COMPOUNDS STABILIZING AMYLASES 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 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 hydrolases (EC 3) and proteases 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 hydrolases (EC 3) and 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 hydrolases (EC 3) and 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 amylolytic activity of at least one amylase 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.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, the method comprising using the compound according to formula (I) as an additive for a composition comprising at least one amylase wherein the compound according to formula (I) and the amylase are solid and wherein amylolytic activity of the amylase is stabilized when the compound according to formula (I) and the amylase are contacted with at least one solvent.

7. A method of using the enzyme preparation of claim 1 to formulate a detergent formulation the method comprising mixing the enzyme preparation in one or more steps with one or more detergent components in effective amounts.

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

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 hydrolases (EC 3) and 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 amylase-sensitive 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 alpha-amylase, and optionally further comprises at least one subtilisin type protease.

12. The method according to claim 11, wherein the amylase-sensitive stain is to be removed from a textile at a cleaning temperature of ≤30° C.

13. A method to increase storage stability of a liquid detergent formulation comprising at least one hydrolase and optionally comprising at least one complexing agent in effective amounts, 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 at least one hydrolase is selected from the group consisting of alpha-amylases (EC 3.2.1.1) and subtilisin type proteases (EC 3.4.21.62), and combinations thereof.

16. The enzyme preparation according to claim 1, wherein the at least one enzyme is selected from the group consisting of amylases, alpha-amylases (EC 3.2.1.1), 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 amylases, alpha-amylases (EC 3.2.1.1), and subtilisin type proteases (EC 3.4.21.62).

18. The method according to claim 6, wherein the at least one amylase is selected from the group consisting of alpha-amylases and subtilisin type proteases.

19. The method according to claim 7, wherein the detergent formulation comprises at least one complexing agent selected from the group consisting of EDTA, DTPA, MGDA, and GLDA.

20. The method according to claim 7, wherein the detergent formulation is a liquid detergent formulation.

Description

EXAMPLES

[0790] The invention will be further illustrated by working examples.

[0791] General remarks: percentages are weight percent unless specifically noted otherwise.

1. Tested Compounds

[0792] A) Compounds according to formula (I)—(component (a)):
A.1 Triethylcitrate—purchased from Sigma Aldrich
A.2 Tripropylcitrate—purchased from Sigma Aldrich
A.3 Tributylcitrate—purchased from Sigma Aldrich
A.4 Acetyltributylcitrate—purchased from Sigma Aldrich
A.5 Acetyltriethylcitrate—purchased from Sigma Aldrich
A.6 Monoethylcitrate—purchased from Sigma Aldrich

A.7 Diethylcitrate

[0793] 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

A.8 Tribenzylcitrate

[0794] Synthesis as described in WO2007/14471 A1, 2007; Location in patent: Page/Page column 19; 27-28

A.9 Trisalicylcitrate

[0795] Synthesis as described in WO2007/14471 A1, 2007; Location in patent: Page/Page column 19; 27-28
B) Comparative compounds:
B.1: citric acid—purchased from Sigma Aldrich
B.2: citric acid trisodiumsalt—purchased from Sigma Aldrich
B.3: diethyloxalate—purchased from Sigma Aldrich
B.4: glyceroltriacetate (triacetine)—purchased from Sigma Aldrich

II. Amylase and Protease Stability

[0796] The storage stability of amylase was assessed at 37° C.

[0797] Base test formulations were manufactured by making base formulations I to V by mixing the components according to Table 1.

[0798] Amylases used: Amy1=Stainzyme, Amy2=Amplify, Amy3=Stainzyme Plus L (12L)

[0799] Protease used: (S) Savinase Ultra 16.0L (CAS-No. 9014-01-1, EC-No. 232-752-2) was purchased from Sigma-Aldrich

[0800] The respective component (a) or comparative compound was added, if applicable, to the respective base formulation in amounts as indicated in Table 1.

[0801] Amylase (component (b)) was added, to the respective base formulation in amounts as indicated in Table 1. The amount of amylase as provided in Table 1 refers to active protein.

[0802] 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.

[0803] Water was added to accomplish the balance to 100.

TABLE-US-00001 TABLE 1 liquid formulation wt % in formulation Ingredients I. II. III. IV V Base formulation: (Comp. 1) 20 15 — 15 10 (Comp. 2) — 5 5 — 5 (Comp. 3) — — 15 10 10 (Comp. 4) 5 2.5 2.5 5 2.5 (Comp. 5) 5 G 5 P 5 P 5 G 5 P (Comp. 6) 2.5 2.0 2.0 2.5 2.5 (Comp. 7) — 1 1 — 1 (Comp. 8) 0.3 0.3 0.3 0.3 0.3 Additives: Protease 2.5 2.5 2.5 3.0 3.0 Amylase * 0.5 Amy1 0.5 Amy3 0.5 Amy2 0.5 Amy1 0.5 Amy2 component 1.5 1.5 1.5 2.0 2.0 (a)** balance Water to 100 after adjust pH 7.5 with citric acid (Comp. 1): Trilon M fl (Max liqu) (Comp. 2): Citric acid (Comp. 3): GLDA 50% solution (Comp. 4): PAA, Polyacrylic acid Mw 5.000 g/mol (Comp. 5): Glycerol (G) or Propanediol (P) (Comp. 6): Dehypon WET (Comp. 7): Na.sub.4HEDP (Comp. 8) Thickener (Rheocare XGN) **for comparative tests without inventive compounds those were replaced by the same amount of glycerol

[0804] Amylase activity at certain points in time as indicated in Table 2 was measured quantitatively by the release of the chromophore para-nitrophenol (pNP) from the substrate (Ethyliden-blocked-pNPG7, Roche Applied Science 10880078103). The alpha-amylase degrades the substrate into smaller molecules and α-glucosidase (Roche Applied Science 11626329103), which is present in excess compared to the amylase, process these smaller products until pNP is released; the release of pNP, measured via an increase of absorption at 405 nm, is directly proportional to the amylase activity of the sample. Amylase standard: Termamyl 120 L (Sigma 3403).

[0805] Table 2 displays amylase activity measured in liquid formulations after storage for 1 to 30 days at 37° C. The amylolytic activity values provided were calculated referring to the value determined in the reference formulation at the time 0.

[0806] 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 amylase activity in the course of time of storage at 37° C. Formulation identifier Base formulation compound T0 3 d 7 d 14 d 21 d 28 d 42 d I. 0 100 76 59 48 34 27 18 I. A.1 100 98 93 88 80 74 61 I. A.2 101 100 96 90 84 79 66 I. A.4 102 103 97 93 86 82 69 I. A.5 97 91 80 72 63 55 37 I. A.6 98 95 84 79 70 61 48 I. A.7 99 96 86 81 74 63 50 II. 0 100 81 65 54 40 32 25 II. A.1 100 100 94 90 84 77 65 II. A.2 102 100 97 93 87 83 69 II. A.4 102 100 95 91 84 80 70 II. A.7 99 94 86 82 72 64 53 II. A.8 99 100 95 90 83 81 71 II. A.9 98 97 94 88 80 77 66 III. 0 98 80 66 56 43 34 27 III. A.1 100 98 95 91 86 78 68 III. A.2 100 102 97 93 87 81 71 III. A.4 99 100 96 93 85 80 70 III. A.5 96 96 92 87 78 71 55 III. A.6 97 96 90 83 74 63 49 III. A.7 98 96 91 85 78 70 60 IV. 0 97 74 58 47 34 25 15 IV. A.1 98 96 92 88 83 73 61 IV. A.3 98 95 90 84 77 68 57 IV. A.7 99 94 88 82 74 63 52 IV. A.8 98 96 92 86 81 70 59 IV. A.9 98 95 89 84 80 72 54 V. 0 99 76 60 51 39 28 19 V. A.1 98 96 91 87 80 73 62 V. A.2 98 95 90 84 76 66 54 V. A.4 100 94 88 83 72 65 52 V. A.7 98 96 87 74 63 55 47 V. A.8 98 93 86 80 77 66 59 V. A.9 97 93 86 75 66 60 54

[0807] Protease activity at certain points in time as indicated in Table 3 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 00405. Measurements were done at 20° C.

[0808] Table 3 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 value determined in the reference formulation at the time 0.

[0809] The nomenclature of formulations is as follows: the Roman number before the full stop characterizes the base formulation, the Arabian number the type of salt (A.#inventive salt (component (a)); B.# comparative compound). Zero (“0”): no salt, but diethylene glycol.

TABLE-US-00003 TABLE 3 protease activity in the course of time of storage at 37° C. Formulation identifier Base formulation compound T0 3 d 7 d 14 d 21 d 28 d 42 d I. 0 100 93 87 75 65 57 43 I. A.1 100 97 94 89 83 79 67 I. A.2 101 100 95 90 85 81 71 I. A.4 102 100 94 90 84 82 70 I. A.5 98 96 90 84 80 76 62 I. A.6 100 94 90 81 73 67 56 I. A.7 100 94 91 85 81 76 63 II. 0 98 93 89 78 68 61 47 II. A.1 100 99 96 90 85 80 69 II. A.2 101 100 96 91 87 83 72 II. A.4 102 100 95 90 85 81 70 II. A.7 100 98 93 88 85 80 71 II. A.8 100 94 93 90 87 83 75 II. A.9 98 96 92 85 81 75 68 III. 0 96 93 86 79 71 65 50 III. A.1 100 99 93 88 85 74 67 III. A.2 101 100 95 93 88 83 73 III. A.4 101 99 95 91 86 81 70 III. A.5 100 98 92 83 79 71 65 III. A.6 100 94 90 84 78 70 62 III. A.7 100 97 95 92 87 74 64 IV. 0 96 91 84 74 68 59 45 IV. A.1 98 96 92 86 80 72 65 IV. A.3 99 98 90 85 78 70 67 IV. A.7 98 96 88 79 74 68 62 IV. A.8 98 96 93 88 83 75 68 IV. A.9 100 97 93 86 79 70 64 V. 0 98 92 87 76 69 62 48 V. A.1 99 94 90 85 81 75 65 V. A.2 100 95 92 87 82 77 70 V. A.4 98 94 90 85 80 76 68 V. A.7 97 93 88 79 72 66 54 V. A.8 100 96 92 86 82 77 69 V. A.9 98 98 93 86 81 74 67

III. Stability in Laundry Formulation

[0810] The storage stability of amylase was assessed at 37° C.

[0811] Base test formulations were manufactured by making base formulations VI to IX by mixing the components according to Table 4.

TABLE-US-00004 TABLE 4 liquid laundry formulations Wt-% in formulation Ingredients VI. VII. VIII. IX. Base formulation: (Comp. 1) — 4 4 — (Comp. 2) 3 2 2 3 (Comp. 3) 14 14 14 14 (Comp. 4) 4 4 4 4 (Comp. 5) 2.0 2.0 2.5 2.5 EDTA EDTA DTPA DTPA (Comp. 6) 4 4 4 4 Glycerol 2.5 2 2.5 2 Ethanol 1.5 1.5 1.5 1.5 Propyleneglycol 3 3.5 3 3.5 Additives: Savinase 16.0 L 0.7 1.0 — 0.7 Amylase 0.5 0.5 0.5 0.5 component (a)** 2.5 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): Tallow oil soap C14-C18 Carbonic acid, sodium salt (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): Complexing agent EDTA or DTPA (Comp. 6) mixture Na-citrate:Na-formiate 9:1 **for comparative tests without inventive compounds those were replaced by the same amount of water.

[0812] Amylase activity at certain points in time as indicated in Table 5 was measured quantitatively by the release of the chromophore para-nitrophenol (pNP) from the substrate (Ethyliden-blocked-pNPG7, Roche Applied Science 10880078103). The alpha-amylase degrades the substrate into smaller molecules and α-glucosidase (Roche Applied Science 11626329103), which is present in excess compared to the amylase, process these smaller products until pNP is released; the release of pNP, measured via an increase of absorption at 405 nm, is directly proportional to the amylase activity of the sample. Amylase standard: Termamyl 120 L (Sigma 3403).

[0813] Table 5 displays amylase activity measured in liquid formulations after storage for 1 to 28 days at 37° C. The amylolytic activity values provided were calculated referring to the value determined in the reference formulation at the time 0.

[0814] 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-00005 TABLE 5 amylase activity in the course of time of storage at 37° C. Formulation identifier Base formulation compound T0 3 d 7 d 14 d 21 d 28 d VI. — 100 71 52 40 28 18 VI. A1 100 98 91 86 81 73 VI. A.2 101 94 86 80 74 65 VI. A.9 100 95 87 82 75 67 VII. — 100 73 55 43 32 23 VII. A.1 100 95 88 79 73 68 VII. A.5 98 92 81 72 66 61 VII. A.7 99 90 82 69 62 56 VIII. — 100 77 56 48 34 25 VIII. A.1 100 98 93 88 83 75 VIII. A.2 99 94 87 83 77 69 VIII. A.5 100 93 83 74 67 64 VIII. A.9 100 96 88 84 78 69 IX. — 100 71 53 40 33 26 IX. A.1 99 98 90 85 78 70 IX. A.2 100 93 85 81 72 63 IX. A.5 98 91 78 69 61 53

[0815] The detergent performance of formulations according to Table 4 in cleaning amylase-sensitive stains can be carried out on applicable types of test fabrics. Pre-soiled test fabrics may be purchased from wfk test fabrics GmbH, Krefeld; EMPA=Swiss Federal Institute of Materials Testing; or CFT=Center for Test Material B.V.

[0816] The test can be performed as follows: a multi stain monitor comprising e.g. 8 standardized soiled fabric patches, each of 2.5×2.5 cm size and stitched on two sides to a polyester carrier is 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 4.

[0817] The conditions may be chosen as follows: Device: Launder-0-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; fabric to liquor ratio 1:12; after the wash cycle, the multi stain monitors are rinsed in water, followed by drying at ambient temperature over a time period of 14 hours.

[0818] The total level of cleaning can be evaluated using color measurements: Reflectance values of the stains on the monitors are 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, are measured before and after washing and averaged for the 8 stains of the monitor. The change of the color value (ΔE) value, can be 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)}

[L* brightness, a* color value on red-green axis, b* color value on blue-yellow axis]
ΔE is a measure of the achieved cleaning effect. All measurements may be repeated six times to yield an average number. Note that higher ΔE values show better cleaning. A difference of 1 unit can be detected by a skilled person. A non-expert can detect 2 units easily.

[0819] The launder-O-meter tests can be executed with freshly prepared formulations according to Table 4 and/or with the same formulations after storage at 37° C. for a defined time such as 3 days, about 7 days, about 14 days, about 21 days, about 28 days, or ≥28 days. As an approximation one week (7 days) at 37° C. is equivalent to 3% weeks at 20° C.