CN109490274B - Experimental device for researching unidirectional mass transfer of enzyme in leather and application method - Google Patents
Experimental device for researching unidirectional mass transfer of enzyme in leather and application method Download PDFInfo
- Publication number
- CN109490274B CN109490274B CN201910007178.1A CN201910007178A CN109490274B CN 109490274 B CN109490274 B CN 109490274B CN 201910007178 A CN201910007178 A CN 201910007178A CN 109490274 B CN109490274 B CN 109490274B
- Authority
- CN
- China
- Prior art keywords
- liquid storage
- storage tank
- leather
- clamp
- enzyme
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000010985 leather Substances 0.000 title claims abstract description 119
- 238000012546 transfer Methods 0.000 title claims abstract description 47
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 41
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 41
- 238000000034 method Methods 0.000 title claims description 21
- 239000007788 liquid Substances 0.000 claims abstract description 169
- 238000003860 storage Methods 0.000 claims abstract description 165
- 239000000243 solution Substances 0.000 claims abstract description 26
- 239000007853 buffer solution Substances 0.000 claims abstract description 23
- 238000002474 experimental method Methods 0.000 claims description 18
- 238000009826 distribution Methods 0.000 claims description 9
- 239000000463 material Substances 0.000 claims description 8
- 241001465754 Metazoa Species 0.000 claims description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims description 7
- 239000000741 silica gel Substances 0.000 claims description 7
- 229910002027 silica gel Inorganic materials 0.000 claims description 7
- 229940088598 enzyme Drugs 0.000 description 34
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 30
- 102000004142 Trypsin Human genes 0.000 description 20
- 108090000631 Trypsin Proteins 0.000 description 20
- 239000012588 trypsin Substances 0.000 description 20
- 238000005520 cutting process Methods 0.000 description 11
- 239000003550 marker Substances 0.000 description 11
- 108090000145 Bacillolysin Proteins 0.000 description 8
- 102000035092 Neutral proteases Human genes 0.000 description 8
- 108091005507 Neutral proteases Proteins 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 229920005654 Sephadex Polymers 0.000 description 6
- 239000012507 Sephadex™ Substances 0.000 description 6
- 238000002073 fluorescence micrograph Methods 0.000 description 6
- 238000004108 freeze drying Methods 0.000 description 6
- 238000005227 gel permeation chromatography Methods 0.000 description 6
- 238000004519 manufacturing process Methods 0.000 description 6
- 239000008055 phosphate buffer solution Substances 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 241000283707 Capra Species 0.000 description 5
- ZMZDMBWJUHKJPS-UHFFFAOYSA-M Thiocyanate anion Chemical compound [S-]C#N ZMZDMBWJUHKJPS-UHFFFAOYSA-M 0.000 description 5
- 238000007796 conventional method Methods 0.000 description 5
- 239000000945 filler Substances 0.000 description 5
- GNBHRKFJIUUOQI-UHFFFAOYSA-N fluorescein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 GNBHRKFJIUUOQI-UHFFFAOYSA-N 0.000 description 5
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N hydrogen thiocyanate Natural products SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 4
- 238000011049 filling Methods 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000010586 diagram Methods 0.000 description 3
- 230000005284 excitation Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 229940079919 digestives enzyme preparation Drugs 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 230000035515 penetration Effects 0.000 description 2
- -1 rhodamine B isothiocyanate Chemical class 0.000 description 2
- 229940043267 rhodamine b Drugs 0.000 description 2
- 238000005070 sampling Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 238000009434 installation Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 239000008176 lyophilized powder Substances 0.000 description 1
- 238000012856 packing Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
Landscapes
- Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Optics & Photonics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention provides an experimental device for researching unidirectional mass transfer of enzyme in leather, which comprises an upper liquid storage tank, a lower liquid storage tank, a base, a horizontal bubble, an adjustable F clamp, a base adjusting bolt and a lower clamp adjusting screw, wherein a leather sample is placed between the upper liquid storage tank and the lower liquid storage tank and is fixed on the upper liquid storage tank through the adjustable F clamp lower clamp and the lower clamp adjusting screw, the lower liquid storage tank is used for containing experimental buffer solution, and the upper liquid storage tank is used for containing solution containing enzyme subjected to fluorescent marking.
Description
Technical Field
The invention belongs to the field of application research of tanning enzyme preparations, and particularly relates to an experimental device for researching unidirectional mass transfer of enzyme in leather and a use method thereof.
Background
The leather industry is an extension of animal husbandry, is an important link of recycling economy, and occupies an important position in the industrial economy of China. With the increasing importance of the national environmental protection, clean tanning technology based on biological enzymes is rapidly developed and widely applied. Unlike the usual enzymatic reactions, animal skin, the catalytic substrate of enzymes in leather production, is a connective tissue with a three-dimensional structure composed of collagen fibers. The nature of the action of enzymes on animal skin is that enzyme molecules enter the fibrous interstices of the skin and hydrolyze the different components of the animal skin, which can be divided into mass transfer and reaction processes. Studies have shown that mass transfer of enzymes accounts for over 70% of the overall reaction time, and therefore mass transfer is a prerequisite and rate limiting step for the effective action of enzymes on animal skins. The mass transfer process of the enzyme in the leather is researched, so that the accurate quantification of the enzyme concentration of different depths of the leather is realized, and the method is a theoretical basis for popularization and application of enzyme preparations in leather manufacturing industry.
At present, the most common research method is to use a fluorescent marking technology to mark enzyme or protein, and then observe and record the fluorescence intensity of a leather longitudinal slice through a fluorescent microscope or a laser scanning confocal fluorescent microscope to obtain the distribution data of the enzyme in the leather. However, since the preparation of the fluorescent marker enzyme or the fluorescent marker protein is very difficult, and the dosage of the fluorescent marker enzyme or the fluorescent marker protein is large in the conventional experimental method, the mass transfer research on the conventional tanning experimental equipment in the laboratory at present is difficult.
The application of enzyme in leather production can be classified into unidirectional mass transfer mode (such as dehairing by dehairing paste coating) and turbulent mass transfer mode (such as soaking process, softening process, enzyme dehairing process in rotary drum, etc.). The experimental device realized by the patent can simulate the unidirectional mass transfer process of the enzyme in the leather.
Disclosure of Invention
Aiming at the problems that the existing mass transfer research technology and experimental equipment of enzyme in leather have large demand on samples, the experimental process is not easy to control and the like, the invention provides the experimental device for researching the unidirectional mass transfer of the enzyme in leather, and the unidirectional mass transfer process of the enzyme in leather can be simulated by using a very small amount of experimental materials under the condition of a laboratory.
The technical problems to be solved by the invention are realized by the following technical scheme: an experimental device for researching unidirectional mass transfer of enzyme in leather comprises an upper liquid storage tank, a lower liquid storage tank, a base, a horizontal bubble, an adjustable F clamp, a base adjusting bolt and a lower clamp adjusting screw;
the upper liquid storage tank and the lower liquid storage tank are of barrel-shaped structures with one ends open, the openings of the upper liquid storage tank and the lower liquid storage tank are placed opposite to each other, and a leather sample is placed between the upper liquid storage tank and the lower liquid storage tank;
the four corners of the bottom surface of the base are provided with base adjusting bolts, the upper surface of the base is provided with horizontal bubbles, and the liquid storage tank and the leather sample are ensured to be horizontal by adjusting the positions of the horizontal bubbles;
the adjustable F clamp is vertically fixed on the base, a lower clamp adjusting screw is arranged on the adjustable F clamp lower clamp, the adjustable F clamp lower clamp slides up and down to be used for coarse adjustment of the distance between the upper clamp and the lower clamp, and the replacement of liquid storage tanks with different specifications and the installation and the disassembly of the liquid storage tanks in the experimental process are convenient; and rotating the lower clamp adjusting screw to fix the leather sample between the upper liquid storage pool and the lower liquid storage pool.
The upper liquid storage pool and the lower liquid storage pool are consistent in material, inner diameter, outer diameter and height, liquid storage pools with different specifications can be selected according to the size of the sample amount and the experiment requirement, leather samples are fixed between the upper liquid storage pool and the lower liquid storage pool in an opening-to-opening manner in the experiment process, the lower liquid storage pool is filled with experiment buffer solution by a medical injector, and then the upper liquid storage pool is injected with a certain amount of solution containing enzyme marked by fluorescence by the medical injector.
The upper liquid storage tank and the lower liquid storage tank are made of elastic silica gel, and the thickness of the silica gel is 3-8 mm. The manufacturing material of the liquid storage tank is elastic silica gel, so that a solution is conveniently injected into the liquid storage tank or a solution sample is conveniently extracted from the liquid storage tank through the injector, and the leather sample can be firmly fixed between the two liquid storage tanks in the experimental process without liquid leakage. The wall thickness of the liquid storage tank is not lower than 3 mm so as to be beneficial to the fixation of the upper liquid storage tank and the leather sample and the rapid healing of the needle eye of the medical injector; the reservoir wall thickness is no greater than 8mm to facilitate penetration of the medical injector.
The invention relates to an experimental device for simulating unidirectional mass transfer of enzyme in leather, which comprises the following using method: adjusting a base adjusting bolt to enable bubbles to be located at the center of a horizontal bubble, taking an upper liquid storage tank and a lower liquid storage tank, respectively, filling the lower liquid storage tank with experimental buffer liquid with the volume of 80%, covering a leather sample on the lower liquid storage tank upwards by grain surfaces, taking an upper liquid storage tank, putting the leather sample on the leather sample in an opposite way, keeping concentricity with the lower liquid storage tank, adjusting the position of a lower clamp of an adjustable F clamp, and fixing the upper liquid storage tank, the leather sample and the lower liquid storage tank by rotating the lower clamp adjusting screw anticlockwise; the lower liquid storage tank is filled with experiment buffer solution through a medical injector, the upper liquid storage tank is filled with enzyme solution containing fluorescent markers through the medical injector, after mass transfer is finished, a leather sample is taken down, and after frozen sections are frozen, the distribution condition of the fluorescence intensity of the leather sections is observed and recorded.
Further, the leather sample is any part of animal skin.
Advantageous effects
(1) Under laboratory conditions, the unidirectional mass transfer process of the enzyme in the leather can be simulated by using a very small amount of experimental materials, and liquid reservoirs with different specifications can be selected according to the size of the sample and the experimental requirements.
(2) The manufacturing material of the liquid storage tank is elastic silica gel, so that a solution is conveniently injected into the liquid storage tank or a solution sample is conveniently extracted from the liquid storage tank through the injector, and the leather sample can be firmly fixed between the two liquid storage tanks in the experimental process without liquid leakage.
(3) The wall thickness of the liquid storage tank is not lower than 3 mm so as to be beneficial to the fixation of the upper liquid storage tank and the leather sample and the rapid healing of the needle eye of the medical injector; the reservoir wall thickness is no greater than 8mm to facilitate penetration of the medical injector.
(4) The adjustable F clamp is adopted to fix the leather sample, the F clamp can be used for coarse adjustment of the distance between the upper clamp and the lower clamp by sliding on the sliding rail, and the replacement of liquid storage tanks with different specifications and the disassembly of the liquid storage tanks in the experimental process are facilitated; the leather sample can be firmly fixed between the two liquid storage tanks by screwing the lower clamp adjusting screw.
Drawings
FIG. 1 is a schematic diagram of the structure of an experimental device for studying unidirectional mass transfer of enzymes in leather;
wherein, 1, an upper liquid storage pool; 2. a leather sample; 3. a lower liquid storage tank; 4. a base; 5. horizontal bubble; 6. an adjustable F clip; 7. a base adjusting bolt; eighthly, clamping an adjusting screw;
FIG. 2 fluorescence micrograph of a longitudinal section of a leather sample after mass transfer for 5min of FITC-Trypsin;
FIG. 3 fluorescence micrograph of a longitudinal section of a leather sample after mass transfer of FITC-Trypsin for 30 min;
FIG. 4 fluorescence intensity distribution diagrams of transverse sections of leather samples at different depths after FITC-Trypsin mass transfer for 30 min;
FIG. 5 fluorescence micrograph of a longitudinal section of a leather sample after mass transfer for 30min with FITC-dispese;
FIG. 6 fluorescence intensity distribution diagrams of transverse sections of leather samples at different depths after FITC-dispese mass transfer for 30 min;
FIG. 7 fluorescence intensity distribution plots of transverse sections of leather samples at different depths after RBITC-Trypsin mass transfer for 30 min.
Detailed Description
The invention is described in further detail below with reference to the attached drawings and specific examples, but the scope of the invention is not limited to the following examples:
the experimental apparatus used in mass transfer experiments in examples 1-6 is shown in FIG. 1, wherein 1. An upper reservoir 2. A leather sample 3. A lower reservoir 4. A base 5. A horizontal bulb 6. An adjustable F clamp 7. A base adjusting bolt 8. A lower clamp adjusting screw;
the upper liquid storage tank 1 and the lower liquid storage tank 3 are of barrel-shaped structures with one ends open, the upper liquid storage tank 1 and the lower liquid storage tank 3 are placed in an opening-to-opening manner, and the leather sample 2 is placed between the upper liquid storage tank 1 and the lower liquid storage tank 3;
the four corners of the bottom surface of the base 4 are provided with base adjusting bolts 7, and the upper surface is provided with a horizontal bubble 5;
the adjustable F clamp 6 is vertically fixed on the base 4, the distance between the upper clamp and the lower clamp is adjusted by vertically sliding the lower clamp of the adjustable F clamp 6, the lower clamp of the adjustable F clamp 6 is provided with a lower clamp adjusting screw 8, and the leather sample 2 is firmly fixed between the upper liquid storage pool 1 and the lower liquid storage pool 3 by rotating the lower clamp adjusting screw 8.
The materials, the inner diameter, the outer diameter and the height of the upper liquid storage tank 1 and the lower liquid storage tank 3 are completely consistent,
the manufacturing materials of the upper liquid storage tank 1 and the lower liquid storage tank 3 are elastic silica gel, and the thickness of the silica gel is 3-8 mm.
The lower liquid storage tank 3 is used for containing experiment buffer solution, and the upper liquid storage tank 1 is used for containing solution containing enzyme marked by fluorescence.
Example 1:
(1) Preparation of fluorescent marker enzyme: dissolving Trypsin (Trypsin) and fluorescein thiocyanate (Fluorescein isothiocyanate, FITC) in a mass ratio of 100:1 in a carbonic acid buffer solution (0.05 mol/L pH9.0), stirring and reacting for 12-14h at 4 ℃ in a dark place, freeze-drying after the reaction is finished, redissolving the freeze-dried powder, removing unreacted FITC by using gel chromatography (Sephadex G25 filler), and measuring the relationship between the fluorescence intensity and concentration of the FITC-labeled Trypsin solution after separation.
(2) Cutting leather samples: the back of the ridge is taken out of the goat skin, dehairing is carried out by a conventional method, and a round cutter is used for cutting into leather samples with the diameter of 50 mm.
(3) Mass transfer experiment: the experimental device is shown in fig. 1, the base adjusting bolt is adjusted to enable bubbles to be positioned at the center of the horizontal bubble, an upper liquid storage tank and a lower liquid storage tank with the outer diameters of 40mm are respectively taken, the lower liquid storage tank is filled with experimental buffer solution with the capacity of 80% (0.05 mol/L phosphate buffer solution with the pH value of 7.4), leather samples are covered on the lower liquid storage tank by grain surfaces, the upper liquid storage tank is placed on the leather samples in an opposite way, the upper liquid storage tank and the lower liquid storage tank are kept concentric with the lower liquid storage tank, the upper liquid storage tank, the lower liquid storage tank and the leather samples are fixed by adjusting the lower clamping position of an adjustable F clamp, and the upper liquid storage tank, the lower liquid storage tank and the leather samples are clamped by rotating the lower clamping adjusting screw anticlockwise. The lower liquid storage tank is filled with experiment buffer solution through a medical injector, the upper liquid storage tank is filled with solution containing FITC marked trypsin through the medical injector, after mass transfer for 5min, the lower clamp adjusting screw is rotated clockwise, and the leather sample is taken down.
(4) The data acquisition is carried out on the center of the leather sample, and the fluorescent intensity distribution condition of the longitudinal slices of the leather sample is observed by a fluorescent microscope. FIG. 2 is a fluorescence micrograph of a longitudinal section of a leather sample after mass transfer of FITC-labeled Trypsin (FITC-Trypsin) for 5 min.
Example 2:
(1) Preparation of fluorescent marker enzyme: dissolving Trypsin (Trypsin) and fluorescein thiocyanate (Fluorescein isothiocyanate, FITC) in a mass ratio of 100:1 in a carbonic acid buffer solution (0.05 mol/L pH9.0), stirring and reacting for 12-14h at 4 ℃ in a dark place, freeze-drying after the reaction is finished, redissolving the freeze-dried powder, removing unreacted FITC by using gel chromatography (Sephadex G25 filler), and measuring the relationship between the fluorescence intensity and the Trypsin concentration of the FITC-labeled Trypsin solution after separation.
(2) Cutting leather samples: the back of the goat skin ridge is taken, dehaired by a conventional method, and a round cutter is used for cutting into leather samples with the diameter of 50 mm.
(3) Mass transfer experiment: adjusting a base adjusting bolt to enable bubbles to be located at the center of a horizontal bubble, taking an upper liquid storage tank and a lower liquid storage tank with the outer diameters of 40mm, respectively, loading the lower liquid storage tank with an experimental buffer solution (0.05 mol/L phosphate buffer solution with pH of 7.4) with the volume of 80%, covering a leather sample on the lower liquid storage tank upwards by grain surfaces, putting the upper liquid storage tank on the leather sample in an opposite way, keeping concentricity with the lower liquid storage tank, adjusting the lower clamping position of an adjustable F clamp to fix the upper liquid storage tank, the lower liquid storage tank and the leather sample, and rotating the lower clamping adjusting screw anticlockwise to clamp the upper liquid storage tank, the lower liquid storage tank and the leather sample. The lower liquid storage tank is filled with experiment buffer solution through a medical injector, the upper liquid storage tank 1 is filled with solution containing FITC marked trypsin through the medical injector, after mass transfer for 30min, the lower clamp adjusting screw is rotated clockwise, and the leather sample is taken down.
(4) And (3) data acquisition: sampling at the center of the leather sample, and observing the distribution of the fluorescence intensity of the longitudinal slices of the leather sample by a fluorescence microscope. FIG. 3 is a fluorescence micrograph of a longitudinal section of a leather sample after mass transfer of FITC-labeled Trypsin (FITC-Trypsin) for 30 min.
Example 3:
(1) Preparation of fluorescent marker enzyme: trypsin (Trypsin) and fluorescein thiocyanate (Fluorescein isothiocyanate, FITC) are taken and dissolved in a carbonic acid buffer solution (0.05 mol/L pH9.0) according to the mass ratio of 100:1, the mixture is stirred and reacted for 12-14 hours at 4 ℃ in a dark place, freeze drying is carried out after the reaction is finished, the freeze-dried powder is redissolved, unreacted FITC is removed by gel chromatography (Sephadex G25 filler), and after separation, the relationship between the fluorescence intensity and the concentration of the solution of the FITC-labeled Trypsin is measured.
(2) Cutting leather samples: the back of the goat skin ridge is taken, dehaired by a conventional method, and a round cutter is used for cutting into leather samples with the diameter of 50 mm.
(3) Mass transfer experiment: as shown in FIG. 1, the experimental device is characterized in that a base adjusting bolt is adjusted to enable bubbles to be positioned at the center of a horizontal bubble, an upper liquid storage tank and a lower liquid storage tank with the outer diameters of 40mm are respectively taken, the lower liquid storage tank is filled with an experimental buffer solution with the capacity of 80% (0.05 mol/L phosphate buffer solution with pH 7.4), leather samples are covered on the lower liquid storage tank by grain surfaces, the upper liquid storage tank is placed on the leather samples in an opposite mode, the upper liquid storage tank and the lower liquid storage tank are kept concentric with the lower liquid storage tank, an upper liquid storage tank, a lower liquid storage tank and the leather samples are fixed at the lower clamping position of an adjustable F clamp, the upper liquid storage tank, the lower liquid storage tank and the leather samples are clamped by rotating the lower clamping adjusting screw anticlockwise, the lower liquid storage tank is filled with the experimental buffer solution through a medical injector, then the upper liquid storage tank 1 is filled with a solution containing FITC marked trypsin through the medical injector for mass transfer for 30min, and then the leather samples are taken down by rotating the lower clamping adjusting screw clockwise.
(4) And (3) data acquisition: the leather sample was sampled at the center, and the leather sample was sequentially sliced from the grain side to the flesh side by a frozen microtome, the slice thickness was 25 μm, and the fluorescence intensity of the slice was detected by a fluorescence spectrophotometer (excitation wavelength 495, emission wavelength 525). FIG. 4 is a graph showing the fluorescence intensity profiles of transverse sections of leather samples at different depths after mass transfer of FITC-labeled Trypsin (FITC-Trypsin) for 30 min.
Example 4:
(1) Preparation of fluorescent marker enzyme: neutral protease (dispese) and fluorescein thiocyanate (Fluorescein isothiocyanate, FITC) are taken and dissolved in a phosphate buffer (0.05 mol/L pH 7.4) according to the mass ratio of 100:1, the mixture is stirred and reacted for 12-14 hours at 4 ℃ in a dark place, freeze drying is carried out after the reaction is finished, the freeze-dried powder is redissolved, unreacted FITC is removed by gel chromatography (Sephadex G25 filler), and after separation, the relationship between the fluorescence intensity and the concentration of a solution of the neutral protease marked by FITC is measured.
(2) Cutting leather samples: the back of the goat skin ridge is taken, dehaired by a conventional method, and a round cutter is used for cutting into leather samples with the diameter of 35 mm.
(3) Mass transfer experiment: the experimental setup is shown in fig. 1, with the base adjusting bolt adjusted so that the bubble is centered in the horizontal bubble 5. Taking an upper liquid storage tank and a lower liquid storage tank with the outer diameter of 25mm, respectively, filling the lower liquid storage tank with an experimental buffer solution (0.05 mol/L phosphate buffer solution with pH 7.4) with the volume of 80%, covering a leather sample on the lower liquid storage tank upwards by grain surfaces, putting the upper liquid storage tank on the leather sample in an opposite way, keeping concentricity with the lower liquid storage tank, adjusting the lower clamping position of an adjustable F clamp to fix the upper liquid storage tank, the lower liquid storage tank and the leather sample, rotating a lower clamping adjusting screw anticlockwise to clamp the upper liquid storage tank, the lower liquid storage tank and the leather sample, filling the experimental buffer solution with the lower liquid storage tank through a medical injector, filling the upper liquid storage tank with a solution containing FITC marked neutral protease through the medical injector, transferring mass for 30min, rotating the lower clamping adjusting screw clockwise, and taking down the leather sample.
(4) And (3) data acquisition: sampling at the center of the leather sample, and observing the distribution of the fluorescence intensity of the longitudinal slices of the leather sample by a fluorescence microscope. FIG. 5 is a fluorescence micrograph of a longitudinal section of a leather sample after mass transfer of FITC-labeled neutral protease (FITC-dispese) for 30 min.
Example 5:
(1) Preparation of fluorescent marker enzyme: neutral protease (dispese) and fluorescein thiocyanate (Fluorescein isothiocyanate, FITC) are taken and dissolved in a phosphoric acid buffer (0.05 mol/L pH 7.4) according to the mass ratio of 100:1, the mixture is stirred and reacted for 12-14 hours at the temperature of 4 ℃ in a dark place, after the reaction is completed, freeze drying is carried out, the freeze-dried powder is redissolved, and then unreacted FITC is removed by gel chromatography (Sephadex G25 filler). After separation, the relationship between fluorescence intensity and concentration of the solution of FITC-labeled neutral protease was measured.
(2) Cutting leather samples: the back of the goat skin ridge is taken, dehaired by a conventional method, and a round cutter is used for cutting into leather samples with the diameter of 50 mm.
(3) Mass transfer experiment: as shown in FIG. 1, the experimental device is characterized in that a base adjusting bolt is adjusted to enable bubbles to be positioned at the center of a horizontal bubble, an upper liquid storage tank and a lower liquid storage tank with the outer diameters of 40mm are respectively taken, the lower liquid storage tank is filled with an experimental buffer solution with the capacity of 80% (0.05 mol/L phosphate buffer solution with pH value of 7.4), leather samples are covered on the lower liquid storage tank by grain surfaces, the upper liquid storage tank is placed on the leather samples in an opposite mode, the upper liquid storage tank and the lower liquid storage tank are kept concentric with the lower liquid storage tank, an upper liquid storage tank, a lower liquid storage tank and the leather samples are fixed at the lower clamping position of an adjustable F clamp, the upper liquid storage tank, the lower liquid storage tank and the leather samples are clamped by rotating the lower clamping adjusting screw anticlockwise, the lower liquid storage tank is filled with the experimental buffer solution through a medical injector, the upper liquid storage tank is filled with a solution containing FITC marked neutral protease through the medical injector, after mass transfer is carried out for 30min, and the leather samples are taken down by rotating the lower clamping adjusting screw clockwise.
(4) And (3) data acquisition: the leather sample was sampled at the center, and the leather sample was sequentially sliced from the grain side to the flesh side by a frozen microtome, the slice thickness was 25 μm, and the fluorescence intensity of the slice was detected by a fluorescence spectrophotometer (excitation wavelength 495, emission wavelength 525). FIG. 6 is a graph showing the fluorescence intensity profiles of transverse sections of leather samples at different depths after mass transfer of FITC-labeled neutral protease (FITC-dispese) for 30 min.
Example 6:
(1) Preparation of fluorescent marker enzyme: trypsin and rhodamine B isothiocyanate (Rhodamine B isothioCyanate, RBITC) are taken and dissolved in a carbonic acid buffer solution (0.05 mol/L pH9.0) according to the mass ratio of 100:1, and the mixture is stirred and reacted for 12 to 14 hours at 4 ℃ in the absence of light. After the reaction was completed, freeze-drying was performed. After redissolving the lyophilized powder, unreacted RBITC was removed by gel chromatography (Sephadex G25 packing). After separation, the relationship between fluorescence intensity and concentration of the RBITC labeled trypsin solution was measured.
(2) Cutting leather samples: the back goatskin is taken, dehaired conventionally, and cut into leather samples with the diameter of 35mm by a round cutter.
(3) Mass transfer experiment: the experimental device is shown in figure 1, a base adjusting bolt is adjusted to enable bubbles to be positioned in the center of a horizontal bubble, an upper liquid storage tank and a lower liquid storage tank with the outer diameters of 25mm are respectively taken, the lower liquid storage tank is filled with an experimental buffer solution with the capacity of 80% (0.05 mol/L phosphate buffer solution with the pH value of 7.4), a leather sample is covered on the lower liquid storage tank by grain surfaces, the upper liquid storage tank is placed on the leather sample in an opposite way, the upper liquid storage tank is kept concentric with the lower liquid storage tank, the upper liquid storage tank, the lower liquid storage tank and the leather sample are fixed by adjusting the lower clamping position of an adjustable F clamp, and the upper liquid storage tank, the lower liquid storage tank and the leather sample are clamped by rotating the lower clamping adjusting screw anticlockwise; the lower liquid storage tank is filled with experiment buffer solution through a medical injector, the upper liquid storage tank is filled with solution containing RBITC marked trypsin through the medical injector, after mass transfer for 30min, the lower clamp adjusting screw is rotated clockwise, and the leather sample is taken down.
(4) Preparation of fluorescent marker enzyme: the leather sample was sampled at the center, and the leather sample was sequentially sliced from the grain side to the flesh side by a frozen microtome, the slice thickness was 25 μm, and the fluorescence intensity of the slice was detected by a fluorescence spectrophotometer (excitation wavelength 495, emission wavelength 525). FIG. 7 is a graph showing the fluorescence intensity profiles of transverse sections of leather samples at different depths after 30min mass transfer of RBITC-labeled Trypsin (RBITC-Trypsin).
Claims (4)
1. An experimental device for researching unidirectional mass transfer of enzyme in leather is characterized by comprising an upper liquid storage tank, a lower liquid storage tank, a base, a horizontal bubble, an adjustable F clamp, a base adjusting bolt and a lower clamp adjusting screw;
the upper liquid storage tank and the lower liquid storage tank are of barrel-shaped structures with one ends open, the openings of the upper liquid storage tank and the lower liquid storage tank are placed opposite to each other, and a leather sample is placed between the upper liquid storage tank and the lower liquid storage tank;
base adjusting bolts are installed at four corners of the lower bottom surface of the base, and horizontal bubbles are installed on the upper plane;
the adjustable F clamp is vertically fixed on the base, the distance between the upper clamp and the lower clamp can be adjusted by sliding the lower clamp of the F clamp up and down, a lower clamp adjusting screw is arranged on the lower clamp of the F clamp, and the leather sample is fixed between the upper liquid storage pool and the lower liquid storage pool by rotating the lower clamp adjusting screw;
the upper liquid storage tank and the lower liquid storage tank are made of elastic silica gel, and the wall thickness is 3-8 mm;
the lower liquid storage tank is used for containing experiment buffer solution, and the upper liquid storage tank is used for containing solution containing enzyme marked by fluorescence.
2. The experimental apparatus for studying unidirectional mass transfer of an enzyme in leather according to claim 1, wherein the upper reservoir and the lower reservoir are identical in material, inner diameter, outer diameter and height.
3. A method of using the experimental apparatus according to claim 1 or 2, wherein the base adjusting bolt is adjusted to position the air bubble at the center of the horizontal bubble, the upper reservoir and the lower reservoir are taken to be respectively filled with 80% of experimental buffer solution, the leather sample is covered on the lower reservoir by grain surface, the upper reservoir is taken to be placed on the leather sample in an opposite way, the leather sample is kept concentric with the lower reservoir, the position of the lower clamp of the adjustable F clamp is adjusted, and the lower clamp adjusting screw is rotated anticlockwise to fix the upper reservoir, the leather sample and the lower reservoir; the lower liquid storage tank is filled with experiment buffer solution through a medical injector, the upper liquid storage tank is filled with solution containing fluorescent marked enzyme through the medical injector, after mass transfer is finished, a leather sample is taken down, and after frozen sections are frozen, the distribution condition of the fluorescence intensity of the leather sections is observed and recorded.
4. The method of claim 3, wherein the leather sample is any part of an animal skin.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910007178.1A CN109490274B (en) | 2019-01-04 | 2019-01-04 | Experimental device for researching unidirectional mass transfer of enzyme in leather and application method |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201910007178.1A CN109490274B (en) | 2019-01-04 | 2019-01-04 | Experimental device for researching unidirectional mass transfer of enzyme in leather and application method |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109490274A CN109490274A (en) | 2019-03-19 |
CN109490274B true CN109490274B (en) | 2023-06-09 |
Family
ID=65713954
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201910007178.1A Active CN109490274B (en) | 2019-01-04 | 2019-01-04 | Experimental device for researching unidirectional mass transfer of enzyme in leather and application method |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109490274B (en) |
Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05223806A (en) * | 1992-02-13 | 1993-09-03 | Hitachi Ltd | Glycoprotein analyzing device |
JPH0961355A (en) * | 1995-08-30 | 1997-03-07 | Mitsubishi Heavy Ind Ltd | Optical axis moving type fluorometer and measuring method |
US5804395A (en) * | 1995-12-01 | 1998-09-08 | The United States Of America As Represented By The Secretary Of The Navy | Fluorescence polarization assays of enzymes and substrates therefore |
CN101011237A (en) * | 2007-02-01 | 2007-08-08 | 中国药科大学 | Pharmaceutical in vivo dynamics characteristic-nondestructive in situ monitoring system and monitoring method |
CN101074952A (en) * | 2007-06-22 | 2007-11-21 | 中国科学院植物研究所 | Immune fluorescent marking method for naked plant pollen tube microtubule skeleton and its use |
CN101271070A (en) * | 2008-05-09 | 2008-09-24 | 东北大学 | Microcurrent controlled capillary tube electrophoresis liquid core waveguide fluorescence testing apparatus |
CN102495037A (en) * | 2011-12-07 | 2012-06-13 | 南京农业大学 | Fluorescent tracing method of assimilation substance unloading path of phloem in pear fruit |
CN103243149A (en) * | 2013-05-20 | 2013-08-14 | 吉林大学 | Method for rapidly determining trypsin and aprotinin combination mechanism |
Family Cites Families (29)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH0697221B2 (en) * | 1987-05-18 | 1994-11-30 | 麒麟麦酒株式会社 | Method for measuring organic concentration and apparatus used for the method |
EP0649476A4 (en) * | 1992-06-29 | 1997-05-07 | Sensor Technologies Inc | Method and device for detecting and quantifying substances in body fluids. |
US7364565B2 (en) * | 2001-07-27 | 2008-04-29 | Ramot At Tel Aviv University Ltd. | Controlled enzymatic removal and retrieval of cells |
JP3896272B2 (en) * | 2001-10-23 | 2007-03-22 | 財団法人日本紡績検査協会 | Judgment method of natural leather products |
AU2003225165A1 (en) * | 2002-04-26 | 2003-11-10 | Board Of Trustees Of The Leland Stanford Junior University | System and method of measuring molecular interactions |
JP2005080535A (en) * | 2003-09-05 | 2005-03-31 | Olympus Corp | Method for detecting reaction between nucleic acid base and helicase |
WO2006114130A1 (en) * | 2005-04-26 | 2006-11-02 | Agilent Technologies, Inc. | Enzymes with modified amino acids |
WO2007035763A2 (en) * | 2005-09-20 | 2007-03-29 | The Arizona Board Of Regents, A Body Corporate Acting On Behalf Of Arizona State University | Methods for detecting and characterizing microorganisms |
JP4763485B2 (en) * | 2006-03-15 | 2011-08-31 | 株式会社日立ハイテクノロジーズ | Fluorescence detection device |
US7498551B2 (en) * | 2006-10-06 | 2009-03-03 | Los Alamos National Security, Llc | Apparatus and method for tracking a molecule or particle in three dimensions |
CN100552434C (en) * | 2007-01-09 | 2009-10-21 | 浙江大学 | The fluorescence spectrum analysing method of fixed enzyme conformation |
EP2129757A2 (en) * | 2007-01-11 | 2009-12-09 | Novozymes A/S | Particles comprising active compounds |
CN101730746A (en) * | 2007-07-06 | 2010-06-09 | 帕普斯特许可有限两合公司 | Determination of the activity of proteases |
CN201186115Y (en) * | 2008-04-11 | 2009-01-28 | 江苏省华建建设股份有限公司 | Drop-prevention wall-clamping device for aloft work |
JP2011047802A (en) * | 2009-08-27 | 2011-03-10 | Olympus Corp | Concentration measuring method of target material |
JP2011169834A (en) * | 2010-02-19 | 2011-09-01 | Kansai Bunri Sogo Gakuen | Method and system for detecting lipid vesicle derived from pathogenic gram-negative bacterium |
DE102010030069A1 (en) * | 2010-06-15 | 2011-12-15 | Agilent Technologies Inc. | Liquid chromatography fluorescence detector for detecting component of fluid sample in e.g. life science apparatus, for separating component of fluid sample, has detection channel detecting fluorescence radiation |
CN102323308B (en) * | 2011-06-14 | 2013-09-11 | 北京科技大学 | Device and method for researching hydrogen permeation behavior of metal subjected to dead-load pulling stress in gaseous medium |
WO2013165232A1 (en) * | 2012-05-02 | 2013-11-07 | Alberto Morales Villagran | Device for on-line measurement of neurotransmitters using enzymatic reactors |
WO2014034275A1 (en) * | 2012-08-30 | 2014-03-06 | 株式会社 日立ハイテクノロジーズ | Nucleic acid analysis device |
CN103439500B (en) * | 2013-08-27 | 2015-01-28 | 陕西科技大学 | Visible tracking and detecting method for protease in leather treatment process |
CN103525954B (en) * | 2013-10-28 | 2015-07-22 | 四川大学 | Immobilized composite unhairing enzyme with leather-making secondary waste as carrier and preparation method thereof |
US20170204451A1 (en) * | 2014-07-17 | 2017-07-20 | Daniel A. Kerschensteiner | Preparation of stabilizer-free gold sols and their configuration into a gelatin-coated protease-indicating homogeneous (one step) assay |
GB201418007D0 (en) * | 2014-10-10 | 2014-11-26 | Xeros Ltd | Animal skin substrate Treatment apparatus and method |
RU2567510C1 (en) * | 2014-11-17 | 2015-11-10 | Федеральное государственное бюджетное образовательное учреждение высшего профессионального образования "Восточно-Сибирский государственный университет технологий и управления" | Method for measuring mass transfer rate in capillary-porous bodies |
EP3073246B1 (en) * | 2015-03-27 | 2019-08-14 | National Institute of Biology | Method and system for simultaneous detection of micro-particle concentration in suspension and their morphological and physiological traits |
CN207326783U (en) * | 2017-07-10 | 2018-05-08 | 上海新月工具有限公司 | A kind of multi-functional F folders |
CN207415218U (en) * | 2017-10-20 | 2018-05-29 | 浙江光辉工具有限公司 | It is a kind of can realizing self disassembling F type fixtures |
CN209460142U (en) * | 2019-01-04 | 2019-10-01 | 齐鲁工业大学 | A kind of experimental provision for studying enzyme unidirectional mass transfer in the leather |
-
2019
- 2019-01-04 CN CN201910007178.1A patent/CN109490274B/en active Active
Patent Citations (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05223806A (en) * | 1992-02-13 | 1993-09-03 | Hitachi Ltd | Glycoprotein analyzing device |
JPH0961355A (en) * | 1995-08-30 | 1997-03-07 | Mitsubishi Heavy Ind Ltd | Optical axis moving type fluorometer and measuring method |
US5804395A (en) * | 1995-12-01 | 1998-09-08 | The United States Of America As Represented By The Secretary Of The Navy | Fluorescence polarization assays of enzymes and substrates therefore |
CN101011237A (en) * | 2007-02-01 | 2007-08-08 | 中国药科大学 | Pharmaceutical in vivo dynamics characteristic-nondestructive in situ monitoring system and monitoring method |
CN101074952A (en) * | 2007-06-22 | 2007-11-21 | 中国科学院植物研究所 | Immune fluorescent marking method for naked plant pollen tube microtubule skeleton and its use |
CN101271070A (en) * | 2008-05-09 | 2008-09-24 | 东北大学 | Microcurrent controlled capillary tube electrophoresis liquid core waveguide fluorescence testing apparatus |
CN102495037A (en) * | 2011-12-07 | 2012-06-13 | 南京农业大学 | Fluorescent tracing method of assimilation substance unloading path of phloem in pear fruit |
CN103243149A (en) * | 2013-05-20 | 2013-08-14 | 吉林大学 | Method for rapidly determining trypsin and aprotinin combination mechanism |
Also Published As
Publication number | Publication date |
---|---|
CN109490274A (en) | 2019-03-19 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Furness et al. | Aqueous aldehyde (Faglu) methods for the fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue | |
CN107300496B (en) | Transparentizing agent for biological material and use thereof | |
DE50010126D1 (en) | METHOD AND DEVICE FOR CHARACTERIZING A CULTURAL FLUID | |
CN101268184A (en) | Method of producing organotypic cell cultures | |
CN101001652A (en) | Cross-linked collagen matrix for producing a skin equivalent | |
CN108578771A (en) | Preparation method and products thereof with the FGF1 sericin gels for promoting cell-proliferation activity | |
CN109490274B (en) | Experimental device for researching unidirectional mass transfer of enzyme in leather and application method | |
Phillips | Monitoring neuron and astrocyte interactions with a 3D cell culture system | |
Siwczak et al. | Culture of vibrating microtome tissue slices as a 3D model in biomedical research | |
EP3477302B1 (en) | Device, system and kit for measuring tension of sheet-like tissue containing cardiomyocytes | |
WO2011135566A1 (en) | Niche system for biological culturing | |
CN209460142U (en) | A kind of experimental provision for studying enzyme unidirectional mass transfer in the leather | |
CN103509711A (en) | Compound type biological tissue and cell chip | |
CN210037612U (en) | Experimental device for be arranged in studying enzyme turbulence mass transfer in leather | |
CN104542505B (en) | A kind of extracorporeal culturing method of parasitic nematode parasitic stages | |
CN109682785B (en) | Experimental device for researching turbulent mass transfer of enzyme in leather and using method | |
CA2230889A1 (en) | Stain and capillary slide to detect animal and plant cells | |
Huang et al. | Versatile on-stage microfluidic system for long term cell culture, micromanipulation and time lapse assays | |
Shnaider et al. | CLARITY and Light-Sheet microscopy sample preparation in application to human cerebral organoids | |
KR20210155870A (en) | A method of pretreatment of transparency of a biological samples with a size of 1 mm or less and a method of transparency of the biological samples comprising the same | |
US6043079A (en) | Cell growth apparatus for histophysiologic culture | |
WO2021192083A1 (en) | Observation sample, method for producing observation sample, and sample production system | |
Angres et al. | 3‐D Life biomimetic hydrogels: A modular system for cell environment design | |
CN111579769A (en) | Method for carrying out immune marking on tissue sample | |
CN107764809A (en) | A kind of quick detection test paper of Sulfide in Water and preparation method thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CP03 | Change of name, title or address |
Address after: 250399 No. 3501 University Road, Changqing District, Jinan City, Shandong Province Patentee after: Qilu University of Technology (Shandong Academy of Sciences) Country or region after: China Address before: 250399 No. 3501 University Road, Changqing District, Jinan City, Shandong Province Patentee before: Qilu University of Technology Country or region before: China |