Engineered pNB Esterase for Imipenem Deprotection
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Solution Overview
Problem
Current methods for synthesizing imipenem, a carbapenem antibiotic, face challenges with low yields and high costs due to the use of palladium or platinum hydrogenation catalysts for removing p-nitrobenzyl protecting groups, and there is a need for an engineered pNB esterase that provides selectivity and high yields under commercially viable conditions.
Innovation Solution
Development of engineered polypeptides with specific amino acid sequence modifications that enhance pNB esterase activity, stability, and selectivity for efficiently converting pNB-protected imipenem precursors to imipenem, using engineered polynucleotides and expression vectors to produce these polypeptides in host cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If palladium or platinum hydrogenation catalysts are used for removing p-nitrobenzyl protecting groups, then the deprotection reaction can proceed, but the cost increases and the yield decreases
Solution Approach 1:
The invention changes the chemical nature of the catalyst from metal-based (palladium or platinum) to enzyme-based (esterase). This parameter change in catalyst type enables the reaction to proceed with both lower cost and higher yield, as the engineered esterase catalyst is both economically viable and highly effective for removing p-nitrobenzyl protecting groups in carbapenem synthesis
Solution Approach 2:
The invention substitutes the mechanical/chemical hydrogenation process using metal catalysts with a biocatalytic process using esterase enzymes. This replacement of the mechanical system with a biological system achieves the same deprotection function while improving both cost efficiency and reaction yield
2Productivity
If wild-type pNB esterase is used for removing p-nitrobenzyl protecting groups, then the reaction can proceed, but the selectivity and yield for carbapenem intermediates are insufficient
Solution Approach 1:
The invention segments the esterase enzyme structure by introducing specific point mutations at key positions (P193, P219, P273, P362) in the active site. These segmented modifications to specific regions of the enzyme enable it to achieve both high yield and high selectivity for carbapenem intermediates, overcoming the limitations of wild-type esterase
Solution Approach 2:
The invention applies local quality changes by modifying specific amino acid residues at critical positions in the esterase active site while leaving the rest of the enzyme structure intact. These localized modifications at positions P193, P219, P273, and P362 enhance the enzyme's selectivity and effectiveness for carbapenem substrate deprotection
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 engineered pNB esterases achieve higher yields and selectivity in converting pNB-protected imipenem intermediates to imipenem, offering improved process conditions and economic viability for carbapenem antibiotic production.
Implementation Method 1
The engineered pNB esterase polypeptides of the present disclosure have been engineered for efficient removal a pNB protecting group from the imipenem precursor compound of compound (2) thereby converting it to the product compound (1), imipenem
Implementation Method 2
pNB esterase activity, comprising an amino acid sequence having at least 90% identity to SEQ ID NO: 2 and a residue difference as compared to SEQ ID NO:2 of X193D or X193E, wherein the engineered polypeptide is capable of converting the substrate of compound (2), a pNB-protected precursor of imipenem, to the corresponding product of compound (1), imipenem
Data Source
AI summary
The present disclosure provides engineered pNB esterase polypeptides useful for the synthesis of the carbapenem antibiotic, imipenem. The disclosure also provides polynucleotides encoding the engineered pNB esterases, host cells capable of expressing the engineered pNB esterases, and methods of using the engineered pNB esterases in the production of imipenem.