Endocellulases and uses thereof
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Solution Overview
Problem
Current cellulases have limited efficiency under industrial conditions, particularly in the enzymatic hydrolysis of cellulose for bioethanol production from lignocellulosic biomass, due to cell wall recalcitrance and the need for improved physicochemical and functional properties.
Innovation Solution
Development of polypeptides comprising endocellulase catalytic domains with sequences selected from SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 69, or functionally equivalent variants, which maintain or improve catalytic activity under acidic pH and high temperatures, enabling efficient hydrolysis of cellulose.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional cellulases are used for cellulose hydrolysis, then the process can proceed under conventional conditions, but the enzymatic hydrolysis efficiency is limited due to cell wall recalcitrance and poor performance under industrial conditions
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of endocellulase through site-directed mutagenesis, specifically changing residues at positions 194, 223, and 247 to enhance thermostability and catalytic activity. This genetic engineering approach alters the physical-chemical parameters of the enzyme to improve its performance under industrial hydrolysis conditions, directly resolving the contradiction between hydrolysis efficiency and enzyme reliability.
2Productivity
If genetic engineering of cellulase strains is performed to increase yield, then production efficiency improves, but the complexity of strain development and optimization increases
Solution Approach 1:
The patent extracts the catalytic domain of endocellulase as a separate functional unit and focuses engineering efforts on this specific domain rather than entire cellulase complexes. By isolating and optimizing the catalytic domain through site-directed mutagenesis, the patent simplifies the genetic engineering process while achieving improved enzyme performance and production yield.
3Reliability
If bacterial cellulases are used to handle harsh industrial conditions, then enzyme stability improves, but catalytic efficiency under optimal conditions may be reduced compared to fungal cellulases
Solution Approach 1:
The patent merges the thermostability characteristics of bacterial endocellulase with the catalytic efficiency of fungal cellulases by creating a chimeric enzyme through site-directed mutagenesis. The engineered enzyme combines beneficial traits from different sources, achieving both high stability under industrial harsh conditions and high catalytic activity for efficient hydrolysis.
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 endocellulase catalytic domains exhibit enhanced catalytic activity under harsh industrial conditions, facilitating efficient conversion of cellulose to glucose for bioethanol production.
Implementation Method 1
contacting a sample containing cellulose with a polypeptide according to the invention under suitable conditions for hydrolysing cellulose
Implementation Method 2
converting the glucose obtained in step (iii) to bioethanol in the presence of a yeast capable of producing bioethanol by fermentation of glucose
Data Source
Figure 1
Figure 2
Figure 3a~3b
AI summary
The invention relates to an endocellulase catalytic domain comprising the sequence of SEQ ID NO: 1 or a functionally equivalent variant of said catalytic domain that substantially maintains or improves its catalytic activity. The invention also relates to a polypeptide, a nucleic acid, an expression cassette, a vector or a host cell. Additionally, the invention relates to the use of an endocellulase catalytic domain or the polypeptide of the invention for hydrolysing cellulose, producing bioethanol or as a detergent. The invention also relates to a method for hydrolysing cellulose and for producing bioethanol.