Engineered Penicillin G Acylase for Insulin Protecting Group Manipulation
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Solution Overview
Problem
Naturally occurring penicillin G acylase (PGA) enzymes are unstable in commercial processes, requiring immobilization on solid substrates, which compromises enzyme activity and selectivity, and limits solute access.
Innovation Solution
Engineered PGA enzymes with specific polynucleotide sequences and mutations that enhance stability and activity, allowing them to produce phenyl acetate mono-protected or di-protected insulin by adding or removing protecting groups from specific positions of insulin.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Duration of action of stationary object
If PGA is immobilized on solid substrates, then enzyme capture and reuse are enabled, but enzyme activity and selectivity are compromised
Solution Approach 1:
The patent applies parameter changes by modifying the enzyme's molecular structure through site-directed mutagenesis. Specific amino acid residues were mutated to improve the enzyme's intrinsic stability and activity, allowing it to function effectively without immobilization. This changes the fundamental parameters of the enzyme's catalytic properties and structural stability.
Solution Approach 2:
The patent extracts the immobilization step from the process by developing a soluble, stable enzyme that does not require attachment to solid substrates. This removes the harmful effect of immobilization (compromised activity and selectivity) while maintaining the benefit of enzyme reuse through improved stability and half-life.
2Duration of action of stationary object
If PGA is immobilized on solid substrates, then enzyme capture is enabled, but solute access is limited
Solution Approach 1:
The patent removes the immobilization requirement entirely by creating a soluble enzyme form. This extraction eliminates the physical barrier that limited solute access, allowing substrates to freely diffuse to the enzyme's active site in solution without being constrained by solid substrate attachment.
3Device complexity
If wild-type PGA is used, then enzyme simplicity is maintained, but stability in commercial processes is insufficient
Solution Approach 1:
The patent systematically changed parameters of the enzyme's amino acid sequence through mutagenesis at specific positions (e.g., residues 71, 74, 129, 253, 254, 256, 348, 352, 372, 373, 374, 380, 457, 467, 470, 474, 616, 623, 704, 706, 708). These parameter changes improved the enzyme's stability and catalytic efficiency while maintaining reasonable structural simplicity.
Solution Approach 2:
The patent created a composite enzyme variant that combines the best features of different amino acid sequences. By integrating beneficial mutations from various positions in the protein structure, the resulting enzyme achieves enhanced stability and activity while maintaining a relatively simple overall structure.
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 PGA enzymes demonstrate improved stability and activity, enabling efficient production of phenyl acetate protected insulin with high yields, surpassing the performance of wild-type PGA enzymes.
Implementation Method 1
Penicillin G acylase (PGA) (penicillin amidase, EC 3.5.1.11) catalyzes the cleavage of the amide bond of penicillin G (benzylpenicillin) side chain
Implementation Method 2
The enzymatic activity of PGA, associated with the phenacetyl moiety, allows the stereospecific hydrolysis of a rich variety of phenacetyl derivatives
Implementation Method 3
engineered penicillin G acylases capable of producing phenyl acetate mono-protected or di-protected insulin by adding the protecting group to the A1, B1 or B29 positions of free insulin
Implementation Method 4
engineered penicillin G acylases capable of removing the A1, B1, or B29 tri-phenyl acetate protecting groups from insulin to produce a di-phenyl acetate protected insulin
Data Source
AI summary
The present invention provides engineered penicillin G acylase (PGA) enzymes having improved properties, polynucleotides encoding such enzymes, compositions including the enzymes, and methods of using the enzymes.