Alpha-amylase variants with 268 and 293 substitutions for thermal stability
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Solution Overview
Problem
Current alpha-amylase variants lack sufficient stability at low pH and high temperatures, limiting their effectiveness in starch liquefaction processes.
Innovation Solution
Development of alpha-amylase variants with specific substitutions at positions 268 and 293, such as Y268G + N293Y, which enhance thermo-stability and maintain sequence identity with parent enzymes, allowing for improved performance in low pH and high temperature conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If alpha-amylase variants are used in starch liquefaction processes, then starch degradation into fermentable sugars is achieved, but the enzyme lacks sufficient stability at low pH and high temperatures
Solution Approach 1:
The patent applies parameter changes by modifying specific amino acid residues at positions 268 and 293 in the alpha-amylase enzyme sequence. These sequence parameter changes result in variants with improved thermal and pH stability while maintaining catalytic activity for starch degradation
Solution Approach 2:
The invention applies local quality by making targeted modifications at specific locations (positions 268 and 293) within the enzyme's protein structure. These localized changes enhance stability without compromising the overall enzymatic function across the entire protein
2Productivity
If alpha-amylase operates at high temperature to increase reaction rate, then starch liquefaction efficiency improves, but enzyme activity decreases due to thermal denaturation
Solution Approach 1:
The patent modifies the enzyme's thermal parameters through amino acid substitutions at positions 268 and 293, enabling the enzyme to maintain structural integrity and catalytic activity at elevated temperatures (80-90°C) without denaturation
Solution Approach 2:
The engineered variants incorporate structural modifications beforehand that cushion against thermal stress. The amino acid changes at positions 268 and 293 pre-strengthen the enzyme's protein fold to resist thermal denaturation during high-temperature operation
3Productivity
If alpha-amylase operates at low pH to optimize starch hydrolysis, then catalytic activity increases, but enzyme stability decreases due to acid denaturation
Solution Approach 1:
The patent changes the enzyme's pH tolerance parameters through amino acid substitutions at positions 268 and 293, allowing the enzyme to maintain stability and activity in acidic conditions (pH 4.5-5.0) without denaturation
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 variants exhibit increased residual activity and improved half-life stability, enhancing their performance in starch liquefaction processes by maintaining activity under heat stress and low pH conditions.
Implementation Method 1
Alpha-amylases (alpha-1,4-glucan-4-glucanohydrolases, E.C. 3.2.1.1) constitute a group of enzymes, which catalyze hydrolysis of starch and other linear and branched 1,4-glucosidic oligo- and polysaccharides
Implementation Method 2
the variant has increased thermo-stability, measured as residual alpha-amylase activity after heat-stress, compared to a parent alpha-amylase
Data Source
AI summary
The present invention relates to alpha-amylase variants comprising substitutions at positions corresponding to positions 268 and 293 of SEQ ID NO: 1, in particular substitutions selected from the group consisting of: 268G+293Y; 268G+293F; 268G+293W; 268G+293H; 268G+293A; 268G+293Q; 268A+293Y; 268A+293F; 268A+293W; 268A+293H; 268A+293A; 268A+293Q; 268P+293Y; 268P+293F; 268P+293W; 268P+293H; 268P+293A; 268P+293Q; 268S+293Y; 268S+293F; 268S+293W; 268S+293H; 268S+293A; 268S+293Q; 268T+293Y; 268T+293F; 268T+293W; 268T+293H; 268T+293A; 268T+293Q; 268V+293Y; 268V+293F; 268V+293W; 268V+293H; 268V+293A; 268V+293Q; 268I+293Y; 268I+293F; 268I+293W; 268I+293H; 268I+293A; 268I+293Q; 268L+293Y; 268L+293F; 268L+293W; 268L+293H; 268L+293A; 268L+293Q; 268M+293Y; 268M+293F; 268M+293W; 268M+293H; 268M+293A; 268M+293Q;, and wherein the variant has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity to a parent alpha amylase selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, and SEQ ID NO: 18. The present invention also relates to polynucleotides encoding the variants; nucleic acid constructs, vectors, and host cells comprising the polynucleotides; and methods of using the variants.