Purification method of beta-glucuronidase

A technology of glucuronidase and purification method, which is applied in the field of in vitro diagnosis, can solve the problems of low recovery rate, slow enzyme production, too many steps, etc., and achieve simple purification method, high development and application value, and good stability Effect

Active Publication Date: 2020-03-17
ZHENGZHOU IMMUNO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, many experts and scholars at home and abroad have obtained a variety of β-glucuronidases from various plant materials and microorganisms, but the activity of plant-derived enzymes is much lower than that of microbial-derived enzymes
As early as the 1980s, foreign scholars began to use the detection of β-glucuronidase to judge the existence of Escherichia coli, indicating that Escherichia coli is rich in β-glucuronidase, and extracting β-glucuronide from Escherichia coli The enzyme method is to use the fractional precipitation of ammonium sulfate on protein combined with column chromatography, but some β-glucuronidases derived from bacteria and fungi generally have low enzyme expression, slow enzyme production, and partial enzyme activity. low level problem
At present, the purification method of β-glucuronidase derived from natural animal extracts uses animal liver or viscera as raw materials, and after homogenization, it is separately precipitated with ammonium sulfate and ethanol, followed by isopoint focusing and molecular sieve chromatography, but the recovery rate is not high. High (only 0.19%), and there are too many steps in the purification process, and the operation is cumbersome

Method used

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  • Purification method of beta-glucuronidase
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  • Purification method of beta-glucuronidase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 The purification method of β-glucuronidase of the present invention comprises the following steps:

[0019] The first step is to take an adult white jade snail with a weight of 35g, and starve it for 1 day. After making it excrete the food in the digestive tract, cut the shell from the shell mouth along the direction of the shell spiral along the suture line until the top of the shell, and then wrap the shell around the shell axis. It should be noted that during the cutting process, it is necessary to constantly remove the broken shells with tweezers, only keep the central shell axis, and then carefully remove the soft body part wound on the shell axis, so as not to damage the inner shell. software structure;

[0020] The second step is to place the cut soft body part in a petri dish, use scissors to cut the body wall wrapped around the viscera mass, lift the body wall, and gently stretch the curled viscera to see the reddish-brown digestion For the digest...

Embodiment 2

[0025] Embodiment 2 The purification method of β-glucuronidase of the present invention comprises the following steps:

[0026] The first step is to take an adult Roman snail with a weight of 45g, starve it for 2 days, make it excrete the food in the digestive tract, cut the shell from the shell mouth along the direction of the shell spiral along the suture line until the top of the shell, and then wrap the shell around the shell axis It should be noted that during the cutting process, it is necessary to constantly remove the broken shells with tweezers, only keep the central shell axis, and then carefully remove the soft body part wound on the shell axis, so as not to damage the inner shell. software structure;

[0027] The second step is to place the cut soft body part in a petri dish, use scissors to cut the body wall wrapped around the viscera mass, lift the body wall, and gently stretch the curled viscera to see the reddish-brown digestion For the digestive glands of the...

Embodiment 3

[0032] Embodiment 3 performance measurement of purified product

[0033] 1. Enzyme Activity Determination Method:

[0034] Use p-nitrophenyl-β-D-glucoside (pNPG) as the substrate for enzymatic hydrolysis, and the p-nitrophenol released after hydrolysis of the substrate has a characteristic absorption peak in the visible light range of 400-420nm, which can be directly in Colorimetric determination between 400-420nm.

[0035] 2. Enzyme Stability Determination Method: 37°C Thermal Acceleration Determination of Stability.

[0036] 3. Measurement results

[0037] 3.1 Enzyme activity assay results

[0038] Measure the β-glucuronidase that embodiment 1 and embodiment 2 purify according to above-mentioned enzyme activity assay method, its result is as shown in table 1 below:

[0039] Table 1

[0040]

[0041] Conclusion: From the data in Table 1, it can be seen that the OD values ​​of the purified β-glucuronidases obtained in Example 1 and Example 2 of the present invention ar...

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Abstract

The invention discloses a purification method of beta-glucuronidase. The method comprises the steps of taking adult snails, cutting off shells, taking down soft parts, cutting off body walls in a culture dish, pulling out digestive glands, and putting digestive glands into a culture dish; puncturing the digestive glands by using a needle head, collecting the flowing viscous liquid and the digestive glands respectively, centrifuging under a certain condition to remove precipitates, combining supernate, and filtering by using a filter membrane to obtain a crude extract containing beta-glucuronidase; enabling the crude extract to pass through a DEAE ion exchange column for linear gradient elution, firstly determining the enzyme activity of the obtained eluents, then combining the eluents withrelatively high enzyme activity to obtain a beta-glucuronidase purified product; adding a preservative and an antioxidant; and performing low-temperature preservation. According to the invention, thepurification method is simple and feasible; the active material (snail) is rich in source, low in price, easy to obtain and short in period; and the purified beta-glucuronidase is high in activity, good in stability and high in recovery rate, can completely meet the requirements of an in-vitro diagnostic kit, and has relatively high development and application values.

Description

technical field [0001] The invention relates to the technical field of in vitro diagnosis, in particular to a method for purifying β-glucuronidase. Background technique [0002] β-Glucuronidase (β-Glucuroidase) is a glycoside hydrolase with the ability to enzymatically / hydrolyze β-glucuronides and sterols. In addition to being found in higher plants and microorganisms, this enzyme also exists in almost all tissues of animals, with the highest content in liver tissue. In higher animals, steroid hormones are transported to target cells in the form of glucuronide, and under the action of β-glucuronidase, they are converted into free steroids, thus showing the role of hormones; this enzyme is also involved in adhesion A decomposition process in polysaccharide metabolism that liberates glucuronic acid residues from the non-reducing ends of oligosaccharide fragments. At present, the enzyme is widely used in the detection of steroids, non-steroids and other substances. [0003] ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24
CPCC12N9/2402C12Y302/01031
Inventor 杨俊华张春鸽赵巧辉李桂林付光宇吴学炜杨增利
Owner ZHENGZHOU IMMUNO BIOTECH
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