Subtilisin BPN' Variant Peptide Synthesis
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Solution Overview
Problem
Current methods for enzymatic peptide synthesis, such as subtiligase and subtilisin BPN', face challenges with low synthesis over hydrolysis ratios and stability issues, particularly in aqueous solutions and the presence of organic co-solvents, leading to inefficient peptide condensation and high hydrolysis side reactions.
Innovation Solution
A subtilisin BPN' variant with specific mutations, including deletion of the calcium binding domain at positions 75-83 and mutations at S221 and P225, is used for the enzymatic synthesis of oligopeptides, offering improved stability and synthesis over hydrolysis ratios, allowing for efficient peptide condensation and cyclization in aqueous solutions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If subtilisin BPN' or subtiligase is used for enzymatic peptide synthesis in aqueous solution, then peptide condensation can be catalyzed, but hydrolysis side reactions occur simultaneously leading to low synthesis over hydrolysis ratios
Solution Approach 1:
The calcium binding domain (amino acids 75-83) is extracted/removed from the subtilisin BPN' structure. This deletion eliminates the calcium binding site that is responsible for stabilizing the hydrolytic conformation, thereby reducing hydrolysis activity while preserving peptide synthesis capability
Solution Approach 2:
The enzyme's structural parameters are changed through site-directed mutagenesis. Specific amino acid substitutions (S221C, P225A, M222G, Y217L, I107V) are introduced to alter the active site geometry and flexibility, shifting the enzyme's preference from hydrolysis to synthesis reactions
2Reliability
If existing subtilisin variants are used for peptide synthesis, then some synthesis activity is achieved, but stability against organic co-solvents and temperature is poor
Solution Approach 1:
Multiple amino acid substitutions are introduced to change the enzyme's physical-chemical parameters. Hydrophobic interactions are enhanced through mutations like M222G and Y217L, while active site flexibility is optimized through S221C and P225A, achieving both stability and activity
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The subtilisin BPN' variant achieves a significantly higher synthesis over hydrolysis ratio compared to existing enzymes, reducing hydrolysis side reactions and enabling high-yield peptide synthesis and cyclization, even with proteins, while maintaining the secondary and tertiary structure of the protein.
Implementation Method 1
a subtilisin BPN' variant, which catalyses the condensation of two (oligo)peptide fragments or the cyclisation of an (oligo)peptide
Implementation Method 2
a major drawback of such enzymatic oligopeptide fragment condensation in aqueous solution is that simultaneous hydrolysis of the peptide bonds within the oligopeptide fragments and of the C-terminal ester functionality takes place leading to low yields and many side products
Data Source
AI summary
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, andwherein 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.