Proline-Specific Endoprotease Variant for Controlled Thermal Inactivation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing proline-specific endoproteases have residual activity issues at elevated temperatures, which is undesirable in food and feed applications where stability and activity retention are crucial.
Innovation Solution
A polypeptide with specific amino acid substitutions, such as those defined by SEQ ID NO: 1 and 2, exhibits reduced residual activity after being kept at 65°C for 15 minutes, making it suitable for applications where minimal activity is desired, and can be used in food or feed processing to hydrolyze gluten or other proteins.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If existing proline-specific endoproteases are used in food and feed applications, then they can effectively hydrolyze proteins and peptides, but they retain residual activity at elevated temperatures which is undesirable
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the proline-specific endoprotease through specific substitutions (such as position 238, 242, 279, 359, 379, 421, 424, 460, 466, 469, 470, 507). These sequence parameter changes result in an enzyme that maintains low residual activity (<70%) after exposure to elevated temperatures (65°C for 15 minutes), thereby resolving the contradiction between reliable enzyme activity control and stability at processing temperatures.
2Reliability
If proline-specific endoprotease is added during beer production to reduce haze, then haze reduction is achieved, but residual enzyme activity may remain in the final beverage which is undesirable
Solution Approach 1:
The patent modifies the enzyme's amino acid sequence parameters to create a variant with reduced thermal stability of activity. This allows the enzyme to effectively perform haze reduction during beer production, then become sufficiently inactive after standard pasteurization temperatures, eliminating residual harmful activity in the final beverage while maintaining processing effectiveness.
3Reliability
If proline-specific endoprotease is used as a medicament or dietary supplement for gluten degradation, then gluten toxicity is reduced, but residual activity in the final food product is undesirable
Solution Approach 1:
The patent applies amino acid sequence modifications to create a proline-specific endoprotease variant that effectively degrades gluten in food products during processing, then becomes sufficiently inactive after standard food processing temperatures. This resolves the contradiction by ensuring complete gluten degradation while eliminating residual enzyme activity that could cause harm in the final food product.
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 polypeptide maintains less than 70% residual activity at elevated temperatures, effectively reducing its activity to desired levels, thereby addressing the stability and activity retention challenges in food and feed applications.
Implementation Method 1
Proline-specific endoproteases are enzymes that hydrolyse a protein or peptide at a position where there is a proline in the protein or peptide
Data Source
Figure 1
AI summary
The present invention relates to a polypeptide having proline-specific endoprotease activity, wherein the polypeptide has less than 70% residual activity when the polypeptide has been kept at a temperature of 65°C for 15 min. The invention further relates to a polypeptide having proline-specific endoprotease activity comprising an amino acid sequence according to SEQ ID NO: 1, wherein SEQ ID NO: 1 comprises at least one combination of amino acid substitutions selected from the group consisting of a combination selected from the group consisting of (K238E, I204V, V460A), (F279S, A242V, N507I), (T145A, K424M), (T359A, F379S), (M170I, A421 T), (N441 S, G484S), (L470H, Q288R), (L470H, E387G), (T281 S, L373I), (P304A, P469A), and (P466T, P469Q), a nucleic acid encoding a polypeptide having proline-specific endoprotease activity, a method of making a variant polypeptide having proline-specific endoprotease activity, a recombinant host cell and a method of producing the polypeptide and a process for the preparation of a food or feed product wherein the polypeptide is used.