Endolysin Variant Overcomes Gram-Negative Outer Membrane Barrier
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Solution Overview
Problem
Current antibacterial agents, including endolysins, face challenges in effectively targeting Gram-negative bacteria due to their outer membrane, which shields against peptidoglycan-degrading enzymes, and there is a growing concern about antibiotic resistance.
Innovation Solution
Development of a polypeptide with specific amino acid sequences exhibiting high sequence identity to Lys68, with modifications such as removal of N-terminal and C-terminal portions and introduction of mutations, enhancing thermal stability and potentially fused with amphipathic, cationic, or hydrophobic peptides to overcome the outer membrane barrier.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If endolysins are used as antibacterials, then they can effectively degrade peptidoglycan and kill bacteria, but they cannot penetrate the outer membrane of Gram-negative bacteria
Solution Approach 1:
The patent introduces a membrane-permeating peptide (such as Lys68 or other cationic amphipathic peptides) as an intermediary that facilitates the delivery of endolysin to the peptidoglycan layer. The membrane-permeating peptide penetrates the outer membrane first, then delivers the endolysin to its target, thus resolving the barrier problem without compromising the antibacterial efficacy of the endolysin.
Solution Approach 2:
The patent creates a composite structure by fusing or complexing the endolysin with a membrane-permeating peptide. This composite molecule combines the peptidoglycan-degrading capability of endolysin with the membrane-penetration ability of the peptide, enabling the system to overcome the outer membrane barrier while maintaining its antibacterial function.
2Reliability
If antibiotics are used to combat bacterial infections, then they can effectively kill bacteria, but antibiotic resistance develops
Solution Approach 1:
The patent replaces the chemical mechanism of antibiotics with a mechanical/enzymatic mechanism. Instead of relying on chemical targets that bacteria can mutate to resist, the endolysin physically degrades the peptidoglycan cell wall through enzymatic hydrolysis, a mechanism that is much harder for bacteria to develop resistance against.
Solution Approach 2:
The patent fundamentally changes the mode of action from chemical inhibition (antibiotics) to enzymatic degradation (endolysin). This parameter change in the mechanism of action transitions from a system where resistance can develop through target modification to a system where the physical breakdown of structural components is much more difficult to resist.
3Stability of the object's composition
If the polypeptide sequence is modified to enhance thermal stability, then thermal stability increases, but sequence identity to the original Lys68 decreases
Solution Approach 1:
The patent deliberately changes the amino acid sequence parameters at positions that do not affect the active site or membrane-permeating domains. By modifying non-critical regions of the polypeptide, the invention achieves improved thermal stability while preserving the essential functional characteristics and maintaining sufficient sequence identity to the original Lys68.
Solution Approach 2:
The patent applies local modifications to specific regions of the polypeptide that are not involved in the core catalytic or membrane-interaction functions. By concentrating stability-enhancing mutations in non-critical areas, the invention improves thermal stability while preserving the local quality and function of the active site and membrane-permeating regions.
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 polypeptide maintains activity and increases thermal stability, enabling effective antibacterial action against Gram-negative bacteria, addressing the resistance issues and membrane permeability challenges.
Implementation Method 1
Endolysins are peptidoglycan hydrolases encoded by bacteriophages... They are synthesized during late gene expression in the lytic cycle of phage multiplication and mediate the release of progeny virions from infected cells through degradation of the bacterial peptidoglycan.
Data Source
AI summary
The present invention relates to the field of antimicrobial enzymes. In particular, the present invention relates to a polypeptide comprising an amino acid sequence exhibiting at least 90% sequence identity with the sequence of SEQ ID NO:1, with the proviso that the polypeptide does neither comprise the sequence according to SEQ ID NO:2, nor the sequence according to SEQ ID NO:3, nor the sequence according to SEQ ID NO:4. The present invention relates also to nucleic acids encoding an inventive polypeptide, vectors or bacteriophages comprising an inventive nucleic acid as well as host cells comprising an inventive polypeptide, nucleic acid, vector, and/or bacteriophage. Similarly, the present invention relates to compositions comprising a polypeptide, nucleic acid, vector, bacteriophage, and/or host cell according to the present invention.