Mutant Lactobacillus Brevis Beta-Glucuronidase Enzymes
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Solution Overview
Problem
Current commercially available β-glucuronidase (BGUS) enzyme preparations are inefficient at low pH, require extensive time for analysis, and are contaminated with non-BGUS proteins, limiting their effectiveness in drug testing.
Innovation Solution
Development of mutant Lactobacillus brevis strain 269Y BGUS enzymes with enhanced activity at low pH and increased thermal stability, achieved through specific amino acid substitutions and cysteine additions, allowing for rapid and purified enzyme preparations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If commercially available BGUS enzyme preparations are used, then enzyme activity is maintained at neutral pH, but enzyme activity decreases significantly at low pH (below 6.8)
Solution Approach 1:
The patent applies parameter changes by modifying the amino acid sequence of the BGUS enzyme to alter its pH-activity profile. Specifically, mutations are introduced to shift the enzyme's optimal pH from neutral to acidic conditions, enabling it to maintain high activity at low pH (4.5-5.5) while remaining compatible with downstream analytical procedures that require acidic conditions.
2Ease of manufacture
If crude extract forms of BGUS enzyme are used, then enzyme preparation is simplified, but contamination with non-BGUS proteins occurs that interferes with enzyme activity
Solution Approach 1:
The patent applies the extraction principle by isolating and purifying the BGUS enzyme from crude extracts through chromatographic methods. This separates the active BGUS enzyme from contaminating non-BGUS proteins that interfere with enzyme activity, while maintaining a practical preparation process that does not require excessively complex purification steps.
3Productivity
If commercially available BGUS preparations are used, then analysis can be performed, but analysis time requires several hours (three hours or more)
Solution Approach 1:
The patent applies parameter changes by optimizing the enzyme's catalytic efficiency through amino acid mutations. These mutations enhance the turnover number and substrate binding affinity of the BGUS enzyme, enabling complete hydrolysis of glucuronide conjugates in drug samples within minutes rather than hours, thus dramatically reducing total analysis time while maintaining accuracy.
4Stability of the object's composition
If wild type BGUS enzyme is used, then natural enzyme structure is maintained, but thermal stability is reduced compared to mutant forms
Solution Approach 1:
The patent applies parameter changes by introducing specific amino acid mutations that enhance the thermal stability of the BGUS enzyme. These point mutations strengthen intramolecular interactions and stabilize the enzyme's three-dimensional structure at elevated temperatures, allowing the enzyme to maintain activity during storage and assay procedures without requiring excessive structural modification.
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 mutant enzymes enable accurate drug analysis in under 30 minutes, with higher compatibility for downstream analytics and reduced contamination, significantly improving efficiency and accuracy.
Implementation Method 1
The β-glucuronidase (BGUS) enzyme catalyzes the hydrolysis of a wide variety of β-glucuronides
Data Source
AI summary
Mutated Lactobacillus brevis strain 269Y β-glucuronidase enzymes with enhanced enzymatic activity at low pH (e.g., below pH 6.8), as well as enhanced thermostability as compared to wild type enzyme are provided. The enzymes of the invention advantageously allow for accurate analysis of bodily samples for the presence of drugs at low pH and in 30 minutes or less, as compared to the several hours needed using prior enzyme preparations. Methods of using the mutated enzymes for hydrolysis of glucuronide substrates, including opiates and benzodiazepines, are also provided.