Carbohydrate Binding Module Variants Protease Resistance

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Solution Overview

Problem

Cellulases in detergents are susceptible to proteolytic cleavage due to exposure in liquid detergents, leading to reduced stability and effectiveness in maintaining textile whiteness and preventing pilling, as proteases degrade cellulases and other glycoside hydrolases.

Innovation Solution

Development of carbohydrate binding module variants, particularly Family 1 variants, which enhance the stability of glycoside hydrolase enzymes by modifying key positions in the carbohydrate binding module and incorporating proline-rich linkers, reducing susceptibility to proteolytic cleavage while maintaining cellulose binding activity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If cellulases are used in liquid detergent compositions, then they provide benefits in laundry processes such as color clarification and anti-pilling, but they are susceptible to proteolytic cleavage by proteases, leading to reduced stability

Engineering Contradiction:

Improvestability of cellulase in detergentVSAvoidproteolytic degradation by proteases

Core Design Contradiction:

ReliabilityVSObject-affected harmful factors

Solution Approach 1:

The cellulase enzyme is divided into separate functional domains: a catalytic domain and a carbohydrate binding module (CBM), connected by a linker region. This segmentation allows the CBM to remain stable and bound to cellulose substrate while the catalytic domain performs hydrolysis, protecting the overall enzyme structure from complete proteolytic degradation

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

The linker region connecting the catalytic domain and CBM is specifically engineered with protease-resistant characteristics, while the CBM maintains its natural cellulose-binding properties. This local modification of the linker's amino acid sequence provides differential protection - the linker resists proteolysis while allowing the CBM to maintain its functional integrity and binding affinity

Inventive Principle:

Principle #3Local quality

2Reliability

If the carbohydrate binding module is modified to increase protease resistance, then stability improves, but there is a risk of reduced cellulose binding activity

Engineering Contradiction:

Improveprotease resistance of CBMVSAvoidcellulose binding affinity

Core Design Contradiction:

ReliabilityVSManufacturing precision

Solution Approach 1:

Specific amino acid residues in the CBM are modified through site-directed mutagenesis to change their chemical properties and increase resistance to proteolytic cleavage. These parameter changes in the amino acid sequence are carefully selected to provide protease resistance while preserving the structural features necessary for cellulose binding

Inventive Principle:

Principle #35Parameter changes

Solution Approach 2:

Only specific residues within the CBM that are accessible to proteases are modified, while the core residues responsible for cellulose binding are left unchanged. This localized modification approach ensures that protease resistance is enhanced without compromising the essential binding function of the CBM

Inventive Principle:

Principle #3Local quality

Data Source

PatentUS20230143128A1Carbohydrate binding module variants

Publication Date: 2023.05.11 NOVOZYMES AS

AI summary

Disclosed are carbohydrate binding module variants, as well as variants of a glycoside hydrolase comprising the carbohydrate binding module variants. The variants having glycoside hydrolase activity comprising the carbohydrate binding module variants have improved stability in the presence of a protease, and are useful in detergent applications, such as laundry or dish wash.