Purification method of beta-glucuronidase

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] ...

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

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