Mutant AlkB Enzyme V129 T136 for Omega-Hydroxy Acid Production
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Solution Overview
Problem
Current methods for producing omega-hydroxy carboxylic acids and their esters are energy-intensive, environmentally damaging, and result in low yields with excessive by-products, limiting their industrial applications.
Innovation Solution
A microbial cell expressing a mutant AlkB enzyme with specific point mutations at amino acid positions V129 and T136 is used to catalyze the conversion of C6-C14 carboxylic acids and esters into omega-hydroxy and omega-oxocarboxylic acids and esters, enhancing yield and reducing by-product formation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Object-affected harmful factors
If wild type AlkB enzyme is used for bioconversion, then the process is environmentally friendly (green process), but the product yield is low and excessive by-products are formed
Solution Approach 1:
The patent applies parameter changes by introducing specific point mutations (V129T/V129A and T136A) in the AlkB enzyme amino acid sequence. These mutations alter the enzyme's catalytic parameters to improve selectivity for omega-hydroxy carboxylic acid production, reducing over-oxidation to dicarboxylic acids and increasing product yield while maintaining the green biocatalytic process
2Ease of manufacture
If wild type AlkB enzyme is used, then the bioprocess remains simple and cost-effective, but the conversion efficiency and product yield are insufficient
Solution Approach 1:
The patent modifies the AlkB enzyme through site-directed mutagenesis at specific positions (V129 and T136), creating variants with improved catalytic efficiency. This approach maintains the simplicity of the bioprocess while enhancing conversion efficiency, as the mutated enzyme can still be produced using standard microbial expression systems without complex process changes
3Ease of manufacture
If wild type AlkB enzyme is used, then the existing fermentation process can be maintained, but the yield of omega-hydroxy carboxylic acid is limited due to over-oxidation to dicarboxylic acids
Solution Approach 1:
The patent addresses over-oxidation by changing the enzyme's amino acid parameters at positions V129 and T136. These mutations reduce the enzyme's ability to catalyze the second oxidation step (omega-hydroxy to dicarboxylic acid), thereby minimizing product loss to by-products while maintaining compatibility with existing fermentation 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 mutant AlkB enzyme significantly increases the bioconversion rate and product yield of omega-hydroxy and omega-oxo carboxylic acids, improving the energy efficiency and economic viability of polyamide 12 precursor production by enhancing the HLAME:DDAME ratio.
Implementation Method 1
The mutant AlkB enzyme comprises at least one point mutation in the wild type sequence of AlkB, wherein the point mutation is at amino acid position V129 and/or T136 of the wild type AlkB enzyme
Implementation Method 2
The monooxygenase AlkB is an enzyme known to catalyse the reaction forming HLAME from LAME. The monooxygenase AlkB is also able to catalyse the oxidation of HLAME to omega-oxolauric acid methylester (OLAME)
Data Source
Figure 1~1D
AI summary
There is provided a microbial cell expressing a mutant AlkB enzyme, the mutant AlkB enzyme comprising at least one point mutation in the wild type sequence of AlkB, wherein the point mutation is at amino acid position V129 and/or T136 of the wild type AlkB enzyme. There is also provided a method for producing omega-hydroxy carboxylic acid and/or ester thereof using this cell.