Microbial extracellular enzyme full-sea depth fidelity enzymology assay device

Abstract

The present invention discloses a full-sea-depth fidelity enzymology assay device for microbial extracellular enzymes, including a pressure-maintaining sampling bottle, a pressure-maintaining transfer device, a pressure-maintaining enzymology reactor, a heat-insulating device and an enzyme activity detection device. The pressure-maintaining enzymology reactor includes a cylinder, a plug, a PTFE gasket, an O-ring, a piston, a high-pressure straight-through valve and a high-pressure interface. The pressure-maintaining enzymology reactor is a closed barrel, a piston is provided inside, and plugs and valves are provided at both ends, and the valve is connected to the pressure-maintaining transfer device with a high-pressure interface. The present invention can realize full-sea-depth (0-11000 meters) sample pressure-maintaining sampling and transfer; sample collection, transfer and enzymology reactions can be carried out under in-situ pressure and temperature conditions. The pressure-maintaining enzymology reactor involved in the present invention has a simple, convenient and reliable structure, can be quickly connected with a pressure-maintaining transfer device and a pressure-maintaining sampling bottle, and has been successfully tested at sea, providing a reliable device for carrying out full-sea-depth microbial extracellular enzyme pressure-maintaining enzyme activity assay, and has good application prospects.

Description

Technical Field

[0001] The invention relates to a fidelity enzymology assay device, in particular to a full-sea-depth fidelity enzymology assay device for microbial extracellular enzymes. Background Art

[0002] The ocean occupies more than 70% of the earth's surface area, of which the hadal area at a depth of 6,000 meters accounts for 45% of the vertical depth of the ocean, with extreme environmental characteristics such as low temperature, high pressure, and oligotrophy. Due to the limitations of survey and detection technology, the hadal is the most difficult environment for humans to reach on earth, and it is also a virgin land for resource discovery and development. Due to the extreme environmental characteristics, hadal microorganisms have special metabolic and physiological adaptation mechanisms compared to the terrestrial or shallow-sea organisms we are familiar with, and have special genes, enzymes and other resources.

[0003] How to obtain in-situ fidelity abyssal samples and how to realize the study of microbial life processes under in-situ conditions are key links to help us analyze the microbial community structure, enzyme activity, life processes and dynamic response of microorganisms to environmental changes in the marine environment. Among them, the device that accurately measures the type and activity of marine microbial extracellular enzymes is an indispensable tool for studying the life processes of marine microorganisms and developing and utilizing microbial enzymology and protein resources. In the past, traditional methods usually measured the extracellular enzyme activity of microorganisms at different depths in the ocean under normal temperature and pressure conditions. The data obtained were not measured under in-situ conditions and could not accurately reflect the actual in-situ conditions, which severely limited our ability to capture, understand and respond to microbial life processes, enzyme activity and dynamic response of microorganisms to environmental changes in the marine environment. It is urgent to invent a fidelity reactor that can measure the extracellular enzyme activity of microorganisms at full sea depth. Summary of the invention

[0004] The purpose of the present invention is to provide a microbial extracellular enzyme fidelity enzymology determination device applicable to the entire ocean depth of 0 to 11,000 meters.

[0005] The present invention is achieved through the following technical solutions:

[0006] A full-sea-depth fidelity enzymological assay device for microbial extracellular enzymes comprises a pressure-maintaining sampling bottle, a pressure-maintaining transfer device, a pressure-maintaining enzymological reactor, a heat-insulating device and an enzyme activity detection device. The pressure-maintaining enzymological reactor is in the shape of a closed barrel and comprises a cylinder, a plug, a polyfluoro gasket, an O-ring, a piston, a high-pressure through valve, a high-pressure interface, an overflow hole and a wrench hole. The cylinder is a container with openings at both ends. A piston is arranged inside the cylinder. Plugs and valves are arranged at both ends of the cylinder. The valve is connected to the pressure-maintaining transfer device using a high-pressure interface. The plug and the piston are both provided with sealing rings for sealing.

[0007] Preferably, the cylinder, plug and piston are all made of titanium alloy TC4, which can minimize the impact on microorganisms.

[0008] Preferably, the high-pressure through valve is made of AISI 304 stainless steel.

[0009] Preferably, the PTFE gasket is made of polytetrafluoroethylene.

[0010] Preferably, the O-ring is made of fluororubber.

[0011] Preferably, the sealing ring is composed of a polytetrafluoroethylene gasket and an O-ring.

[0012] Preferably, both open ends of the cylinder are provided with internal threads, and the plug is provided with external threads, and the two are connected by threads.

[0013] Preferably, an external thread is provided on the outside of the plug, and internal threads are provided on both sides of the high-pressure through valve, and the two are connected by threads.

[0014] Preferably, overflow holes are provided at both open ends of the cylinder, and the overflow holes are located outside the corresponding polytetrafluoroethylene gasket and O-ring on the plug.

[0015] Preferably, a wrench hole is provided on the outer side of the plug.

[0016] Beneficial effects: The pressure-maintaining enzymology reactor of the present invention can be quickly connected with the pressure-maintaining transfer equipment and the pressure-maintaining sampling bottle through the high-pressure interface. The pressure-maintaining enzymology reaction of microbial extracellular enzymes can be carried out by pre-placing the enzymology reaction fluorescent substrate in the pressure-maintaining enzymology reactor and placing it in a constant temperature water bath, that is, sample collection, transfer and enzymology reaction are all carried out under in-situ pressure and temperature conditions. The device has important scientific significance and good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a schematic diagram of the overall appearance and structure of the pressure-maintaining enzymatic reactor.

[0018] Figure 2 Schematic diagram of the cross-sectional structure of a pressure-maintaining enzymatic reactor.

[0019] Figure 3 Schematic diagram of the principle of transferring the in-situ high-pressure sample to the pressure-maintaining reactor.

[0020] Figure 4 Schematic diagram of the principle of sampling and measuring the pressurized enzymatic reactor.

[0021] The symbols in the figure represent:

[0022] 1. Pressure-maintaining enzymology reactor; 2. Pressure-maintaining sampling bottle; 3. Pressure-maintaining transfer equipment; 4. Waste liquid collection device; 5. Insulation equipment and enzyme activity detection equipment; 11. Cylinder; 12. Plug; 13. Piston; 14. High-pressure straight-through valve; 15. Sealing ring. DETAILED DESCRIPTION

[0023] The following is a detailed description of an embodiment of the present invention in conjunction with the accompanying drawings: This embodiment is implemented on the premise of the technical solution of the present invention, and a detailed implementation method and a specific operation process are given, but the protection scope of the present invention is not limited to the following embodiments.

[0024] Reference Figure 1-4 The full-sea-depth fidelity enzymological assay device for microbial extracellular enzymes of the present invention comprises a pressure-maintaining sampling bottle 2, a pressure-maintaining transfer device 3, a pressure-maintaining enzymological reactor 1, a heat preservation device and an enzyme activity detection device 5. The pressure-maintaining enzymological reactor comprises a cylinder 11, two plugs 12, a piston 13, two high-pressure through valves 14 and six pairs of sealing rings 15 composed of polytetrafluoroethylene gaskets and O-rings.

[0025] like Figure 3 , 4 As shown, when the present invention is working, firstly, the enzymatic reaction fluorescent substrate is preset in the C chamber of the pressure-maintaining enzymatic reactor 1, and the D chamber of the pressure-maintaining enzymatic reactor 1 is filled with sterile tap water (in-situ pressure); the pressure-maintaining transfer device 3 injects ddH2O into the A chamber of the pressure-maintaining sampling bottle at in-situ pressure, and the pressure-maintaining seawater sample ( Figure 2 , Chamber B) is transferred to Chamber C of the pressure-maintaining enzymology reactor 1 at the in-situ pressure, at which time the enzymology reaction fluorescent substrate is mixed with the pressure-maintaining seawater, and the enzymology reaction begins. Close the high-pressure through valves at both ends, and place the pressure-maintaining enzymology reactor 1 in an in-situ temperature water bath for heat preservation and pressure incubation.

[0026] When the enzymatic reaction reaches the predetermined time, the sample is transferred from the pressure-maintaining transfer device 3 to the heat-maintaining device and enzyme activity detection device 5 for fluorescence measurement and enzyme activity calculation. Figure 3 ) The sample pressure remains unchanged (in-situ pressure), and the fluorescence measurement of the samples in the heat preservation equipment and the enzyme activity detection equipment 5 is carried out under normal pressure conditions.

[0027] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. A full-sea-depth fidelity enzymological assay device for microbial extracellular enzymes, characterized in that: It includes a pressure-maintaining sampling bottle, a pressure-maintaining transfer device, a pressure-maintaining enzymology reactor, a heat preservation device and an enzyme activity detection device. The pressure-maintaining enzymology reactor is a closed barrel. The pressure-maintaining enzymology reactor includes a cylinder, a plug, a polytetrafluoroethylene gasket, an O-ring, a piston, a high-pressure straight-through valve, a high-pressure interface, an overflow hole and a wrench hole. The cylinder is a container with two ends open. A piston is arranged inside the cylinder. Both ends of the cylinder are provided with a plug and a valve. The valve is connected to the pressure-maintaining transfer device using a high-pressure interface. The plug and the piston are provided with sealing rings. The cylinder, plug and piston are all made of titanium alloy TC4; The high-pressure straight-through valve is made of stainless steel AISI 304; The PTFE gasket is made of polytetrafluoroethylene; The O-ring is made of fluororubber; The sealing ring is composed of a PTFE gasket and an O-ring; Both open ends of the cylinder are provided with internal threads, and the plug is provided with external threads, and the two are connected by threads; The plug is provided with an external thread on the outside, and the high-pressure through valve is provided with internal threads on both sides, and the two are connected by threads; Both open ends of the cylinder are provided with overflow holes, and the overflow holes are located outside the corresponding PTFE gasket and O-ring on the plug; A wrench hole is provided on the outer side of the plug; When the device is in operation, firstly, an enzymatic reaction fluorescent substrate is pre-placed in the C cavity of the pressure-maintaining enzymatic reactor, and the D cavity of the pressure-maintaining enzymatic reactor is filled with sterile tap water; ddH2O is injected into the A cavity of the pressure-maintaining sampling bottle by the pressure-maintaining transfer device at the in-situ pressure, and the pressure-maintaining seawater sample is transferred to the C cavity of the pressure-maintaining enzymatic reactor at the in-situ pressure, at which time the enzymatic reaction fluorescent substrate is mixed with the pressure-maintaining seawater, and the enzymatic reaction starts, and the high-pressure straight-through valves at both ends are closed, and the pressure-maintaining enzymatic reactor is placed in an in-situ temperature water bath for heat preservation and pressure-maintaining incubation; When the enzymatic reaction reaches the predetermined time, the pressure-maintaining transfer device transfers the sample to the heat preservation device and enzyme activity detection device for fluorescence measurement and enzyme activity calculation. During this process, the sample pressure in cavity C remains unchanged, and the samples in the heat preservation device and enzyme activity detection device are subjected to fluorescence measurement under normal pressure conditions.

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