Device for deeply concentrating bacillus subtilis plasmin by refrigerating and thawing method

Abstract

The deep concentration device comprises a shady and cool box and a box body, the box body is arranged in the shady and cool box, a containing cavity is formed in the box body, an opening is formed in the top of the box body, a sealing cover is hinged to the opening, a buckle is arranged at the free end of the sealing cover, a lock catch meshed with the buckle is arranged at the opening of the box body, and the box body is arranged in the containing cavity. A scale is arranged on the outer edge face of the box body, and a clamping edge is arranged on the inner side face of the box body. After a bacillus subtilis plasmin fermentation concentrated solution filled in a plastic bag is frozen, the solid and the plastic bag are wrapped together and placed on the partition plate of the device, a plurality of small holes are cut in the bottom of the plastic bag, the refrigeration temperature is regulated, the liquid scale below the partition plate is recorded, and refrigeration thawing is completed. Or the plastic bag is integrally removed, the plastic bag is placed on a partition plate to complete defrosting through a refrigeration method, the high-concentration part is firstly melted, the whole still keeps the ice appearance, the part which is firstly melted is independently leached and collected, and then the part which is not melted is concentrated and abandoned.

Description

Technical Field

[0001] The utility model relates to the technical field of enzyme preparation concentration, and more specifically to a Bacillus subtilis fibrinolytic enzyme deep concentration device using a refrigeration and thawing method. Background Art

[0002] After the Bacillus subtilis fibrinolytic enzyme fermentation broth is centrifuged to remove the bacteria, hollow fiber column ultrafiltration is generally used to further remove small molecules and a large amount of water.

[0003] The working principle of ultrafiltration membrane is a membrane separation process based on screening process and pressure as the driving force. Under a certain pressure, when the fermentation liquid flows through the membrane surface, the many tiny micropores densely distributed on the surface of the ultrafiltration membrane only allow water and small molecules to pass through and become permeate, while the substances in the fermentation liquid with a volume larger than the micropore diameter of the membrane surface are retained on the liquid inlet side of the membrane and become concentrated liquid. During the ultrafiltration process, when the concentration of Bacillus subtilis fibrinolytic enzyme on the membrane surface reaches their saturation concentration, the viscosity of the fermentation liquid increases, and a sediment layer or gel layer will be formed on the membrane surface, increasing the permeation resistance and causing serious concentration polarization. As the volume of the fermentation liquid increases with the workload, the filtration speed slows down. Even if it is diluted and then ultrafiltered, the working time will be extended. Therefore, a hollow fiber column that is too small will not be selected.

[0004] Since the volume of the hollow fiber column will not decrease and it has many pores and dead volume, the volume of the fermentation concentrate eluted after ultrafiltration of Bacillus subtilis plasmin macromolecules is the fixed volume and cannot be smaller, resulting in the ratio of the volume of the fermentation concentrate to the volume of the fermentation liquid cannot be reduced any further.

[0005] In short, the ultrafiltration concentration multiple, that is, the ratio of the volume of fermentation broth to the volume of fermentation concentrate, cannot be increased any further, resulting in the inability to increase the enzyme activity of the fermentation concentrate, which restricts the efficiency of subsequent chromatographic protein purification.

[0006] Therefore, it is necessary to select a gentle method that does not inactivate the enzyme to further concentrate the Bacillus subtilis fibrinolytic enzyme. Utility Model Content

[0007] The utility model provides a deep concentration device for Bacillus subtilis fibrinolytic enzyme by refrigeration thawing method, which has high total recovery rate, reduces the volume of recovered concentrated liquid, solves the problem of reduced concentration efficiency in the late stage of ultrafiltration concentration and achieves the purpose of deep concentration.

[0008] According to one aspect of the utility model, a deep concentration device for Bacillus subtilis fibrinolytic enzyme by refrigerated thawing method is provided, comprising a cool box and a box body, wherein the box body is installed in the cool box, wherein a accommodating cavity is arranged in the box body and the top is open, and a sealing cover is hinged at the opening, a buckle is arranged at the free end of the sealing cover, and a lock buckle engaged with the buckle is arranged at the opening of the box body, a ruler is arranged on the outer edge surface of the box body, a card edge is arranged on the inner side surface of the box body, and a partition for carrying Bacillus subtilis fibrinolytic enzyme is arranged on the card edge.

[0009] Preferably, based on the above scheme, the temperature in the cool box is 4°.

[0010] Preferably, based on the above solution, the cool box has a built-in temperature sensor.

[0011] Preferably, based on the above solution, the partition has a mesh structure.

[0012] Preferably, based on the above scheme, the length of the box is 60 cm and the width is 50 cm.

[0013] Preferably, based on the above solution, square holes with a width of 2 cm are arranged on the surface of the partition.

[0014] Preferably, based on the above solution, the box is transparent.

[0015] The utility model discloses a deep concentration device for Bacillus subtilis fibrinolytic enzyme by refrigeration thawing method. During operation, after the fermentation concentrate of Bacillus subtilis fibrinolytic enzyme is frozen in a plastic bag, the solid is placed on the partition of the device together with the plastic bag, several small holes are cut in the bottom of the plastic bag, the refrigeration temperature is adjusted, and the liquid scale under the partition is recorded to complete the thawing by refrigeration method. Alternatively, the plastic bag is removed as a whole and placed on the partition to complete the thawing by refrigeration method. The high-concentration part melts first, and the whole still maintains the shape of ice. The first thawed part is leached and collected separately, and the concentration is completed, and the unthawed part is discarded.

[0016] After testing, it was found that the enzyme activity of the part that was thawed first was extremely high, while the enzyme activity of the unthawed part was extremely low after being separated and thawed separately for testing. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the technical solutions of the embodiments of the present utility model, the following is a brief introduction to the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present utility model. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work. In the drawings:

[0018] Figure 1 It is a three-dimensional schematic diagram of a deep concentration device of Bacillus subtilis fibrinolytic enzyme by refrigeration and thawing method of the utility model;

[0019] Figure 2 It is a three-dimensional diagram of the box body of the utility model;

[0020] Figure 3 It is a cross-sectional view of the box body of the utility model;

[0021] Description of Figure Numbers:

[0022] The cool box 1, the box body 2, the accommodating cavity 21, the opening 22, the sealing cover 23, the buckle 24, the lock buckle 25, the scale 26, the card edge 27, and the partition 28. DETAILED DESCRIPTION

[0023] The following is a further detailed description of the specific implementation of the present invention in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0024] It should be understood that when used in this specification and the appended claims, the term "comprising" indicates the presence of described features, integers, steps, operations, elements and / or components, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or collections.

[0025] In order to simplify the drawings, only the parts related to the utility model are schematically shown in each figure, and they do not represent the actual structure of the product. In addition, in order to simplify the drawings and facilitate understanding, in some figures, only one of the parts with the same structure or function is schematically drawn or marked. In this article, "one" not only means "only one", but also means "more than one".

[0026] It should be further understood that the term “and / or” used in the specification and appended claims refers to any combination and all possible combinations of one or more of the associated listed items, and includes these combinations.

[0027] In the embodiments shown in the drawings, the directions (such as up, down, left, right, front and back) used to explain the structure and movement of the various components of the present invention are not absolute but relative. These descriptions are appropriate when the components are in the positions shown in the drawings. If the description of the positions of the components changes, the directions also change accordingly.

[0028] In addition, in the description of the present application, the terms "first", "second", etc. are only used to distinguish the description and cannot be understood as indicating or implying relative importance.

[0029] In order to more clearly illustrate the embodiments of the utility model or the technical solutions in the prior art, the specific implementation methods of the utility model will be described below with reference to the accompanying drawings. Obviously, the accompanying drawings described below are only some embodiments of the utility model. For ordinary technicians in this field, other drawings and other implementation methods can be obtained based on these drawings without creative work.

[0030] See also Figure 1 , and combined with Figure 2 and Figure 3 As shown, a deep concentration device of Bacillus subtilis fibrinolytic enzyme by refrigerated thawing method of the utility model comprises a cool box 1 and a box body 2, wherein the box body 2 is installed in the cool box 1, a accommodating cavity 21 is arranged in the box body 2 and a top opening 22, and a sealing cover 23 is hinged at the opening 22, a buckle 24 is arranged at the free end of the sealing cover 23, and a lock buckle 25 engaged with the buckle 24 is arranged at the opening 22 of the box body 2, a scale 26 is arranged on the outer edge surface of the box body 2, a card edge 27 is arranged on the inner side surface of the box body 2, and a partition 28 for carrying Bacillus subtilis fibrinolytic enzyme is arranged on the card edge 27, wherein the temperature in the cool box 1 is 4°, the length of the box body 2 is 60 cm, the width is 50 cm, the surface of the partition 28 is arranged with a square hole with a width of 2 cm, the box body 2 is transparent, and preferably, the cool box 1 has a built-in temperature sensor, and the partition 28 has a mesh structure.

[0031] The principle of the refrigerated thawing method of the utility model is: the fermentation substance and ice form a eutectic. During the solidification process of the liquid fermentation liquid, the middle solidifies first to become an ice cube, while the surrounding solution concentrates until it reaches the eutectic point, and the remaining fermentation concentrate begins to solidify (the temperature of the solution remains unchanged at this time). It can be seen that the ice cube in the middle is white, and the solid part of the surrounding fermentation liquid is dark. The melting process is the opposite. At a certain fixed temperature below 0 degrees Celsius, the fermentation substance and a part of the ice in the solid phase are liquefied, and the temperature remains unchanged; the dark fermentation liquid melts and flows out of the ice body, and the ice skeleton remains completely white. When only ice is left in the solid phase, the temperature resumes and rises until it is completely liquefied. Refrigerated thawing can maintain the approximate shape of the ice cube, which cannot be achieved by thawing at room temperature, and most of the ice cube has also melted.

[0032] In order to verify the effect of the utility model, it is necessary to use an enzyme activity determination method to test the activity of Bacillus subtilis fibrinolytic enzyme. The enzyme activity determination method of the utility model is operated as follows: the determination method of Bacillus subtilis fibrinolytic enzyme activity refers to the Astrup method to prepare agarose fibrin plate, and different concentrations of urokinase standard (20, 40, 80, 160, 620, 640 IU / mL) are spotted on the newly prepared plate, placed at 37°C for 18h, and the vertical diameter of the fibrinolytic circle is determined. The area of ​​the fibrinolytic circle is calculated to prepare a standard curve. Take the separated Bacillus subtilis fibrinolytic enzyme and spot the fibrin plate, place it at 37°C for 18h, calculate the area of ​​the fibrinolytic circle, and use the urokinase standard curve to obtain the corresponding enzyme activity.

[0033] During use, 50 liters of fermentation tanks, harvest 30 liters of Bacillus subtilis fibrinolytic enzyme fermentation liquid, remove bacteria through tubular centrifuge, and then concentrate through hollow fiber column ultrafiltration. After ultrafiltration, the minimum volume is fixed at about 3.0 liters. Take 3 liters of concentrated solution, and it is 28319IU / mL to measure the enzyme activity. Use 30*40 cm ziplock bags for bagging, put them in the refrigerator freezer and freeze them to a solid state, take them out, cut the ziplock bags, put them on the utility model partition 28, move the casing 21 into the refrigerated environment 6, and the temperature of the cool box 1 is set at 4-8 degrees, and the humidity is 40-60%. Along with the extension of the low-temperature thawing time, brown liquid gradually flows out from the bottom of the ice cube until the ice cube all turns white, and the brown liquid flowing out contains fibrinolytic enzyme. The unthawed ice cube is white and transparent, and the upper white ice cube is taken out and thawed at room temperature separately, and the volume is 1.1L, and the detection enzyme activity is 1422IU / mL. The volume of the thawed brown liquid was 1.9 L, and the enzyme activity was 42316 IU / mL.

[0034] The results showed that: recovery rate, total enzyme activity of lower fermentation broth (enzyme activity multiplied by volume) / total enzyme activity of mixed liquid = 98.1%; volume reduction multiple, mixed liquid volume / lower fermentation broth volume = 1.58; enzyme activity increase multiple, lower fermentation broth enzyme activity / mixed liquid enzyme activity = 1.49.

[0035] The utility model discloses a deep concentration device for Bacillus subtilis fibrinolytic enzyme by refrigeration thawing method. When in operation, after the fermentation concentrate of Bacillus subtilis fibrinolytic enzyme is frozen in a plastic bag, the solid is placed on the partition 28 of the device together with the plastic bag, several small holes are cut in the bottom of the plastic bag, the refrigeration temperature is adjusted, and the liquid scale below the partition 28 is recorded to complete the refrigeration thawing, or the plastic bag is removed as a whole and placed on the partition 28 to complete the refrigeration thawing, the high-concentration part is melted first, and the whole still keeps the ice shape, the first thawed part is separately leached and collected, that is, the concentration is completed, and the unthawed part is discarded.

[0036] After testing, it was found that the enzyme activity of the part that was thawed first was extremely high, while the enzyme activity of the unthawed part was extremely low after being separated and thawed separately for testing.

[0037] Finally, the method of this application is only a preferred implementation scheme and is not intended to limit the protection scope of the utility model. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the utility model should be included in the protection scope of the utility model.

Claims

1. A deep concentration device for Bacillus subtilis fibrinolytic enzyme by refrigeration and thawing method, characterized in that: The invention comprises a cool box and a box body, wherein the box body is installed in the cool box, wherein a accommodating cavity is arranged inside the box body and the top is opened, and a sealing cover is hinged at the opening, a buckle is arranged at the free end of the sealing cover, and a lock buckle engaged with the buckle is arranged at the opening of the box body, a ruler is arranged on the outer edge surface of the box body, a card edge is arranged on the inner side surface of the box body, and a partition for carrying Bacillus subtilis fibrinolytic enzyme is arranged on the card edge.

2. The deep concentration device of Bacillus subtilis fibrinolytic enzyme by refrigeration and thawing method as claimed in claim 1, characterized in that: The temperature in the cool box is 4°.

3. The deep concentration device of Bacillus subtilis fibrinolytic enzyme by refrigeration and thawing method as claimed in claim 1, characterized in that: The cool box has a built-in temperature sensor.

4. The deep concentration device of Bacillus subtilis fibrinolytic enzyme by refrigeration and thawing method as claimed in claim 1, characterized in that: The partition is in a mesh structure.

5. The deep concentration device of Bacillus subtilis fibrinolytic enzyme by refrigeration and thawing method as claimed in claim 1, characterized in that: The length of the box is 60 centimeters and the width is 50 centimeters.

6. The deep concentration device of Bacillus subtilis fibrinolytic enzyme by refrigeration and thawing method as claimed in claim 5, characterized in that: The surface of the partition is provided with square holes with a hole width of 2 cm.

7. The deep concentration device of Bacillus subtilis fibrinolytic enzyme by refrigeration and thawing method as claimed in claim 5, characterized in that: The box body is transparent.

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