 to about 45 TV' is particularly satisfactory. The enzyme material mayTbe pre-cooled outside the treating chamber or zone or it may be pfe-cooled within the treating chamber or zone by preliminary flow of the coolant gas before generation of the microwave energy.

"~" Another feature of the present invention is holding the enzyme material being treated in a sealed container during the treatment. The walls of the container may be conven-tional, substantially gas-Impermeable packaging materials like glass, polymethylmethacrylate, polystyrene and polyethylene, as in bottles, flasks, and pouches. The container will be essentially gas tight.

The container holding the enzyme material during exposure will be held in a larger treating chamber or zone into which the microwave energy is directed to penetrate the microwave-permeable wall of the container and permeate the enzyme.

Still another feature of the invention is the cir-culation of a coolant gas through the treating chamber or zone and around the walls of the container holding the enzyme material. The coolant gas employed may be any substantially inert (non-reactive with the environment in the presence of microwave energy) gas existing as a gas at the temperatures employed, especially air, nitrogen or carbon dioxide. While gases like argon, helium, neon, krypton, xenon, ethylene oxide, and mixtures thereof, and the like, are equivalent^ they are less desirable at the present because of their cost.

The temperature of the coolant gas entering the treating zone should be below about 60°F., and is preferably below about 55° F. While the temperature thereof may go as low as 0°P., there is no advantage in it going below about 20°F. and at such lower temperatures there may be freezing problems if an enzyme material is left in the treating zone containing the cold gas for extended periods after the source of microwave energy has been turned off. A temperature for the incoming gas between about 30 and about 50° F. has been found to be particularly suitable. The coolant gas will become warmed during its travel through the treating zone, particularly from contact with the walls of the container v ® holding the enzyme material, and the warmed gas is removed from the treating zone making way for incoming coolant gas. When the gas is recirculated for reuse, the temperature thereof must be reduced back to the desired temperature for admission to the treating zone.

Since the principal nction of the coolant gas is to keep the walls of the container at a temperature well below that of the enzyme material being treated, forcing the coolant gas into the treating chamber and past the walls of the container under at least some positive pressure (at least slightly above atmospheric pressure) provides more efficient overall cooling without some area or areas of the walls becoming insufficiently cooled. Pressures as low as 0.03 psig. have been used and pressures as high as 50 psig. may be desirable. Air is particularly satisfactory at low positive pressures, whereas a substantially oxygen-free gas, especially nitrogen, is preferred at higher pressures.

The precise time of treatment with microwave energy according to the present invention may depend somewhat upon the particular enzyme being treated, the volume of the enzyme material, the concentration of enzyme in the material being treated and the power of the microwave generating means. In general, the time required is directly proportional to the volume of enzyme-containing material and concentration of enzyme therein and is inversely proportional to the power of the microwave-generating means. It has been found that the exposure time, in any case, will be at least about one second. It has also been found that overexposure results in complete inactivation of the enzyme. Since this is undesirable in accordance with the present invention, the total exposure time will be short of that producing such complete inactivation. Since this time will differ, for reasons stated above, it may be necessary to run a prelimin-ary test or tests to note the extent to which the particular enzyme undergoing treatment can be subjected to the microwave energy without becoming completely inactivated. Each enzyme has its own assay so that it can readily be determined whether or not a treated sample thereof has become completely inactiva-ted. In any event, the time is short of that causing a temperature rise in the enzyme material to the temperature of permanent inactivation of the enzyme; generally the time is not longer than that causing a temperature rise in the enzyme o material above about 125-130 F., and in most cases the tem- o perature rises to from about 100 to about 120 P. The enzyme material may be subjected, according to the present invention, to a single exposure or to a plurality of exposures to the microwave energy.

A feature of the preferred process of the present invention is the presentation of all the major walls of the container holding the enzyme material directly to the microwave energy during the stated exposure. This is most conveniently done, using an upright bottle as. the container, by rotating the bottle about its longitudinal (vertical) axis at least once (360°) , and preferably a plurality of times, during exposure of the bottle to the microwave energy directed toward the bottle in a generally horizontal direction, i.e. in a direction generally normal to the major walls of the container. This presents all major side walls of the container, whether it be circular, square, rectangular or polygonal in cross section, directly to the microwave radiation.

After exposure to the microwave energy for the required period of time, exposure to microwave energy is discontinued and the enzyme material is quickly cooled. It is preferred, in this regard, to continue the cooling effect of the coolant gas after exposure to the microwave energy has been discontinued in order to cool or chill the treated enzyme material, preferably down at least to about 85°Ρ· This can be accomplished by leaving the container of enzyme material to the treating zone, through which is circulated the coolant gas, after the source of microwave energy has been turned off, or, in the case of a continuously moving line of containers of enzyme material, by extending the movement beyond the field of direct microwave exposure while continuing the flow of coolant gas in contact with the containers. On the other hand, the enzyme material may be cooled by other means, supplemental to or instead of, the foregoing means, as by contacting the walls of the container with a cold liquid, like cold water, or by placing the con-tainer in a refrigerator. In any event, it is preferable to cool the treated enzyme material to below 50°P.

The present invention will be more readily understood from a consideration of the following specific examples which are given for the purpose of illustration only and are not to be considered as limiting the scope of the invention in any way.

Example 1 Lactic dehydrogenase ("LDH") was dissolved In a phosphate-buffered aqueous non-serum base (pH about 7.2) to provide 627 units/ml., and the solution was filtered through a bacterial filter. Small sterile glass bottles (7 ml. nominal capacity) were filled with the solution, capped and the bottles were cooled to 40°F. Forty-eight of the bottles were then placed in a pressure chamber equipped with a 2 kw magnetron connected to a 220 volt source of alternating current and capable of delivering microwave energy into the chamber at about 50 megacycles per second. The bottles were divided into groups of three, and each group was held on a small individual turntable. All small individual turntables were held on a larger turntable, so that while the large turntable was rotating at 24 rpm., the small individual turntables were rotating at 60 rpm.; that is, the small individual turntables rotated 2 .5 times for each revolution of the large turntable. The turntable assembly was made of polymethylmethacrylate. Cold nitrogen gas was flowed into, through and out of the chamber, at a pressure of 2.5 psig., its inlet temperature being about 35°P. The magnetron was next turned on for 62 seconds, the magnetron and associated wave guide being positioned to direct the microwave energy in a horizontal direction toward the side walls of the bottles. The maximum temperature reached by the solution o was about 120 p. After the magnetron was turned off (after the 62 seconds exposure), circulation of the cold nitrogen gas was continued for a short time until the solution had _ o returned to about 80 P. Rotation of the turntable assembly was stopped, the chamber was opened and the bottles were removed from the chamber and immediately cooled to 40°P. in an icewater bath. The treated samples, along with untreated controls, were stored at various temperatures and assayed from time to time with the following results: treated material: 40°F.- after 13 days, 583 units/ml. ; after 20 days, 375 units/ml . 70°P.- after 13 days, 497 units/ml. ; after 20 days, 375 units/ml . 100°F.- after 13 days, 520 units/ml.; after 20 days, 400 units/ml. control: 40°F.-"after 1 week, 315 units/ml.; after 13 days, 0. 70°P.- after 1 week, 0. 100 F.- after 48 hours, 0.

Example 2 Serum glutamic oxalacetic transaminase ("SGOT") was dissolved in a phosphate-buffered aqueous non-serum base (pH about 7.2) to provide 37 units/ml., and filtered through a bacterial filter. The solution was bottled, treated, stored and assayed as in Example 1 with the following results: treated material: 40°F.- after 13 days, 2(5.7 units/ml.; after 20 days, 20 units/ml. 70°P.- after 13 days, 13 units/ml. ; after 20 days, 10 units/ml. 100°F.- after 13 days, 16 units/ml . i . after 20 days, 3 units/ml. control: 40°P*. - after 1 week, 0. 70°P." - after 4 days, 0.

IOOV. - after 48 hours, 0.

Example 3 Five milligrams of deoxyribonuclease, from bovine pancreas, were reconstituted with 1 ml. of distilled water and then diluted to 800 ml. with distilled water. The solution was s filtered through a bacterial filter. The starting potency after filtration was 786 units/mg. Small sterile glass bottles (Tml. nominal capacity) were filled with the solution, capped and cooled to 40°F. Various samples (24 bottles per lot) were then treated as in Example 1 but for different exposure times as follows: Sample Exposure time Peak temperature of sample (seconds) A 60 about 112°P.

B 68 about 115°F.

C 7 about 122°F.

Upon removal from the chamber at about 80°P. and cooling to 40°P. in an icewater bath, the treated samples, along with untreated controls, were stored at 77°P. for 21 days. All samples were then assayed by optical density in a Beckman DU spectrophotometer with ultraviolet range wavelength using the assay method according to Kunitz, M., J. Gen. Physiol.. 33* 349 (1950) .

The results were as follows: Sample Assay* (units/mg. ) A 475 ""*"" B 520 C 450 Control 200 ♦Averages of two bottles from each treatment, three separate samples from each bottle.

Example 4 Liver fraction lactic dehydrogenase isoenzyme, extracted from bovine heart, was dissolved in sterile distilled water to 1 unit/ml., and the solution was filtered through a V capacity) were filled with the solution, capped and cooled to 40°P. Forty-eight of the bottles were then exposed to microwave energy in the presence of cold flowing nitrogen gas as in Example 1 but for an exposure time of 56 seconds. After removal from the chamber at a temperature of about 80°F., and cooling to 40°F., the treated samples, along with untreated controls, were stored* at room temperature. The controls became completely inactive on standing overnight, whereas the treated material was 'as active as the original after standing for three weeks At the end of three months, there was less than 10 loss in activity in the treated material.

Example 5 Heart "fraction lactic dehydrogenase Isoenzyme, extracted from rabbltf muscle, was dissolved in sterile dis-tilled water to 1' unit/ml., and the solution was filtered through a bacterial filter. After bottling and capping, the material was treated" and then stored as in Example 4. The controls became completely inactive on standing overnight, whereas the treated" material was as active as the original after standing for"'three weeks. At the end of three months, there was less than 10$ loss in activity in the treated materials . * "· ■"■ Example 6 An aqueous' solution of amylase (1000 units/ml.) was prepared and filtered "through a bacterial filter, and 10 ml. portions thereof were placed in six small sterile glass bottles (10 ml. nominal capacity) which were then capped. The six bottles were then cooled to 40°F., and three of them were placed in a pressure chamber equipped with a 2 w magnetron connected to a 220 ' volt 'source of alternating current and capable of delivering microwave energy into the chamber at about 2450 megacycles per second. Each bottle was held on a small individual turntable, each of which was held on a larger turntable, so while the large turntable was rotating the small individual turntables, and the bottles thereon, were also rotating. The turntable assembly was made of polymethylmethacrylate. The large turntable and the small individual turntables were rotated at 30 rmp. Cold nitrogen gas was then flowed into, through and out of the chamber, at a pressure in the chamber of 2.5 psig., its inlet tempera- o c ture being about 35 F« The magnetron was next turned on for 7 seconds, the magnetron and associated waveguide being positioned to direct ¾he microwave energy in a horizontal direction toward the side' alls of the bottles. After the magnetron was turned o'ff" (after the 7 seconds exposure) circulation of the cold" nitrogen was continued for a short time until the amylase solution in the exposed bottles had returned to about 800FT " The turntable assembly was stopped, and the bottles were removed from the chamber, immediately cooled to 40°F. in an icewater bath and, along with the three untreated control bottles, rheld at 40°F. for 48 hours. All samples were then assayed for amylase activity with the following results (figures are averages): 'treated material 800 units/ml. control 200 units/ml.

""Exam le 7 Fresh human blood serum was filtered through a bacterial filter and pla ed' n sterile glass bottles, 30 ml. per bottle, and the bottles were capped and cooled to 40°F. Three of the bottles were then exposed to microwave energy In the presence of flowing nitrogen gas under pressure as in Example 6 but for 15 seconds. Upon removal from the chamber at about 72-75°P.5 the bottles were immediately cooled to 40°P. in an icewater bath. The bottles, along with untreated controls, were then stored at 40°F., and assayed from time to time. By the end of two weeks, the biochemical assay values of the controls had changed so that they were no longer within the normal ranges for the enzymes, The treated material, upon continued storage at 40°F.f was assayed, from time to time over a period of nine months with the following results: Table 1 Values after designated days Constituent Days : 30 90 150 210 270 Upper Limit 145 143 142 142.5 142 Sodium Mean 141.8 140.8 140.9 l4l 141.5 Lower Limit 140 139 139.5 139.7 , 139 Upper Limit 4.3 4.4 4.3 4.4 4.4 Potassium Mean 4.0 4.0 4.0 4.3 Lower Limit 3.9 3.8 3.92 3.94 3.8 Upper Limit 101 100.4 102 100 100 Glucose Mean 98.8 99.0 98.8 97.5 97.5 Lower Limit 9Cj.,5 93.6 93.5 93.2 91.4 Upper Limit 34. I 34.5 35.0 34.Ο 34.1 Alkaline Mean 32.9 33.2 33.4 33.0 33.0 phosphatase Lower Limit 30.0 30.2 31.0 : » , . ' 30.9 30.5 Upper Limit 27.Ο 25.7 25.0 24.0 23.5 SOOT* Mean 25. I 24.5 24.0 ,23.8 22.8 Lower Limit 22.5 21.8 21.0 20.0 20.0 Upper Limit 21.0 20.7 20.0 20.0 19.0 SQPT* Mean 20.2 19.0 19.2 I9.O I0.7 Lower Limit I9. 1 .4 18.3 18.0 17.8 Upper Limit 210 204 204 201 198 LDH* (total ) Mean 200 I96 196 I96 190 Lower Limit I90 I92.2 192 I90 188 LDH fracUpper Limit 23.4 22.6 22.8 22.1 22.5 tionation Mean 22.5 21.3 21.4 21.2 Isoenzymes Lower Limit 21.2 20.4 20.0 19.4 I9.O liver fraction (LD¾ ) heart Upper Limit 46.5 45.8 44.0 43.6 43. I fraction Mean 44.6 44.0 43.2 42.9 42.6 (LDH5 ) Lower Limit 40.8 41.8 41.0 40.2 4o.3 remaining Upper Limit 134 134 134 I32 132 fractions Mean 132.5 132.4 132 I3I.4 130.2 (LD¾> 3 j ) Lower Limit 130.6 130.2 129. I29.2 128 - Up er Limit 68 67 65 66.5 65 Amylase Mean 65 64 63.6 63.7 62.6 Lower Limit 62.5 61.8 61.3 61.6 61.4 Creatine Upper Limit 6.1 5.8 5.7 *# #* phospho- Mean 5.3 Jp.O kinase Lower Limit 4.8 4.5 4.2 (C. P. K. ) Upper Limit 7.5 7.5 7.5 7.5 7.4 Total Mean 7.2 7.4 7.3 7.3 7.3 Protein Lower Limit 6.9 7.0 7.1 7.P , 7.0 Table 1 (continued) Values after designated days Constituent Days; 30 90 150 210 270 Upper Limit 4.00 4.25 4.12 4.19 4.11 Albumin Mean 3.82 3.87 3.90 3.87 3.92 Lower Limit 3.42 3.44 3.56 3.51 3.70 Upper Limit 3.00 3.02 3.00 2.94 2.98 Globulin Mean 2.80 2.90 2.88 2.91 2.90 Lower Limit 2.55 2.66 2.61 2.60 2.70 *SG0T is serum glutamic oxala'cetic transaminase SGPT is serum glutamic pyruvic transaminase LDH is lactic dehydrogenase ** no further assays made The treated material showed normal electrophoresis throughout the testing period.

Normal values for these constituents are as follows: Table 2 Constituent Normal values Sodium 135 - 145 meq./l.

Potassium 3.5 - 5.0 meq./l.

Glucose 6 - 110 mg# Alkaline phosphatase 5 - 35 units (International SOOT 5 - 40 units [International SOPT 5 - 35 units (international LDH (total) 150 - 500 units (International) LDH liver fraction 10 - 20$ of. total LDH heart fraction 20 - 4056 of total LDH remaining fractions 35 - 55# of total Amylase 50 - 160 units (Diatase) Creatine phosphokinase up to 35 units (inter- . national) Total protein 6. 8 - 8.0 mg.# Albumin 3.25 - 5.70 gm.# Globulin 1.5 - 3.0 gm.# Considerable modification is possible in the enzyme materials treated as well as in the particular techniques employed without departing from the scope of the invention.

Claims (13)

U.S. 817,181

1. A method of improving the stability of an enzyme which comprises cooling the enzyme in aqueous medium to below 50°P., and then, in quick succession, (a) subjecting the enzyme in a treating zone, while held in a closed, microwave-permeable container within said treating zone, to microwave energy through a moving atmosphere of coolant gas, for a period short of inactivation of the enzyme, said moving atmosphere being in direct contact with microwave-permeable walls of said container but out of direct contact with said enzyme and being at a temperature below about 60° P. upon its admission to said treating zone; (b) discontinuing exposure of said enzyme to said microwave energy at the end of said period and (c) cooling said enzyme.

2. A method according to claim 1, wherein said microwave energy has a frequency of from about 1000 to about 5000 megacycles per second.

3. A method according to claim 2, wherein said microwave energy has a frequency of from about 2000 to about 3000 megacycles per second.

4. A method according to claim 3* wherein said microwave energy has a frequency of from about 2400 to about 2500 megacycles per second.

5. A method according to any one of the preceding claims, wherein after exposure to the microwave energy the treated enzyme in the container is continued to be subjected to the action of said coolant gas.

6. A method according to any one of the preceding claims, wherein said enzyme in aqueous medium is at least one of those in blood serum.

7. A method according to any one of claims 1-5* wherein the enzyme in aqueous medium is blood serum.

8. A method according to claim 7, wherein the blood serum is cooled to a temperature of from about 35 to about 45°P. before exposure to said microwave energy.

9. A method according to claim 7 or 8, wherein the blood serum does not exceed a temperature of about 125°F. during said exposure to said microwave energy.

10. A method according to any one of claims 7-9* wherein, after discontinuing the exposure of said blood serum to said microwave energy, said blood serum is cooled to below about 50°P.

11. A method according to any one of the preceding claims, wherein substantially all of the major microwave-permeable walls of said container are presented directly to said microwave energy during said exposure.

12. . Blood serum treated in accordance with the method of any one of claims 7-11 and in which enzymes thereof possess improved stability against deterioration as compared to similar blood serum not so treated.

13. A method of improving the stability of an enzyme substantially as herein described with reference to the Examples.