COMPOUNDS STABILIZING HYDROLASES IN LIQUIDS

Abstract

Described herein is an enzyme preparation including component (a): at least one compound according to general formula (I)

##STR00001## wherein R.sup.1 is; R.sup.2, R.sup.3, R.sup.4 are independently from each other selected from the group consisting of H, linear C.sub.1-C.sub.8 alkyl, and branched C.sub.3-C.sub.8 alkyl, C.sub.6-C.sub.10-aryl, non-substituted or substituted with one or more carboxylate or hydroxyl groups, and C.sub.6-C.sub.10-aryl-alkyl, wherein an alkyl of the C.sub.6-C.sub.10-aryl-alkyl is selected from the group consisting of linear C.sub.1-C.sub.8 alkyl and branched C.sub.3-C.sub.8 alkyl, wherein at least one of R.sup.2, R.sup.3, and R.sup.4 is not H, component (b): at least one enzyme selected from the group consisting of hydrolases (EC 3); and optionally component (c): at least one compound selected from the group consisting of solvents, enzyme stabilizers different from component (a), and compounds stabilizing the enzyme preparation.

Claims

1. An enzyme preparation comprising component (a): at least one compound according to general formula (I) ##STR00018## wherein the variables in formula (I) are defined as follows: R.sup.1 is H; R.sup.2, R.sup.3, R.sup.4 are independently from each other selected from the group consisting of H, linear C.sub.1-C.sub.8 alkyl, and branched C.sub.3-C.sub.8 alkyl, C.sub.6-C.sub.10-aryl, non-substituted or substituted with one or more carboxylate or hydroxyl groups, and C.sub.6-C.sub.10-aryl-alkyl, wherein an alkyl of the C.sub.6-C.sub.10-aryl-alkyl is selected from the group consisting of linear C.sub.1-C.sub.8 alkyl and branched C.sub.3-C.sub.8 alkyl, wherein at least one of R.sup.2, R.sup.3, and R.sup.4 is not H; component (b): at least one enzyme selected from the group consisting of proteases, and optionally component (c): a compound selected from the group consisting of at least one solvent, at least one enzyme stabilizer different from component (a), and at least one compound stabilizing the enzyme preparation.

2. The enzyme preparation according to claim 1, wherein said enzyme preparation comprises component (a) in amounts in a range of 0.1 to 30% by weight relative to a total weight of the enzyme preparation.

3. The enzyme preparation according to claim 1, characterized in that the at least one enzyme comprised in component (b) is stabilized when compared to an enzyme preparation lacking component (a).

4. A process for making a stable enzyme preparation, said process comprising the steps of mixing at least component (a): at least one compound according to general formula (I) ##STR00019## wherein the variables in formula (I) are defined as follows: R.sup.1 is H; R.sup.2, R.sup.3, R.sup.4 are independently from each other selected from the group consisting of H, linear C.sub.1-C.sub.8 alkyl, and branched C.sub.3-C.sub.8 alkyl, C.sub.6-C.sub.10-aryl, non-substituted or substituted with one or more carboxylate or hydroxyl groups, and C.sub.6-C.sub.10-aryl-alkyl, wherein an alkyl of the C.sub.6-C.sub.10-aryl-alkyl is selected from the group consisting of linear C.sub.1-C.sub.8 alkyl and branched C.sub.3-C.sub.8 alkyl, wherein at least one of R.sup.2, R.sup.3, and R.sup.4 is not H, component (b): at least one enzyme selected from the group consisting of proteases, and optionally component (c): a compound selected from the group consisting of at least one solvent, at least one enzyme stabilizer different from component (a), and at least one compound stabilizing the enzyme preparation.

5. A method of reducing loss of proteolytic activity of at least one protease comprised in a liquid enzyme preparation during storage, the method comprising the step of adding to the liquid enzyme preparation at least one compound according to formula (I): ##STR00020## wherein the variables in formula (I) are defined as follows: R.sup.1 is H; R.sup.2, R.sup.3, R.sup.4 are independently from each other selected from the group consisting of H, linear C.sub.1-C.sub.8 alkyl, and branched C.sub.3-C.sub.8 alkyl, C.sub.6-C.sub.10-aryl, non-substituted or substituted with one or more carboxylate or hydroxyl groups, and C.sub.6-C.sub.10-aryl-alkyl, wherein an alkyl of the C.sub.6-C.sub.10-aryl-alkyl is selected from the group consisting of linear C.sub.1-C.sub.8 alkyl and branched C.sub.3-C.sub.8 alkyl, wherein at least one of R.sup.2, R.sup.3, and R.sup.4 is not H.

6. A method of using a compound according to formula (I): ##STR00021## wherein the variables in formula (I) are defined as follows: R.sup.1 is H; R.sup.2, R.sup.3, R.sup.4 are independently from each other selected from the group consisting of H, linear C.sub.1-C.sub.8 alkyl, and branched C.sub.3-C.sub.8 alkyl, C.sub.6-C.sub.10-aryl, non-substituted or substituted with one or more carboxylate or hydroxyl groups, and C.sub.6-C.sub.1-aryl-alkyl, wherein an alkyl of the C.sub.6-C.sub.10-aryl-alkyl is selected from linear C.sub.1-C.sub.8 alkyl and branched C.sub.3-C.sub.8 alkyl, wherein at least one of R.sup.2, R.sup.3, and R.sup.4 is not H, the method comprising using the compound according to formula (I) as an additive for at least one protease, wherein the compound according to formula (I) and the protease are solid and wherein proteolytic activity of the protease is stabilized when the compound according to formula (I) and the protease are contacted with at least one solvent [component (c)].

7. A method of using the enzyme preparation of claim 1 to formulate detergent formulations, the method comprising mixing the enzyme preparation in one or more steps with one or more detergent components.

8. A detergent formulation comprising the enzyme preparation of claim 1 and at least one detergent component.

9. A method of preparing a detergent formulation comprising the steps of mixing at least component (a): at least one compound according to general formula (I) ##STR00022## wherein the variables of formula (i) are as follows: R.sup.1 is H; R.sup.2, R.sup.3, R.sup.4 are independently from each other selected from the group consisting of H, linear C.sub.1-C.sub.8 alkyl, and branched C.sub.3-C.sub.8 alkyl, C.sub.6-C.sub.10-aryl, non-substituted or substituted with one or more carboxylate or hydroxyl groups, and C.sub.6-C.sub.10-aryl-alkyl, wherein an alkyl of the C.sub.6-C.sub.10-aryl-alkyl is selected from the group consisting of linear C.sub.1-C.sub.8 alkyl and branched C.sub.3-C.sub.8 alkyl, wherein at least one of R.sup.2, R.sup.3, and R.sup.4 is not H, component (b): at least one enzyme selected from the group consisting of proteases, and at least one detergent component in effective amounts.

10. A method of preparing a detergent formulation, comprising the steps of mixing the enzyme preparation of claim 1 and at least one detergent component in effective amounts.

11. A method for removing stains, comprising the step of contacting at least one stain with the detergent formulation according to claim 8, wherein component (b) of said detergent formulation comprises at least one protease, and optionally further comprises at least one lipase.

12. The method according to claim 11, wherein the stain is to be removed from a textile and the stain comprises fatty compounds having a melting temperature of >30° C., and the removal is done at a cleaning temperature of ≤30° C.

13. A method to increase storage stability of a liquid detergent formulation comprising at least one protease, the method comprising adding at least one compound according to formula (I) to the detergent formulation: ##STR00023## wherein the variables of formula (I) are as follows: R.sup.1 is H; R.sup.2, R.sup.3, R.sup.4 are independently from each other selected from the group consisting of H, linear C.sub.1-C.sub.8 alkyl, and branched C.sub.3-C.sub.8 alkyl, C.sub.6-C.sub.10-aryl, non-substituted or substituted with one or more carboxylate or hydroxyl groups, and C.sub.6-C.sub.10-aryl-alkyl, wherein an alkyl of the C.sub.6-C.sub.10-aryl-alkyl is selected from the group consisting of linear C.sub.1-C.sub.8 alkyl and branched C.sub.3-C.sub.8 alkyl, wherein at least one of R.sup.2, R.sup.3, and R.sup.4 is not H.

14. The method according to claim 13, wherein the detergent formulation is stored at 37° C. for at least 20 days.

15. The method according to claim 13, wherein the protease is selected from the group consisting of subtilisin type proteases (EC 3.4.21.62), and wherein the liquid detergent formulation optionally comprises at least one lipase.

16. The enzyme preparation according to claim 1, wherein the at least one enzyme is selected from the group consisting of serine endopeptidases (EC 3.4.21) and subtilisin type proteases (EC 3.4.21.62).

17. The process according to claim 4, wherein the at least one enzyme is selected from the group consisting of serine endopeptidases (EC 3.4.21) and subtilisin type proteases (EC 3.4.21.62).

18. The method according to claim 7, wherein the detergent formulations are liquid detergent formulations.

19. The method according to claim 9, wherein the at least one enzyme is selected from the group consisting of serine endopeptidases (EC 3.4.21) and subtilisin type proteases (EC 3.4.21.62).

20. The method according to claim 15, wherein the lipase is selected from the group consisting of Thermomyces lanuginosa lipase and variants thereof.

Description

EXAMPLES

[0424] The invention will be further illustrated by working examples.

[0425] General remarks: percentages are weight percent unless specifically noted otherwise.

I. Tested Compounds

A) Compounds According to Formula (I)—(Component (a)):

[0426] A.1 Triethylcitrate—purchased from Sigma Aldrich

[0427] A.2 Tripropylcitrate—purchased from Sigma Aldrich

[0428] A.3 Tributylcitrate—purchased from Sigma Aldrich

[0429] A.4 Acetyltributylcitrate—purchased from Sigma Aldrich

[0430] A.5 Acetyltriethylcitrate—purchased from Sigma Aldrich

[0431] A.6 Monoethylcitrate—purchased from Sigma Aldrich

[0432] A.7 Diethylcitrate [0433] Synthesis of as described in: Journal of Chemical & Engineering Data 2018, DOI: 10.1021/acs.jced.7b01060, C. Berdugo, A. Suaza, M. Santaella, O. Sanchez

[0434] A.8 Tribenzylcitrate [0435] Synthesis as described in WO2007/14471 A1, 2007; Location in patent: Page/Page column 19; 27-28

[0436] A.9 Trisalicylcitrate [0437] Synthesis as described in WO2007/14471 A1, 2007; Location in patent: Page/Page column 19; 27-28

B) Comparative Compounds:

[0438] B.1: citric acid—purchased from Sigma Aldrich

[0439] B.2: citric acid trisodium salt—purchased from Sigma Aldrich

[0440] B.3: diethyloxalate—purchased from Sigma Aldrich

[0441] B.4: glyceroltriacetate (triacetine)—purchased from Sigma Aldrich

II. Protease Stability

[0442] The storage stability of protease was assessed at 37° C.

[0443] Base test formulations were manufactured by making base formulations I to V by mixing the components according to Table 1.

[0444] Protease used: Savinase® 16.0L (CAS-No. 9014-01-1, EC-No. 232-752-2) was purchased from Sigma-Aldrich.

[0445] The respective component (a) or comparative compound was added, if applicable, to the respective base formulation in amounts as indicated in Table 1.

[0446] Protease (component (b)) was added, to the respective base formulation in amounts as indicated in Table 1. The amount of protease as provided in Table 1 refers to active protein.

[0447] Water was added to accomplish the balance to 100.

TABLE-US-00001 TABLE 1 liquid formulations wt % in formulation Ingredients I. II. III. IV. V. Base formulation: (Comp. 1) 15 8 — 6 6 (Comp. 2) — 6 8 8 8 (Comp. 3) 6 4 — 4 4 (Comp. 4) 2 — — 2 — (Comp. 5) — 4 8 4 4 (Comp. 6) — 2.5 — — 2.5 Sorbitol 3 — — 2 — PEI-EO20 3 5 3 5 5 Propyleneglycol — 4 — 2 4 Glycerol — — 6 — — Ca-formiate 1 — 1 — — Additives: Savinase 16.0 L 0.7 0.7   0.7 0.7 0.7 component (a)** 2.5 2.5   2.0 2.0 2.0 balance Water to 100 (Comp. 1): n-C.sub.18-alkyl-(OCH.sub.2CH.sub.2).sub.25—OH (Comp. 2): C.sub.10-C.sub.18-alkylpolygycoside blend (Comp. 3): Sodium C.sub.10-C.sub.12-alkyl benzenesulfonate (Comp. 4): Sodium cumenesulfonate (Comp. 5): Sodium laurethsulfate - n-C.sub.12H.sub.25—O—(CH.sub.2CH.sub.2O).sub.3—SO.sub.3Na (Comp. 6): n-C.sub.12H.sub.25(CH.sub.3).sub.2N.fwdarw.O **for comparative tests without inventive compounds those were replaced by the same amount of water.

[0448] Savinase activity at certain points in time as indicated in Table 2 was be determined by employing Succinyl-Ala-Ala-Pro-Phe-p-nitroanilide (Suc-AAPF-pNA, short AAPF) as substrate. pNA is cleaved from the substrate molecule by proteolytic cleavage, resulting in release of yellow color of free pNA which was determined by measuring OD.sub.405. Measurements were done at 20° C.

[0449] Table 2 displays protease activity measured in liquid formulations after storage for 1 to 30 days at 37° C. The proteolytic activity values provided in Table 3 were calculated referring to the 100% value determined in the reference formulation at the time 0.

[0450] The nomenclature of formulations is as follows: the Roman number before the full stop characterizes the base formulation, the Arabian number the type of compound (A. # compound according to invention (component (a)); (B. #) comparative compound).

TABLE-US-00002 TABLE 2 protease activity in the course of time of storage at 37° C. Formulation identifier Base com- formulation pound T0 3 d 7 d 14 d 21 d 28 d 35 d I. 0  100 93 87 78 69 64 59  .sup. I. (A.1) 100 96 93 89 83 79 72  .sup. I. (A.2) 101 100 94 91 85 80 76  .sup. I. (A.4) 102 100 96 92 87 83 79  .sup. I. (A.5) 98 96 94 90 85 82 78  .sup. I. (A.7) 100 95 91 85 81 76 69  .sup. I. (B.1) 98 94 86 76 67 62 57  .sup. I. (B.2) 97 95 84 68 64 59 55  .sup. I. (B.3) 99 90 86 70 65 62 58  .sup. I. (B.4) 100 91 84 70 66 61 56 II. 0 .sup.   98 93 89 74 69 64 59  II. (A.1) 100 100 96 93 89 83 78  II. (A.5) 102 101 100 94 90 85 81  II. (A.6) 101 102 100 95 92 87 83  II. (A.7) 98 98 96 94 90 87 83  II. (A.8) 100 94 91 88 85 83 77  II. (A.9) 98 96 92 87 84 80 76  II. (B.2) 98 95 82 75 66 63 60  II. (B.4) 97 87 80 73 58 57 54 III. 0  .sup.  96 93 85 75 64 60 55 III. (A.1) 100 98 94 91 87 84 81 III. (A.2) 102 100 100 96 91 88 84 III. (A.5) 99 97 95 91 86 81 77 III. (A.6) 99 96 92 86 82 79 76 III. (A.7) 100 96 93 84 83 76 72 III. (A.9) 102 98 97 95 91 87 83 III. (B.1) 100 92 84 75 65 60 53 III. (B.2) 101 93 83 73 62 57 50 III. (B.3) 98 92 85 74 60 55 51 IV. 0 .sup.    96 90 85 77 68 60 55 IV. (A.1) 98 96 93 88 82 79 74 IV. (A.3) 99 100 96 91 84 81 77 IV. (B.2).sup.  98 91 86 70 64 58 52 V. 0 .sup.  98 93 89 74 68 62 57  V. (A.1) 97 94 90 86 83 79 75  V. (A.3) 101 98 93 88 85 80 74  V. (A.8) 96 96 92 86 84 80 73  V. (B.1) 97 93 86 75 65 57 50

III. Textile Cleaning Tests

[0451] The detergent performance of formulations in cleaning two types of test fabrics was carried out. Testing cloth samples comprised a complex soil comprising proteinaceous and fatty components due to CFT process as well as test cloth samples comprised a fatty/particulate type of soil.

[0452] The test was performed as follows: a multi stain monitor comprising 8 standardized soiled fabric patches, each of 2.5×2.5 cm size and stitched on two sides to a polyester carrier was washed together in a launder-O-meter with 2.5 g of cotton fabric and 5 g/L of the liquid test laundry detergent, Table 3.

[0453] The conditions were as follows: Device: Launder-O-Meter from SDL Atlas, Rock Hill, USA. Washing liquor: 250 ml, washing time: 60 minutes, washing temperature: 30° C. Water hardness: 2.5 mmol/L; Ca:Mg:HCO.sub.3 4:1:8

[0454] Fabric to liquor ratio 1:12 After the wash cycle, the multi stain monitors were rinsed in water, followed by drying at ambient temperature over a time period of 14 hours.

[0455] The following pre-soiled test fabrics were used:

[0456] CFT C-S-10: butter on cotton

[0457] CFT C-S-62: lard, colored on cotton

[0458] CFT C-S-68: chocolate ice-cream on cotton

[0459] EMPA 112: cocoa on cotton

[0460] EMPA 141/1: lipstick on cotton

[0461] EMPA 125: monitor for surfactant

[0462] wfk20D: pigment and sebum-type fat on polyester/cotton mixed fabric

[0463] CFT C-S-70: chocolate mousse

[0464] wfk=wfk test fabrics GmbH, Krefeld

[0465] EMPA=Swiss Federal Institute of Materials Testing

[0466] CFT=Center for Test Material B.V.

[0467] The total level of cleaning was evaluated using color measurements. Reflectance values of the stains on the monitors were measured using a sphere reflectance spectrometer (SF 500 type from Datacolor, USA, wavelength range 360-700 nm, optical geometry d/8°) with a UV cutoff filter at 460 nm. In this case, with the aid of the CIE-Lab color space classification, the brightness L*, the value a* on the red-green color axis and the b* value on the yellow-blue color axis, were measured before and after washing and averaged for the 8 stains of the monitor. The change of the color value (ΔE) value, defined and calculated automatically by the evaluation color tools on the following equation:

ΔE*.sub.ab=√{square root over (ΔL*.sup.2+Δa*.sup.2+Δb*.sup.2)}

[0468] [L* brightness, a* color value on red-green axis, b* color value on blue-yellow axis]

[0469] ΔE is a measure of the achieved cleaning effect. All measurements were repeated six times to yield an average number. Note that higher A E values show better cleaning. A difference of 1 unit can be detected by a skilled person. A non-expert can detect 2 units easily. The results are shown in Table 4.

[0470] R.sub.w=washed soil reflectance

[0471] R.sub.o=unsoiled reflectance

[0472] The detergency was calculated as: A total of 6 replications of each cloth were run during this study; a statistical confidence level of 90-95% was calculated.

[0473] Test formulations were manufactured by making formulations VI to X by mixing the components according to Table 3.

[0474] The respective component (a) or comparative compound was added, if applicable, to the respective base formulation in amounts provided in Table 3.

[0475] Lipolase® 100L was added, if applicable, to the respective base formulation in amounts provided in Table 3.

[0476] Savinase® 16.0L was added, if applicable, to the respective base formulation in amounts provided in Table 3.

[0477] Water was added to accomplish the balance to 100.

TABLE-US-00003 TABLE 3 liquid laundry formulations Wt-% in formulation Ingredients VI. VII. VIII. IX. X. Base formulation: (Comp. 1) 8 8 8 8 8 (Comp. 2) 6 6 6 6 6 (Comp. 3) 4 4 4 4 4 (Comp. 4) 4 4 4 4 4 (Comp. 5) 2.5 2.5 2.5 2.5 2.5 PEI-EO20 5 5 5 5 5 Propyleneglycol 4 4 4 4 4 Additives: Savinase 16.0 L — — — 0.7 0.7 Lipolase — — 0.2 0.2 0.2 component (a)** — 2.5 2.5 — 2.5 balance Water to 100 (Comp. 1): n-C.sub.18-alkyl-(OCH.sub.2CH.sub.2).sub.25—OH (Comp. 2): C.sub.10-C.sub.18-alkylpolygycoside blend (Comp. 3): Sodium C.sub.10-C.sub.12-alkyl benzenesulfonate (Comp. 4): Sodium laurethsulfate - n-C.sub.12H.sub.25—O—(CH.sub.2CH.sub.2O).sub.3—SO.sub.3Na (Comp. 5): n-C.sub.12H.sub.25(CH.sub.3).sub.2N.fwdarw.O **for comparative tests without inventive compounds those were replaced by the same amount of water.

[0478] The launder-O-meter tests were executed with freshly prepared formulations and with formulations stored at 37° C. during a 2-month storage (1 week [7 days], 2 weeks [14 days], 4 weeks [28 days], 6 weeks [42 days], 8 weeks [56 days]). As an approximation one week at 37° C. is equivalent to 3½ weeks at 20° C.

TABLE-US-00004 TABLE 4 Results of launder-O-meter tests: sum of ΔE of the above mentioned multi-stain monitor Formulation identifier Base ΔE ΔE ΔE ΔE ΔE formulation compound ΔE T0 1 week 2 weeks 4 weeks 6 weeks 8 weeks VI. — 152 154 153 151 153 153 VII. A.1 154 153 154 152 152 153 VII. A.2 152 152 154 152 152 153 VII. A.5 154 155 153 153 152 153 VII. A.8 153 153 152 152 152 151 VIII. 0 183 184 181 179 179 175 VIII. A.3 185 185 181 178 176 173 VIII. A.4 185 185 183 181 182 181 VIII. A.7 182 179 179 175 173 170 IX. — 187 183 176 172 165 159 X. A.1 191 188 187 184 184 180 X. A.2 189 187 184 182 182 177 X. A.5 190 187 187 183 180 180 X. A.8 191 189 185 186 182 176 X. B.1 190 186 180 175 168 160 X. B.3 190 187 182 177 169 162 X. B.4 188 185 181 173 166 159