Enzyme Specificity Modifiers via Binding Polypeptides
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Solution Overview
Problem
Current methods for modifying enzyme substrate specificity are limited by the need for detailed knowledge of the enzyme's three-dimensional structure, which is often unavailable, especially for industrial enzymes like β-galactosidase and lipase 1.
Innovation Solution
Development of novel enzyme substrate specificity modifiers in the form of polypeptides that bind to β-galactosidase and lipase 1, including inert binders, inhibitors, and specificity modifiers, without requiring structural information, using combinatorial libraries of FN3 domain variants.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If standard enzyme-engineering approaches (structure-guided design or directed evolution) are used, then enzyme catalytic properties can be modified, but detailed knowledge of the enzyme's three-dimensional structure is required which is often unavailable for industrial enzymes
Solution Approach 1:
The patent introduces binding proteins as intermediary molecules that bind to the enzyme at sites distinct from the active site. These binding proteins serve as mediators to modulate enzyme-substrate interactions without requiring direct modification of the enzyme structure or knowledge of the enzyme's three-dimensional structure. The binding proteins act as external regulators that can enhance or inhibit enzyme activity and alter substrate specificity through non-covalent interactions.
Solution Approach 2:
The approach segments the enzyme system into two independent components: the enzyme itself and the separately engineered binding proteins. This segmentation allows the binding proteins to be designed and optimized independently using combinatorial libraries, bypassing the need for detailed enzyme structural information. The binding proteins can then be combined with the enzyme to achieve desired catalytic property modifications.
2Quantity of substance
If industrial enzymes are purified from native hosts, then they can be obtained, but their 3D structures are seldom known and assays are labor intensive
Solution Approach 1:
Binding proteins serve as intermediaries that can be developed using combinatorial libraries without requiring knowledge of the target enzyme's three-dimensional structure. This approach decouples the availability of the enzyme from the need for its structural information, allowing binding proteins to be engineered against purified industrial enzymes even when their structures are unknown.
Solution Approach 2:
The binding protein development system is self-sufficient in that it does not rely on external structural information about the target enzyme. The combinatorial library approach allows the binding proteins to 'find' their binding sites through selection processes, making the system independent of pre-acquired structural knowledge and reducing the need for labor-intensive assays.
3Adaptability or versatility
If combinatorial libraries of FN3 domain variants are used, then enzyme substrate specificity modifiers can be generated without structural knowledge, but the process requires development of novel binding proteins
Solution Approach 1:
The FN3 domain serves as a universal scaffold that can be used to generate binding proteins against multiple different target enzymes. The combinatorial library of FN3 domain variants provides a versatile platform that can be applied to various industrial enzymes regardless of their specific structures or functions, making the approach universally applicable across different enzyme types while maintaining ease of manufacture through standardized domain architecture.
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
Enables the modification of enzyme substrate specificity without structural knowledge, maintaining enzyme activity and binding affinity, thus overcoming the limitations of traditional enzyme engineering approaches.
Implementation Method 1
polypeptides that bind to β-galactosidase or lipase 1
Implementation Method 2
β-galactosidase catalyzes lactose hydrolysis and trans-galactosylation reactions
Data Source
AI summary
Aspects of the disclosure relate to compositions of enzyme-binding polypeptides (EBPs) that modify the substrate specificity of an enzyme and a method for identifying an EBP that modifies substrate specificity of an enzyme binding at least one substrate, the method comprising: contacting the enzyme with a polypeptide library comprising a plurality of EBPs that bind different epitopes of the enzyme; identifying EBPs that bind to the enzyme to form an EBP-enzyme complex; assaying for the activity level and substrate specificity of the EBP-enzyme complex; and identifying EBPs that modify the substrate specificity of the enzyme by identifying EBPs that, when in an EBP-enzyme complex, have a different substrate specificity than un-complexed EBP; wherein the catalytic rate constant of the EBP-enzyme complex is ≥50% of the un-complexed enzyme for at least one substrate and/or wherein the EBP-enzyme complex retains binding to a substrate.