CN112322603A - Method for rapidly extracting alpha-glucosidase from fresh small intestine of rabbit - Google Patents
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
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Abstract
The invention discloses a method for quickly extracting alpha-glucosidase from fresh small intestine of rabbit, which comprises the steps of collecting intestinal mucus and intestinal mucosa of the small intestine by using the fresh small intestine of rabbit as raw material, adding weak acid salt buffer solution, homogenizing at high speed, standing at low temperature, centrifuging and collecting supernatant; adding a weak acid salt buffer solution into the supernatant, standing at low temperature, performing centrifugal separation, and taking the supernatant to obtain an extracting solution containing the alpha-glucosidase; the specific activity of the enzyme extract prepared by the method is 6U/mg, is close to that of commercial enzyme, and the enzyme activity of the enzyme which is preserved for 3 months at 0 ℃ and-80 ℃ is basically kept stable; compared with commercial enzyme, acarbose has higher inhibitory activity to the enzyme extract, and the alpha-glucosidase extraction system from rabbit small intestine is proved to be effective; compared with commercial enzymes with high price and long shelf life, the alpha-glucosidase prepared by the invention is safe and environment-friendly, has simple extraction process and low cost, and is suitable for industrial production and market popularization and application.
Description
Technical Field
The invention belongs to the technical field of enzyme product preparation, and particularly relates to a method for quickly extracting alpha-glucosidase from fresh small intestine of a rabbit.
Background
The glucosidase is a large class of enzymes in a glycoside hydrolase large family, has the main functions of hydrolyzing a glucoside bond and releasing glucose, and is an indispensable enzyme in a sugar metabolic pathway of an organism. Glucosidases can be classified into alpha-glucosidase and beta-glucosidase depending on the type of glycosidic bond hydrolyzed. The alpha-glucosidase mainly comprises enzymes such as maltase, sucrase, maltulase, lactase and the like, is widely distributed in the brush border of villous mucosa cells of the small intestine, and participates in a plurality of biological processes such as digestion of body food, biosynthesis of glycoprotein, synthesis and catabolism of polysaccharide and glycoconjugate and the like; α -glucosidase is widely present in various organisms in nature, for example: grapes, sweet corn, rice, fruit flies, aspergillus niger and saccharomyces cerevisiae.
At present, the common method for extracting alpha-glucosidase mainly comprises the step of extracting alpha-glucosidase from wheat or mature human clone colon adenocarcinoma cells (Caco-2 cells). In addition, an expression vector is constructed by utilizing a gene engineering technology to increase the copy number of the alpha-glucosidase gene, so that a recombinant strain for efficiently expressing the alpha-glucosidase is obtained. At present, alpha-glucosidase required in China needs to be imported, various alpha-glucosidase in the market is mainly derived from metabolites of microorganisms, has certain difference with the alpha-glucosidase in intestinal tracts of mammals, and is expensive, long in shelf life and inconvenient.
A large number of studies show that the animal small intestine has rich alpha-glucosidase, and has good practical prospect when being used for extracting the alpha-glucosidase; the extraction of alpha-glucosidase from the small intestine of mammals has not been much studied.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides a method for quickly extracting alpha-glucosidase from fresh small intestine of rabbit, which adopts centrifugal separation technology to achieve the aims of simple process, high extraction efficiency, small enzyme activity loss and no environmental pollution.
The method adopts fresh small intestines of rabbits as raw materials, collects intestinal mucus and intestinal mucosa of the small intestines, adds a weak acid salt buffer solution, homogenizes at a high speed, stands at a low temperature, centrifugalizes and collects supernatant; adding weak acid salt buffer solution into the supernatant, standing at low temperature, centrifuging, and collecting the supernatant to obtain the extractive solution containing alpha-glucosidase.
The method comprises the following specific operations:
(1) fresh small intestine pretreatment of rabbits: selecting rabbit small intestine, scraping intestinal mucosa and intestinal mucus with sterile scalpel at low temperature of 0-10 deg.C, and collecting;
the small intestine is obtained within 0-4 h after death of the rabbit and is transported and stored at 0-10 deg.C;
(2) adding 0.1-0.6mol/L weak acid salt buffer solution with volume 1-10 times of intestinal mucosa and intestinal mucus, homogenizing at high speed for 2-30min, standing at 0-10 deg.C for 2-20 h, centrifuging at 4 deg.C for 5-30min at 4000-10000g, and collecting supernatant;
(3) mixing the supernatant with 0.1mol/L weak acid salt buffer solution according to the volume ratio of 1:1-5, standing for 2-20 h at 0-10 ℃, centrifuging for 5-30min at 4000-10000g at 4 ℃, removing precipitates, and collecting the supernatant to obtain an extracting solution containing alpha-glucosidase;
(4) and (3) determining the enzyme activity of the alpha-glucosidase of the extracting solution in the step (3) and the stability of the extracting solution at different temperatures, and detecting the inhibition rate of acarbose on the enzyme extracting solution prepared by the method, wherein the experimental result shows that the activity of the enzyme solution extracted by the method is close to that of a commercial enzyme, the enzyme activity stability is good, and the experimental result also shows that the method is simple and effective.
The invention has the advantages and technical effects that:
the method adopts the weak acid salt buffer solution to extract the fresh small intestine of the rabbit, uses the weak acid salt buffer solution for storage, is safe and environment-friendly, has simple process route and low cost, and the obtained alpha-glucosidase product has high extraction rate and high enzyme activity, and can be used for screening alpha-glucosidase inhibitors.
The method adopts weak acid salt buffer solution for extraction, adopts a high-speed homogenization method to enable small intestine mucosal cells to release alpha-glucosidase, removes impurities through centrifugal separation, adopts weak acid salt buffer solution for preservation, reduces the loss of alpha-glucosidase activity, is simple and easy to operate, and is suitable for industrial production and market popularization and application.
Drawings
FIG. 1 is a schematic diagram showing the results of the enzyme activity changes of alpha-glucosidase after the enzyme extract is diluted by different times;
FIG. 2 is a diagram showing the results of the change in the enzyme activity of alpha-glucosidase in different time periods of the reaction between the enzyme extract and the substrate;
FIG. 3 is a graph showing the stability results of enzyme extracts at different storage temperatures;
FIG. 4 is a graph of the inhibition of a-glucosidase from different sources by acarbose.
Detailed Description
The technical scheme of the present invention is further described in detail by the following examples, but the content of the present invention is not limited thereto, and the methods in the examples are all conventional methods unless otherwise specified, and materials, reagents and the like used therein are commercially available unless otherwise specified.
Example 1: the method for rapidly extracting alpha-glucosidase from fresh small intestine of rabbit in the embodiment is as follows:
(1) pretreatment of fresh rabbit small intestine: taking duodenum of the rabbit within 0.5 h after the rabbit dies, and scraping intestinal mucosa and intestinal mucus by using a sterile scalpel at the low temperature of 0 ℃;
(2) adding 1-time volume of 0.6mol/L Tris-HCl buffer solution into the intestinal mucosa and intestinal mucus, homogenizing at high speed for 5min (Vortex-Genie 2, MOBIO), standing at 4 ℃ for 6 h, centrifuging at 4 ℃ for 25 min at 4000 g, and collecting supernatant;
(3) mixing the supernatant obtained in the step (2) with 0.1mol/L Tris-HCl buffer solution according to the volume ratio of 1:1, standing for 6 h at 4 ℃, centrifuging for 25 min at 4000 g at 4 ℃, removing precipitates, collecting the supernatant, and storing at 0 ℃, 20 ℃ and 80 ℃ respectively.
Example 2: the method for rapidly extracting alpha-glucosidase from fresh small intestine of rabbit in the embodiment is as follows:
(1) pretreatment of fresh rabbit small intestine: taking the small intestine of the rabbit immediately after the rabbit is killed, and scraping the intestinal mucosa and the intestinal mucus by using a sterile scalpel at a low temperature of 4 ℃;
(2) adding 2 times of Tris-HCl buffer solution with the volume of 0.3mol/L into the intestinal mucosa and the intestinal mucus, homogenizing at high speed for 15min (Vortex-Genie 2, MOBIO), standing for 2 h at 0 ℃, then centrifuging for 15min at 6000g at 4 ℃, and collecting supernatant;
(3) mixing the supernatant obtained in the step (2) with 0.1mol/L Tris-HCl buffer solution according to the volume ratio of 1:2, standing at 5 ℃ for 5h, centrifuging at 4 ℃ for 15min at 7000g, removing precipitate, collecting supernatant, and storing at 0 ℃.
Example 3: the method for rapidly extracting alpha-glucosidase from fresh small intestine of rabbit in the embodiment is as follows:
(1) pretreatment of fresh rabbit small intestine: taking small intestine of the rabbit within 1h after the rabbit dies, and scraping intestinal mucosa and intestinal mucus by using a sterile scalpel at the low temperature of 8 ℃;
(2) adding 0.2mol/L Tris-HCl buffer solution with the volume 4 times of that of the intestinal mucosa and intestinal mucus, homogenizing at high speed for 10min (Vortex-Genie 2, MOBIO), standing for 3h at 8 ℃, centrifuging for 6min at 8000g at 4 ℃, and collecting supernatant;
(3) mixing the supernatant obtained in the step (2) with 0.1mol/L Tris-HCl buffer solution according to the volume ratio of 1:2, standing for 5h at 8 ℃, centrifuging for 5min at 10000g at 4 ℃, removing precipitates, collecting the supernatant, and storing at-80 ℃.
Example 4: example 1 enzyme Activity detection experiment of alpha-glucosidase after dilution with different fold of enzyme extract
(1) Diluting the enzyme extract by different times: diluting the enzyme extract with sterile water by 10 times, 20 times, 30 times, 40 times and 50 times respectively;
(2) adding 50 μ L of substrate PNPG (p-nitrophenyl alpha-D-glucopyranoside, 2.5mmol/L, pH 6.8) and 50 μ L of PBS buffer (100 mmol/L, pH 6.8) to 100 μ L of the enzyme solution diluted in step (1), respectively, and reacting at 37 ℃ for 40 min; separately, 100. mu.L of 0.1M Na was added2CO3The reaction is stopped by the solution, and the value of absorbance at 405 nm is measured by a microplate reader.
The results are shown in FIG. 1, and it can be seen from FIG. 1 that the value of absorbance gradually decreased with increasing dilution factor, the value of absorbance 0.172 with 50-fold dilution and the value of absorbance 0.43 with 10-fold dilution, and the results show that the enzyme activity gradually decreased with increasing dilution gradient.
Example 5: example 1 detection of enzyme Activity of alpha-glucosidase by reacting enzyme extract with substrate for various periods of time
(1) Diluting the enzyme extract of example 1 with sterile water by a factor of 10;
(2) to 100. mu.L of the enzyme solution diluted 10 times in step (1), 50. mu.L of the substrate PNPG (p-nitrophenyl α -D-glucopyranoside, 2.5mmol/L, pH 6.8) and 50. mu.L of PBS buffer (100 mmol/L, pH 6.8), Nos. 1-6 were added; reacting at 37 deg.C for 0min, 10min, 20 min, 30min, 40 min, and 50 min, respectively, adding 100 μ L0.1M Na2CO3Stopping the reaction of the solution, and measuring the value of absorbance at 405 nm by using an enzyme-labeling instrument;
the results are shown in FIG. 2, and it can be seen from FIG. 2 that the absorbance value gradually increases with the increase of the reaction time of the enzyme extract and the substrate, and the absorbance value at 40 min was higher by 0.35 and then becomes flat, indicating that the optimum reaction time of the enzyme extract and the substrate was 40 min.
Example 6: example 1 measurement of enzyme Activity and specific Activity of alpha-glucosidase in enzyme extract
(1) Determination of protein content: protein content was measured by a kit (Bradford, 100T, Solarbio), and protein content of the enzyme extract and the extract diluted 30 times was measured;
(2) adding 50 μ L substrate PNPG (p-nitrophenyl alpha-D-glucopyranoside, 2.5mmol/L, pH 6.8) and 50 μ L PBS buffer (100 mmol/L, pH 6.8) into 100 μ L enzyme solution diluted 30 times, and reacting at 37 deg.C for 40 min;
(3) add 100. mu.L of 0.1M Na2CO3The reaction is stopped by the solution, and the value of absorbance at 405 nm is measured by a microplate reader.
The enzyme activity of the alpha-glucosidase was determined to be 0.1U/mL, the total protein content was 16 mg/mL, the specific activity was 6U/mg protein, which is close to that of the commercial enzyme (10U/mg protein, Sigma, G5003 MSDS), see Table 1;
TABLE 1
Example 7: example 1 stability analysis of enzyme extracts at different storage temperatures
(1) Taking out enzyme extract stored at 0 deg.C, -20 deg.C, -80 deg.C for 3 months, and diluting with sterile water by 30 times respectively;
(2) adding 50 μ L substrate PNPG (p-nitrophenyl alpha-D-glucopyranoside, 2.5mmol/L, pH 6.8) and 50 μ L PBS buffer (100 mmol/L, pH 6.8) into 10 μ L of the enzyme solution diluted 30 times in step (1) respectively, and reacting at 37 deg.C for 40 min;
(3) separately, 100. mu.L of 0.1M Na was added2CO3The reaction is stopped by the solution, and the value of absorbance at 405 nm is measured by a microplate reader.
As a result, as shown in FIG. 3, it can be seen from FIG. 3 that the initial absorbance values were all 0.35, the enzyme stability at 0 ℃ was the best, the absorbance value at 90 days was 0.314, the absorbance value at 90 days was 0.297 for the enzyme at-80 ℃ and the enzyme activity was substantially stable, and the enzyme activity at-20 ℃ was unstable, indicating that the suitable storage temperatures were 0 ℃ and-80 ℃.
Example 8: example 1 measurement of in vitro inhibitory Activity of enzyme extract
(1) Respectively diluting a positive medicament (acarbose, Sigma) into 0.1mg/mL, 0.5mg/mL, 1mg/mL, 1.5mg/mL and 2mg/mL, and diluting an alpha-glucosidase extracting solution into 0.2U/mL;
(2) taking 50 mu L of acarbose with different concentrations in the step (1), respectively adding 100 mu L of 0.2U/mL alpha-glucosidase, and reacting at 37 ℃ for 10 min; adding 50 μ L substrate PNPG (p-nitrophenyl alpha-D-glucopyranoside 2.5mmol/L, pH 6.8) and reacting at 37 deg.C for 30 min;
(3) separately, 100. mu.L of 0.1M Na was added2CO3The reaction is stopped by the solution, and the absorbance at 405 nm is measured by a microplate readerA value of (d);
(4) and (3) calculating an inhibition rate: inhibition (%) =1- (a)Sample (I)-ASample controls）/（AEnzyme activity-AEnzyme blank) X 100% where ASample (I)(enzyme + sample + substrate), ASample controls(PBS buffer + sample + substrate), AEnzyme activity(PBS buffer + enzyme + substrate), AEnzyme blank(PBS buffer + substrate), PBS buffer 100mmol/L, pH 6.8.
As a result, see fig. 4, it can be seen from fig. 4 that acarbose is logarithmically related to the inhibition ratio of the enzyme extract in a certain concentration range, and equation y = 0.1186ln (x) + 0.6466 (R = 0.9958) is obtained, so that the half-number effective inhibition concentration (IC 50) of the enzyme extract is calculated to be 0.3 mg/mL; similarly, acarbose has a logarithmic relationship with the inhibition rate of the commercial enzyme in a certain concentration range, and the equation y = 0.1505ln (x) + 0.5909 is obtained (R = 0.9925), so that the half-effective inhibition concentration (IC 50) of the commercial enzyme is calculated to be 0.55mg/mL, which indicates that acarbose has higher inhibition activity on the enzyme extract of the invention, and proves that the rabbit intestine-derived alpha-glucosidase extraction system is effective.
Claims (6)
1. A method for rapidly extracting alpha-glucosidase from fresh small intestine of rabbit is characterized in that: collecting intestinal mucus and intestinal mucosa of rabbit, adding weak acid salt buffer solution, homogenizing at high speed, standing at low temperature, centrifuging, and collecting supernatant; adding weak acid salt buffer solution into the supernatant, standing at low temperature, centrifuging, and collecting the supernatant to obtain the extractive solution containing alpha-glucosidase.
2. The method for rapidly extracting alpha-glucosidase from fresh small intestine of rabbit as claimed in claim 1, wherein: the small intestine is obtained within 0-4 h after death of the rabbit and is transported and stored at 0-10 ℃.
3. The method for rapidly extracting alpha-glucosidase from fresh small intestine of rabbit as claimed in claim 1, wherein: the weak acid salt buffer mixed with the small intestine is 0.1-0.6mol/L Tris-HCl buffer, and the weak acid salt buffer mixed with the supernatant is 0.1mol/L Tris-HCl buffer.
4. The method for rapidly extracting alpha-glucosidase from fresh small intestine of rabbit as claimed in claim 1, wherein: the volume ratio of the intestinal mucus to the intestinal mucosa to the weak acid salt buffer solution is 1: 1-10.
5. The method for rapidly extracting alpha-glucosidase from fresh small intestine of rabbit as claimed in claim 1, wherein: the volume ratio of the supernatant to the weak acid salt buffer solution is 1: 1-5.
6. The method for rapidly extracting alpha-glucosidase from fresh small intestine of rabbit as claimed in claim 1, wherein: centrifugation is carried out at 4 ℃ and 4000-.
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