Modified Lipolytic Enzyme 499EST for Enantioselective Resolution
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Solution Overview
Problem
Current methods for obtaining enantiomers of medicaments like ibuprofen and naproxen often rely on costly racemic mixture separation, and there is a need for biologically efficient mechanisms to produce pure S-enantiomers, which are more beneficial than racemic mixtures.
Innovation Solution
A modified lipolytic enzyme, 499EST, is developed from a thermoacidophilus organism, which exhibits enantioselective activity and is capable of hydrolyzing triacylglycerols and profens, with enhanced stability and activity across a wide pH and temperature range, including the addition of a histidine tail for improved purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If racemic mixture separation methods are used to obtain enantiomers, then pure S-enantiomers can be produced, but the production cost is high
Solution Approach 1:
The patent replaces mechanical/chemical separation methods with a biological catalysis system. The modified esterase enzyme selectively catalyzes the hydrolysis of racemic ester substrates to produce pure S-enantiomers, substituting complex separation processes with a specific enzymatic reaction that inherently produces enantiomerically pure products.
Solution Approach 2:
The patent modifies the enzyme's physical and chemical parameters through site-directed mutagenesis, specifically changing amino acid residues in the binding pocket to enhance enantioselectivity. This allows the enzyme to discriminate between R and S enantiomers based on subtle steric and electronic parameter differences, achieving high purity without expensive separation.
2Manufacturing precision
If a modified esterase enzyme is used to resolve racemic mixtures, then enantioselectivity is improved, but the enzyme purification process becomes more complex
Solution Approach 1:
The patent incorporates a histidine tag sequence at the N-terminus of the esterase enzyme during gene construction. This preliminary modification enables affinity purification through metal-chelating resins, simplifying the purification process despite the enzyme's modified structure and enhancing enantioselectivity.
3Productivity
If the enzyme is modified with a histidine tail for better purification, then purification efficiency is improved, but the enzyme structure becomes more complex
Solution Approach 1:
The patent segments the enzyme molecule into two functional parts: the catalytic domain (residues 1-305) that performs ester hydrolysis with enantioselectivity, and the histidine tag (residues 1-10) that facilitates purification. This segmentation allows the small terminal modification to provide purification benefits without significantly affecting the overall structure or function of the large catalytic domain.
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 enzyme effectively resolves racemic mixtures to produce a higher proportion of S-enantiomers, improving the efficacy of non-steroidal anti-inflammatory drugs and demonstrating stability in organic solvents and detergents, thus facilitating more efficient production of valuable enantiomers.
Implementation Method 1
The modified esterase enzyme, 499EST, is capable of hydrolyzing lipid substrates (triacylglycerols) combined medium chain fatty acids (C6-C10) such as tributyrine, tricapryln, among others. It can also carry out other reactions inverse to hydrolysis, as synthesis reactions. On the other hand, this enzyme has enantioselective preference over (S) substrates of profens esters
Implementation Method 2
The enzyme effectively resolves racemic mixtures to produce a higher proportion of S-enantiomers, improving the efficacy of non-steroidal anti-inflammatory drugs
Data Source
AI summary
This invention refers to the obtainment of a modified lipolytic enzyme that was isolated, expressed and purified from the heterologous expression. The gene sequence that codifies for the basal enzyme was obtained based on a thermo acidophilus organism of the acidobacteraceae family. This basal enzyme that comes from a thermo acidophilus organism, it is able to hydrolyze lipid substrates (triacylglycerols) united to middle chain fatty acids (C.sub.6-C.sub.10) such as tributyrine and tricapryln, among others. It also can carry out other inverse reactions to the hydrolysis such as synthesis reactions. On the other hand, this enzyme has enantioselective preference on (S) substrates of profens esters such as ibuprofen, naproxen and others. The enantioselective lipolytic basal enzyme was modified in its terminal C end to add an amino acid histidine tail that gives a higher efficiency in its purification process. The invention therefore refers to a method for making a pure, active polypeptide, which is called lipolytic enzyme 499EST obtained through the host E. coli BL 21 (DE3).