Subtilisin Variants With Targeted Charge Tuning for Detergent Stability
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Solution Overview
Problem
There is a need for improved protease variants, particularly subtilisins, with enhanced stability and soil removal capabilities for cleaning applications.
Innovation Solution
Development of subtilisin variants with specific amino acid substitutions, such as X9T, X17H, X45R, X68S, X78I, X86E, X87A, X96D, X100E, X100N, X103F, X103I, X108Q, X115L, X117R, X127S, X127T, X128K, X128P, X128R, X129Q, X155E, X161Q, X181E, X181Q, X202V, X203E, X203N, X217S, X221Q, X260W, and X264H, which exhibit at least 75% identity to SEQ ID NO: 1 and a net charge of −4 to +2 at pH 8, resulting in improved stability and cleaning performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If subtilisin variants with specific amino acid substitutions are developed, then stability and cleaning performance are improved, but protein structure complexity increases
Solution Approach 1:
The patent applies parameter changes by systematically modifying amino acid substitutions at specific positions (e.g., X9T, X17H, X45R, X68S, X78I, X86E, X87A, X96D, X100E, X100N, X103F, X103I, X108Q, X115L, X117R, X127S, X127T, X128K, X128P, X128R, X129Q, X155E, X161Q, X181E, X181Q, X202V, X203E, X203N, X217S, X221Q, X260W, and X264H) to optimize the balance between stability improvement and structural complexity. These targeted substitutions change local chemical properties (charge, hydrophobicity, size) to enhance detergent stability while maintaining catalytic function.
Solution Approach 2:
The patent applies local quality by making specific amino acid substitutions at defined positions rather than uniform modifications throughout the protein. Each substitution is strategically placed to affect local properties such as charge distribution (achieving net charge of -4 to +2 at pH 8), hydrophobicity, or steric constraints, thereby improving stability without globally complicating the protein structure.
2Productivity
If subtilisin variants with specific amino acid substitutions are developed, then cleaning performance is improved, but manufacturing complexity increases
Solution Approach 1:
The patent applies parameter changes by defining specific amino acid substitutions at predetermined positions to achieve desired cleaning performance characteristics. The substitutions are designed to optimize detergent stability and cleaning efficacy while maintaining a manageable number of variants for industrial production.
Solution Approach 2:
The patent applies local quality by implementing targeted amino acid substitutions at specific positions rather than comprehensive protein redesign. This localized approach allows for improved cleaning performance through focused modifications while simplifying manufacturing compared to complete protein synthesis or extensive purification protocols.
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 variants demonstrate at least 25% improved stability and enhanced cleaning performance compared to parent subtilisins, making them suitable for use in cleaning compositions and methods.
Implementation Method 1
A protease (also known as a proteinase) is an enzyme that has the ability to break down other proteins. A protease has the ability to conduct proteolysis, which begins protein catabolism by hydrolysis of peptide bonds that link amino acids together in a peptide or polypeptide chain
Implementation Method 2
where the variant has at least 75% identity to the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 8 and has a net charge of −4 to +2 at pH 8 relative to the subtilisin having the amino acid sequence of SEQ ID NO: 1
Data Source
AI summary
Disclosed herein is one or more subtilisin variant, nucleic acid encoding same, and compositions and methods related to the production and use thereof, including one or more subtilisin variant that has improved stability and/or soil removal compared to one or more reference subtilisin.