Method for improving thermal stability of liquid fructosaminase

A fructose amino acid, thermal stability technology, applied in the directions of enzyme stabilization, biochemical equipment and methods, enzymes, etc., can solve the problems of high viscosity of determination reagents, poor thermal stability of liquid glycated amino acid oxidase, etc., and achieves improved method, The effect of improved thermal stability and easy handling

Active Publication Date: 2015-05-13
浙江夸烨生物科技有限公司
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Benefits of technology

This patented technology provides an improvement over existing methods used to determine glucosamine-6-phosphate dehydrogenases (GAPs) such as alpha-2 adrenal cortex protein 33 or beta-34. It allows for more accurate analysis even with less time than previously possible due to its simplicity compared to other techniques like enzyme immunoassays. Overall this new technique enhances the performance of these proteins while maintain their original function when exposed to heat stress during manufacturing processes.

Problems solved by technology

This patented technical problem addressed in this patent relates to achieving stable liquified albumin solutions while maintaining their ability to detect various metabolites associated with lipid binding compounds like fatty acids or carboxylic acids released from cells through cell membranes. Existing techniques involve modifying the structure of lysericcase basic ammonia adhesion factor (GAL), sacrificial protection layers, and chemistry modifications to enhance thermostability. However, none provide adequately satisfying results due either to reduced heat stability caused by excessive alkali proteases or low levels of specific peptides added without affecting the performance of the analysis process itself.

Method used

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  • Method for improving thermal stability of liquid fructosaminase

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Experimental program
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Embodiment 1

[0020] (1) Prepare 100ml of pH7.5 tris-hydrochloric acid (Tris-HCL) buffer solution, the concentration of Tris-HCL contained is 30mmol / L, the concentration of fructose amino acid oxidase (derived from recombinant E.coli) 1KU / L, the concentration of sodium azide is 1g / L;

[0021] (2) Add 0.2 g of 1-butylsulfonic acid-3-methyltrifluoromethanesulfonate ([ BSMIM]OTf), 1g trehalose, 0.05ml TritonX-100, stir to dissolve.

[0022] The solution obtained in step (2) was placed in a water bath at 37° C. for 30 minutes, and then the enzyme retention rate was measured. The enzyme activity retention rate (%) placed at 4°C without heat treatment and without protective agent was taken as 100%, and treated under the same experimental conditions without protective agent as a control group, and the enzyme activity retention rate (%) was calculated and compared. The enzyme activity retention rates measured by the inventive group and the control group are 98.2% and 45.7% respectively. It can be...

Embodiment 2

[0024] (1) Prepare 100ml of pH8.0 tris-hydrochloric acid (Tris-HCL) buffer solution, the concentration of Tris-HCL contained is 65mmol / L, the concentration of fructose amino acid oxidase (derived from recombinant E.coli) 5.5KU / L, the concentration of sodium azide is 1g / L;

[0025] (2) Add 0.6 g of 1-butylsulfonic acid-3-methyltrifluoromethanesulfonate ([ BSMIM]OTf), 3g trehalose, 0.3ml TritonX-100, stir to dissolve.

[0026] The solution obtained in step (2) was placed in a water bath at 37° C. for 30 minutes, and then the enzyme retention rate was measured. The enzyme activity retention rate (%) placed at 4°C without heat treatment and without protective agent was taken as 100%, and treated under the same experimental conditions without protective agent as a control group, and the enzyme activity retention rate (%) was calculated and compared. The enzyme activity retention rates measured by the inventive group and the control group are 98.5% and 47.4% respectively. It can b...

Embodiment 3

[0028] (1) Prepare 100ml of pH8.5 tris-hydrochloric acid (Tris-HCL) buffer solution, the concentration of Tris-HCL contained is 100mmol / L, the concentration of fructose amino acid oxidase (derived from recombinant E.coli) 10KU / L, the concentration of sodium azide is 1g / L;

[0029] (2) Add 1 g of 1-butylsulfonic acid-3-methyltrifluoromethanesulfonate ([BSMIM ]OTf), 5g trehalose, 0.5ml TritonX-100, stir to dissolve.

[0030]The solution obtained in step (2) was placed in a water bath at 37° C. for 30 minutes, and then the enzyme retention rate was measured. The enzyme activity retention rate (%) placed at 4°C without heat treatment and without protective agent was taken as 100%, and treated under the same experimental conditions without protective agent as a control group, and the enzyme activity retention rate (%) was calculated and compared. The enzyme activity retention rates measured by the invention group and the control group are 98.0% and 46.3% respectively. It can be s...

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Abstract

The invention discloses a method for improving thermal stability of liquid fructosaminase. The method comprises the following steps: 1) preparing a tris(hydroxymethyl) aminomethane-hydrochloric acid buffer solution comprising fructosaminase and sodium azide and having a pH value of 7.5-8.5; 2) under a low-temperature stirring condition, adding 1-butyl sulfonate-3-methyl trifluoromethanesulfonate, trehalose and TritonX-100 into the buffering solution prepared in the step (1). With adoption of the method disclosed by the invention, the thermal stability of the fructosaminase is obviously improved, and the fructosaminase can be applied to develop a determination reagent for glycated albumin determined by an FAOD (fructosaminase) clearance method; the method for improving thermal stability of liquid fructosaminase cannot interfere GA content determination by the FAOD clearance method, can obviously improve the thermal stability of the liquid for fructosaminase, enables the liquid fructosaminase to be still enough in activity 12 months after preservation at the temperature of 4 DEG C.

Description

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Claims

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Application Information

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Owner 浙江夸烨生物科技有限公司
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