Thermostable Protease Mutations for High-Temperature Detergent Activity
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Solution Overview
Problem
Current industrial proteases lack stability and activity under high temperature and high alkaline conditions, particularly in the presence of surfactants, limiting their practical application in detergents and other industrial processes.
Innovation Solution
A novel protease with an amino acid sequence capable of maintaining high activity at temperatures above 100°C and pH levels above 11.5, exhibiting 40% residual activity after 90 minutes at 100°C and 80% residual activity in the presence of sodium dodecyl sulfate, along with a pro-form and encoding polynucleotides, which do not require calcium ions for activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional proteases are used, then they can perform basic proteolytic functions, but they lack stability and activity under high temperature and high alkaline conditions
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the protease through site-directed mutagenesis. Specific amino acid residues are changed to alter the enzyme's physicochemical properties, enabling it to maintain stability and activity under high temperature (100°C or higher) and high alkaline (pH 11.5 or higher) conditions that conventional proteases cannot withstand.
Solution Approach 2:
The patent creates a composite enzyme system by combining multiple amino acid mutations within the protease structure. The synergistic effect of multiple point mutations (e.g., in the substrate binding pocket and catalytic domain) produces a protease with enhanced extreme condition stability that exceeds the sum of individual mutation effects.
2Temperature
If proteases are designed for high temperature stability, then thermostability improves, but activity under high alkaline conditions and in the presence of surfactants deteriorates
Solution Approach 1:
The patent achieves multi-functionality by engineering the protease to simultaneously perform multiple functions: maintaining catalytic activity at high temperature, resisting high alkaline conditions, and tolerating surfactant presence. The mutated protease structure incorporates features that address all three challenges concurrently, making it universally effective in complex detergent formulations.
Solution Approach 2:
The patent applies local quality changes by introducing specific amino acid mutations at critical locations within the protease structure. Mutations are strategically placed in the substrate binding pocket, catalytic domain, and surface regions to locally enhance properties such as alkaline resistance and surfactant tolerance while preserving overall thermostability.
3Productivity
If protease activity is enhanced for detergent applications, then cleaning efficiency improves, but stability to protein denaturants and surfactants worsens
Solution Approach 1:
The patent applies preliminary action by pre-engineering the protease structure through site-directed mutagenesis before exposing it to extreme conditions. The amino acid mutations are introduced in advance to create a robust structure that can withstand subsequent exposure to protein denaturants, surfactants, and extreme pH and temperature conditions during detergent application.
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 protease demonstrates enhanced stability and activity under extreme conditions, effectively degrading substrates at low concentrations and maintaining functionality in the presence of protein denaturants and surfactants, making it suitable for use in high-temperature and high-alkaline environments.
Implementation Method 1
Protease is a collective term for enzymes catalyzing hydrolysis of peptide bonds
Data Source
Figure 1(a)~1(b)
Figure 2~3
Figure 4~6
AI summary
A novel protease comprising any one of (a) the amino acid sequence of SEQ ID NO: 1, (b) an amino acid sequence having deletion, substitution or addition of one to several amino acids in the amino acid sequence of SEQ ID NO: 1, (c) the amino acid sequence of SEQ ID NO: 3, and (d) an amino acid sequence having deletion, substitution or addition of one to several amino acids in the amino acid sequence of SEQ ID NO: 3, has a high activity under high temperature and high alkaline conditions, a high stability to protein denaturants and surfactants and high effectiveness as a protease used in detergents.