Chimeric Cellobiohydrolase I Enzymes for Stable Biomass Hydrolysis

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Solution Overview

Problem

Current cellulase mixtures used in biomass conversion processes lack stability and efficiency, leading to high costs and limited effectiveness in hydrolyzing cellulose, particularly due to the difficulty in expressing thermostable CBH I enzymes in heterologous hosts and the need for diverse enzyme formulations for various applications and feedstocks.

Innovation Solution

Development of a chimeric polypeptide comprising segments from different parental cellobiohydrolase I (CBH I) polypeptides with specific sequence identities and conservative amino acid substitutions, enhancing thermostability, pH stability, and expression, using the SCHEMA recombination method to create a diverse set of thermostable CBH I chimeras.

Engineering Contradictions & Design Principles

VSEngineering Contradiction Analysis

1Reliability

If chimeric polypeptides are constructed using SCHEMA recombination method, then thermostability and pH stability are improved, but protein expression difficulty increases due to complex chimeric structure

Engineering Contradiction:

ImprovethermostabilityVSAvoidexpression difficulty

Core Design Contradiction:

ReliabilityVSEase of manufacture

Solution Approach 1:

The CBH I enzyme is divided into eight segments (segment 1 to segment 8) corresponding to specific amino acid residues, allowing systematic recombination of parental sequences to create chimeric polypeptides with improved thermostability while maintaining expression capability

Inventive Principle:

Principle #1Segmentation

Solution Approach 2:

Chimeric polypeptides are constructed by combining segments from different parental CBH I sequences (SEQ ID NO:2, 4, 6, 8, or 10) to create composite enzyme structures that exhibit enhanced thermostability and pH stability compared to parental enzymes

Inventive Principle:

Principle #40Composite materials

2Productivity

If higher enzyme loading is used to compensate for stability issues, then cellulose hydrolysis efficiency improves, but operational costs increase

Engineering Contradiction:

Improvecellulose hydrolysis efficiencyVSAvoidenzyme loading

Core Design Contradiction:

ProductivityVSQuantity of substance

Solution Approach 1:

The patent creates stable enzymes that can be used at lower loading amounts, replacing the need for high enzyme loading with stable, reusable chimeric polypeptides that maintain activity over extended periods at elevated temperatures

Inventive Principle:

Principle #27Cheap short-living objects (Disposable)

3Adaptability or versatility

If diverse cellulase formulations are developed for different applications, then adaptability to various feedstocks improves, but formulation complexity increases

Engineering Contradiction:

Improveadaptability to feedstocksVSAvoidformulation complexity

Core Design Contradiction:

Adaptability or versatilityVSDevice complexity

Solution Approach 1:

The chimeric CBH I polypeptides provide universal enzyme activity across different cellulose substrates with improved stability properties, reducing the need for multiple specialized formulations while maintaining adaptability to various feedstocks and processing conditions

Inventive Principle:

Principle #6Universality (Multi-functionality)

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 resulting chimeric cellulases exhibit improved thermostability, pH stability, and expression, enabling more efficient cellulose hydrolysis at higher temperatures and reducing enzyme loading requirements, thus lowering operational costs and increasing biomass conversion efficiency.

Implementation Method 1

the chimeric polypeptide has cellobiohydrolase activity and improved thermostability, pH stability and/or expression compared to a CBH I polypeptide

Methodology Applied

Scientific EffectEnzymatic hydrolysis: Enzyme

Implementation Method 2

cellulose hydrolysis

Methodology Applied

Scientific EffectHydrolysis: Hydrolysis

Data Source

PatentUS9708633B2Stable, functional chimeric cellobiohydrolase class I enzymes

Publication Date: 2017.07.18 CALIFORNIA INST OF TECH

AI summary

The present disclosure relates to CBH I chimera fusion polypeptides, nucleic acids encoding the polypeptides, and host cells for producing the polypeptides.