US5202235A
Expired / Lapsed
US
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Enzymatic method for the synthesis and separation of peptides
Assignee: Coca-Cola Co
•
Filing: 1990-12-28
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Granted: 1993-04-13
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Expires: 2010-12-28
Abstract
Abstract The present invention provides a method for the enzymatic synthesis of peptides accomplished by shifting the chemical equilibrium that exists in a reaction mixture between charged or ionized reacting amino acids and uncharged or non-ionized peptide product in the presence of a proteolytic enzyme such as thermolysin. The equilibrium is shifted by diffusion of the uncharged peptide product across an ion-rejection membrane which removes the uncharged peptide from the reaction mixture and preferably the diffused uncharged peptide is quickly converted to a charged species that cannot back-diffuse into the reaction mixture so that the uncharged peptide is effectively "pulled" across the membrane.
Inventors
- 👤 Guillermo A. Iacobucci
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-29 03:36
CPC Classifications
C; C07; C07K; C07K5/00; C07K5/04; C07K5/06; C07K5/06104; C07K5/06113; C07K5/06121; C07K5/0613; C07K1/00; C12; C12P; C12P21/00; C12P21/02
Claims Analysis
Total Claims: 15
Claim 1
Independent Claim
67 words
A method for the enzymatic synthesis of peptides, comprising the steps of: condensing a N-acyl-β-substituted-L-aspartic acid having an α-carboxylate group with a phenylalanine lower alkyl ester having an α-ammonium group, in an aqueous reaction mixture including a condensation enzyme to form N-acyl-L-aspartyl-(β-substituted)-L-phenylalanine lower alkyl ester) an uncharged peptide; and transporting the uncharged peptide from the aqueous reaction mixture across an ion rejection membrane into a product mixture.
Claim 2
Independent Claim
67 words
A method for the enzymatic synthesis of peptides, comprising the steps of: condensing a N-acyl-β-substituted-L-aspartic acid having an α-carboxylate group with a phenylalanine lower alkyl ester having an α-ammonium group, in an aqueous reaction mixture including a condensation enzyme to form N-acyl-L-aspartyl-(β-substituted)-L-phenylalanine lower alkyl ester) an uncharged peptide; and transporting the uncharged peptide from the aqueous reaction mixture across an ion rejection membrane into a product mixture.
Claim 3
Dependent Claim
28 words
The method recited in claim 1 wherein the condensation enzyme is a peptide forming acidic or neutral protease active in a pH range of about 4.0 to 8.0.
Claim 4
Dependent Claim
27 words
The method recited in claim 1 wherein the condensation enzyme is a protease selected from the group consisting of serine proteinases, thiol proteinases, carboxyl proteinases and metalloproteinases.
Claim 5
Dependent Claim
41 words
The method recited in claim 1 wherein the condensation enzyme is selected from the group consisting of chymotrypsin, trypsin, substilisin BNP' Achromobacter protease, papain, pepsin, thermolysin and prolisin; and the pH of the reaction mixture is between about 4.0 to 8.0.
Claim 6
Dependent Claim
22 words
The method recited in claim 1 wherein the condensation enzyme is thermoylsin and the pH of the reaction mixture is about 5.5.
Claim 7
Dependent Claim
33 words
The method recited om claim 4 wherein the membrane comprises a water-immiscible organic liquid immobilized by capillarity in pores in a microporous sheet the organic liquid being a solvent for the uncharged peptide.
Claim 8
Dependent Claim
44 words
The method recited in claim 6 wherein the microporous sheet is made from a material selected from a group consisting of polytetrafluoroethylene and polypropylene and the solvent comprises at least one compound selected from a group consisting of n-1-decanol, n-hexadecanol, n-dodecanol and mixtures thereof.
Claim 9
Dependent Claim
19 words
The method recited in claim 6 wherein the membrane is a Bend, Type 1 Hollow Fiber Selective Dialysis Membrane.
Claim 10
Dependent Claim
19 words
The method recited in claim 3 wherein the pH of the aqueous solution is between about 4.0 and 6.0.
Claim 11
Dependent Claim
20 words
The method recited in claim 4 wherein the pH of the aqueous solution is between about pH 4.0 and 6.0.
Claim 12
Dependent Claim
25 words
The method recited in claim 5 wherein the first compound is D,L-phenylalanine methyl ester, and the second compound is N-acyl-β-substituted-L-aspartic acid having an α-carboxylate group.
Claim 13
Dependent Claim
45 words
The process recited in claim 11 wherein the N-acyl group is selected from the group consisting of φCH 2 OCO--, --CH═O and φCH 2 CO--; and the β-substituent is selected form the group consisting of φCH 2 O--, ter BuO--, CH 3 --NH 2 --.
Claim 14
Independent Claim
74 words
A method for the enzymatic synthesis of peptides comprising th steps of: condensing first and second amino acid compounds in an aqueous initial reaction mixture to form an uncharged compound; transporting the uncharged compound into an aqueous second reaction mixture accross a membrane that will not transport substantial amounts of the amino acid compounds; and converting the transported uncharged compound to a form that cannot be retransported across the membrane to the initial mixture.
Claim 15
Independent Claim
74 words
A method for the enzymatic synthesis of peptides comprising th steps of: condensing first and second amino acid compounds in an aqueous initial reaction mixture to form an uncharged compound; transporting the uncharged compound into an aqueous second reaction mixture accross a membrane that will not transport substantial amounts of the amino acid compounds; and converting the transported uncharged compound to a form that cannot be retransported across the membrane to the initial mixture.
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USPTO Annuity & Maintenance Windows
| Annuity Stage | Window Opens | Payment Deadline | Surcharge Period Until | Estimated Fee | Status |
|---|---|---|---|---|---|
| 3.5 Years (Year 4) | 1996-04-12 | 1996-10-12 | 1997-04-13 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 2000-04-12 | 2000-10-12 | 2001-04-13 | $3760.00 | Window Closed / Lapsed |
| 11.5 Years (Year 12) | 2004-04-12 | 2004-10-12 | 2005-04-13 | $7700.00 | Window Closed / Lapsed |