WO2021077456A1 - Filter device and method used for microbial enzymatic activity testing - Google Patents

Abstract

The present invention provides a filter device and method used for microbial enzymatic activity testing; the device comprises a liquid supply mechanism used for supplying a liquid to be filtered, and a collection mechanism used for collecting the filtrate; the liquid supply mechanism comprises an upper connecting platform; the collection mechanism comprises a collection container; the collection container is provided with a lower connecting platform used for placing a filter membrane; the upper and lower connecting platforms may be detachably connected; at least one filtration passageway runs through the upper connecting platform and the lower connecting platform; the liquid to be filtered in the liquid supply mechanism passes through the filtration passageway and the filter membrane, and enters the liquid collection chamber of the collection mechanism, and the retentate is enriched on the filter membrane. The device of the present invention enriches cells by means of filtering; the amount of cell liquid required to be filtered is significantly reduced, the number of samples filtered at one time is significantly increased, and in turn, the substrate required for subsequent enzymatic activity tests is significantly reduced; the device and method achieve high-throughput filtration, operation is simple, and costs are low, facilitating promotion and application.

Description

Filtering device and method for microbial enzyme activity test Technical field

The invention relates to the technical field of microbial enzyme activity testing, in particular to a filtering device and method for microbial enzyme activity testing.

Background technique

Halogenated organic pollutants are highly persistent and difficult to biodegrade. They mainly include perfluorinated or partially fluorinated compounds (PFCs), chlorinated organic compounds (COCs) and brominated organic compounds (BOCs). Although organic halides are often used as raw materials, intermediates, solvents, etc. in organic synthesis, they play a significant role in human production and life. However, many organic halogenated substances are arbitrarily or inevitably discharged into the environment, causing serious harm to the ozone layer, ecological safety and human health.

The extensive use and discharge of halogenated organic matter has made the pollution of halogenated substances in water bodies increasingly serious, threatening ecological safety and human health. In addition, halogenated organic pollutants have the characteristics of environmental persistence, refractory biodegradability, bioaccumulation, high toxicity and long-distance migration ability. They are also distributed in the field environment such as soil and atmosphere. Therefore, how to effectively solve halogenated organic pollutants Pollutants have become the focus of attention in the environmental field. At present, the main methods for remediation of soil and groundwater contaminated by halogenated organic pollutants are physical remediation, chemical remediation and microbial remediation. The anaerobic microbial dehalogenation process has the advantages of no secondary pollution, lower cost, relatively physical and chemical dehalogenation methods, and higher environmental friendliness, making it one of the most potential in-situ remediation methods for environmental halogenated organic pollution. . The nature of the process of dehalogenation using anaerobic microorganisms is an enzymatic reaction catalyzed by dehalogenase, so a large number of experiments are needed to test the activity of dehalogenase.

Microbial in vitro enzyme activity experiment is one of the methods to study the catalytic reaction of microbial enzymes. It can test the catalytic activity of microbial enzymes on substrates in a short time, and study the functional characteristics of specific enzymes that catalyze the conversion of substrates; by adjusting the reaction conditions, Study the mechanism of microbial substrate utilization. Take the existing in vitro enzyme activity experiment of microbial cells as an example, as shown in Figure 1. Specifically, 1 liter of cell bacteria liquid is aliquoted into a centrifuge tube and centrifuged (10000×g, 5 minutes, 4°C), and the supernatant is removed. Make the remaining 3mL supernatant in each centrifuge tube, mix the precipitate, and perform a second centrifugation (15000×g, 5 minutes, 4°C), remove the supernatant, and repeat the cycle (usually more than 20 centrifugation is required). It takes about 1-2h or longer) until 1 liter of cell bacteria liquid is used up. Finally, the cell liquid in each centrifuge tube is combined, and the final volume is about 0.1mL, and the enzyme concentration is 100-150μg /mL of cell bacteria solution, and then react with 4mL of reaction solution for enzyme activity test. This method is cumbersome to operate, limited cells collected by centrifugation, time-consuming, low throughput, large test reaction system, and large amount of consumables. Therefore, it is necessary to develop an enzyme activity test technology with convenient operation, low cost and high cell recovery rate for further development. Study microbial enzymatic reactions.

The existing vacuum filtration device has an unreasonable structure design and cannot be used for cell separation in the experiment of in vitro enzyme activity of microorganisms.

Summary of the invention

The technical problem to be solved by the present invention is to overcome the defects and deficiencies of the prior art, and provide a filtering device and method for microbial enzyme activity test. The present invention overcomes the complicated operation of enzyme activity test in the existing microbial dehalogenation experiment. , The centrifugal collection of cells has the disadvantages of low recovery rate, long time consumption, low throughput, large test reaction system, and large consumption of consumables. Especially for dehalogenated bacteria with extremely low microbial growth, the present invention uses suction membrane filtration to filter Each glass fiber membrane enriched with cells reacts with a small amount of substrate (0.4mL), which can achieve a very small amount (≤10mL per tube) of cell fluid and rapid enrichment of different microbial extracellular enzymes at the same time to test the different substrates. Enzyme activity.

The above objectives of the present invention are achieved through the following technical solutions:

A filtering device for testing microbial enzyme activity, comprising a liquid supply mechanism for providing liquid to be filtered and a collection mechanism for collecting filtrate. The liquid supply mechanism includes an upper connection platform, and the collection mechanism includes a collection container, The collection container is provided with a lower connection platform for placing the filter membrane, the upper connection platform and the lower connection platform are detachably connected, and the upper connection platform and the lower connection platform are provided with at least one filter channel through them, and the supply The liquid to be filtered in the liquid mechanism passes through the filter channel and the filter membrane, and enters the liquid collection chamber of the collection mechanism, and the filter membrane is enriched with retentate. The number of filter channels can be designed according to needs. Compared with the existing centrifuge centrifugal separation method, the number of centrifuge tubes that can be placed during each centrifugation is limited. The filter device of the present invention overcomes the above-mentioned defects and has high throughput. The characteristics of filtration, the number of samples filtered each time is much larger than that of a centrifuge.

Optionally, the upper connecting platform is provided with a liquid supply chamber above the filter membrane, the lower connecting platform is provided with a lower channel located below the filter membrane, and the liquid supply chamber and the lower channel are combined The filtration channel is formed.

Optionally, a groove located outside the filter membrane is provided on the lower connecting platform, and a flange that can be inserted into the groove is provided on the upper connecting platform.

Optionally, a first sealing ring is provided in the groove, and the flange is inserted into the groove to compress the first sealing ring.

Optionally, it further includes a liquid supply container, the liquid supply container is provided with a liquid outlet, and the liquid outlet is inserted into the liquid supply cavity as shown.

Optionally, it further includes a second sealing ring, the upper connecting platform is provided with a limiting platform located at the lower part of the liquid supply chamber, and the limiting platform presses the second sealing ring against the edge of the filter membrane .

Optionally, at least one support platform for supporting the filter membrane is provided on the lower connecting platform, and the support platform is provided on the inner wall of the lower channel.

Optionally, the connection mode between the upper connection platform and the lower connection platform is adhesive or snap connection.

Optionally, a negative pressure device is communicated with the collection container.

Optionally, it further includes an engaging member, the edge of the lower connecting platform is provided with a downward lower boss, a lower clamping groove is formed between the lower boss and the side wall of the lower connecting platform, and the upper connecting platform The edge is provided with an upward upper boss, an upper card slot is formed between the upper boss and the side wall of the upper connection platform, and after the upper connection platform and the lower connection platform are attached, the upper boss, The shape formed by the combination of the lower boss matches the groove of the engaging piece, and the engaging piece is inserted from the sides of the upper boss and the lower boss to lock the upper boss and the lower boss .

The present invention also provides a method for testing microbial enzyme activity, which includes the following steps:

(1) Prepare a cell liquid containing the microorganisms to be detected, and use the above-mentioned filter device to suction and filter to obtain a filter membrane enriched with the cells to be detected;

(2) Providing a reaction solution containing a substrate, placing the filter membrane in the reaction solution, after the reaction is completed, detecting the ratio of the product to the substrate, and calculating the dehalogenation efficiency.

Optionally, in the step (1), the volume of the cell fluid sucked and filtered by each filter channel is 2-10 mL, and specifically can be 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, etc., It is preferably 3-9 mL, still preferably 3-7 mL, still preferably 4-7 mL, still preferably 4-6 mL, and still preferably 5 mL.

Optionally, in the step (1), the time required for suction filtration of the cell fluid is 10-60s, specifically 10s, 20s, 30s, 40s, 50s, 60s, etc., preferably 20-60s, and more preferably 20s. -50s, more preferably 20-40s, more preferably 30s.

Optionally, in the step (2), the ratio of the material peak area of the product to the substrate is calculated to obtain the dehalogenation efficiency.

Optionally, in the step (2), the product and the substrate are detected by a gas chromatography electron capture detector, and the ratio of the peak area of the substance is calculated, which is the dehalogenation efficiency.

Optionally, in the step (2), the substrate is selected from PCB-180, and the product is PCB-153.

Optionally, in the step (2), in the aqueous solution containing the substrate, the concentration of the substrate is 0.5 mg/L.

Optionally, in the step (2), each filter membrane is independently placed in 0.4 mL of the aqueous solution containing the substrate for reaction.

The present invention also provides applications of the above-mentioned device in microbial enzyme activity testing, cell collection, and cellular enzyme collection. The device can also be used to collect filtrate, preferably for microbial enzyme activity testing.

The present invention has the following beneficial effects:

Aiming at the disadvantages of the existing cell enrichment device and method, such as low efficiency, cumbersome operation, large consumption of consumables, etc., the present invention provides a small and efficient cell enrichment device. The device of the present invention enriches cells by filtration, and requires The amount of filtered cell sap is significantly reduced. Compared with the existing centrifuge, the number of single filtered samples of this device is significantly increased, which significantly reduces the substrate required for subsequent enzyme activity tests. The device and method achieve high throughput Filtration is simple and quick to operate, low in cost, significantly improves the enrichment efficiency, significantly reduces the volume of cell fluid required for filtration, and further reduces the volume of reaction solution required for subsequent enzyme activity tests, which is convenient for popularization and application.

Description of the drawings

Figure 1 shows a flow chart of the existing in vitro enzyme activity experiment of microbial cells.

Fig. 2 is a schematic diagram showing the structure of a filtering device according to an embodiment of the present invention.

Fig. 3 is a schematic diagram showing the cross-sectional structure of the upper connecting station in Fig. 2.

Fig. 4 is a schematic diagram showing the cross-sectional structure of the lower connecting platform in Fig. 2.

Fig. 5 is a schematic diagram showing a structure to be combined of a filtering device according to another embodiment of the present invention.

Fig. 6 is a schematic diagram showing the structure of the combined filtering device in Fig. 5.

Fig. 7 shows a schematic diagram of the filtering process using the filtering device in Fig. 6.

Fig. 8 shows a comparison diagram of the reaction efficiency of the dehalogenase pcbA1 after using the enzyme activity testing device and method of the present invention and the prior art after enriching cells.

Number description:

1—Collection agency

2—Collection container

21—Liquid chamber

3—Vent

4—The first sealing ring

5—groove

6—Filter membrane

7—liquid supply mechanism

8—Flange

9—Second sealing ring

10—liquid supply cavity

11—liquid supply container

110—Liquid outlet

12—down channel

13—support table

14—Get off the connection platform

15—Upper connecting station

16—Get off the card table

161-lower card slot

17—Upper card table

171—Upper card slot

18—Clamping pieces

19—Limiting table

Detailed ways

The present invention will be further described below with reference to the drawings and specific embodiments of the specification, but the embodiments do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.

Unless otherwise specified, the reagents and materials used in the following examples are all commercially available.

As shown in Figures 2 to 4, a filtering device for microbial enzyme activity testing is provided, which includes a liquid supply mechanism 7 for providing liquid to be filtered, a collection mechanism 1 for collecting filtrate, and the liquid supply mechanism 7 includes upper Connecting platform 15. The collection mechanism 1 includes a collection container 2. The collection container 2 is provided with a lower connecting platform 14 for placing the filter membrane 6. The upper connecting platform 15 and the lower connecting platform 14 can be detachably connected. The detachable connecting structure is convenient for filtering. On the other hand, it is convenient to separate the upper connection stage 15 and the lower connection stage 14 after the filtration is completed, and remove the filter membrane 6 from the lower connection stage 14 for microbial enzyme activity test. At least one filter channel runs through the connecting platform 14, and the number of filter channels can be any number, such as 1, 2, 3, 4, 5, 6, 7, 8, 9 Each filter membrane corresponds to a filter channel. The number of filter channels is determined according to the needs of the experiment. The liquid to be filtered in the liquid supply mechanism 7 passes through the filter channel and the filter membrane 6, and enters the collection of the collection mechanism 1. In the liquid chamber 21, the retentate is enriched on the filter membrane 6. When the filtered object is cell fluid, the cells are enriched on the filter membrane 6, which is the test substance.

In one embodiment, the upper connecting platform 15 is provided with a liquid supply chamber 10 located above the filter membrane 6, and the lower connecting platform 14 is provided with a lower channel 12 located below the filter membrane 6. In some embodiments, the lower channel 12 The diameter can be 10-12mm, specifically 10mm, 11mm, 12mm, etc., preferably 11mm. The liquid supply chamber 10 and the lower channel 12 combine to form a filter channel. The liquid supply chamber 10 is located above the filter membrane 6, under the action of a vacuum pump. , The liquid in the liquid supply container 11 passes through the liquid supply chamber 10, and after being filtered by the filter membrane 6, the filtrate enters the liquid collection chamber 21, and the filter membrane 6 is enriched with retentate. In some embodiments, the inner diameters of the liquid supply chamber 10 and the lower channel 12 can be kept the same, and the lower channel 12 is located directly below the liquid supply chamber 10 so that the cell fluid is fully filtered.

In one embodiment, the lower connecting platform 14 is provided with a groove 5 located outside the filter membrane 6, the upper connecting platform 15 is provided with a flange 8 that can be inserted into the groove 5, and the groove 5 is provided with a first sealing ring 4. The flange 8 is inserted into the groove 5 to compress the first sealing ring 4 so that the cell fluid on the filter membrane 6 is fully separated during filtration, and the cells are trapped on the filter membrane as much as possible, thereby improving the dehalogenation efficiency.

In one embodiment, it further includes a liquid supply container 11. The bottom of the liquid supply container 11 is provided with a liquid outlet 110, which is inserted into the liquid supply cavity 10, so that the liquid in the liquid supply container 11 smoothly flows into the liquid supply cavity. 10, so as to carry out the subsequent filtering step. The liquid supply container 11 may specifically be an existing syringe. The injection nozzle at the bottom is the liquid outlet 110. After the required amount of cell fluid is sucked by the piston handle, the injection nozzle at the bottom is inserted into the liquid supply chamber 10, and the vacuum pump is started. Can be filtered.

In one embodiment, it further includes a second sealing ring 9. The upper connecting platform 15 is provided with a limiting platform 19 located at the lower part of the liquid supply chamber 10, and the limiting platform 19 presses the second sealing ring 9 against the filter membrane 6 Edge, effectively enhance the airtightness of the device and improve filtration efficiency.

In some embodiments, the diameter of the liquid supply cavity 10 is 4-6 mm, and specifically may be 4 mm, 4.5 mm, 5 mm, 5.5 mm, 6 mm, etc.

In an embodiment, the lower connection platform 14 is provided with a support platform 13 for supporting the filter membrane 6. The support platform 13 may be specifically ring-shaped, and the center of the support platform 13 is hollowed out, so that the filtrate can enter the collection from the filter membrane 6.液室21。 Liquid chamber 21.

In one embodiment, the supporting table 13 is arranged on the inner wall of the lower channel 12, and the supporting surface of the supporting table 13 may be slightly lower than the upper surface of the lower connecting table 14, which is convenient for placing the filter membrane 6 and plays a good supporting role for the filter membrane 6 At the same time, it is convenient for the second sealing ring 9 to be pressed to the edge of the filter membrane 6 to avoid air leakage, so that the filtrate flows smoothly into the liquid collection chamber 21 through the lower channel 12.

As shown in Figure 2, the liquid supply chamber 10 can be cylindrical, with an inner diameter slightly larger than the outer diameter of the liquid outlet 110. The liquid outlet 110 of the liquid supply container 11 is inserted into the liquid supply chamber 10, the vacuum pump is turned on, and the vacuum pump passes through the vent hole. 3 The air in the liquid collection chamber 21 and the liquid supply chamber 10 connected to it is sucked away, so that the liquid in the liquid supply container 11 flows to the filter membrane 6 to achieve filtration.

As in one embodiment, as shown in Figures 5 and 6, the bottom of the liquid supply chamber 10 can be inverted cone shape. After the liquid to be filtered is injected into the liquid supply chamber 10, the vacuum pump is turned on, and the liquid supply chamber 10 is under the action of vacuum. The liquid in it flows to the filter membrane 6 to achieve filtration.

In an embodiment, the connection between the liquid supply mechanism 7 and the collection mechanism 1 is by bonding. Specifically, the upper connection platform 15 of the liquid supply mechanism 7 and the lower connection platform 14 of the collection mechanism 1 can be glued to each other. At the same time, a closed structure is formed, which is convenient for fully filtering the cell liquid. After the filtering is completed, the connection point can be pried to remove the upper connection platform 15 from the lower connection platform 14, and then take out the filter membrane for subsequent enzyme activity test.

In another embodiment, the connection between the liquid supply mechanism 7 and the collection mechanism 1 is a snap connection. Specifically, as shown in FIGS. 5 and 6, the edge of the lower connecting platform 14 is provided with a downward convex A lower card slot 161 is formed between the lower boss 16 and the side wall of the lower connecting platform 14, the edge of the upper connecting platform 15 is provided with an upward upper boss 17, and the upper boss 17 is on the side of the upper connecting platform 15 An upper clamping slot 171 is formed between the walls. The upper boss 17 and the lower boss 16 are connected by a clamping piece 18. The upper and lower bosses 17 and 16 are provided on the left and right sides of the collection container 2 to act as the upper connecting platform. 15. After the lower connecting platform 14 is attached, the upper boss 17 and the lower boss 16 are combined to form a T-shape, the engaging piece 18 has a T-shaped groove, and the lugs of the engaging piece 18 are inserted into the upper slot 171 and the lower card transversely. In the groove 161, the upper boss 17 and the lower boss 16 are locked, so that the upper connecting platform 15 and the lower connecting platform 14 are locked. The shape formed by the combination of the upper boss 17 and the lower boss 16 can also be other shapes. The shape of the engaging member 18 matches with it, so that the engaging member 18 can be inserted from the side, and the upper boss 17 and the lower boss 16 can be smoothly inserted. Lock tightly.

The collection container 2 is provided with a vent 3, and the vent 3 is connected to a negative pressure device. The negative pressure device may specifically be a vacuum pump. The vent 3 is usually higher than the highest liquid level in the collection container 2 to prevent liquid from being sucked into the pipe.

The vacuum pump can be purchased from the market, specifically it can be Jiangsu Danyang Danhao Electromechanical Equipment Co., Ltd., the model is OL90A. The installation process of the device is as follows: Place the first sealing ring 4 on the groove 5 of the lower connection platform 14 in turn, place the filter membrane 6 on the support platform 13, place the second sealing ring 9 on the filter membrane 6, and connect the upper connection platform 15 is buckled on the lower connecting platform 14 so that the flange 8 is locked into the groove 5, and the entire device is fixed with the external engaging member 18, so that the upper connecting platform 15 is further pressed on the lower connecting platform 14, and the negative pressure device is connected The air hole 3 is connected with a vacuum pump, and 3-5 mL of cell liquid is poured into the cell liquid container, namely the liquid supply chamber 10, for suction filtration, and the filtrate flows to the liquid collection chamber 21 of the filtrate collection container, and the cells are trapped on the filter membrane 6. Take out the external clamping piece 18 to separate the upper connection platform 15 and the lower connection platform 14, take out the filter membrane 6, put the filter membrane 6 enriched with bacteria into the reaction vessel, add the reaction solution, and test for appropriate enzyme activity. Enzyme-catalyzed reactions occur in the environment.

The material of the above-mentioned filtering device can be specifically PLA, that is, polylactic acid. Polylactic acid has good thermal stability, and the processing temperature is 170-230℃, and it has good solvent resistance. It can be processed in a variety of ways, such as extrusion and spinning. , Biaxial stretching, injection blow molding, etc. In addition to being biodegradable, products made of polylactic acid have good biocompatibility, gloss, transparency, hand feel and heat resistance, as well as certain bacteria resistance, flame retardancy and UV resistance.

Through experiments, it was found that only 5 mL of cell sap needs to be filtered, and the filtered solid reacts with a reaction solution with a substrate concentration of 0.5 mg/L and a volume of 0.4 mL, and 95.68% of the substrate can be converted into a product.

In the following examples, the obligate anaerobic organic halide respiratory bacteria Dehalococcoides mccartyi CG1 is selected for experiments. The characteristics of the other bacteria are similar to it, and the present invention can also be used for the enzyme activity test of other bacteria.

Example 1

In this example, the obligate anaerobic organic halide respiratory bacteria Dehalococcoides mccartyi CG1 was used to test the in vitro enzyme activity of the dehalogenase pcbA1. The bacterium was isolated and purified by our laboratory. You can refer to the literature: Genomic characterization of three unique Dehalococcoides that respire on persistent polychlorinated biphenyls. Authors: Shanquan Wang, Kern Rei Chng, Andreas Wilm, etc., DOI: 10.1073/pnas.1404845111.

This embodiment uses the high-throughput microbial in vitro enzyme activity experiment device shown in Figures 5-6. The operation process is shown in Figure 7. The lower end of the cell fluid container and the upper end of the filtrate collection container are detachably connected together by a snap mechanism. , The filter element (ie filter membrane) is installed between the cell fluid container and the filtrate collection container. The external fixing mechanism is fitted with the flanges at both ends of the cell fluid container and the filtrate collection container, and the buckle is disassembled by pushing and pulling; the cell fluid is injected into the cell fluid solution, and the connection port of the filtrate collector is connected to the negative pressure device to start the negative After pressing, the liquid will flow into the filtrate collector after being filtered by the filter element, and the cells will remain on the glass fiber; separate the cell fluid container from the filtrate collector, then the glass fiber membrane can be removed and put into the container containing the reaction solution , The glass fiber membrane is completely immersed, and incubated for 48 hours in a dark environment at 30°C. The filter element is a glass fiber filter membrane with a pore size of 0.22 μm and a diameter of 13 mm, purchased from Tianjin Keyilong Experimental Equipment Co., Ltd. The pore size of this filter membrane is determined according to the cell size in the experiment. If you filter other materials, you can freely choose other pore sizes. The diameter of the filter membrane is determined according to the design of the device. In this set of devices, the diameter of each filter membrane should be kept the same size to fit the device.

For the preparation method of the cell fluid, refer to the document "Genomic characterization of three unique Dehalococcoides that respire on persistent polychlorinated biphenyls" (Shanquan Wanga, Kern Rei Chng, Andreas Wilm, Siyan Zhao, Kun-Lin Yang, Nijan, Henan, China and Environmental Engineering and Chemical and Biomolecular Engineering, National University of Singapore, Singapore 117576; and Computational and Systems Biology, Genome Institute of Singapore, Singapore 138672; Edited by James M. Tiedje, Michigan, State approved, East University 13, 2014 (received for review March 15, 2014)), page 12108 "Materials and Methods" section.

The amount of cell fluid taken for each sample is 5mL, the maximum working pressure of the oil-free vacuum pump used is -92Kpa, and the pumping rate is 3.3L/s. It takes 30s to completely filter the cell fluid. There are 9 filtration channels on the filter device. Filter 9 samples at a time.

For the preparation method of the reaction solution containing the substrate, refer to the document "Genomic characterization of three unique Dehalococcoides that respire on persistent polychlorinated biphenyls" in "Supporting Information" (Wang et al. 10.1073/pnas.1404845111), see "Enzymatic Analysis" for details , The substrate concentration in the prepared reaction solution is 0.5mg/L, that is, 0.5ppm, and the concentration of other components is the same as the above reference, specifically containing 100mM Tris·HCl (pH 7.0), 20mM methylviologen, 15mM lemon Titanium(III) oxide.

The specific name of the dehalogenase pcbA1 can be found in the literature: "Genomic characterization of three unique Dehalococcoides that respire on persistent polychlorinated biphenyls".

The filter membrane does not need special treatment, just immerse the filter membrane completely in the reaction solution. The reaction substrate of the dehalogenase pcbA1 is 2,2′,3,4,4′,5,5′-heptachlorobiphenyl (I.e. PCB-180, also known as polychlorinated biphenyl-180), the amount of the reaction solution is 0.4mL, sealed at room temperature, after 48 hours of catalytic reaction, the product and the bottom are detected by GC-ECD (Gas Chromatography Electron Capture Detector) The dehalogenation efficiency is calculated. The reaction result is shown in Figure 8. PCB-153 (ie polychlorinated biphenyl-153) is the product, and the dehalogenation efficiency is 95.68% (the original data is the substance peak measured on the chromatograph Area, the dehalogenation efficiency is calculated from the ratio of the peak area of the product to the substrate). 2,2',3,4,4',5,5'-Heptachlorobiphenyl was purchased from Dr. Ehrenstorfer GmbH, catalog number: C20018000, Lot: G164639.

References for the calculation method of dehalogenation efficiency: Phylogenetically distinct bacteria involve extensive dechlorination of aroclor 1260 in sediment-free cultures. (Wang Shanquan; He Jianzhong; Department of Civil and Environmental Engineering; National; Singapore; University. of Singapore;

The publication information of the literature is as follows:

JOURNAL: PloS one;

DOI: 10.1371/journal.pone.0059178;

SOURCE: PubMed journal; DOI: 10.1371/journal.pone.0059178; YEAR: 2013; PAGES: e59178;

PAGES: e59178; PUBLISHER: PubMed.

Literature: "Genomic characterization of three unique Dehalococcoides that respire on persistent polychlorinated biphenyls" uses the existing centrifuge tube to separate the cell sap. From the figure on page 16 of this document, it can be seen in Fig.S4 (CG-1 2345-245-CB). Knowing the experimental results, according to the coordinate concentration estimation in the figure, the highest dehalogenation efficiency is about 78%. In the document, 2345-245-CB is PCB-153. The substrate, product, and calculation method of this embodiment are the same as those in the document, and the device used is different. The results show that the dehalogenation efficiency of this embodiment is as high as 95.68%, which is significantly higher than the 78% in the prior art.

From the centrifugal method in the literature "Genomic characterization of three unique Dehalococcoides that respire on persistent polychlorinated biphenyls", it can be found that in the existing microbial enzyme activity testing methods, centrifuge centrifugation is usually used. On the one hand, the centrifuge can be used every time The number of centrifuge tubes is limited. On the other hand, after each centrifugation, the cells enriched on the wall of the centrifuge tube are limited. On the other hand, each centrifugation takes 3-5 minutes. After removing the supernatant, Add the cell sap again, and perform multiple centrifugation in this cycle, which takes a long time. Even with repeated centrifugation, the final dehalogenation efficiency measured is still far lower than that of the present invention. It can be seen that increasing the number of centrifugation will result in a limited number of cells that can be enriched. The present invention successfully overcomes the above-mentioned defects. It does not need to be repeated, the enrichment efficiency is significantly higher than that of the prior art, and it has the characteristics of high throughput. Moreover, the volume of cell fluid required to obtain a sample by filtration is only about 2-10 mL. The time required is only 10-60s, and the required reaction solution volume is only 0.4mL. In the existing centrifugation method, the volume of cell fluid required to filter a sample is about 1L. It takes more than 20 centrifugation, which is time-consuming. In about 1-2 hours, the volume of the reaction solution is as high as 4 mL (the type and molar amount of the substrate in the reaction solution of Example 1 of the present invention are the same as those in the aforementioned literature).

In summary, it has at least the following beneficial effects:

(1) The device can set the number of filter channels as required, and the cell fluid containers are independent of each other and can be taken out at the same time, eliminating the need for repeated steps of operation one by one, making it more convenient to use;

(2) While maintaining a high cell recovery rate, the present invention significantly shortens the time required for cell enrichment, simplifies the operation steps, and reduces the amount of experimental consumables;

(3) The present invention greatly reduces the amount of cell fluid and reaction solution while maintaining high reaction efficiency;

(4) The buckle mechanism of the device includes flanges arranged at both ends of the cell fluid container and the filtrate collection container, and external clamping parts, which embed the cell fluid container and the filtrate collection container with the flanges at the two ends of the cell fluid container and the filtrate collection container. The buckle can be assembled and disassembled in a push-pull manner to achieve a close fit between the cell fluid container and the filtrate collector.

(5) The filter element structure of the device is a sealing ring-filter membrane-seal ring, the filter membrane is arranged between the two sealing rings, and the filter membrane is tightly sealed. The filter membrane is made of glass fiber membrane, etc. During the suction filtration process, The filter membrane is not easy to deform, and the cell fluid will not leak along the gap between the device and the membrane, thereby improving the cell recovery rate.

(6) The filtrate collection container of the device is provided with a supporting table for supporting the filter element, and the groove of the outer ring of the supporting table is provided with a sealing ring, which can ensure that there is no air leakage during the filtering process and the filtering effect is good.

(7) The material of the cell sap container and the filtrate collection container of this device can be PLA, that is, polylactic acid. Polylactic acid has good thermal stability, and the processing temperature is 170～230℃. It has good solvent resistance and can be used in a variety of ways. Processing methods, such as extrusion, spinning, biaxial stretching, injection blow molding. In addition to being biodegradable, products made of polylactic acid have good biocompatibility, gloss, transparency, hand feel and heat resistance, as well as certain bacteria resistance, flame retardancy and UV resistance.

(8) The cell-enriched filter membrane of this device can directly react with the reaction liquid containing the substrate.

(9) After the cell fluid passes through the filter membrane, all cells can be trapped on the filter membrane;

(10) The reaction system used in the device of the present invention is about 10 times smaller than the traditional reaction system.

The above-mentioned embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, etc. made without departing from the spirit and principle of the present invention Simplified, all should be equivalent replacement methods, and they are all included in the protection scope of the present invention.

Claims (10)

1. A filter device for testing microbial enzyme activity, which is characterized by comprising a liquid supply mechanism (7) for providing liquid to be filtered, a collection mechanism (1) for collecting filtrate, and the liquid supply mechanism (7) It includes an upper connection platform (15), the collection mechanism (1) includes a collection container (2), the collection container (2) is provided with a lower connection platform (14) for placing the filter membrane (6), and the upper The connecting platform (15) and the lower connecting platform (14) are detachably connected, and at least one filtering channel is penetrated through the upper connecting platform (15) and the lower connecting platform (14). The liquid to be filtered passes through the filter channel and the filter membrane (6), and enters the liquid collection chamber (21) of the collection mechanism (1), and the filter membrane (6) is enriched with retentate.
2. The filter device according to claim 1, characterized in that: the upper connection platform (15) is provided with a liquid supply chamber (10) located above the filter membrane (6), and the lower connection platform (14) A lower channel (12) located below the filter membrane (6) is provided on the upper side, and the liquid supply cavity (10) and the lower channel (12) are combined to form the filter channel.
3. The filter device according to claim 1, characterized in that: the lower connecting platform (14) is provided with a groove (5) located outside the filter membrane (6), and the upper connecting platform (15) is A flange (8) that can be inserted into the groove (5) is provided.
4. The filter device according to claim 3, characterized in that: the groove (5) is provided with a first sealing ring (4), the flange (8) is inserted into the groove (5), and the The first sealing ring (4) is compressed.
5. The filter device according to claim 2, characterized in that it further comprises a liquid supply container (10), the liquid supply container (11) is provided with a liquid outlet (110), and the liquid outlet (110) is inserted Shown in the liquid supply chamber (10).
6. The filter device according to claim 1, characterized in that it further comprises a second sealing ring (9), and the upper connection platform (15) is provided with a limit platform (19) located at the lower part of the liquid supply chamber (10) The limit table (19) presses the second sealing ring (9) against the edge of the filter membrane (6).
7. The filter device according to claim 2, characterized in that: at least one support platform (13) for supporting the filter membrane (6) is provided on the lower connection platform (14), and the support platform (13) is provided with On the inner wall of the lower channel (12).
8. The filter device according to claim 1, characterized in that: the connection between the upper connection platform (15) and the lower connection platform (14) is adhesive or snap connection, and the collection container (2) ) There is a negative pressure device connected.
9. The filter device according to claim 1, characterized in that it further comprises an engaging member (18), the edge of the lower connecting platform (14) is provided with a downward lower boss (16), the lower boss (16) A lower card slot (161) is formed between the side wall of the lower connecting platform (14), and the edge of the upper connecting platform (15) is provided with an upward upper boss (17), and the upper boss ( 17) An upper card slot (171) is formed between the side wall of the upper connecting platform (15), and after the upper connecting platform (15) and the lower connecting platform (14) are attached, the upper boss (17) ), the lower boss (16) is combined into a shape that matches the groove of the engaging member (18), and the engaging member (18) moves from the upper boss (17), the lower boss (16) ), and lock the upper boss (17) and lower boss (16) tightly.
10. A method for testing microbial enzyme activity, which is characterized in that it comprises the following steps:

    (1) Prepare a cell liquid containing the microorganisms to be detected, and perform suction filtration with the filtering device according to any one of claims 1-9 to obtain a filter membrane enriched with the cells to be detected;

    (2) Providing a reaction solution containing a substrate, placing the filter membrane in the reaction solution, after the reaction is completed, detecting the ratio of the product to the substrate, and calculating the dehalogenation efficiency.

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