Stabilization of perhydrolases
View Patent ↗Disclosed herein is a method for stabilization of the perhydrolase activity of the CE-7 esterase in a formulation with a carboxylic acid ester that employs the addition of a buffering agent, substantially undissolved, to the mixture of the CE-7 esterase and the carboxylic acid ester. Further, disinfectant and laundry care formulations comprising the peracids produced by the processes described herein are provided.
1. A process to stabilize the perhydrolysis activity of an enzyme when present in a formulation comprised of said enzyme and a carboxylic acid ester, the process comprising:
(a) providing an aqueous formulation comprising
(i) at least one enzyme structurally classified as a CE-7 enzyme having perhydrolysis activity and a signature motif comprising:
(A) an RGQ motif at amino acid residues 118-120,
(B) a GXSQG motif at amino acid residues 179-183, and
(C) an HE motif at amino acid residues 298-299 when aligned to reference sequence SEQ ID NO:1 using CLUSTALW,
(ii) at least one excipient, and
(iii) optionally at least one surfactant;
(b) spray-drying the aqueous formulation of (a) to produce an enzyme powder comprising said at least one enzyme, said at least one excipient, and optionally said at least one surfactant; and
(c) combining the enzyme powder of (b) with at least one buffer and a carboxylic acid ester to form a formulation, wherein the addition of the at least one buffer to the formulation enhances the stability of the perhydrolysis activity of said at least one enzyme when present in said formulation as compared to the perhydrolase activity of said at least one enzyme when the at least one excipient is not present.
2. The process of claim 1 , wherein the at least one excipient ranges from about 95 wt % to about 25 wt % of the enzyme powder.
3. The process of claim 2 , wherein the at least one excipient is selected from the group consisting of maltodextrin, trehalose, xylan, mannan, fucoidan, galactomannan, chitosan, raffinose, stachyose, pectin, inulin, levan, graminan, amylopectin, and mixtures thereof.
4. The process of claim 3 , wherein the at least one excipient is maltodextrin.
5. The process of claim 3 , wherein the at least one excipient is trehalose.
6. The process of claim 1 , wherein the carboxylic acid ester is selected from the group consisting of monoacetin, diacetin, triacetin, monopropionin, dipropionin, tripropionin, monobutyrin, dibutyrin, tributyrin, and mixtures thereof.
7. The process of claim 6 , wherein the carboxylic acid ester is triacetin.
8. The process of claim 1 , wherein the at least one surfactant is present and is polysorbate 80.
9. The process of claim 2 , wherein the at least one excipient is an oligosaccharide excipient having a number average molecular weight of at least about 1250 and a weight average molecular weight of at least about 9000.
10. The process of claim 9 , wherein the oligosaccharide excipient has a number average molecular weight of at least about 1700 and a weight average molecular weight of at least about 15000.
11. The process of claim 1 , wherein the formulation of step (c) is substantially free of water.
12. The process of claim 11 , wherein the at least one buffer is sodium bicarbonate, potassium bicarbonate, a mixture of sodium bicarbonate and potassium bicarbonate, sodium phosphate, potassium phosphate, or a mixture of sodium phosphate and potassium phosphate.
13. The process of claim 12 , wherein the at least one buffer has a buffering capacity in a pH range of from about 5.5 to about 9.5.
14. The process of claim 12 , wherein:
(a) the at least one enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:19, and SEQ ID NO:20, wherein amino acid residue 277 of SEQ ID NO: 19 or SEQ ID NO: 20 is selected from the group consisting of alanine, valine, serine, and threonine, and
(b) the carboxylic acid ester is selected from the group consisting of diacetin, triacetin, and mixtures thereof.
15. The process of claim 14 , wherein said at least one excipient is maltodextrin.
16. The process of claim 12 , wherein:
(a) the at least one enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO:6 and SEQ ID NO:7;
(b) the at least one excipient is maltodextrin;
(c) the at least one buffer is selected from the group consisting of sodium bicarbonate, potassium bicarbonate, and mixtures thereof; and
(d) said carboxylic acid ester is triacetin.
17. The process of claim 16 , wherein the at least one buffer is sodium bicarbonate.