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Method for quickly extracting [alpha]-glucosidase from fresh small intestines of rabbit

A glucosidase and small intestine technology, applied in the directions of glycosylase, biochemical equipment and methods, enzymes, etc., can solve the problems of high price, inconvenience, long delivery period, etc., and achieve simple method, simple process route and low cost. Effect

Inactive Publication Date: 2021-02-05
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the α-glucosidase required in the country needs to rely on imports. Many α-glucosidases on the market are mainly derived from microbial metabolites, which are somewhat different from the α-glucosidase in the intestinal tract of mammals. And the price is expensive, the delivery time is long, causing inconvenience

Method used

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  • Method for quickly extracting [alpha]-glucosidase from fresh small intestines of rabbit
  • Method for quickly extracting [alpha]-glucosidase from fresh small intestines of rabbit
  • Method for quickly extracting [alpha]-glucosidase from fresh small intestines of rabbit

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Effect test

Embodiment 1

[0021] Embodiment 1: In this embodiment, the method for rapidly extracting α-glucosidase from the fresh small intestine of rabbits is as follows:

[0022] (1) Fresh rabbit small intestine pretreatment: the duodenum was collected within 0.5 h after the rabbit was sacrificed, and the intestinal mucosa and intestinal mucus were scraped with a sterile scalpel at a low temperature of 0°C;

[0023] (2) Add 1 volume of 0.6 mol / L Tris-HCl buffer solution to the intestinal mucosa and mucus, homogenize at high speed for 5 minutes (Vortex-Genie®2, MOBIO), let stand at 4°C for 6 hours, and then Centrifuge at 4000 g for 25 min at 4°C and collect the supernatant;

[0024] (3) Mix the supernatant from step (2) with 0.1 mol / L Tris-HCl buffer at a volume ratio of 1:1, let stand at 4°C for 6 h, and centrifuge at 4,000 g for 25 min at 4°C , remove the precipitate, collect the supernatant, and store at 0°C, -20°C, and -80°C respectively.

Embodiment 2

[0025] Embodiment 2: In this embodiment, the method for rapidly extracting α-glucosidase from the fresh small intestine of rabbits is as follows:

[0026] (1) Pretreatment of fresh rabbit small intestine: the small intestine of the rabbit was taken immediately after sacrifice, and the intestinal mucosa and intestinal mucus were scraped with a sterile scalpel at a low temperature of 4°C;

[0027] (2) Add 2 times the volume of 0.3mol / L Tris-HCl buffer to the intestinal mucosa and intestinal mucus, homogenize at high speed for 15 minutes (Vortex-Genie®2, MOBIO), let stand at 0°C for 2 hours, and then Centrifuge at 6000g for 15min at 4°C and collect the supernatant;

[0028] (3) Mix the supernatant from step (2) with 0.1 mol / L Tris-HCl buffer at a ratio of 1:2 by volume, let stand at 5°C for 5h, and centrifuge at 7000g for 15min at 4°C to remove the precipitate , collect the supernatant and store it at 0°C.

Embodiment 3

[0029] Embodiment 3: In this embodiment, the method for rapidly extracting α-glucosidase from the fresh small intestine of rabbits is as follows:

[0030] (1) Fresh rabbit small intestine pretreatment: take the small intestine within 1 hour after the rabbit is sacrificed, scrape the intestinal mucosa and intestinal mucus with a sterile scalpel at a low temperature of 8°C;

[0031] (2) Add 4 times the volume of 0.2mol / L Tris-HCl buffer solution to the intestinal mucosa and intestinal mucus, homogenize at a high speed for 10 minutes (Vortex-Genie®2, MOBIO), let stand at 8°C for 3 hours, and then Centrifuge at 8000g for 6min at 4°C and collect the supernatant;

[0032] (3) Mix the supernatant from step (2) with 0.1 mol / L Tris-HCl buffer at a volume ratio of 1:2, let stand at 8°C for 5 hours, and centrifuge at 10,000g for 5 minutes at 4°C to remove the precipitate , collect the supernatant and store at -80°C.

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Abstract

The invention discloses a method for quickly extracting [alpha]-glucosidase from the fresh small intestines of a rabbit. The method comprises the following steps of: adopting the fresh small intestines of the rabbit as a raw material, collecting the intestinal mucus and the intestinal mucosa of the small intestines, adding weak acid salt buffer solution, after high-speed homogenization is carriedout, standing at a low temperature, and carrying out centrifugation to collect liquid supernatant; and adding the weak acid salt buffer solution into the liquid supernatant, standing at the low temperature, and after centrifugation separation is carried out, taking the liquid supernatant to obtain extraction liquid containing the [alpha]-glucosidase. The specific activity of the enzyme extractionliquid prepared by the method disclosed by the invention is 6U / mg and is similar to the specific activity of commercial enzyme, and in addition, the enzyme activity of the enzyme preserved at the temperature of 0-80DEg C for 3 months is almost kept stable. Compared with the commercial enzyme, acarbose has higher inhibition activity for the extraction liquid of the invention to prove that an [alpha]-glucosidase extraction system of the small intestine source of the rabbit is effective. Compared with the commercial enzyme which has an expensive price and a long date of delivery, the [alpha]-glucosidase prepared by the method disclosed by the invention is safe and environmentally friendly, has a simple extraction technology and a low cost, and is suitable for industrial production and marketpopularization and application.

Description

technical field [0001] The invention belongs to the technical field of preparation of enzyme products, in particular to a method for rapidly extracting α-glucosidase from fresh small intestine of rabbits. Background technique [0002] Glucosidase is a large class of enzymes in the glycoside hydrolase family. Its main function is to hydrolyze glucosidic bonds and release glucose. It is an indispensable class of enzymes in the carbohydrate metabolism pathway of organisms. According to the type of hydrolysis of glycosidic bonds, glucosidase can be divided into α-glucosidase (α-glucosidase) and β-glucosidase (β-glucosidase). α-glucosidase mainly includes enzymes such as maltase, sucrase, isomerized maltase, and lactase, which are widely distributed in the brush border of small intestinal villous mucosa cells, and participate in the digestion of food, the biosynthesis of glycoproteins, polysaccharides and Synthesis and catabolism of carbohydrate complexes and other biological pr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/24
CPCC12N9/2402C12Y302/0102
Inventor 伊日布斯高茜毛泽楠刘彦敏王康
Owner KUNMING UNIV OF SCI & TECH