Modified Metallo-beta-lactamase N-terminal Truncation for Enzyme Homogeneity
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Solution Overview
Problem
Current beta-lactamase therapies face challenges due to batch variations and heterogeneity in metallo-beta-lactamase production, which affect their robustness and clinical use, particularly in hydrolyzing cephalosporins, carbapenems, and penicillins, and are sensitive to intestinal proteases and pH variations.
Innovation Solution
Modified metallo-beta-lactamases with a truncated amino terminal end and post-translational modifications, such as the insertion of a dipeptide, are produced to reduce heterogeneity and enhance stability and activity, allowing for the production of a substantially pure form suitable for pharmaceutical use.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If metallo-beta-lactamase is produced using conventional methods, then enzyme activity is achieved, but batch variations and heterogeneity occur affecting robustness
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence at the N-terminus of metallo-beta-lactamase through site-directed mutagenesis. Specific residues (Glu1, Lys2, Thr3, Glu4, Lys5) are mutated to create a more stable and homogeneous enzyme product that reduces batch variations while maintaining catalytic activity against beta-lactam antibiotics.
Solution Approach 2:
The patent creates a standardized copy of the metallo-beta-lactamase enzyme with a defined mutant sequence that can be reproducibly produced across different batches. This standardized enzyme copy eliminates heterogeneity by ensuring consistent amino acid composition at the N-terminus, thereby improving manufacturing precision and batch reliability.
2Object-affected harmful factors
If beta-lactamase therapy is administered, then adverse effects of antibiotics are reduced, but the enzyme must withstand intestinal proteases and pH variations
Solution Approach 1:
The patent modifies physical and chemical parameters of the enzyme by changing the N-terminal amino acid sequence through mutagenesis. These parameter changes enhance the enzyme's stability against proteolytic degradation and pH variations in the intestinal environment, ensuring reliable therapeutic function.
Solution Approach 2:
The patent applies preliminary anti-action by pre-modifying the enzyme structure at the N-terminus to resist degradation before the enzyme encounters intestinal proteases. The mutant sequence is designed to be resistant to proteolytic cleavage, providing preliminary protection that maintains enzyme activity throughout the gastrointestinal tract.
3Adaptability or versatility
If broad spectrum beta-lactamase is used, then hydrolysis of cephalosporins, carbapenems, and penicillins is achieved, but substrate profile complexity increases
Solution Approach 1:
The patent applies parameter changes by making targeted mutations at the N-terminus of the enzyme that broaden substrate specificity without requiring complex structural modifications elsewhere. The mutant enzyme maintains a relatively simple overall structure while achieving enhanced adaptability to hydrolyze diverse beta-lactam substrates including cephalosporins, carbapenems, and penicillins.
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 modified metallo-beta-lactamases achieve a single, stable enzyme form with broad beta-lactam spectrum activity, resistant to intestinal proteases and pH variations, effectively eliminating beta-lactam antibiotic-induced adverse effects in the intestinal tract.
Implementation Method 1
Beta-lactamase enzymes catalyse the irreversible hydrolysis of the amide bond of the beta-lactam ring
Implementation Method 2
hydrolysis of the amide bond of the beta-lactam ring
Implementation Method 3
Classes B beta-lactamases are metallo-enzymes that require one or two zinc ions as a cofactor for enzyme activity. Subgroup B1 possesses three histidines and one cysteine as the key zinc coordinating residues
Data Source
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AI summary
The invention relates to targeted post translational modification of metallo-beta-lactamase by truncation and insertion of a dipeptide at the amino terminal end to reduce amino terminal heterogeneity in a recombinant DNA production system. A protein K-T-E-?BL is expressed, and modified by host proteases to E-?BL. Appropriate nucleotide molecules, vectors and hosts are also described. E-?BL is useful in a pharmaceutical composition for treating antibiotic induced adverse effects in the intestine of patients treated with beta-lactam antibiotics.