TEMPERATURE CHANGE INDICATOR EMPLOYING ENZYMES

Abstract: Enzyme reactions may be carried out in a non-aqueous medium comprising a C12-C20 aliphatic alcohol such as lauryl alcohol. The medium may be a mixture of such an alcohol with a C6-C30 aliphatic hydrocarbon such as hexane or decane. The principle may be used to produce a temperature change indicator, comprising a solid non-aqueous medium comprising a C12-C20 aliphatic alcohol such as lauryl alcohol and a C6-C30 aliphatic hydrocarbon such as hexane or decane, together with an enzyme, a substrate for the enzyme, and a development reagent capable of producing a detectable change, on reaction of the substrate with the enzyme. The medium is chosen to melt at a specific temperature, or in a specific temperature range, to permit reaction to occur between the enzyme, the substrate, and the development reagent. Preferably, the enzyme is mushroom phenol oxidase, the enzyme substrate is o-dihydroxyphenol, and the development reagent is an ester of an amino acid with a C4-C20 alcohol, for example the butyl ester of proline.

Description: TEMPERATURE CHANGE INDICATOR EMPLOYING ENZYMES This invention relates to enzymatic reactions, and in particular to the carrying out of enzymatic reaction in non-aqueous solvents. It is known that enzymatic reactions may be carried out in non-aqueous solvents, for example in liquid and solid paraffins. Examples of such reactions are disclosed, for example, in US Patent 4826762, and in an Article by Klibanof et al (Biotechnology Volume 4, November 1986, page 997). These references also disclose the application of such reactions, in the preparation of enzyme-based temperature abuse sensors. Although paraffins are useful for certain types of enzyme reactions, they have a number of practical disadvantages. In particular, because of their highly non-polar nature, they are very poor solvents for a number of materials which one might otherwise wish to employ in enzyme reactions, for example, certain enzyme substrates or development reagents. We have now discovered that enzyme reactions may be carried out in a non-aqueous reaction medium, comprising an aliphatic alcohol, containing at least twelve carbon atoms. It has also been discovered that the use of such a reaction ..nedium can enable certain enzyme reactions to be carried out more advantageously than is possible in other solvent systems, and/or using reagents which would not otherwise be possible. Accordingly, in a first embodiment, the invention provides a method of carrying out an enzyme reaction in a non-aqueous medium, characterised in that the medium comprises an aliphatic alcohol containing at least twelve carbon atoms. The fact that an aliphatic alcohol may be employed as or as part of the reaction medium is a surprising one, since it is well known that low molecular weight alcohols rapidly lead to deactivation of most enzymes. We have discovered however that by employing an aliphatic alcohol containing twelve carbon atoms or more, substantial enzyme deactivation may be avoided, whilst at the same time desirable properties are conferred upon the reaction medium, for example the ability to solubilise various components of the enzyme reaction. The term "aliphatic alcohol" as used herein is intended to mean any alcohol having at least twelve carbon atoms, in which the alcoholic moiety is not attached directly to an aromatic ring, and includes within its scope alcohols which contain an aromatic structure, for example benzene ring, provided that the hydroxyl group is not phenolic. The method of the invention finds particular application in the production of indicators which may be employed to measure historical changes in temperature, and has particular application to indicators which can be used to monitor whether a particular items have exceeded a specified temperature for a specified period, during for example, storage or transportation. Indicators of this general kind, are disclosed in US Patent 4826762. The indicators disclosed in this US Patent comprise an enzyme together with a substrate for the enzyme disposed in a medium which is solid below the temperature which it is desired to indicate, but which melts at or around the said temperature. When the temperature of the indicator is permitted to rise above the melting point of the medium, the enzyme reacts with the substrate. As a result of this reaction a modification of the substrate occurs such that the colour of the medium changes. A particular enzyme of choice in the said US patent is horseradish peroxidase, which is employed with hydrogen peroxide and p-anisidine as substrate. A colour change is produced as a result of the reaction of the substrate with horseradish peroxidase enzyme. The possible use of mushroom polyphenol oxidase is also mentioned, but only in connection with p-anisidine as a substrate. p-anisidine is, for practical purposes, not an effective substrate for mushroom polyphenol oxidase, and the reference in US 4826762 to mushroom polyphenol oxidase can only be seen therefore as a speculative one, without any practical utility. The system described in US 4826762 suffers from a number of substantial disadvantages. Chief among these is the fact that the choice of substrates and colour forming agents which it is possible to employ is severely limited because of the hydrophobic nature of the long chain alkanes employed. Furthermore, in practical terms, virtually all of the reagent combinations which would dissolve in a medium consisting of aliphatic hydrocarbon solvents include at least one member which is hazardous to health. For example, the material p-anisidine, which is employed in US Patent 4826762 is thought to be a carcinogen. In accordance with the present invention enzyme reactions can be carried out in a medium comprising substantial amounts of an aliphatic alcohol containing at least twelve carbon atoms preferably and C ] _2~ C 20 aϋP 1"13" ^ 0 alcohol. Such materials may be incorporated into a solid medium used as the support medium for a temperature change indicator incorporating mushroom phenol oxidase, and o- dihydroxyphenol as an enzyme substrate. The use of high molecular weight alcohols as part of the support medium for such enzyme reactions permits alternative development reagents to be used for the enzyme reaction, which are non-carcinogenic, and also have a number of advantageous properties. We have also discovered that the presence of long chain aliphatic alcohols in the reaction medium, as described above, can, with certain colour development reagents, lead to a two-stage change in colour development, which permits the production of a temperature sensor able to indicate for how long the exposure to specific temperature has occurred. Accordingly, in a preferred aspect of the invention, there is provided a temperature change indicator, comprising a solid medium, containing an enzyme, a substrate for the enzyme, and a development reagent capable of producing a detectable change on reaction of the substrate with the enzyme, wherein the said medium is chosen to melt at a specific temperature, or in a specific temperature range, to permit reaction to occur between the enzyme, the substrate, and the development reagent, characterised in that the solid medium comprises an aliphatic alcohol comprising at least twelve carbon atoms, preferably a -^-C^g aliphatic alcohol, for example lauryl alcohol, the development reagent is a material compatible with the solid medium, and is preferably one capable of reacting to produce a coloured product with a material produced on reaction of the enzyme with the enzyme substrate. The enzyme employed in accordance with the aspect of the present invention is preferably mushroom phenol oxidase. This material is widely available as a waste product in mushroom culture, and may conveniently be employed in the form of a mushroom extract obtained from low grade mushroom waste, although if desired the purified enzyme may alternatively be employed. The enzyme substrate for mushroom phenol oxidase is preferably o-dihydroxyphenol, and the development reagent is preferably one which is capable of producing a two-stage colour change, on prolonged exposure to the oxidation products of o-dihydroxyphenol, in a medium comprising a long chain aliphatic alcohol. It has been found that amino acid esters, preferably esters of amino acids such as cysteine and, particularly preferably, proline with a C Λ - C 2 g alcohol, and in particular the butyl ester, are particularly suitable. The reaction medium preferably comprises at least 10%, more preferably at least 20%, more preferably still at least 40%, most preferably at least 50% by weight, of the long chain aliphatic alcohol, although the level chosen will depend upon the particular application and, in the case of the temperature indicator, on the desired melting point. For use in the temperature indicator in accordance with the invention, the solid medium preferably comprises a ' mixture of a 2~ c 20 aliphatic alcohol, with a suitable .lydrocarbon, for example, a Cg-Cgg hydrocarbon, preferably hexane. The medium preferably consists only or consists essentially of the said two materials, although, if desired, other materials which do not adversely affect the melting point properties may be incorporated. A medium having any desired melting point (and thus any desired colour change temperature) may be formulated by appropriate mixing of different long chain aliphatic alcohols with other suitable materials of varying melting points, and in particular with aliphatic hydrocarbons. The solid medium employed in the temperature preferably comprises from 10 to 75 percent by weight of the long chain aliphatic alcohol, and from 25 to 90 percent by weight of aliphatic hydrocarbon. In particular a mixture of 75 percent by weight lauryl alcohol and 25 percent by weight hexane is found to have a melting point of approximately 15°C. The oxidation of o-dihydroxyphenol by mushroom phenol oxidase produces an intermediate quinone structure which reacts with a number of materials, particularly esters of amino acids, to produce a coloured material typically of a deep red colour. The use of amino acid esters is particularly desirable because of their low toxicity. Aromatic amines, for example, naphthylamine and the like may be also employed, although these are not preferred, as many of them present a health hazard. Temperature change indicators in accordance with the present invention can be produced in the form of a laminated strip, or in any other desired form, as is appropriate for the particular application. Such laminated strips may preferably include a removable oxygen- impermeable cover layer, effective to inactivate the temperature indicator, by the exclusion of oxygen, until a desired time of activation, when the removable oxygen impermeable strip is detached. In a particularly preferred embodiment, a temperature change indicator may comprise the following layers: a) an impermeable base layer, b) a temperature sensitive layer comprising the aliphatic alcohol and the enzyme, c) an oxygen-permeable protective cover layer, and d) a removable oxygen-impermeable film. In an alternative embodiment, the indicator may comprise a bubble pack having an oxygen-impermeable element which may be ruptured so as to enable ingress of oxygen into a compartment containing the enzyme and substrate dispersed in the solid medium. Temperature change indicators in accordance with the invention may be attached to, for example, a food item, drug packaging, vaccines, photographic materials (eg., photographic films or the like), or to anything else where it is desired to have an historical record of whether an item has exceeded a particular temperature during its history. Using the particular combination described above, a temperature change indicator may be produced which has a two-stage indication process, for example employing butyl esters of amino acids as development reagents. When such materials are employed, a first colour change, typically from colourless to red, take place after a relatively short exposure to a temperature above the melting point of the medium (typically one to two minutes), and a subsequent colour change, typically from red to yellow, takes place after an extended exposure to a temperature in excess of the melting point. The time taken for the second colour change will depend upon the particular materials employed, and the amount by which the temperature exceeds the melting point of the medium. However, a time of twenty-five minutes is typical. The time needed for the second colour change to take place can be affected by, for example, the amounts of enzyme or colour development reagent included in the formulation. An alternative application for the method of the invention is in the carrying out of enzyme reaction in circumstances in which, for some reason, it is desirable not to have water present, and hitherto proposed solvents, such as paraffins, are for some reason unsuitable. Examples of such reactions are preparative reactions, in which the presence of water would have an adverse effect on the position of the reaction equilibrium, and the reactants are substances of transitional polarity, such as esters of amino acids. In particular, the method of the invention may be utilised in the preparation of esters such as dopa esters, by the action of hydrolytic enzymes such as chymotrypsin or lipase, on a tyrosine ester. Tyrosine is an example of a material which is poorly soluble in both water and paraffins, and therefore the preparative reaction cannot readily be carried out in either of those solvents. Furthermore, the presence of water tends to hydrolyse the esters produced. When the method of the invention is used preparatively, a desired product is generally isolated after the enzyme reaction has taken place in the specified medium. A number of preferred embodiments of the invention are described in the following examples. Example 1 Various mixtures were formed of lauryl alcohol and hexane. The mixtures had the compositions and melting points shown in Table 1. TABLE I EXAMPLE lauryl alcohol wt % n-hexane wt% M. Pt.(C°) I 25 75 3 II 50 50 6 III 75 25 15 Into a 5ml sample of each of the mixtures shown in Table I was incorporated ImM o-dihydroxyphenol, 1.5mM proline butyl ester, 0.02ml of mushroom extract containing 5mg/ml mushroom phenol oxidase, and lOO M phosphate buffer (pH 6.0). The mixing was carried out in the following way: All of the components of each mixture except mushroom extract, were mixed together at a temperature just above the melting point of the respective mixture. Each mixture was then cooled to a temperature below its melting point and the mushroom extract was added. The mixtures obtained were then stored at a temperature below their respective melting points. All of the compositions produced showed no colour change when maintained below their respective melting points for a period of 48 hours. Upon warming to a temperature above the melting point however, a red coloration appeared within a period of 1 to 2 minutes, and when allowed to stand at this temperature for a further period of 25 minutes, the red colour changed to a yellow colour. Example 2 Example 1 was repeated, except that naphthylamine (ImM) was substituted for the 1.5mM of proline butyl ester. The temperature indicators produced again remained colourless, when stored for extended periods below their respective melting points. When the mixtures were warmed to a temperature above their melting points, a purple colour developed in each case within a period of from one to three minutes. No further colour change was observed, when the indicators were maintained at this temperature for an extended period. Example 3 Example 1 was repeated except that n-decane was substituted for hexane as the hydrocarbon component of the medium. The mixtures had the compositions and melting points shown in Table 2. TABLE 2 EXAMPLE lauryl alcohol wt % n-hexane wt% M. Pt.(C°) IV 50 50 13 V 75 25 17 It will of course be appreciated that various other specific compositions are contemplated, within the scope of the appended Claims.

Claims: CLAIMS 1. A method of carrying out an enzyme reaction in a non-aqueous medium, characterised in that the medium comprises an aliphatic alcohol containing at least 12 carbon atoms. 2. A method as claimed in Claim 1, wherein the aliphatic alcohol is a a l c °h°l• 3. A method as claimed in Claim 2, wherein the ^i2~ C ' 20 alcohol is lauryl alcohol. 4. A method as claimed in any one of the preceding claims, wherein the reaction medium also comprises a g-Cgg aliphatic hydrocarbon. 5. A method as claimed in Claim 4, wherein the Cg-C βQ aliphatic hydrocarbon is hexane. 6. A method as claimed in any one of the preceding Claims, wherein the enzyme is mushroom phenol oxidase. 7. A method as claimed in Claim 6, wherein the substrate upon which the enzyme acts is o-dihydroxyphenol. 8. A method as claimed in any one of the preceding Claims, wherein the reaction is such as to produce a visible colour change when the reaction medium exceeds a predetermined threshold temperature. 9. A method as claimed in any one of Claims 1 to 7, wherein the method includes the further step of isolating a desired product. 10. A temperature change indicator, comprising a solid non-aqueous medium containing an enzyme, a substrate for the enzyme, and a development reagent capable of producing a detectable change, on reaction of the substrate with the enzyme, wherein the said medium is chosen to melt at a specific temperature, or in a specific temperature range, to permit reaction to occur between the enzyme, the substrate, and the development reagent, characterised in that the solid medium comprises an aliphatic alcohol containing at least 12 carbon atoms, and the development reagent is a material compatible with the solid aliphatic alcohol-containing medium. 11. An indicator as claimed in Claim 1, wherein the enzyme is mushroom phenol oxidase, and the enzyme substrate is o-dihydroxyphenol. 12. An indicator as claimed in Claim 10 or Claim 11, wherein the solid medium is as defined in any one of Claims 2 to 5. 13. An indicator as claimed in any one of Claims 10 to 12, wherein the development reagent is a material capable of reacting so as to produce a coloured product with a material produced on reaction of the enzyme with the enzyme substrate. 14. An indicator as claimed in Claim 13, wherein the development reagent is an amino acid ester. 15. An indicator as claimed in Claim 3, wherein the development reagent is an ester of an amino acid with a C 4 -C 20 alcoho1 - 16. An indicator as claimed in Claim 5, wherein the development reagent is the butyl ester of proline. 17. The use of a non-aqueous medium comprising an aliphatic alcohol having at least 12 carbon atoms as a reaction medium for an enzyme reaction. 18. A method of producing a temperature change indicator, which comprises incorporating into a solid non- aqueous medium having a melting point corresponding to a desired temperature of measurement an enzyme, an enzyme substrate, and a development reagent, characterised in that the a) the enzyme is mushroom phenol oxidase b) the enzyme substrate is o-dihydroxyphenol, c) the solid medium comprises a C i 2~ < - ' 20 aliphatic alcohol, and, d) the development reagent is a material compatible with the solid aliphatic alcohol-containing medium.

Inventors: KOZLOV IGOR ANATOLIEVICH, PARTON ADRIAN, KOZLOV, IGOR, ANATOLIEVICH, PARTON, ADRIAN
Applicants: RAYBOURNE LIMITED
Application year: 1991
Main classification: G01N31
IPC codes: C12Q1, G01N31