CN113308452A - Purification method of venuosus digestive protease - Google Patents
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Images
Classifications
-
- 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)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6402—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals
- C12N9/6405—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from non-mammals not being snakes
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K61/00—Culture of aquatic animals
- A01K61/50—Culture of aquatic animals of shellfish
- A01K61/51—Culture of aquatic animals of shellfish of gastropods, e.g. abalones or turban snails
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention relates to a purification method of venosal digestive protease, belonging to the field of sampling of digestive protease. Comprises the following steps: the rapana venosa adult snail is artificially cultured for not less than 20 days by using a single variety of bivalve shellfish; the whelk adult snail is fasted for 3-7 days, the fasting time is adjusted along with the water temperature, the water temperature is 21-25 ℃ and is fasted for 3 days, the water temperature is 18-20 ℃ and is fasted for 4 days, the water temperature is 14-17 ℃ and is fasted for 5 days, the water temperature is 10-13 ℃ and is fasted for 6 days, and the water temperature is 6-7 ℃ and is fasted for 7 days; feeding the same variety of bivalve shellfish, and observing the predation process of the rapana venosa; when the foot part is extended and wrapped by the rapana venosa, the rapana venosa is taken out to quickly break the shell of the rapana venosa; rapidly dissecting the soft part of the venosa; the salivary glands were removed and frozen at-20 ℃. The invention ensures the concentration of the crude enzyme solution of the cercospora venosa digestive protease and provides enough material basis for the later purification of the protease.
Description
Technical Field
The invention relates to a protease sampling method, in particular to a purification method of venosal digestive protease.
Background
Leven published The Cleavage Products of proteins from 1/10.1905, and to date, proteases have been studied for over 100 years, with 35 million documents relating to proteases. Proteases are involved in biological processes such as the transmission of DNA genetic information, angiogenesis, wound repair, stem cell mobilization, hemostasis, coagulation, inflammatory responses, immune responses, autophagy, senescence, necrosis and apoptosis [3-4 ]. Many proteases have become biomarkers for disease prediction and diagnosis, and provide new directions for people in terms of drug treatment. The venosa is a large carnivorous mollusk, has strong locomotor organs and sensitive sensory organs, is mainly eaten by bivalve shellfish such as oysters, scallops, Chinese clams, philippinarum and the like, and can digest and absorb high protein in a short time, so the digestive juice of the venosa contains protease with strong activity, however, the venosa has a plurality of digestive glands including salivary glands, lebulin glands and livers, and the digestive glands have different material compositions in different ingestion modes, and the secretion of the digestive protease is different in different stages of ingestion, so the sampling method is proper, and the obtained enough crude protease liquid is the material basis for later protease separation and purification.
Disclosure of Invention
In order to effectively obtain the young rapana venosa digestive protease and avoid the conditions of small protease obtaining amount and low activity caused by improper sampling, the invention provides an optimal method for obtaining the crude enzyme liquid of the rapana venosa high-efficiency digestive protease, and provides guarantee for further purification of the later-stage protease.
The technical scheme adopted by the invention for solving the technical problems is that the purification method of the venosal digestive protease comprises the following steps: a. the rapana venosa adult snail is artificially cultured for not less than 20 days by using a single variety of bivalve shellfish; b. the whelk adult snail is fasted for 3-7 days, the fasting time is adjusted along with the water temperature, the water temperature is 21-25 ℃ and is fasted for 3 days, the water temperature is 18-20 ℃ and is fasted for 4 days, the water temperature is 14-17 ℃ and is fasted for 5 days, the water temperature is 10-13 ℃ and is fasted for 6 days, and the water temperature is 6-7 ℃ and is fasted for 7 days; c. feeding the same variety of bivalve shellfish, and observing the predation process of the rapana venosa; d. when the foot part is extended and wrapped by the rapana venosa, the rapana venosa is taken out to quickly break the shell of the rapana venosa; e. rapidly dissecting the soft part of the venosa; f. the salivary glands were removed and frozen at-20 ℃.
The method has the advantages that the concentration of the crude enzyme solution of the cervus venosus digestive protease is ensured, an enough material basis is provided for the later purification of the protease, and 1) the phenomenon that the secretion of cervus venosus digestive gland mucus is more unfavorable for the separation and purification of the protease due to the various types of baits in the early stage is effectively avoided; 2) the digestive gland of the praemasal red snail ingested by the bivalves does not synthesize protease; 3) the situation that the protease in the digestive gland of the venosal is secreted into the digestive gland of food after the bivalve shellfish is ingested is low in protease content; 4) the secretion of protease from the Robushel gland and liver of the Nexus venosa as auxiliary digestive glands is small, and the content of foreign proteins in the crude enzyme solution is increased by mixing with salivary glands. Meanwhile, when the protease content in the salivary gland of the rapana venosa is the highest, the rapana venosa is quickly dissected and frozen, so that the secretion or the self-degradation of the protease is prevented.
Drawings
FIG. 1 is a photograph of a living wild Rapana venosa.
FIG. 2 is a photograph of dissecting the soft part of the Rapana venosa after crushing the shell of the Rapana venosa to remove the salivary gland.
FIG. 3 is a photograph showing the detection of enzyme activity of the crude enzyme solutions of the Lebulin gland, salivary gland and liver.
FIG. 4 is a relative specific activity diagram of salivary gland proteases in different feeding stages of the Nepalustris.
Detailed Description
The first embodiment is as follows:
obtaining 50 wild rapana venosa from the sea area of the double island bay of Weihai city, feeding mussels for 30 days with water temperature of 14-15 ℃, starving for 5 days, feeding the mussels, observing that when the rapana venosa is wrapped by the mussels with feet (as shown in figure 1), immediately taking out the rapana venosa and smashing the snail shell, quickly dissecting the soft part of the rapana venosa, taking out the salivary gland and putting the salivary gland into the sea area of the double island bay of Weihai city for freezing (as shown in figure 2).
Mixing 10 glands of salivary gland, lebulin gland and liver collected in the same ingestion period, respectively, cleaning with pure water, cutting, grinding in a mortar, adding a proper amount of precooled 0.02M phosphate buffer solution with pH of 7.0, transferring to a glass homogenizer, and homogenizing with a high-speed homogenizer until the solution is uniform. The homogenate was transferred to a centrifuge tube and centrifuged at 5000r/min for 15 min. The precipitate is discarded, and the supernatant is the crude enzyme solution. Accurately weighing 7.0g of casein agar, heating and dissolving in 200ml of distilled water, uniformly pouring 30ml of casein agar into a flat plate, cooling and solidifying, punching, adding 30 mu L of crude enzyme liquid of the Lebulin gland, the salivary gland and the liver, reacting for 8 hours at room temperature, pouring 0.4mol/L of trichloroacetic acid, and observing the size of a hydrolysis ring. Phosphate buffer pH7.0 was used as a negative control, and trypsin at 0.5mg/ml was used as a positive control. The results of enzyme activity detection of the crude enzyme solutions of the Lebulin gland, salivary gland and liver are shown in FIG. 3.
Example two:
taking 50 temporary cultured Rapana venosa of Wihai Shengshui obstetrics and gynecology, feeding with oyster for 23 days with water temperature of 22-23 ℃, after hungry for 3 days, feeding the oyster, observing, respectively taking Rapana venosa which begins to move close to the oyster (during ingestion), wraps the oyster with feet (before ingestion), scrapes the soft part of the oyster with kiss (during ingestion) and discards the oyster shell (after ingestion) after eating the oyster, smashing the snail shell, quickly dissecting the soft part of the Rapana venosa, taking out the salivary gland and putting into a freezer with the temperature of-20 ℃. Mixing 10 collected salivary glands of different ingestion periods, cleaning with pure water, cutting, grinding in a mortar, adding appropriate amount of pre-cooled 0.02M phosphate buffer solution with pH of 7.0, transferring to a glass homogenizer, and homogenizing with a high speed homogenizer until the solution is uniform. The homogenate was transferred to a centrifuge tube and centrifuged at 5000r/min for 15 min. The precipitate is discarded, and the supernatant is the crude enzyme solution. 1mL of each of the tyrosine solutions having concentrations of 100. mu.g/mL, 200. mu.g/mL, 300. mu.g/mL, 400. mu.g/mL, and 500. mu.g/mL was added with 5mL of 0.4mol/L Na2CO3And (3) solution. Placing in a constant temperature water bath at 40 deg.C, keeping the temperature for 20min, and measuring absorbance value A680nm with a spectrophotometer. The standard curve is plotted with tyrosine content as abscissa and a680nm as ordinate. Preheating the crude enzyme solution and casein solution in 40 deg.C water bath for 15min, adding 1mL of 1% casein solution into 1mL of enzyme solution, mixing, and reacting in 40 deg.C water bath for 40 min. Accurately timing, adding 2mL of 0.4mol/L trichloroacetic acid into each tube at once after the reaction is finished,mix well to terminate the reaction. Standing for 10min, centrifuging at 5000r/min for 10min, and taking 1mL of supernatant in a test tube. 5ml of 0.4mol/L Na is added2CO3The solution was mixed with 1mL of Folin's phenol working solution. Mixing, keeping in 40 deg.C water bath for 20min, and measuring OD at 680 nm. Protease activity was measured according to the Folin phenol method.
Control group: adding 2mL of 0.4mol/L trichloroacetic acid into 1mL of enzyme solution which is preserved in advance, reacting for 40min, adding 1mL of 1% casein solution, shaking up, centrifuging, and taking supernatant as a control.
Unit of enzyme activity: it is stated that hydrolysis of casein produces 1ug of tyrosine per minute as one unit of enzyme activity, denoted by U, at 40 ℃ and pH 7.0.
The unit of enzyme activity contained in 1mL of sample is: (sample A-pair A). times.KxV.times.N/t.
Wherein sample a is the absorbance value of the sample; pair a is the absorbance value of the control; k is the tyrosine content when the absorbance on the standard curve is 1; t is the reaction time, and in the experiment, t is 10 min; v is the total volume of the reaction sample, and in the experiment, V is 4 mL; n is the dilution multiple of the enzyme solution, and corresponding dilution is carried out according to the condition of the sample each time.
The specific activity is the number of enzyme activity units contained in 1mL of sample divided by the protein content of 1mL of sample, expressed as U/mg.
The protein content of the enzyme solution was measured by Coomassie Brilliant blue G-250 method (Bradford method).
The preparation method of the Coomassie brilliant blue reagent comprises the following steps: 100mg of Coomassie Brilliant blue G-250 was weighed, dissolved in 50mL of 95% ethanol, added with 100mL of 85% phosphoric acid, diluted to 1000mL with distilled water, and filtered through a filter paper.
100 mu L of the saliva gland grinding liquid at each period after being diluted properly is added with 5mL of Coomassie brilliant blue G-250 reagent, and after the mixture is mixed uniformly, the absorbance value of the sample at 595nm is measured by a spectrophotometer. Blank control replaced the sample with 100. mu.L of water. A standard curve was prepared using 1mg/mL of bovine serum albumin as a standard protein solution.
The relative activities of salivary gland proteases in different feeding stages of the venorula are shown in fig. 4.
It can be seen that the relative ratio activity of the salivary gland protease is the lowest in the predation process of the rapana venosa, the relative ratio activity of the salivary gland protease is the highest when the rapana venosa is wrapped by the bivalve shellfish before the food intake.
Claims (1)
1. A purification method of a venuosus digestive protease is characterized by comprising the following steps:
a. the rapana venosa adult snail is artificially cultured for not less than 20 days by using a single variety of bivalve shellfish;
b. the whelk adult snail is fasted for 3-7 days, the fasting time is adjusted along with the water temperature, the water temperature is 21-25 ℃ and is fasted for 3 days, the water temperature is 18-20 ℃ and is fasted for 4 days, the water temperature is 14-17 ℃ and is fasted for 5 days, the water temperature is 10-13 ℃ and is fasted for 6 days, and the water temperature is 6-7 ℃ and is fasted for 7 days;
c. feeding the same variety of bivalve shellfish, and observing the predation process of the rapana venosa;
d. when the foot part is extended and wrapped by the rapana venosa, the rapana venosa is taken out to quickly break the shell of the rapana venosa;
e. rapidly dissecting the soft part of the venosa;
f. the salivary glands were removed and frozen at-20 ℃.
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080219970A1 (en) * | 1999-06-09 | 2008-09-11 | Henogen S.A. | Identification and molecular characterisation of proteins, expressed in the ixodes ricinus salivary glands |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080219970A1 (en) * | 1999-06-09 | 2008-09-11 | Henogen S.A. | Identification and molecular characterisation of proteins, expressed in the ixodes ricinus salivary glands |
Non-Patent Citations (2)
| Title |
|---|
| 周永灿等: "毛嵌线螺的研究Ⅱ.捕食行为与捕食习性", 《海洋学报(中文版)》 * |
| 王茜: "脉红螺唾液腺转录组部分未注释序列的功能预测", 《中国硕士学位论文全文数据库》 * |
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