Variant Endoglucanases for Enhanced Enzyme Stability and Activity

Solution Overview

Problem

Current endoglucanases used in textile, detergent, and pulp and paper industries lack improved total activity, specific activity, temperature activity, pH activity, stability, and pH tolerance, necessitating the development of variant enzymes with enhanced properties.

Innovation Solution

Variant endoglucanases with specific amino acid substitutions at positions such as 12, 25, 40, 44, 65, 138, 139, 160, 161, 171, 172, 175, 182, 184, 194, 212, 214, 55, 80, 86, 104, 126, 128, 136, 145, 152, 164, 165, 169, 173, 176, 179, 181, 187, 205, 207, and 218, which exhibit at least 90% identity to parent enzymes and demonstrate improved activity and stability across various temperatures and pH levels.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If naturally occurring endoglucanases are used, then the enzyme can perform basic cellulose hydrolysis, but the total activity, specific activity, temperature activity, pH activity, stability, and pH tolerance are insufficient for industrial applications

Engineering Contradiction:

Improveenzyme stability and activityVSAvoidperformance across various temperatures and pH levels

Core Design Contradiction:

ReliabilityVSAdaptability or versatility

Solution Approach 1:

The patent applies parameter changes by systematically modifying amino acid sequences of endoglucanases to alter their biochemical properties. Specific amino acid substitutions at defined positions (e.g., positions 12, 25, 40, 44, 65, 138, 139, 160, 161, 171, 172, 175, 182, 184, 194, 212, 214, 55, 80, 86, 104, 126, 128, 136, 145, 152, 164, 165, 169, 173, 176, 179, 181, 187, 205, 207, and 218) are designed to enhance total activity, specific activity, temperature activity, pH activity, stability, and pH tolerance, thereby improving enzyme performance across various industrial conditions

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

The patent applies local quality by making targeted amino acid substitutions at specific positions within the enzyme structure rather than uniform modifications. Each substitution at defined positions is designed to locally improve specific properties such as thermostability or pH tolerance while maintaining overall enzyme function, allowing precise optimization of enzyme characteristics for different industrial applications

Inventive Principle:

Principle #3Local quality

2Productivity

If enzyme activity is increased through amino acid substitutions, then total activity and specific activity improve, but the complexity of enzyme production and characterization increases

Engineering Contradiction:

Improvetotal activity and specific activityVSAvoidproduction and characterization complexity

Core Design Contradiction:

ProductivityVSDevice complexity

Solution Approach 1:

The patent applies segmentation by dividing the enzyme optimization process into discrete, manageable components: specific amino acid positions are identified and modified independently, each substitution is characterized separately, and combinations of substitutions are systematically evaluated. This segmented approach allows complex enzyme optimization to be broken down into individual mutagenesis events that can be produced and characterized in a structured manner

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The patent systematically changes amino acid parameters at specific positions to improve productivity metrics such as total activity and specific activity. By focusing changes on defined positions rather than throughout the entire sequence, the complexity of production and characterization is managed while achieving significant improvements in enzyme performance

Inventive Principle:

Principle #35Parameter changes

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 variant endoglucanases show increased total activity, pH tolerance, and thermostability, offering improved performance in biostoning and biofinishing processes, with enhanced stability and efficiency in industrial applications.

Implementation Method 1

Endoglucanases of the present invention mean enzymes classified as E.C. 3.2.1.4 and are one type of cellulases generally needed for the biological conversion of cellulose to glucose. Endoglucanases cut internal beta-1,4-glucosidic bonds

Methodology Applied

Scientific EffectEnzymatic hydrolysis: Enzyme

Implementation Method 2

Variant endoglucanases with specific amino acid substitutions at positions such as 12, 25, 40, 44, 65, 138, 139, 160, 161, 171, 172, 175, 182, 184, 194, 212, 214, 55, 80, 86, 104, 126, 128, 136, 145, 152, 164, 165, 169, 173, 176, 179, 181, 187, 205, 207, and 218, which exhibit at least 90% identity to parent enzymes and demonstrate improved activity and stability across various temperatures and pH levels

Methodology Applied

Scientific EffectThermostability enhancement through protein engineering:

Implementation Method 3

Variant endoglucanases with specific amino acid substitutions at positions such as 12, 25, 40, 44, 65, 138, 139, 160, 161, 171, 172, 175, 182, 184, 194, 212, 214, 55, 80, 86, 104, 126, 128, 136, 145, 152, 164, 165, 169, 173, 176, 179, 181, 187, 205, 207, and 218, which exhibit at least 90% identity to parent enzymes and demonstrate improved activity and stability across various temperatures and pH levels

Methodology Applied

Scientific EffectpH stability enhancement through protein engineering:

Data Source

PatentUS10927358B2Endoglucanase compositions and methods

Publication Date: 2021.02.23 FORNIA BIOSOLUTIONS INC

AI summary

The invention is directed to novel variant endoglucanases.