Subtilisin BPN' Variants for Selective Peptide Coupling in Water
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Solution Overview
Problem
Existing subtilisin BPN' variants and homologues face challenges in achieving high reaction rates and coupling efficiencies for peptide synthesis, particularly in aqueous environments, with issues such as undesirably high hydrolytic activity and side reactions.
Innovation Solution
Development of subtilisin BPN' variants with specific mutations in the acyl donor binding pockets, including deletions and substitutions at positions 75-83 and S221, combined with mutations at L96, D99, A223, and S224, to enhance reaction rate and coupling efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If subtilisin BPN' variants with mutations S221C and P225A are used to increase synthesis over hydrolysis ratio, then coupling efficiency is improved, but hydrolytic activity remains undesirably high in aqueous environment
Solution Approach 1:
The patent applies parameter changes by introducing multiple specific mutations (S221C, P225A, and additional mutations at positions 75-83, 96, 99, 223, and 224) to alter the enzyme's catalytic properties. These mutations change the physical-chemical parameters of the active site, reducing hydrolytic activity while maintaining or enhancing synthesis activity, thereby resolving the contradiction between improving synthesis efficiency and reducing harmful hydrolysis.
2Productivity
If chemical coupling of side-chain protected peptide fragments is used, then peptide synthesis can be achieved, but racemisation occurs upon activation of the acyl donor
Solution Approach 1:
The patent replaces chemical coupling mechanisms with enzymatic coupling mechanisms. Instead of using chemical reagents that cause racemisation, the engineered subtilisin variant catalyzes the coupling reaction through a biological mechanism that preserves stereochemical integrity. This substitution of chemical mechanism with enzymatic mechanism eliminates racemisation while maintaining peptide synthesis capability.
3Productivity
If chemical peptide synthesis is used, then peptide coupling can be achieved, but side reactions on side-chain functionalities occur
Solution Approach 1:
The patent changes the reaction parameters from chemical to enzymatic catalysis. The engineered subtilisin variant provides a specific catalytic environment that recognizes and processes only the target peptide bonds, while the enzyme's active site geometry and catalytic mechanism prevent unwanted side reactions on side-chain functionalities. This parameter change from chemical to biological catalysis achieves selective coupling without side reactions.
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 modified enzymes exhibit increased reaction rates, improved coupling efficiencies, and selectivity in aqueous media, making them suitable for efficient peptide synthesis.
Implementation Method 1
the coupling is catalysed by a subtilisin BPN' variant or homologue thereof
Implementation Method 2
which has ligase and/or cyclase activity with an increased reaction rate and coupling efficiency
Data Source
AI summary
The invention relates to a subtilisin BPN′ variant or homologue thereof, having new mutations compared to subtilisin BPN′ represented by SEQ ID NO: 2 or a homologue sequence thereof. Such mutations may occur at amino acid positions L96, D99, A223 or S224. The invention further relates to a method for enzymatically synthesizing a peptide by coupling peptide fragments, wherein the coupling is catalyzed by the subtilisin BPN′ variant or homologue thereof.