Thermolysin Stabilization via Salt Concentration Control
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Solution Overview
Problem
Thermolysin, a thermostable neutral metalloproteinase, is unstable in aqueous solutions, leading to precipitation and reduced solubility, which complicates its use and storage due to its tendency to form autoproteolytic fragments and low solubility, especially when used in blends with other enzymes for tissue dissociation.
Innovation Solution
Thermolysin is first dissolved in a buffer with low ion concentration, followed by the addition of a further salt to stabilize the enzyme, forming a solution that remains clear and homogeneous for an extended period by reducing the tendency of thermolysin to precipitate, allowing for higher concentrations and prolonged stability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of operation
If thermolysin is dissolved in aqueous solution, then the enzyme becomes available for use, but it precipitates and loses stability
Solution Approach 1:
The patent applies parameter changes by systematically varying salt concentration, pH, and temperature to identify optimal conditions for thermolysin stability. Specifically, it uses buffers at pH 7.5-8.5 with salt concentrations of 0.5-2.0 M (particularly NaCl or KCl) to maintain the enzyme in solution and prevent precipitation, thereby resolving the contradiction between availability and stability.
2Loss of time
If thermolysin is stored in aqueous solution, then it can be used immediately, but it forms autoproteolytic fragments and reduces solubility
Solution Approach 1:
The patent applies preliminary anti-action by pre-establishing protective conditions before autoproteolysis can occur. It uses specific buffer systems with controlled pH (7.5-8.5) and high salt concentrations (0.5-2.0 M) to prevent the formation of autoproteolytic fragments in advance, allowing the enzyme to be stored in solution without degradation and ready for immediate use.
3Productivity
If thermolysin is used in blends with other enzymes, then tissue dissociation effectiveness increases, but stability and solubility decrease
Solution Approach 1:
The patent applies universality by creating a buffer system that serves multiple functions simultaneously: it maintains pH (7.5-8.5), provides ionic strength (0.5-2.0 M salt), prevents precipitation, and stabilizes thermolysin in enzyme blends. This multi-functional buffer composition enables thermolysin to maintain stability and solubility when combined with other enzymes like collagenase for tissue dissociation applications.
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 method allows for the preparation of thermolysin solutions that remain stable and homogeneous for several hours, enabling effective storage, transport, and blending with other enzymes, such as collagenase, for tissue dissociation applications.
Implementation Method 1
Thermolysin is first dissolved in a buffer with low ion concentration
Implementation Method 2
followed by the addition of a further salt to stabilize the enzyme, forming a solution that remains clear and homogeneous
Data Source
AI summary
The present invention deals with the proteolytic enzyme thermolysin which tends to be unstable in aqueous solution. The invention provides methods and compositions to enhance the stability of dissolved thermolysin in aqueous solution. Thermolysin, crude thermolysin or a lyophilisate containing thermolysin and one or more salts, is contacted with an aqueous buffer with a low salt concentration and a first solution is formed. Subsequently, a further salt in solid form is added and dissociated, thereby forming a second solution comprising thermolysin in a stabilized form.