US5002871A
Expired / Lapsed
US
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Enzymatic membrane method for the synthesis and separation of peptides
Assignee: Coca-Cola Co
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Filing: 1987-07-28
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Granted: 1991-03-26
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Expires: 2007-07-28
Abstract
Abstract The present invention provides a membrane 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 unchanged 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. An enzymatic conversion of the uncharged species utilizing an esterase having proteolytic activity such as aminoacylase I is disclosed. Copermeating reactants can be separated from the product mixture and returned to the reaction mixture. Also, the ion-rejection membrane can be utilized to resolve enantiomers of racemic carboxylic acids including D,L-amino acids.
Inventors
- 👤 Guillermo A. Iacobucci
Bibliographic Details
Application Number
—
Jurisdiction
US
Data Source
google_patents
Last Synchronized
2026-10-28 10:16
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; Y; Y10; Y10S; Y10S435/00; Y10S435/813; Y10S435/819
Claims Analysis
Total Claims: 28
Claim 1
Independent Claim
84 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; transporting the uncharged peptide from the aqueous reaction mixture across an ion rejection membrane into a product mixture; and converting the uncharged peptide to a charged species by cleavage with an esterase enzyme having proteolytic activity.
Claim 2
Independent Claim
84 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; transporting the uncharged peptide from the aqueous reaction mixture across an ion rejection membrane into a product mixture; and converting the uncharged peptide to a charged species by cleavage with an esterase enzyme having proteolytic activity.
Claim 3
Dependent Claim
25 words
The method recited in claim 1 wherein the condensation enzyme is a peptide forming protease active in a pH range of about 4.0 to 9.0.
Claim 4
Dependent Claim
28 words
The method recited in claim 2 further comprising the step of converting the uncharged peptide in the product mixture to a species that cannot back-diffuse across the membrane.
Claim 5
Dependent Claim
21 words
The method recited in claim 1 wherein the esterase enzyme is active in a pH range of about 6.0 to 9.0.
Claim 6
Dependent Claim
23 words
The method recited in claim 1 wherein the esterase enzyme is selected from the group consisting of aminoacylase I, α-chymotrypsin and subtilisin A.
Claim 7
Dependent Claim
23 words
The method recited in claim 1 wherein the esterase enzyme is aminoacylase I and the pH of the reaction mixture is about 7.0.
Claim 8
Dependent Claim
33 words
The method recited in claim 3 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 9
Dependent Claim
47 words
The method recited in claim 7 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 N,N-diethyl-dodecanamide, dodecane, 2-undecanone and mixtures thereof.
Claim 10
Dependent Claim
21 words
The method recited in claim 7 wherein the microporous sheet comprises polypropylene hollow fibers and the solvent comprises N,N-diethyl-dodecanamide and dodecane.
Claim 11
Dependent Claim
19 words
The method recited in claim 7 wherein the membrane is a Bend, Type 2 Hollow Fiber Selective Dialysis Membrane.
Claim 12
Dependent Claim
25 words
The method recited in claim 3 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 from the group consisting of φCH 2 O--, ter BuO--, CH 3 O--,NH 2 --.
Claim 14
Dependent Claim
12 words
The process recited in claim 3 wherein the condensation enzyme is immobilized.
Claim 15
Dependent Claim
22 words
The process recited in claim 13 wherein the condensation enzyme is thermolysin and the pH of the reaction mixture is about 7.0.
Claim 16
Dependent Claim
39 words
The process recited in claim 13 further comprising the step of readjusting the pH of the reaction mixture to a pH of about 5.0 prior to transporting the uncharged peptide from the reaction mixture across the ion rejection membrane.
Claim 17
Dependent Claim
23 words
The process recited in claim 3 further comprising the step of separating N-acyl-β-substituted-L-aspartic acid copermeating the ion rejection membrane into the product mixture.
Claim 18
Dependent Claim
25 words
The process recited in claim 3 further comprising the step of separating phenylalanine lower alkyl ester copermeating the ion rejection membrane into the product mixture.
Claim 19
Independent Claim
97 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; transporting the uncharged peptide from the aqueous reaction mixture across an ion rejection membrane into a product mixture; converting the uncharged in the product mixture to a species that cannot back-diffuse across the membrane; and removing the species that cannot back-diffuse across the membrane from the product mixture.
Claim 20
Independent Claim
97 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; transporting the uncharged peptide from the aqueous reaction mixture across an ion rejection membrane into a product mixture; converting the uncharged in the product mixture to a species that cannot back-diffuse across the membrane; and removing the species that cannot back-diffuse across the membrane from the product mixture.
Claim 21
Dependent Claim
21 words
The process recited in claim 18 further comprising the step of separating N-acyl-β-substituted-L-aspartic acid copermeating the ion rejection membrane product mixture.
Claim 22
Dependent Claim
25 words
The process recited in claim 19 further comprising the step of separating phenylalanine lower alkyl ester copermeating the ion rejection membrane into the product mixture.
Claim 23
Independent Claim
91 words
A method for the enzymatic synthesis peptides comprising the steps of: enzymatically 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 across a membrane that will not transport substantial amounts of the amino acid compounds; converting the transported uncharged compound to a form that cannot be retransported across the membrane to the initial reaction mixture; and removing the compound form that cannot be retransported across the membrane to the initial reaction mixture.
Claim 24
Independent Claim
91 words
A method for the enzymatic synthesis peptides comprising the steps of: enzymatically 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 across a membrane that will not transport substantial amounts of the amino acid compounds; converting the transported uncharged compound to a form that cannot be retransported across the membrane to the initial reaction mixture; and removing the compound form that cannot be retransported across the membrane to the initial reaction mixture.
Claim 25
Dependent Claim
36 words
The process recited in claim 21 further comprising the step of separating the first amino acid compound copermeating the membrane with the uncharged compound into the aqueous second reaction mixture from the aqueous second reaction mixture.
Claim 26
Dependent Claim
36 words
The process recited in claim 21 further comprising the step of separating the second amino acid compound copermeating the membrane with the uncharged compound into the aqueous second reaction mixture from the aqueous second reaction mixture.
Claim 27
Dependent Claim
29 words
The process recited in claim 22 further comprising the step of returning first amino acid compound copermeating the membrane with the uncharged compound to the aqueous initial reaction mixture.
Claim 28
Dependent Claim
29 words
The process recited in claim 22 further comprising the step of returning second amino acid compound copermeating the membrane with the uncharged compound to the aqueous initial reaction mixture.
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| 3.5 Years (Year 4) | 1994-03-25 | 1994-09-24 | 1995-03-26 | $2000.00 | Window Closed / Lapsed |
| 7.5 Years (Year 8) | 1998-03-25 | 1998-09-24 | 1999-03-26 | $3760.00 | Window Closed / Lapsed |
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