Engineered Penicillin G Acylase for Stable Immobilized Catalysis
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Solution Overview
Problem
Naturally occurring penicillin G acylase enzymes are unstable in commercial processes, leading to reduced activity and selectivity when immobilized on solid substrates, limiting their effectiveness in producing 6-APA and other phenacetyl derivatives.
Innovation Solution
Engineered penicillin G acylase enzymes with specific mutations or substitutions, achieving high sequence identity to SEQ ID NOs: 2, 4, 6, 8, 34, 46, 54, 74, and/or 88, capable of removing A1/B1/A1′/B1′-tetra-phenyl acetate or A1/A1′-di-phenyl acetate protecting groups from insulin-dimers to produce free insulin-dimers, enhancing stability and activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If naturally occurring PGA enzyme is used, then the enzyme can catalyze the cleavage of penicillin G to produce 6-APA, but the enzyme shows instability in commercial processes
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the PGA enzyme through site-directed mutagenesis. Specific residues are mutated to alter the enzyme's physical and chemical properties, thereby improving its stability while maintaining catalytic activity. This direct modification of molecular parameters resolves the contradiction between stability and activity.
Solution Approach 2:
The patent implements local quality by making targeted mutations at specific positions within the enzyme structure rather than global modifications. By focusing changes on particular amino acid residues that are critical for stability, the patent maintains the overall catalytic function while locally improving structural stability to address the contradiction.
2Duration of action of stationary object
If PGA is immobilized on solid substrates, then enzyme capture and reuse is enabled, but enzyme properties such as activity and selectivity are compromised
Solution Approach 1:
The patent applies preliminary action by pre-modifying the enzyme's amino acid sequence through mutagenesis before immobilization. This preliminary structural optimization ensures that the enzyme maintains high activity and selectivity even after being attached to solid substrates, thereby resolving the contradiction between reuse capability and enzyme performance.
Solution Approach 2:
The patent uses parameter changes by altering the enzyme's molecular structure through specific amino acid substitutions. These changes improve the enzyme's intrinsic properties, allowing it to retain high catalytic activity and selectivity during immobilization and subsequent reuse cycles, thus overcoming the activity loss typically associated with solid substrate attachment.
3Reliability
If PGA is immobilized on solid substrates, then enzyme capture is improved, but solute access to the enzyme is limited
Solution Approach 1:
The patent applies parameter changes by modifying the enzyme's surface properties through amino acid mutations. These changes can affect the enzyme's hydrophobicity, charge distribution, and overall conformation, thereby improving solute accessibility to the active site while maintaining effective capture on solid substrates. This resolves the contradiction between capture efficiency and substrate access.
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 enzymes maintain high stability and activity, effectively producing free insulin-dimers by removing protecting groups, addressing the limitations of naturally occurring PGA enzymes.
Implementation Method 1
Penicillin G acylase (PGA) (penicillin amidase, EC 3.5.1.11) catalyzes the cleavage of the amide bond of the penicillin G (benzylpenicillin) side chain
Implementation Method 2
The enzyme is used commercially in the manufacture of 6-amino-penicillanic acid (6-APA) and phenyl-acetic acid (PAA)
Data Source
AI summary
The present invention provides engineered penicillin G acylase (PGA) enzymes, polynucleotides encoding the enzymes, compositions comprising the enzymes, and methods of using the engineered PGA enzymes.