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
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- 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
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- 238000002474 experimental method Methods 0.000 claims description 18
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- 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
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- 239000012588 trypsin Substances 0.000 description 20
- 238000005520 cutting process Methods 0.000 description 11
- 239000003550 marker Substances 0.000 description 11
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- 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
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- 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
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- 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
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- -1 rhodamine B isothiocyanate Chemical class 0.000 description 2
- 229940043267 rhodamine b Drugs 0.000 description 2
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- 102000008186 Collagen Human genes 0.000 description 1
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- 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
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- General Health & Medical Sciences (AREA)
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- 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.
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