Peptide fragment condensation and cyclization with subtilisin variants having improved synthetic hydrolysis ratio

By specifically mutating the BPN' protease from Bacillus subtilis, the problems of low yield and hydrolysis side reactions in the condensation and cyclization of oligopeptide fragments in aqueous solution were solved, achieving efficient oligopeptide synthesis and improving the synthesis hydrolysis ratio (S/H ratio), which is applicable to the synthesis of various oligopeptides and proteins.

CN107002110BActive Publication Date: 2026-06-12FRESENIUS KABI GMBH
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Patent Information

Authority / Receiving Office
CN · China
Patent Type
Patents(China)
Current Assignee / Owner
FRESENIUS KABI GMBH
Filing Date
2015-10-09
Publication Date
2026-06-12

AI Technical Summary

Technical Problem

Existing technologies for synthesizing oligopeptides in aqueous solutions suffer from low yields and numerous byproducts. In particular, the hydrolysis side reaction of subtilisin during oligopeptide fragment condensation is severe, resulting in a poor synthesis hydrolysis ratio (S/H ratio). Furthermore, organic co-solvents and high temperatures are required to dissolve oligopeptide fragments, leading to poor stability.

Method used

By specifically mutating the BPN' protease subtilisin, deleting amino acid positions 75-83 and introducing the S221C or S221 selenocysteine ​​mutation at S221, combined with other optimized mutations, an improved enzymatic method is formed for catalyzing the condensation and cyclization of oligopeptide fragments in aqueous solution.

Benefits of technology

It achieves high-yield oligopeptide condensation and cyclization in aqueous solution, significantly improves the synthesis hydrolysis ratio (S/H ratio), reduces hydrolysis side reactions, and is suitable for synthesizing various oligopeptide fragments and proteins while maintaining the secondary and tertiary structures of proteins.

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Abstract

The present invention relates to a method for the enzymatic synthesis of (oligo)peptides comprising coupling (a) a (oligo)peptide C-terminal ester or thioester and (b) a (oligo)peptide nucleophile having an N-terminal unprotected amine, wherein the coupling is performed in a fluid comprising water, and wherein the coupling is catalyzed by a subtilisin BPN' variant or a homolog thereof comprising the following mutations compared to subtilisin BPN' as set forth in SEQUENCE ID NO: 2: deletion of the amino acids corresponding to positions 75-83; a mutation at the amino acid position corresponding to S221 which is S221C or S221 selenocysteine; preferably a mutation at the amino acid position corresponding to P225, wherein the amino acid positions are defined according to the sequence of subtilisin BPN' as set forth in SEQUENCE ID NO: 2. In addition, the present invention relates to enzymes suitable for use as catalysts in the method of the present invention.
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Claims

1. A method for enzymatically synthesizing a peptide, comprising coupling (a) a C-terminal ester or thioester of the peptide and (b) a peptide nucleophilic group having an N-terminal deprotected amine. The coupling is performed in a fluid containing water, and The coupling is catalyzed by a subtilisin BPN' variant, the mutation of which, compared to the subtilisin BPN' shown in SEQUENCE ID NO: 2, is as follows: - Corresponds to the deletion of amino acids at positions 75-83; -S221C; and -Optionally, select a group of mutations from the following groups: (1) P225A or P225G; (2) P225A and N218S; (3) P225A, N218S and M50F; (4) P225A, N218S, M50F, S3C, Q206C, Q2K, A73L and P5S; or (5) P225A, N218S, M50F, S3C, Q206C, Q2K, A73L, P5S and Y217L; The amino acid positions are defined according to the sequence of BPN' of subtilisin, as shown in SEQUENCE ID NO:

2.

2. A method for enzymatically synthesizing a peptide, comprising coupling (a) a C-terminal ester or thioester of the peptide and (b) a peptide nucleophilic group having an N-terminal deprotected amine. The coupling is performed in a fluid containing water, and The coupling is catalyzed by a subtilisin BPN' variant, the mutation of which, compared to the subtilisin BPN' shown in SEQUENCE ID NO: 2, is as follows: - Corresponding to the deletion of amino acids at positions 75-83; and Q2K, S3C, P5S, S9A, 131L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S, S221C, P225A, T254A, and Q271E, and -Optionally, select a group of mutations from the following groups: (1) M222G, M222P, Y104S, I107V, L135N, L135S or L135D; (2) I107V and L135S; (3) I107V and L135N; (4) I107V and M222G; (5) M222P and mutations at the amino acid positions corresponding to L217, wherein the mutations are selected from L217N, L217T, L217E, L217I, L217V, L217A, L217G, L217H or L217F; (6) M222G and the mutation at the amino acid position corresponding to L217, wherein the mutation is selected from L217G, L217F, L217N, L217E, L217I, L217H, L217A, L217R, L217T or L217S; The amino acid positions are defined according to the sequence of BPN', the subtilisin shown in SEQUENCE ID NO:

2.

3. A method for the enzymatic synthesis of a cyclic peptide of at least 12 amino acids, comprising a cyclization step of a C-terminal C-terminal ester or thioester of a peptide having an N-terminal deprotected amine, wherein the cyclization is carried out in a fluid containing water, and The cyclization is catalyzed by a subtilisin BPN' variant, and the mutation of the subtilisin BPN' variant compared to the subtilisin BPN' shown in SEQUENCE ID NO: 2 is as follows: - Corresponds to the deletion of amino acids at positions 75-83; -S221C; and -Optionally, select a group of mutations from the following groups: (1) P225A or P225G; (2) P225A and N218S; (3) P225A, N218S and M50F; (4) P225A, N218S, M50F, S3C, Q206C, Q2K, A73L and P5S; or (5) P225A, N218S, M50F, S3C, Q206C, Q2K, A73L, P5S and Y217L; The amino acid positions are defined according to the sequence of BPN', the subtilisin shown in SEQUENCE ID NO:

2.

4. A method for the enzymatic synthesis of a cyclic peptide of at least 12 amino acids, comprising a cyclization step of a C-terminal C-terminal ester or thioester of a peptide having an N-terminal deprotected amine, wherein the cyclization is carried out in a fluid containing water, and The cyclization is catalyzed by a subtilisin BPN' variant, and the mutation of the subtilisin BPN' variant compared to the subtilisin BPN' shown in SEQUENCE ID NO: 2 is as follows: - Corresponding to the deletion of amino acids at positions 75-83; and Q2K, S3C, P5S, S9A, 131L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S, S221C, P225A, T254A, and Q271E, and -Optionally, select a group of mutations from the following groups: (1) M222G, M222P, Y104S, I107V, L135N, L135S or L135D; (2) I107V and L135S; (3) I107V and L135N; (4) I107V and M222G; (5) M222P and mutations at the amino acid positions corresponding to L217, wherein the mutations are selected from L217N, L217T, L217E, L217I, L217V, L217A, L217G, L217H or L217F; (6) M222G and the mutation at the amino acid position corresponding to L217, wherein the mutation is selected from L217G, L217F, L217N, L217E, L217I, L217H, L217A, L217R, L217T or L217S; The amino acid positions are defined according to the sequence of BPN', the subtilisin shown in SEQUENCE ID NO:

2.

5. The method according to any one of claims 1-4, characterized in that, The C-terminal ester of the peptide is defined as peptide-(C=O)O-CX2-C(=O)N-R1R2, where each X independently represents a hydrogen atom or an alkyl group; and R1 represents a hydrogen atom or an alkyl group and R2 represents a peptide residue or hydrogen atom or alkyl group or amino acid having a C-terminal carboxamide or carboxylic acid functional group, optionally protected on a side chain functional group of an amino acid or on one or more of the side chain functional groups of a plurality of amino acids.

6. An enzyme, wherein the enzyme is a variant of subtilisin BPN', the mutation of which, compared to the subtilisin BPN' shown in SEQUENCE ID NO: 2, is: - Corresponds to the deletion of amino acids at positions 75-83; -S221C; -Optionally, select a group of mutations from the following groups: (1) P225A or P225G; (2) P225A and N218S; (3) P225A, N218S and M50F; (4) P225A, N218S, M50F, S3C, Q206C, Q2K, A73L and P5S; or (5) P225A, N218S, M50F, S3C, Q206C, Q2K, A73L, P5S and Y217L; The amino acid positions are defined according to the sequence of BPN', the subtilisin shown in SEQUENCE ID NO:

2.

7. An enzyme, wherein the enzyme is a variant of subtilisin BPN', the mutation of which, compared to the subtilisin BPN' shown in SEQUENCE ID NO: 2, is: - Corresponding to the deletion of amino acids at positions 75-83; and Q2K, S3C, P5S, S9A, 131L, K43N, M50F, A73L, E156S, G166S, G169A, S188P, Q206C, N212G, Y217L, N218S, S221C, P225A, T254A, and Q271E, and -Optionally, select a group of mutations from the following groups: (1) M222G, M222P, Y104S, I107V, L135N, L135S or L135D; (2) I107V and L135S; (3) I107V and L135N; (4) I107V and M222G; (5) M222P and mutations at the amino acid positions corresponding to L217, wherein the mutations are selected from L217N, L217T, L217E, L217I, L217V, L217A, L217G, L217H or L217F; (6) M222G and the mutation at the amino acid position corresponding to L217, wherein the mutation is selected from L217G, L217F, L217N, L217E, L217I, L217H, L217A, L217R, L217T or L217S; The amino acid positions are defined according to the sequence of BPN', the subtilisin shown in SEQUENCE ID NO:

2.

8. The enzyme as described in claim 7, characterized in that, The mutation of the BPN' variant of the subtilisin is further as follows: - Corresponds to M222P and L217H; - Corresponds to M222P and L217G; - Corresponds to M222G and L217F; or - Corresponds to M222G and L217G.

9. The enzyme as described in claim 8, characterized in that, The mutation of the BPN' variant of the subtilisin is further as follows: - Corresponds to M222P and L217H; or - Corresponds to M222G and L217F.