A method for improving the thermal stability of liquid fructose amino acid oxidase

A fructose amino acid and thermostability technology, applied in the directions of enzyme stabilization, biochemical equipment and methods, enzymes, etc., can solve the problems of poor thermostability of liquid glycated amino acid oxidase and high viscosity of determination reagents, and improve thermostability. , improve the method, improve the effect of GA content

Active Publication Date: 2016-09-07
浙江夸烨生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In order to improve the thermal stability of fructose amino acid oxidase in the determination reagent of FAOD clearance method, the existing technology is to add a large amount of non-specific protective agent that does not interfere with GA detection in most cases, and fails to solve the problem of liquid glycosylated amino acid oxidase. The disadvantage of poor thermal stability, on the other hand, the addition of a large amount of protective agent leads to high viscosity of the determination reagent, which is not conducive to the application in automatic biochemical analyzers

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  • A method for improving the thermal stability of liquid fructose amino acid oxidase
  • A method for improving the thermal stability of liquid fructose amino acid oxidase

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Experimental program
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Effect test

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

technical field [0001] The invention relates to the technical field of liquid enzyme stability, in particular to a method for improving the thermal stability of liquid fructose amino acid oxidase. Background technique [0002] Glycated serum albumin (GA) is the product of serum albumin being glycosylated by glucose, that is, the ε-amino group on the lysine residue of serum albumin is glycosylated. The half-life of glycosylated albumin is short, and the determination of the concentration of glycosylated albumin can effectively reflect the average blood sugar level of the patient in the past 2 to 3 weeks, and is not affected by temporary blood sugar concentration fluctuations. At present, the fructose amino acid oxidase (FAOD) method (hereinafter referred to as the FAOD removal method) is widely used clinically to determine the content of GA. The resulting fructose amino acids are oxidized to produce amino acids, glucosone and hydrogen peroxide. Since the liquid fructose ami...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/96C12N9/06
CPCC12N9/0022C12N9/96
Inventor 连国军
Owner 浙江夸烨生物科技有限公司
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