Peptide fragment condensation and cyclisation using a subtilisin variant with improved synthesis over hydrolysis ratio
Abstract:
The invention relates to a method for enzymatically synthesizing an (oligo)peptide, comprising coupling (a) an (oligo)peptide C-terminal ester or thioester and (b) an (oligo)peptide nucleophile having an N-terminally unprotected amine, wherein the coupling is carried out in a fluid comprising water, and wherein the coupling is catalyzed by a subtilisin BPN′ variant or a homolog thereof, which comprises the following mutations compared to subtilisin BPN′ represented by SEQUENCE ID NO: 2 or a homolog sequence thereof: a deletion of the amino acids corresponding to positions 75-83; a mutation at the amino acid position corresponding to S221, the mutation being 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′ represented by SEQUENCE ID NO: 2. Further, the invention relates to an enzyme suitable for use as a catalyst in a method of the invention.
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