Alpha-Amylase Variants Stable in Chelating Agent Formulations
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Solution Overview
Problem
Existing alpha-amylases are sensitive to chelating agents, which impair their stability and activity in applications like detergents and ethanol production from whole grains, where calcium-dependent enzymes face challenges due to the presence of chelating compounds.
Innovation Solution
Development of alpha-amylase variants with specific amino acid alterations at positions 193, 195, 196, 200, 200, 203, 206, 210, 212, and 213, and optionally 243, combined with chelating agents that reduce free calcium ion concentration from 2.0 mM to 0.10 mM at pH 8 and 21°C, enhancing stability and wash performance.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If calcium-dependent alpha-amylases are used in detergents containing chelating agents, then enzyme activity is maintained through calcium binding, but enzyme stability deteriorates due to chelating agents removing calcium ions
Solution Approach 1:
The invention extracts the calcium dependency from the enzyme system by creating calcium-independent alpha-amylase variants through site-directed mutagenesis. Specific amino acid residues involved in calcium binding (such as Asp195, Glu210, Asp212) are mutated to eliminate calcium coordination, allowing the enzyme to function without calcium and thus remain stable in the presence of chelating agents like EDTA and NTA.
Solution Approach 2:
The invention changes the biochemical parameters of the enzyme by modifying its amino acid sequence to alter calcium binding affinity. Through systematic mutation of calcium-coordinating residues, the enzyme transitions from calcium-dependent to calcium-independent operation, fundamentally changing its stability profile in chelating agent-containing environments.
2Stability of the object's composition
If calcium is added to detergents to improve enzyme stability, then enzyme stability improves, but stain removing effect deteriorates due to calcium interfering with chelating agents
Solution Approach 1:
The invention removes the requirement for calcium by creating calcium-independent enzyme variants. This eliminates the need to add calcium to detergents for enzyme stability, thereby preserving the effectiveness of chelating agents in stain removal while maintaining enzyme activity through genetic modification rather than chemical supplementation.
3Object-generated harmful factors
If strong chelating compounds are present in detergent formulations, then water hardness reduction and stain removal improve, but enzyme stability deteriorates due to calcium ion sequestration
Solution Approach 1:
The invention converts the harmful effect of chelating agents (which normally destabilize calcium-dependent enzymes) into a beneficial situation by creating enzymes that thrive in chelating agent environments. The calcium-independent variants not only tolerate but are specifically designed to operate optimally in the presence of strong chelators, allowing full utilization of chelating agents for water hardness reduction and stain removal without compromising enzyme stability.
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 maintain or improve amylolytic activity and stability in the presence of chelating agents, ensuring effective performance in detergent and ethanol production processes.
Implementation Method 1
comprising a variant of a parent alpha-amylase, wherein the variant comprises a substitution at one or more positions selected from the group consisting of 195, 193, 197, 198, 200, 203, 206, 210, 212, 213 and 243, using the numbering according to SEQ ID NO: 6, and further comprising at least one chelating agent wherein said chelating agent at a concentration below 10 mM is capable of reducing the concentration of free calcium ions from 2.0 mM to 0.10 mM
Data Source
AI summary
The present invention relates to variants of an alpha-amylase having improved stability to chelating agents relative to its parent enzyme, compositions comprising the variants, nucleic acids encoding the variants, methods of producing the variants, and methods for using the variants.