Protease Variant Composition for Low-Temperature Detergent Cleaning
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Solution Overview
Problem
Existing proteases in liquid washing and cleaning agents exhibit insufficient catalytic activity and stability under standard washing conditions, leading to suboptimal cleaning performance on protease-sensitive stains, particularly in the temperature range of 20° C. to 40° C.
Innovation Solution
A protease from Bacillus pumilus with specific amino acid substitutions at positions 9, 89, 130, 133, 144, 189, 217, 224, 252, and 271, and optionally additional substitutions at positions 63, 99, 156, 166, and 170, enhancing its proteolytic activity and stability, making it suitable for improved cleaning performance in liquid agents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild-type protease from Bacillus pumilus is used in liquid washing agents, then the formulation is simple and cost-effective, but the proteolytic activity and stability under standard washing conditions are insufficient
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid positions (9, 89, 130, 133, 144, 189, 217, 224, 252, 271) in the protease sequence to alter its catalytic and stability properties. This systematic modification of sequence parameters transforms the wild-type protease into variants with enhanced performance in liquid washing agents.
Solution Approach 2:
The invention implements local quality by introducing specific amino acid substitutions at predetermined positions in the protease sequence while maintaining the overall structure. This localized modification approach optimizes specific functional regions (catalytic activity and stability) without completely redesigning the entire enzyme, balancing improvement with structural integrity.
2Productivity
If protease variants with multiple amino acid substitutions are developed to improve cleaning performance, then the catalytic activity and stability increase, but the production and characterization complexity increases
Solution Approach 1:
The patent segments the protease optimization process into two distinct groups of amino acid substitutions: Group I (positions 9, 89, 130, 133, 144, 189, 217, 224, 252, 271) for fundamental performance enhancement, and Group II (positions 63, 99, 156, 166, 170) for additional optimization. This segmentation allows systematic development and testing of variants with different substitution combinations, managing complexity through structured approach.
Solution Approach 2:
The invention creates protease variants that serve multiple functions simultaneously: improved catalytic activity for stain removal, enhanced stability under washing conditions, and maintained compatibility with liquid surfactant-containing formulations. The multi-functional design addresses various performance requirements through a unified enzyme variant platform.
3Reliability
If existing proteases are used in liquid surfactant-containing preparations, then the formulation is straightforward, but the catalytic performance and storage stability are not sufficient
Solution Approach 1:
The patent modifies physical-chemical parameters of the protease through amino acid substitutions, specifically targeting stability parameters (resistance to denaturation, storage stability) and catalytic parameters (activity in surfactant-containing environments). These parameter changes enable the protease to maintain functionality in liquid surfactant preparations where wild-type enzymes fail.
Solution Approach 2:
The invention creates protease variants that are cost-effective alternatives to commercially available proteases, achieving comparable or superior performance in liquid washing agents. By optimizing the Bacillus pumilus protease in-house, the patent avoids reliance on expensive commercial enzymes while maintaining or improving catalytic performance and stability.
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 protease demonstrates enhanced catalytic activity and stability, resulting in improved removal of protease-sensitive stains such as blood, egg yolk, and milk stains on textiles and hard surfaces, with increased performance up to 130% compared to wild-type variants.
Implementation Method 1
They act as unspecific endopeptidases and hydrolyze any acid amide bonds within peptides or proteins
Implementation Method 2
Proteases are among the technically most important enzymes... they cause the degradation of protein-containing stains on the articles to be cleaned
Data Source
AI summary
The invention relates to proteases that exhibits proteolytic activity and comprises an amino acid sequence which, over its entire length, is at least 70% and increasingly preferably at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 90.5%, 91%, 91.5%, 92%, 92.5%, 93%, 93.5%, 94%, 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5% and 98% identical to the amino acid sequence specified in SEQ ID NO. 1, the protease having, in each case based on the numbering according to SEQ ID NO:1, (i) at the positions corresponding to positions 9, 89, 130, 133, 144, 189, 217, 224, 252 and 271, the amino acid substitutions P9T, S89A, N130D, T133A, N144K, S189T, Y217M, S224A, N252T and Q271E, and (ii) at at least one and increasingly preferably two, three, four or five of the positions corresponding to positions 63, 99, 156, 166 and 170, at least one and increasingly preferably two, three, four or five amino acid substitution(s) selected from the group consisting of S63Q, N99H, S156R, G166A and K170R, and the production and use thereof. Such proteases exhibit very good cleaning performance.