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Method for preparing Beta-glucuronidase crude enzyme preparation

A crude aldolase and aldolase technology, which can be applied in microorganism-based methods, biochemical equipment and methods, hydrolase and other directions, and can solve problems such as low enzyme activity

Active Publication Date: 2013-10-02
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Solved the problem of low enzyme activity to a large extent

Method used

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  • Method for preparing Beta-glucuronidase crude enzyme preparation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, only using glycyrrhizic acid monoammonium salt as inducer to produce enzyme implementation method:

[0031] Inoculate the Penicillium purpurea Li-3 strain on the slant medium, and cultivate it at a constant temperature of 30°C for 3 days. The composition of the slant medium is: glucose 0.5g, NH 4 NO 3 0.3g, KH 2 PO 4 0.1g, KCl0.05g, MgSO 4 ·7H 2 O0.05g, FeSO 4 ·7H 2 O0.001g, distilled water 100mL, agar 1.5g, adjust the pH to 5.0, sterilize at 121°C for 20min, and cool to room temperature.

[0032] Take the spores on the slant medium, inoculate them into the seed medium, and culture them on a shaker at 30°C at 170r / min for 72h, then transfer 5% of the inoculum into the secondary seed medium at 30°C on a shaker at 170r / min Cultivate for 24 hours to obtain secondary seed liquid, the composition of the seed medium is: glucose 0.5g, NH 4 NO 3 0.3g, KH 2 PO 4 0.1g, KCl0.05g, MgSO 4 ·7H 2 O0.05g, FeSO 4 ·7H 2 O0.001g, 100mL of distilled water, adju...

Embodiment 2

[0034] Example 2, the implementation method of adding exogenous accelerator to produce enzymes of monoammonium glycyrrhizinate:

[0035] Slope culture and seed culture are the same as in Example 1.

[0036] Put the secondary seed liquid into the enzyme-producing medium according to the inoculation amount of 10% by volume, and culture it on a shaker at 30°C and 170r / min for 72 hours to reach the maximum specific enzyme activity of 157U / mg. solution as a crude enzyme preparation. The composition of the enzyme production medium: monoammonium glycyrrhizinate 0.6g, licorice total extract 0.4g, NH 4 NO 3 0.3g, KH 2 PO 4 0.1g, KCl0.05g, MgSO 4 ·7H 2 O0.05g, FeSO 4 ·7H 2 O0.001g, 100mL of distilled water, adjust the pH to 5.0, sterilize at 121°C for 20min, and cool to room temperature.

Embodiment 3

[0038] Slope culture, seed culture, and enzyme production culture are the same as in Example 1.

[0039] Put the secondary seed solution into the enzyme-producing medium according to the inoculation amount of 10% by volume, and cultivate it on a shaker at 30°C and 170r / min for 60 hours to reach the maximum specific enzyme activity of 135U / mg. The fermentation broth is centrifuged to collect the bacteria. 47.8 mg of crude enzyme freeze-dried powder was obtained as crude enzyme preparation after the body was freeze-dried and ground. Composition of enzyme production medium: 0.6g monoammonium glycyrrhizinate, 0.25g licorice total polysaccharide, NH 4 NO 3 0.3g, KH 2 PO 4 0.1g, KCl0.05g, MgSO 4 ·7H 2 O0.05g, FeSO 4 ·7H 2 O0.001g, 100mL of distilled water, adjust the pH to 5.0, sterilize at 121°C for 20min, and cool to room temperature.

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Abstract

The invention relates to a method for preparing a Beta-glucuronidase crude enzyme preparation, and belongs to the field of biotechnology (fermentation engineering). The method comprises the following steps: adding an exogenous accelerant into an enzyme production culture medium containing glycyrrhizic acid or glycyrrhetate, wherein the exogenous accelerant is total liquorice extractives, or total liquorice polysaccharides and / or total liquorice flavone; promoting penicillium purpurogenum Li-3 (Collection No.: CGMCC No. 5446) of strains to induce the generation of Beta-glucuronidase. Compared with the method without the accelerant adding step, the method has the advantages that glucuronidase is generated 5 to 48 hours earlier, and the activity of glucuronidase is improved by 10 to 200 percent.

Description

technical field [0001] The invention relates to a method for preparing crude beta-glucuronidase enzyme preparation, which belongs to the field of biotechnology (fermentation engineering). Background technique [0002] β-glucuronidase (β-glucuronidase, EC: 3.2.1.31, referred to as GUS) is a glycoside hydrolase and a typical glycosylase, which can catalyze various types of β-glucuronide Hydrolysis, as early as the 1950s, foreign scholars have done research on enzymes derived from Escherichia coli and animals. , Tobacco, rice, corn and other higher plants detected the activity of the enzyme. [0003] The monomeric molecular weight of β-glucuronidase of Escherichia coli is 68.2kDa, and it often exists in the form of homopolymeric tetravalent. It is very stable and still has activity under the condition of large changes in environmental conditions, such as showing extremely high activity in the presence of thiol reducing agent β-mercaptoethanol or DTT (dithiothreitol). β-Glucu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24C12R1/80
Inventor 刘桂艳李春王栋高自强刘冬羽曹欠
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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