Alpha-amylase variants for low pH starch liquefaction
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Solution Overview
Problem
Current alpha-amylases used in starch liquefaction processes lack stability at low pH and low calcium concentrations, limiting process efficiency and chemical savings.
Innovation Solution
Development of alpha-amylase variants with specific substitutions, including proline at position 185 and additional substitutions at various positions, enhancing stability at low pH and high temperature, particularly at low calcium concentrations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If wild type alpha-amylases are used for starch liquefaction, then the process can be carried out with standard enzyme formulations, but the enzymes lack stability at low pH and low calcium concentrations, requiring higher chemical usage and limiting process efficiency
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of alpha-amylase through specific substitutions (e.g., positions 15, 48, 49, 50, 107, 116, 133, 138, 156, 176, 181, 187, 188, 190, 197, 201, 205, 209, 213, 239, 241, 255, 264, 299, 360, 375, 416, 437, 474, 475) to enhance enzyme stability at low pH and low calcium concentrations, thereby reducing the quantity of chemicals required in the starch liquefaction process
2Reliability
If protein engineered variants are developed to improve stability, then enzyme performance at low pH and high temperature can be enhanced, but the complexity of enzyme development and characterization increases
Solution Approach 1:
The patent systematically modifies specific amino acid positions in the alpha-amylase sequence to achieve improved stability properties, using rational protein engineering approaches that target key residues known to influence pH and thermal stability while managing development complexity through focused mutagenesis strategies
Solution Approach 2:
The patent creates composite enzyme variants by combining multiple amino acid substitutions within a single protein molecule, where the synergistic effect of multiple mutations at different positions (e.g., combinations involving positions 15, 48, 49, 50, 107, 116, 133, 138, 156, 176, 181, 187, 188, 190, 197, 201, 205, 209, 213, 239, 241, 255, 264, 299, 360, 375, 416, 437, 474, 475) produces enhanced stability that exceeds individual mutations
3Quantity of substance
If starch liquefaction is performed at reduced pH to save chemicals, then chemical savings are achieved, but the enzyme requires higher stability at low pH which wild type enzymes lack
Solution Approach 1:
The patent enables reduced pH operation by engineering alpha-amylase variants with enhanced low pH stability through specific amino acid substitutions, allowing the starch liquefaction process to be conducted at lower pH values (e.g., pH 4.0-6.0) which reduces chemical usage while maintaining enzyme activity and stability throughout the process
4Reliability
If alpha-amylase variants with multiple substitutions are created, then stability at low pH, high temperature, and low calcium concentrations is improved, but the manufacturing and characterization process becomes more complex
Solution Approach 1:
The patent improves ease of manufacture by developing alpha-amylase variants with multiple stabilizing substitutions that can be produced using standard recombinant expression systems, with the engineered proteins maintaining high stability at low pH, high temperature, and low calcium concentrations while being amenable to conventional purification and formulation processes
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 demonstrate increased stability and residual activity half-life at high temperatures and low pH, even at low calcium levels, improving process efficiency and reducing chemical usage.
Implementation Method 1
Alpha-amylases (E.C. 3.2.1.1) constitute a group of enzymes which catalyze hydrolysis of starch, glycogen and related polysaccharides and oligosaccharides
Data Source
AI summary
The present invention relates to alpha-amylase variants. 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.