Modified Cellulase Linker Peptides Reduce Lignin Binding

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Solution Overview

Problem

Current cellulase enzymes are hindered by lignin, leading to reduced enzymatic hydrolysis efficiency in converting lignocellulosic biomass to sugars, as they bind to lignin, causing inactivation and decreased activity.

Innovation Solution

Modified cellulase enzymes with altered linker peptides that decrease lignin binding and increase cellulose hydrolyzing activity, featuring amino acid substitutions and modifications that lower the isoelectric point and enhance the threonine:serine ratio, thereby reducing lignin interaction while maintaining cellulose binding affinity.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Productivity

If cellulase enzymes are used to hydrolyze cellulose in the presence of lignin, then cellulose conversion to sugars can occur, but the enzymes bind to lignin causing inactivation and reduced activity

Engineering Contradiction:

Improvecellulose hydrolysis efficiencyVSAvoidenzyme activity stability

Core Design Contradiction:

ProductivityVSReliability

Solution Approach 1:

The patent applies parameter changes by modifying the amino acid composition of the linker peptide region of cellulase enzymes. Specifically, it increases the threonine:serine ratio and decreases the isoelectric point through amino acid substitutions, insertions, or deletions. These parameter changes in the enzyme structure reduce lignin binding affinity while maintaining cellulose hydrolysis activity, thereby resolving the contradiction between productivity and reliability in lignocellulosic biomass conversion.

Inventive Principle:

Principle #35Parameter changes

2Ease of operation

If cellulase enzymes bind to lignin, then they can access cellulose in lignocellulosic biomass, but binding to lignin causes inactivation and decreased enzymatic activity

Engineering Contradiction:

Improveaccess to cellulose substrateVSAvoidlignin binding-induced inactivation

Core Design Contradiction:

Ease of operationVSObject-generated harmful factors

Solution Approach 1:

The patent applies local quality by making specific modifications only to the linker peptide region of the cellulase enzyme while leaving the catalytic domain and carbohydrate-binding module unchanged. This localized modification approach allows the enzyme to maintain its cellulose-binding capability and catalytic function while reducing harmful interactions with lignin, thus resolving the contradiction between substrate accessibility and enzyme stability.

Inventive Principle:

Principle #3Local quality

3Stability of the object's composition

If natural cellulase enzymes are used, then they maintain native structure and function, but they exhibit high lignin binding affinity leading to reduced hydrolysis efficiency

Engineering Contradiction:

Improveenzyme native structureVSAvoidsugar production efficiency

Core Design Contradiction:

Stability of the object's compositionVSProductivity

Solution Approach 1:

The patent applies segmentation by dividing the cellulase enzyme into distinct functional regions (catalytic domain, linker peptide, and carbohydrate-binding module) and modifying only the linker peptide segment. This segmentation strategy allows the enzyme to retain its native overall structure and function while improving productivity through targeted modifications that reduce lignin binding affinity in the linker region.

Inventive Principle:

Principle #1Segmentation

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 cellulase enzymes exhibit increased cellulose hydrolyzing activity and reduced lignin binding, enhancing the efficiency of sugar production from lignocellulosic substrates, such as ethanol production from cellulose.

Implementation Method 1

enzymatic hydrolysis to sugars and the fermentation of those sugars to ethanol or other high-value organic molecules

Methodology Applied

Scientific EffectEnzymatic hydrolysis: Hydrolysis

Implementation Method 2

modified cellulase enzymes exhibit increased cellulose hydrolyzing activity

Methodology Applied

Scientific EffectEnzyme catalysis: Enzyme

Implementation Method 3

amino acid substitutions and modifications that lower the isoelectric point and enhance the threonine:serine ratio, thereby reducing lignin interaction

Methodology Applied

Scientific EffectElectrostatic interaction: Electrostatics

Data Source

PatentUS10364422B2Cellulase enzymes having a modified linker and reduced lignin binding

Publication Date: 2019.07.30 IOGEN ENERGY CORP

AI summary

Provided are modified cellulase enzymes exhibiting increase cellulose-hydrolyzing activity in the presence of lignin and/or reduced binding to lignin comprising modified linker peptides comprising one or more amino acid substitutions, insertions, or deletions that result in (a) a decrease in the calculated isoelectric point of the linker peptide and/or (b) an increase in the ratio of threonine:serine in the linker peptide relative to a parental linker peptide from which said modified linker peptide is derived. Also provided are genetic constructs comprising nucleic acid sequences encoding for modified cellulase enzymes, methods for the production of the modified cellulase enzymes from host strains and a process for hydrolysing cellulose with the modified cellulases in the presence of lignin.