Glycosylated oxalate decarboxylase as well as preparation and application of glycosylated oxalate decarboxylase

A technology of oxalate decarboxylase and glycosylation, which is applied in the direction of biochemical equipment and methods, applications, enzymes, etc., can solve the problems of inactivity and instability, and achieve the effects of excellent stability, high activity, and high degradation efficiency

Pending Publication Date: 2018-04-03
WUHAN KANGFUDE BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0012] Aiming at the technical defect in the prior art that oxalate decarboxylase preparations are inactive or unstable at low pH values ​​and cannot truly reduce urinary oxalate, the present invention provides a glycosylated oxalate decarboxylase that can effectively reduce urinary oxalate content

Method used

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  • Glycosylated oxalate decarboxylase as well as preparation and application of glycosylated oxalate decarboxylase
  • Glycosylated oxalate decarboxylase as well as preparation and application of glycosylated oxalate decarboxylase
  • Glycosylated oxalate decarboxylase as well as preparation and application of glycosylated oxalate decarboxylase

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Embodiment 1

[0035] In this example, the fermentation production process of glycosylated oxalate decarboxylase is described by taking Agrocybe abacus as an example. The conclusions of the experiments carried out by the inventors with Agrocybe arvenifera, Agrocybe sp. , except for some comparison results, the present application will not repeat the description.

Embodiment 2

[0037] Embodiment 2: Purification and identification of oxalate decarboxylase

[0038] The supernatant containing oxalate decarboxylase obtained by the method in Example 1 was purified by Q sepharose chromatographic column to obtain purified oxalate decarboxylase. The specific operation process is as follows: adjust the pH value of the supernatant containing oxalate decarboxylase to 6.0 with 0.5M NaOH, then remove the precipitated impurities by high-speed centrifugation at 15000g, and concentrate the supernatant through a 50KDa ultrafiltration membrane; NaH 2 PO 4 Buffer (pH 6.0) equilibrates 5 column volumes (CV), loads the concentrated oxalate decarboxylase solution onto a well-balanced Q sepharose column, and then equilibrates with 2CV of equilibration buffer (25mM NaH 2 PO 4 , pH value 6.0) to wash away the impurity protein not hanging on the column, and then through the elution buffer (elution buffer A: 25mM NaH 2 PO 4 , pH 6.0; Elution buffer B: 25mMNaH 2 PO 4 , 1...

Embodiment 3

[0039] Embodiment 3 Oxalate decarboxylase activity and enzymatic property determination

[0040] In the present invention, an HPLC (High Performance Liquid Chromatography, high performance liquid chromatography) method is used to measure the activity of oxalate decarboxylase. The specific operation process is as follows: 1.0mL 5mM (millimolar concentration) oxalate solution (containing 25mM citrate buffer, pH value 3.0), after preheating at 37°C for 10 minutes, add 0.01-0.1ml (according to enzyme The concentration is determined by adding the volume) to the solution or bacterial powder suspension containing oxalate decarboxylase to start the reaction. After reacting for 30 minutes, add 50 μL of 2.5M (molar concentration) sulfuric acid to inactivate the enzyme. Centrifuge rapidly and take the supernatant, and measure the residual oxalate concentration by HPLC. One enzyme activity unit (U) is defined as the amount of enzyme required to degrade 1 micromole of oxalate per minute ...

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Abstract

The invention discloses glycosylated oxalate decarboxylase. The oxalate decarboxylase is derived from edible basidiomycete; the glycosylated oxalate decarboxylase has enzymatic activity in an environment that pH value is 1.5-7.5; and the activity is 10% greater than optimum activity in an environment that pH value is 1.5-2.0, which is 2U / mg greater than an activity unit. The invention also discloses a preparation method and an application of the glycosylated oxalate decarboxylase. The glycosylated oxalate decarboxylase provided by the invention can keep activity under a low-pH condition; and the glycosylated oxalate decarboxylase can be used for effectively preventing and treating kidney stone.

Description

technical field [0001] The present invention relates to glycosylated oxalate decarboxylase and its preparation and application. Background technique [0002] Kidney stones, 80% of which are calcium oxalate stones, bring pain to tens of millions of people around the world and cause tens of billions of dollars in economic losses every year. In the United States, about 5 million people enter the emergency room every year because of kidney stones. One of the seven major hospitalization diseases, the annual economic loss is more than 5 billion US dollars, and the annual growth rate is 7-11%. The situation in China is similar to that of the United States; in addition, kidney stones are still many modern diseases (such as diabetes, obesity, etc. ) complications. [0003] Oxalic acid is a dicarboxylic acid linked by C-C bonds. Under physiological pH conditions, its sodium and potassium salts are soluble in water, but the solubility of its calcium salt in water is lower than 0.06mM....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/88A23L31/00A23K20/189A23L29/00A23L33/10A61K38/51A61P13/04A61P13/00
CPCA23V2002/00A61K38/00A61K38/51A23K10/30A23K20/189A23L29/06A23L31/00A23L33/10C12N1/14C12N9/88C12Y401/01002A23V2200/30A23V2250/208A23V2300/14A61P13/00A61P13/04
Inventor 刘海峰宋保平陈先桥陈火晴童佩汪卫汪小锋
Owner WUHAN KANGFUDE BIOTECH CO LTD
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