Patent US5336601A successfully refreshed from Google Patents.
US5336601A
Expired / Lapsed
US
⏱️ 0.0 years remaining
Enzymatic membrane method for the snythesis and separation of peptides
Assignee: Coca-Cola Co
•
Filing: 1991-12-10
•
Granted: 1994-08-09
•
Expires: 2011-12-10
Abstract
Abstract The present invention discloses a method for the enzymatic synthesis of a peptide. A protected peptide having a C-terminal carboxylate group or a protected, N-acyl amino acid having an alpha carboxylate group is reacted with a protected peptide having an N-terminal ammonium group or a protected amino acid having an alpha ammonium group in the presence of a condensation enzyme under conditions in which the carboxylate group and the ammonium group condense to form a protected, uncharged, peptide product. This peptide product is transported across a water-immiscible hydrophobic phase into an aqueous product phase. This peptide product is removed from the aqueous product phase to prevent back diffusion across the water-immiscible hydrophobic phase. Reverse osmosis and other separation techniques may be utilized to remove the peptide product. The water-immiscible hydrophobic phase is an ion rejection membrane separating the aqueous reaction phase from the product phase creating oil/water interfaces with each of the aqueous phases.
Inventors
- 👤 Guillermo A. Iacobucci
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 03:09
CPC Classifications
C; C07; C07K; C07K5/00; C07K5/04; C07K5/06; C07K5/06104; C07K5/06113; C07K5/06121; C07K5/0613; C07K1/00; C07K14/00; C07K14/435; C07K14/665; C07K14/70; C12; C12M; C12M1/00; C12M1/40; C12M21/00; C12M21/18; C12P; C12P21/00; C12P21/02
Claims Analysis
Total Claims: 21
Claim 1
Independent Claim
241 words
A method for the enzymatic synthesis of a peptide, in and oil/water contactor, comprising the steps of: in a first hollow fiber membrane module reacting a protected, peptide first reactant having a C-terminal carboxylate group with a protected peptide, second reactant having an N-terminal ammonium group, in the presence of a condensation enzyme in an aqueous reaction phase under conditions in which the C-terminal carboxylate group and the N-terminal ammonium group condense forming a protected, uncharged, peptide product; transporting the protected, uncharged, peptide product from the first hollow fiber membrane module into a water-immiscible hydrophobic phase; transporting the protected, uncharged, peptide product from the water-immiscible hydrophobic phase into an aqueous product phase in a second hollow fiber membrane module; and wherein in the first membrane module the hollow fibers have an inner lumen side and an outer surface side connected by pores and the aqueous reaction phase circulates on one side of the fibers and the water immiscible hydrophobic phase circulates on the other side the fibers creating an oil/water interface between the aqueous reaction phase and the water-immiscible hydrophobic phase; and wherein in the second membrane module the hollow fibers have an inner lumen and an outer surface connected by pores and the water-immiscible hydrophobic phase circulates on one side of the fibers and the aqueous product phase ciruclates on the other side of the fibers creating an oil/water interface between the water-immiscible hydrophobic phase and the aqueous product phase.
Claim 2
Independent Claim
241 words
A method for the enzymatic synthesis of a peptide, in and oil/water contactor, comprising the steps of: in a first hollow fiber membrane module reacting a protected, peptide first reactant having a C-terminal carboxylate group with a protected peptide, second reactant having an N-terminal ammonium group, in the presence of a condensation enzyme in an aqueous reaction phase under conditions in which the C-terminal carboxylate group and the N-terminal ammonium group condense forming a protected, uncharged, peptide product; transporting the protected, uncharged, peptide product from the first hollow fiber membrane module into a water-immiscible hydrophobic phase; transporting the protected, uncharged, peptide product from the water-immiscible hydrophobic phase into an aqueous product phase in a second hollow fiber membrane module; and wherein in the first membrane module the hollow fibers have an inner lumen side and an outer surface side connected by pores and the aqueous reaction phase circulates on one side of the fibers and the water immiscible hydrophobic phase circulates on the other side the fibers creating an oil/water interface between the aqueous reaction phase and the water-immiscible hydrophobic phase; and wherein in the second membrane module the hollow fibers have an inner lumen and an outer surface connected by pores and the water-immiscible hydrophobic phase circulates on one side of the fibers and the aqueous product phase ciruclates on the other side of the fibers creating an oil/water interface between the water-immiscible hydrophobic phase and the aqueous product phase.
Claim 3
Dependent Claim
30 words
The method as recited in claim 1, further comprising the step of preventing the protected, uncharged, peptide product in the aqueous product phase from back-diffusing into the water-immiscible hydrophobic phase.
Claim 4
Dependent Claim
22 words
The method recited in claim 1, wherein the first reactant is a protected, N-acyl amino acid first reactant having an α-carboxylate group.
Claim 5
Dependent Claim
19 words
The method recited in claim 1, wherein the second reactant is a protected, amino acid having an α-ammonium group.
Claim 6
Dependent Claim
33 words
The method recited in claim 1, wherein the first reactant is a protected, N-acyl amino acid having an α-carboxylate group and the second reactant is a protected, amino acid having an α-ammonium group.
Claim 7
Dependent Claim
32 words
The method recited in claim 1, wherein: the first reactant is an N-carbobenzoxy aspartic acid having an α-carboxylate group and the second reactant is a L-phenylalanine methyl ester having an ammonium group.
Claim 8
Dependent Claim
40 words
The method recited in claim 1, wherein the first reactant is a N-acyl-(β-substituted) aspartic acid and the second reactant is a L-phenylalanine lower alkyl ester derived from a secondary alcohol having 3 to 6 carbon atoms and an α-ammonium group.
Claim 9
Dependent Claim
18 words
The method recited in claim 1, wherein the peptide product includes from three to eight amino acid residues.
Claim 10
Dependent Claim
17 words
The method recited in claim 7, wherein the protected, amino acid second reactant is L-phenylalanine isopropyl ester.
Claim 11
Dependent Claim
12 words
The method recited in claim 9, wherein the condensing enzyme is thermolysin.
Claim 12
Dependent Claim
22 words
The method recited in claim 1, wherein the protected, peptide first reactant is N-formyl-(O-Bzl)-tyr-gly-OH and the protected, peptide second reactant is H-gly-phe-leu-OMe.
Claim 13
Dependent Claim
30 words
The method recited in claim 1, wherein the protected, peptide first reactant is N-formyl-(β-methyl)-asp-phe-OH and the protected, amino acid second reactant is H-trp-OMe and wherein the condensing enzyme is pepsin.
Claim 14
Dependent Claim
28 words
The method recited in claim 7, wherein the aqueous reaction phase is maintained at a temperature in a range of from about 20° C. to about 65° C.
Claim 15
Dependent Claim
30 words
The method recited in claim 6, wherein the N-acyl-(β-substituted) aspartic acid first reactant is N-formyl-(β-methyl) aspartic acid and wherein the L-phenylalanine lower alkyl ester second reactant is L-phenylalanine isopropyl ester.
Claim 16
Dependent Claim
24 words
The method recited in claim 14, wherein the condensing enzyme is thermolysine and the temperature of the aqueous reaction phase is about 50° C.
Claim 17
Dependent Claim
17 words
The method recited in claim 1, wherein circulation of the phases at the oil/water interfaces is countercurrent.
Claim 18
Dependent Claim
22 words
The method recited in claim 15, further comprising the steps of processing the aqueous product phase through a plurality of membrane contactors.
Claim 19
Dependent Claim
21 words
The method recited in claim 1, wherein the protected, uncharged, peptide product is converted to a charged species by chemical means.
Claim 20
Dependent Claim
22 words
The method recited in claim 1, further comprising the step of separating the protected, uncharged peptide product from the aqueous product phase.
Claim 21
Dependent Claim
20 words
The method recited in claim 1, wherein the aqueous reactant phase and the aqueous product phase wet the hollow fibers.
References Cited (Backward Citations)
FR902474A
Cited
US3625734A
Cited
US3779907A
Cited
US4215631A
Cited
US3813317A
Cited
US3933781A
Cited
US4119493A
Cited
US4022417A
Cited
US4187086A
Cited
US4119408A
Cited
GB2047564A
Cited
EP0017485A1
Cited
US4786597A
Cited
EP0075160A2
Cited
US4455210A
Cited
US4670151A
Cited
US4443414A
Cited
US4664808A
Cited
US4563337A
Cited
EP0121265A2
Cited
EP0124313A2
Cited
EP0149594A2
Cited
US4572824A
Cited
EP0188342A2
Cited
US4743547A
Cited
US4778688A
Cited
US4795704A
Cited
US4921612A
Cited
WO1988001298A2
Cited
US5002871A
Cited
US5037741A
Cited
US5202235A
Cited
US4754089A
Cited
US4956289A
Cited
USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1997-08-08 | 1998-02-07 | 1998-08-09 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2001-08-08 | 2002-02-07 | 2002-08-09 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2005-08-08 | 2006-02-07 | 2006-08-09 | $7700.00 | Window Closed / Lapsed |