Thermostabile beta-glucuronidase formulations
A technology of aldonid glucosidase and glucose, applied in the direction of enzyme stabilization, glycosylase, enzyme, etc., can solve the problems of not showing enzyme stability, etc., and achieve the effect of long storage life and low aggregation tendency
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[0052] III. Preparation of preparations
[0053] Aqueous and lyophilized formulations of the invention can be prepared using methods well established in the art. Typically, aqueous formulations are prepared by combining the desired concentration of BGUS enzyme and excipients. Lyophilized formulations can be prepared by freeze-drying aqueous formulations using techniques well established in the art. Non-limiting examples of suitable conditions for freeze-drying (lyophilization) of aqueous solutions are given in Example 6. All excipients used in the formulations are commercially available.
[0054] Furthermore, it has been found that when preparing aqueous formulations, first combining the BGUS enzyme with an amphoteric compound (e.g. betaine), L-histidine and β-alanine prior to the addition of sugar (e.g. sucrose) results in the most beneficial thermostabilization and additional beneficial properties. Therefore, in one aspect, the invention provides a kind of method for p...
Embodiment 1
[0068] Example 1 : Analysis of Amino Acids as Heat Stabilizers
[0069] In this example, a set of amino acid pairs was examined Effect of melting temperature of genetically modified (mutated) recombinant E. coli β-glucuronidase enzyme. For all experiments described in the Examples, BGUS enzyme (commercially available from Integrated Micro-Chromatography Systems, LLC) was used in the formulation.
[0070] Check the melting temperature of the enzyme in formulations containing different amino acids using protein thermal shift assays. For this analysis, the enzyme protein was purified using standard chromatographic techniques to achieve at least >99% purity based on SDS PAGE. The purified recombinant enzyme was first dialyzed against pure water (18.2 Mohm) with a protein concentration level of at least 2 mg / ml. Stock solutions of this enzyme in water were mixed 1:1 with various buffer solutions to achieve the final concentrations listed in Table 1 below. All samples were ...
Embodiment 2
[0077] Example 2 : Analysis of Polyols as Thermal Stabilizers
[0078] In this example, a panel of polyol sugar pairs was examined using the protein thermal shift assay described in Example 1. Effect of melting temperature of genetically modified β-glucuronidase enzymes.
[0079] The results for the polyol panel tested are shown in Table 2 below:
[0080] Table 2:
[0081]
[0082]
[0083] While most of the sugars tested as excipients initially showed an increase in Tm values when compared to water, only two compounds, xylitol (Formulation #15) and sucrose (Formulation #17), were effective for Tm showed a consistent increase. Glycerol (Formulations #19 and #20) also improved stability. Complex sugars such as (2-hydroxypropyl)-β-cyclodextrin (Formulation #27) and 16 mM α-cyclodextrin (Formulation #28) also have consistent efficacy in stabilizing the enzyme by raising the Tm value by 1.3 °C. improve.
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