CN109490274A - A kind of experimental provision and application method for studying enzyme unidirectional mass transfer in the leather - Google Patents
A kind of experimental provision and application method for studying enzyme unidirectional mass transfer in the leather Download PDFInfo
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- CN109490274A CN109490274A CN201910007178.1A CN201910007178A CN109490274A CN 109490274 A CN109490274 A CN 109490274A CN 201910007178 A CN201910007178 A CN 201910007178A CN 109490274 A CN109490274 A CN 109490274A
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- 239000010985 leather Substances 0.000 title claims abstract description 109
- 238000012546 transfer Methods 0.000 title claims abstract description 48
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 40
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 23
- 239000007788 liquid Substances 0.000 claims abstract description 164
- 238000003860 storage Methods 0.000 claims abstract description 157
- 239000000872 buffer Substances 0.000 claims abstract description 19
- 238000012360 testing method Methods 0.000 claims abstract description 18
- NJPPVKZQTLUDBO-UHFFFAOYSA-N novaluron Chemical compound C1=C(Cl)C(OC(F)(F)C(OC(F)(F)F)F)=CC=C1NC(=O)NC(=O)C1=C(F)C=CC=C1F NJPPVKZQTLUDBO-UHFFFAOYSA-N 0.000 claims abstract description 17
- 239000003550 marker Substances 0.000 claims abstract description 16
- 239000000463 material Substances 0.000 claims abstract description 12
- 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
- 238000009826 distribution Methods 0.000 claims description 7
- 239000000741 silica gel Substances 0.000 claims description 7
- 229910002027 silica gel Inorganic materials 0.000 claims description 7
- 238000009434 installation Methods 0.000 claims description 2
- 238000003825 pressing Methods 0.000 claims 1
- 229940088598 enzyme Drugs 0.000 description 30
- 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 25
- 239000000243 solution Substances 0.000 description 14
- 239000012588 trypsin Substances 0.000 description 14
- 238000002474 experimental method Methods 0.000 description 13
- 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 10
- ZMZDMBWJUHKJPS-UHFFFAOYSA-N thiocyanic acid Chemical compound SC#N ZMZDMBWJUHKJPS-UHFFFAOYSA-N 0.000 description 10
- 101000693530 Staphylococcus aureus Staphylokinase Proteins 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000035617 depilation Effects 0.000 description 7
- 239000008363 phosphate buffer Substances 0.000 description 7
- 241000283707 Capra Species 0.000 description 6
- 229920005654 Sephadex Polymers 0.000 description 6
- 239000012507 Sephadex™ Substances 0.000 description 6
- 102000004142 Trypsin Human genes 0.000 description 6
- 108090000631 Trypsin Proteins 0.000 description 6
- 239000000945 filler Substances 0.000 description 6
- 238000005227 gel permeation chromatography Methods 0.000 description 6
- 150000002540 isothiocyanates Chemical class 0.000 description 6
- 239000000843 powder Substances 0.000 description 6
- 238000005070 sampling Methods 0.000 description 6
- 238000007796 conventional method Methods 0.000 description 5
- 238000004108 freeze drying Methods 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 239000007853 buffer solution Substances 0.000 description 4
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 238000002073 fluorescence micrograph Methods 0.000 description 3
- 235000012149 noodles Nutrition 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
- 108010007093 dispase Proteins 0.000 description 2
- 230000035876 healing Effects 0.000 description 2
- 229940043267 rhodamine b Drugs 0.000 description 2
- -1 Rhodamine B isothiocyanate Chemical class 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003139 buffering effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 230000002500 effect on skin Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000001215 fluorescent labelling Methods 0.000 description 1
- 238000011010 flushing procedure Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000010422 painting Methods 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 230000035484 reaction time Effects 0.000 description 1
- PYWVYCXTNDRMGF-UHFFFAOYSA-N rhodamine B Chemical compound [Cl-].C=12C=CC(=[N+](CC)CC)C=C2OC2=CC(N(CC)CC)=CC=C2C=1C1=CC=CC=C1C(O)=O PYWVYCXTNDRMGF-UHFFFAOYSA-N 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
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
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- 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 present invention provides a kind of experimental provisions for studying enzyme unidirectional mass transfer in the leather, including upper liquid storage tank, lower liquid storage tank, pedestal, spirit bubble, adjustable F folder, pedestal adjusts bolt and lower folder adjusting screw, leather sample is placed on, between lower liquid storage tank, and by fixed in the adjustable lower folder of F folder and lower folder adjusting screw, wherein, lower liquid storage tank is for containing test buffer, upper liquid storage tank is used to contain the solution containing the enzyme by fluorescent marker, use experimental provision provided by the invention, it may be implemented in laboratory conditions, it can the unidirectional mass transport process of analogue enztme in the leather using minute quantity experimental material, it is easy to operate, save experimental material.
Description
Technical field
The invention belongs to the Applied research fields of process hides enzyme preparation, and in particular to one kind is unidirectional in the leather for studying enzyme
The experimental provision and application method of mass transfer.
Background technique
Extension of the leather industry as animal husbandry is the important link of circular economy, and weight is occupied in China's industrial economy
Want position.As Environmental protection is increasingly paid attention to, the clean leather-making technology based on biological enzyme is obtained rapidly
Develop and is widely applied.Different from common enzymatic reaction, the catalysis substrate of enzyme in tanning production --- Animal Skin is a kind of
The connective tissue with three-dimensional structure being made of collagenous fibres.Enzyme and the essence of Animal Skin effect are that enzyme molecule enters skin
Fibre gap is simultaneously hydrolyzed Animal Skin different component, can be divided into two processes of mass transfer and reaction.Research shows that enzyme
Mass transfer accounts for 70% or more of entire reaction time, therefore mass transfer is prerequisite and rate-limiting step of the enzyme to Animal Skin useful effect.
Mass transport process, realization are enzyme preparations in leather industry to the accurate quantitative analysis of leather different depth enzyme concentration to studying enzyme in the leather
The theoretical basis of promotion and application.
Currently, the most common research method is using fluorescent labelling techniques by after enzyme or protein labeling, then pass through fluorescence
The fluorescence intensity of microscope or laser scanning co-focusing fluorescence microscope and record leather longitudinal section obtains enzyme in leather
Middle distributed data.But since fluorescent marker enzyme or fluorescent marker protein prepare extremely difficult, and routine experiment method fluorescence mark
Remember that the dosage of enzyme or fluorescent marker protein is big, therefore, it is difficult to carry out mass transfer on the common process hides experimental facilities in current laboratory
Research.
The application that enzyme is produced in leather, difference can be divided into unidirectional mass transfer mode (as using painting unhairing paste method in a manner of mass transfer
Depilation) and turbulent mass transfer mode (such as soak process, softening process, the enzymatic depilation process in rotary drum).What this patent was realized
Experimental provision can be with the unidirectional mass transport process of analogue enztme in the leather.
Summary of the invention
For existing enzyme mass transfer investigative technique and experimental facilities is big to sample requirements, experimentation is not easily-controllable in the leather
The problems such as processed, the present invention provides a kind of experimental provisions for studying enzyme unidirectional mass transfer in the leather, realize in laboratory item
It, can the unidirectional mass transport process of analogue enztme in the leather using minute quantity experimental material under part.
The technical problems to be solved by the invention are realized by the following technical solution: one kind being used for studying enzyme in the leather
The experimental provision of unidirectional mass transfer, by upper liquid storage tank, lower liquid storage tank, pedestal, spirit bubble, adjustable F folder, pedestal adjust bolt and under
Adjusting screw is pressed from both sides to constitute；
The upper liquid storage tank and lower liquid storage tank are barrel-like structure open at one end, and upper liquid storage tank and lower liquid storage tank are put mouth to mouth
It sets, leather sample is placed between upper and lower liquid storage tank；
The base end face quadrangle is equipped with pedestal and adjusts bolt, is equipped with spirit bubble above, by the position for adjusting spirit bubble
To ensure the level of liquid storage tank and leather sample；
The adjustable F folder is vertical to be fixed on the base, and the adjustable lower folder of F folder is equipped with lower folder adjusting screw, and F folder is adjusted
Lower folder slides up and down the distance between folder above and below coarse adjustment, convenient for liquid storage in the replacement of different size liquid storage tank and experimentation
The installation and removal in pond；Leather sample is fixed between upper liquid storage tank and lower liquid storage by the lower folder adjusting screw of rotation.
Upper liquid storage tank is consistent with lower liquid storage tank material, internal diameter, outer diameter and height, can be according to the size and experiment of sample size
Demand selects the liquid storage tank of different size, and leather sample is fixed between upper and lower liquid storage tank mouth to mouth in experimentation, first
Lower liquid storage tank is filled into test buffer with injector for medical purpose, then by upper liquid storage tank by injector for medical purpose inject it is a certain amount of containing
By the solution of the enzyme of fluorescent marker.
The making material of upper liquid storage tank and lower liquid storage tank is flexible silica gel, and silica gel is with a thickness of 3 ~ 8mm.Liquid storage tank
Making material is flexible silica gel, in order to inject solution to liquid storage tank by syringe or extract solution from liquid storage tank
Sample, and can be firmly fixed to leather sample among two liquid storage tanks during the experiment, without generating the phenomenon of leakage.Storage
Liquid pool wall thickness is not less than 3 mm in favor of the fixation of upper and lower liquid storage tank and leather sample and the quick healing of injector for medical purpose pinprick；
Liquid storage tank wall thickness is no more than 8 mm in favor of the piercing of injector for medical purpose.
A kind of experimental provision of heretofore described analogue enztme unidirectional mass transfer in the leather, application method are as follows: adjust
Pedestal adjusts bolt, and bubble is made to be located at the center of spirit bubble, takes liquid storage tank and lower liquid storage tank each one, and lower liquid storage tank is packed into
The test buffer of 80% capacity, grain upwards cover leather sample on lower liquid storage tank, and liquid storage tank is taken to be placed on skin mouth to mouth
It removes from office on sample, keeps concentric with lower liquid storage tank, adjust the lower position pressed from both sides of adjustable F folder, the lower folder adjusting screw of rotation will counterclockwise
Upper liquid storage tank, leather sample and lower liquid storage tank are fixed；Lower liquid storage tank is first filled into test buffer by injector for medical purpose, then will
Upper liquid storage tank fills the enzyme solutions containing fluorescent marker by injector for medical purpose, after mass transfer, removes leather sample, freezing is cut
After piece, the distribution situation of observation and record leather slice fluorescence intensity.
Further, the leather sample is any part of Animal Skin.
Beneficial effect
(1) in laboratory conditions, using minute quantity experimental material can the unidirectional mass transport process of analogue enztme in the leather, and can
According to the liquid storage tank of the size of sample size and the demand of experiment selection different size.
(2) making material of liquid storage tank is flexible silica gel, in order to inject solution to liquid storage tank by syringe
Or solution example is extracted from liquid storage tank, and leather sample can be firmly fixed in two liquid storage tanks during the experiment
Between, without generating the phenomenon of leakage.
(3) liquid storage tank wall thickness is not less than fixation and injector for medical purpose needle of 3 mm in favor of upper and lower liquid storage tank and leather sample
The quick healing of eye；Liquid storage tank wall thickness is no more than 8 mm in favor of the piercing of injector for medical purpose.
(4) realize the fixation of leather sample using adjustable F folder, the sliding of the lower folder of F folder on the slide rail can be with coarse adjustment above and below
The distance between folder, convenient for the disassembly of liquid storage tank in the replacement of different size liquid storage tank and experimentation；It is adjusted by screwing lower folder
Leather sample can be firmly fixed between two liquid storage tanks by section screw.
Detailed description of the invention
The experimental provision structural schematic diagram of the unidirectional mass transfer of Fig. 1 studying enzyme in the leather；
Wherein, liquid storage tank on 1.；2. leather sample；3. lower liquid storage tank；4. pedestal；5. spirit bubble；6. adjustable F folder；7. pedestal
Adjust bolt；8. lower press from both sides adjusting screw；
The fluorescence micrograph of leather sample longitudinal section after Fig. 2 FITC-Trypsin mass transfer 5min；
The fluorescence micrograph of leather sample longitudinal section after Fig. 3 FITC-Trypsin mass transfer 30min；
The fluorescence intensity profile of leather sample different depth transverse section after Fig. 4 FITC-Trypsin mass transfer 30min；
The fluorescence micrograph of leather sample longitudinal section after Fig. 5 FITC-Dispase mass transfer 30min；
The fluorescence intensity profile of leather sample different depth transverse section after Fig. 6 FITC-Dispase mass transfer 30min；
The fluorescence intensity profile of leather sample different depth transverse section after Fig. 7 RBITC-Trypsin mass transfer 30min.
Specific embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail, but protection scope of the present invention
It is not limited in following embodiments:
The experimental provision that mass transfer experiment uses in embodiment 1 ~ 6 is as shown in Figure 1, wherein 2. leather sample of liquid storage tank on 1.
3. lower 4. pedestal of liquid storage tank, 5. spirit bubble 6. is adjusted F and presss from both sides the lower folder adjusting screw of 7. pedestals adjusting bolt 8.；
Upper liquid storage tank 1 and lower liquid storage tank 3 are barrel-like structure open at one end, and upper liquid storage tank 1 and lower liquid storage tank 3 are put mouth to mouth
It sets, leather sample 2 is placed between liquid storage tank 1 and lower liquid storage tank 3；
The bottom surface quadrangle of pedestal 4 is equipped with pedestal and adjusts bolt 7, is equipped with spirit bubble 5 above；
Adjustable F folder 6 is vertical to be fixed on pedestal 4, and the lower folder of F folder 6 is adjusted and slides up and down coarse adjustment the distance between folder up and down,
The lower folder of adjustable F folder 6 is equipped with lower folder adjusting screw 8, rotates lower folder adjusting screw 8 for leather sample 2 and is secured firmly to upper storage
Between liquid pool 1 and lower liquid storage tank 3.
The material of upper liquid storage tank 1 and lower liquid storage tank 3, internal diameter, outer diameter and height are completely the same,
The making material of upper liquid storage tank 1 and lower liquid storage tank 3 is flexible silica gel, and silica gel is with a thickness of 3 ~ 8mm.
Lower liquid storage tank 3 is used to contain containing the molten of the enzyme by fluorescent marker for containing test buffer, upper liquid storage tank 1
Liquid.
Embodiment 1:
(1) trypsase (Trypsin), thiocyanic acid fluorescein (Fluorescein the preparation of fluorescent marker enzyme: are taken
Isothiocyanate, FITC) it is dissolved in carbonic acid buffer (0.05mol/L pH9.0) according to the mass ratio of 100:1,4 DEG C
Under be protected from light and be stirred to react 12 ~ 14h, after the reaction was completed, freeze-dried powder is re-dissolved, utilizes gel chromatography by freeze-drying
(Sephadex G25 filler) removes unreacted FITC, after having separated, the fluorescence of the trypsin solution of measurement FITC label
The relationship of intensity and concentration.
(2) leather sample is cut: goat skin takes ridge, and conventional method depilation is cut into the skin of diameter 50mm with round cut-off knife
Remove from office sample.
(3) mass transfer experiment: experimental provision is located at bubble in spirit bubble as shown in Figure 1, adjustment base adjusting bolt
The heart, take outer diameter 40mm upper liquid storage tank and lower liquid storage tank each one, lower liquid storage tank is packed into the test buffer (0.05 of 80% capacity
The phosphate buffer of mol/L pH7.4), grain upwards covers leather sample on lower liquid storage tank, mouth to mouth by upper liquid storage tank
It is placed on leather sample, keeps concentric with lower liquid storage tank, the lower clamp position for adjusting adjustable F folder sets fixed upper and lower liquid storage tank and skin
Sample is removed from office, the lower folder adjusting screw of rotation clamps upper and lower liquid storage tank and leather sample counterclockwise.First lower liquid storage tank is passed through medical
Syringe fills test buffer, then upper liquid storage tank is filled by injector for medical purpose and marks the molten of trypsase containing FITC
Liquid after 5 min of mass transfer, rotates clockwise lower folder adjusting screw, removes leather sample.
(4) sampling core of the data acquisition in leather sample, fluorescence microscope leather sample longitudinal section fluorescence intensity
Distribution situation.The fluorescence of leather sample longitudinal section is aobvious after Fig. 2 is FITC label 5 min of trypsase (FITC-Trypsin) mass transfer
Micro- photo.
Embodiment 2:
(1) trypsase (Trypsin), thiocyanic acid fluorescein (Fluorescein the preparation of fluorescent marker enzyme: are taken
Isothiocyanate, FITC) it is dissolved in carbonic acid buffer (0.05mol/L pH9.0) according to the mass ratio of 100:1,4 DEG C
Under be protected from light and be stirred to react 12 ~ 14h, after the reaction was completed, freeze-dried powder is re-dissolved, utilizes gel chromatography by freeze-drying
(Sephadex G25 filler) removes unreacted FITC, after having separated, the fluorescence of the trypsin solution of measurement FITC label
The relationship of intensity and trypsinase concentration.
(2) leather sample is cut: taking goat skin ridge, conventional method depilation is cut into the skin of diameter 50mm with round cut-off knife
Remove from office sample.
(3) mass transfer experiment: adjustment base adjusts bolt, so that bubble is located at the center of spirit bubble, takes the upper storage of outer diameter 40mm
Liquid pool and lower liquid storage tank each one, lower liquid storage tank is packed into the test buffer of 80% capacity, and (phosphoric acid of 0.05 mol/L pH7.4 is slow
Fliud flushing), grain upwards covers leather sample on lower liquid storage tank, and upper liquid storage tank is placed on leather sample mouth to mouth, keeps
Concentric with lower liquid storage tank, the lower clamp position for adjusting adjustable F folder sets fixed upper and lower liquid storage tank and leather sample, counterclockwise under rotation
Adjusting screw is pressed from both sides to clamp upper and lower liquid storage tank and leather sample.Lower liquid storage tank is first filled into experiment buffering by injector for medical purpose
Liquid, then upper liquid storage tank 1 is filled to the solution that trypsase is marked containing FITC by injector for medical purpose, it is suitable after 30 min of mass transfer
Hour hands rotation is lower to press from both sides adjusting screw, removes leather sample.
(4) data acquire: in the sampling core of leather sample, fluorescence microscope leather sample longitudinal section fluorescence intensity
Distribution situation.Fig. 3 is the fluorescence that FITC marks leather sample longitudinal section after 30 min of trypsase (FITC-Trypsin) mass transfer
Microphoto.
Embodiment 3:
(1) trypsase (Trypsin), thiocyanic acid fluorescein (Fluorescein the preparation of fluorescent marker enzyme: are taken
Isothiocyanate, FITC) it is dissolved in carbonic acid buffer (0.05mol/L pH9.0) according to the mass ratio of 100:1,4 DEG C
Under be protected from light and be stirred to react 12-14h, after the reaction was completed, freeze-drying after re-dissolving freeze-dried powder, utilizes gel chromatography
(Sephadex G25 filler) removes unreacted FITC, after having separated, measures the glimmering of the solution of the trypsase of FITC label
The relationship of luminous intensity and concentration.
(2) leather sample is cut: taking goat skin ridge, conventional method depilation is cut into the skin of diameter 50mm with round cut-off knife
Remove from office sample.
(3) mass transfer experiment: experimental provision is located at bubble in spirit bubble as shown in Figure 1, adjustment base adjusting bolt
The heart, take outer diameter 40mm upper liquid storage tank and lower liquid storage tank each one, lower liquid storage tank is packed into the test buffer (0.05 of 80% capacity
The phosphate buffer of mol/L pH7.4), grain upwards covers leather sample on lower liquid storage tank, mouth to mouth by upper liquid storage tank
It is placed on leather sample, keeps concentric with lower liquid storage tank, the lower clamp position for adjusting adjustable F folder sets fixed upper and lower liquid storage tank and skin
Sample is removed from office, the lower folder adjusting screw of rotation clamps upper and lower liquid storage tank and leather sample counterclockwise, first passes through lower liquid storage tank medical
Syringe fills test buffer, then upper liquid storage tank 1 is filled by injector for medical purpose and marks the molten of trypsase containing FITC
Liquid after 30 min of mass transfer, rotates clockwise lower folder adjusting screw, removes leather sample.
(4) data acquire: suitable from the grain of leather sample to flesh noodles with freezing-microtome in the sampling core of leather sample
Sequence slice, 25 μm of slice thickness, it is strong that sepectrophotofluorometer detects (exciting long light wave 495, launch wavelength 525) slice fluorescence
Degree.Leather sample different depth transverse section is glimmering after Fig. 4 is FITC label 30 min of trypsase (FITC-Trypsin) mass transfer
Light intensity distributions figure.
Embodiment 4:
(1) neutral proteinase (Dispase), thiocyanic acid fluorescein (Fluorescein the preparation of fluorescent marker enzyme: are taken
Isothiocyanate, FITC) it is dissolved in phosphate buffer (0.05mol/L pH7.4) according to the mass ratio of 100:1,4 DEG C
Under be protected from light and be stirred to react 12-14h, after the reaction was completed, freeze-drying after re-dissolving freeze-dried powder, utilizes gel chromatography
(Sephadex G25 filler) removes unreacted FITC, after having separated, the solution of the neutral proteinase of measurement FITC label
The relationship of fluorescence intensity and concentration.
(2) leather sample is cut: taking goat skin ridge, conventional method depilation is cut into the skin of diameter 35mm with round cut-off knife
Remove from office sample.
(3) mass transfer experiment: experimental provision is located at bubble in spirit bubble 5 as shown in Figure 1, adjustment base adjusting bolt
The heart.Take outer diameter 25mm upper liquid storage tank and lower liquid storage tank each one, lower liquid storage tank is packed into the test buffer (0.05 of 80% capacity
The phosphate buffer of mol/L pH7.4), grain upwards covers leather sample on lower liquid storage tank, mouth to mouth by upper liquid storage tank
It is placed on leather sample, keeps concentric with lower liquid storage tank, the lower clamp position for adjusting adjustable F folder sets fixed upper and lower liquid storage tank and skin
Sample is removed from office, the lower folder adjusting screw of rotation clamps upper and lower liquid storage tank and leather sample counterclockwise, first passes through lower liquid storage tank medical
Syringe fills test buffer, then upper liquid storage tank is filled by injector for medical purpose and marks the molten of neutral proteinase containing FITC
Liquid after mass transfer 30min, rotates clockwise lower folder adjusting screw, removes leather sample.
(4) data acquire: in the sampling core of leather sample, fluorescence microscope leather sample longitudinal section fluorescence intensity
Distribution situation.Leather sample longitudinal section is glimmering after Fig. 5 is FITC label 30 min of neutral proteinase (FITC-Dispase) mass transfer
Light microphoto.
Embodiment 5:
(1) neutral proteinase (Dispase), thiocyanic acid fluorescein (Fluorescein the preparation of fluorescent marker enzyme: are taken
Isothiocyanate, FITC) it is dissolved in phosphate buffer (0.05mol/L pH7.4) according to the mass ratio of 100:1,4 DEG C
Under be protected from light and be stirred to react 12-14h, after the reaction was completed, freeze-drying after re-dissolving freeze-dried powder, utilizes gel chromatography
(Sephadex G25 filler) removes unreacted FITC.After having separated, the solution of the neutral proteinase of measurement FITC label
The relationship of fluorescence intensity and concentration.
(2) leather sample is cut: taking goat skin ridge, conventional method depilation is cut into the skin of diameter 50mm with round cut-off knife
Remove from office sample.
(3) mass transfer experiment: experimental provision is located at bubble in spirit bubble as shown in Figure 1, adjustment base adjusting bolt
The heart, take outer diameter 40mm upper liquid storage tank and lower liquid storage tank each one, lower liquid storage tank is packed into the test buffer (0.05 of 80% capacity
The phosphate buffer of mol/L pH7.4), grain upwards covers leather sample on lower liquid storage tank, mouth to mouth by upper liquid storage tank
It is placed on leather sample, keeps concentric with lower liquid storage tank, the lower clamp position for adjusting adjustable F folder sets fixed upper and lower liquid storage tank and skin
Sample is removed from office, the lower folder adjusting screw of rotation clamps upper and lower liquid storage tank and leather sample counterclockwise, first passes through lower liquid storage tank medical
Syringe fills test buffer, then upper liquid storage tank is filled by injector for medical purpose and marks the molten of neutral proteinase containing FITC
Liquid after mass transfer 30min, rotates clockwise lower folder adjusting screw, removes leather sample.
(4) data acquire: in the sampling core of leather sample, with freezing-microtome from leather sample grain to flesh noodles sequence
Slice, 25 μm of slice thickness, sepectrophotofluorometer detects (exciting long light wave 495, launch wavelength 525) and is sliced fluorescence intensity.
Fig. 6 is the fluorescence that FITC marks leather sample different depth transverse section after 30 min of neutral proteinase (FITC-Dispase) mass transfer
Intensity distribution.
Embodiment 6:
(1) trypsase (Trypsin), Rhodamine B isothiocyanate (Rhodamine B the preparation of fluorescent marker enzyme: are taken
IsothioCyanate, RBITC) it is dissolved in carbonic acid buffer (0.05mol/L pH9.0) according to the mass ratio of 100:1,4
It is protected from light at DEG C and is stirred to react 12-14h.After the reaction was completed, it is freeze-dried.After freeze-dried powder is re-dissolved, gel chromatography is utilized
(Sephadex G25 filler) removes unreacted RBITC.After having separated, the solution of the trypsase of measurement RBITC label
The relationship of fluorescence intensity and concentration.
(2) leather sample is cut: ridge goat skin is taken, it is conventional to lose hair or feathers, and the leather of diameter 35mm is cut into round cut-off knife
Sample.
(3) mass transfer experiment: experimental provision is located at bubble in spirit bubble as shown in Figure 1, adjustment base adjusting bolt
The heart, take outer diameter 25mm upper liquid storage tank and lower liquid storage tank each one, lower liquid storage tank is packed into the test buffer (0.05 of 80% capacity
The phosphate buffer of mol/L pH7.4), grain upwards covers leather sample on lower liquid storage tank, mouth to mouth by upper liquid storage tank
It is placed on leather sample, keeps concentric with lower liquid storage tank, the lower clamp position for adjusting adjustable F folder sets fixed upper and lower liquid storage tank and skin
Sample is removed from office, the lower folder adjusting screw of rotation clamps upper and lower liquid storage tank and leather sample counterclockwise；First lower liquid storage tank is passed through medical
Syringe fills test buffer, then upper liquid storage tank is filled by injector for medical purpose and marks the molten of trypsase containing RBITC
Liquid after 30 min of mass transfer, rotates clockwise lower folder adjusting screw, removes leather sample.
(4) preparation of fluorescent marker enzyme: in the sampling core of leather sample, with freezing-microtome from the grain of leather sample
It is sliced to flesh noodles sequence, 25 μm of slice thickness, sepectrophotofluorometer detects (exciting long light wave 495, launch wavelength 525) slice
Fluorescence intensity.Fig. 7 is that RBITC marks leather sample different depth after 30 min of trypsase (RBITC-Trypsin) mass transfer horizontal
The fluorescence intensity profile of slice.
Claims (6)
1. a kind of experimental provision for studying enzyme unidirectional mass transfer in the leather, which is characterized in that by upper liquid storage tank, lower liquid storage
Pond, pedestal, spirit bubble, adjustable F folder, pedestal adjusts bolt and lower folder adjusting screw is constituted；
The upper liquid storage tank and lower liquid storage tank are barrel-like structure open at one end, and upper liquid storage tank and lower liquid storage tank are put mouth to mouth
It sets, leather sample is placed between liquid storage tank and lower liquid storage tank；
The bottom surface quadrangle of the pedestal is equipped with pedestal and adjusts bolt, and upper Plane Installation has spirit bubble；
The adjustable F folder is vertically fixed on pedestal, and the lower folder of F folder is adjusted and slides up and down coarse adjustment the distance between folder up and down,
Folder is equipped with lower folder adjusting screw under adjustable F folder, rotates lower folder adjusting screw for leather sample and is fixed on upper liquid storage tank under
Between liquid storage.
2. a kind of experimental provision for studying enzyme unidirectional mass transfer in the leather according to claim 1, which is characterized in that
The upper liquid storage tank is consistent with the material of lower liquid storage tank, internal diameter, outer diameter and height.
3. a kind of experimental provision for studying enzyme unidirectional mass transfer in the leather according to claim 1, which is characterized in that
The making material of the upper liquid storage tank and lower liquid storage tank is flexible silica gel, and wall thickness is 3 ~ 8mm.
4. a kind of experimental provision for studying enzyme unidirectional mass transfer in the leather according to claim 1, which is characterized in that
The lower liquid storage tank is used to contain the solution containing the enzyme by fluorescent marker for containing test buffer, upper liquid storage tank.
5. a kind of application method of the described in any item experimental provisions of claim 1 ~ 4, which is characterized in that adjustment base adjusts spiral shell
Bolt makes bubble be located at the center of spirit bubble, takes liquid storage tank and lower liquid storage tank each one, and lower liquid storage tank is packed into the reality of 80% capacity
Buffer is tested, grain upwards covers leather sample on lower liquid storage tank, and liquid storage tank is taken to be placed on leather sample mouth to mouth, protects
Position concentric with lower liquid storage tank, to press from both sides under the adjustable F folder of adjusting is held, the lower adjusting screw of pressing from both sides of rotation counterclockwise is by upper liquid storage tank, skin
Leather sample and lower liquid storage tank are fixed；Lower liquid storage tank is first filled into test buffer by injector for medical purpose, then upper liquid storage tank is led to
It crosses the solution that injector for medical purpose fills the enzyme containing fluorescent marker, after mass transfer, removes leather sample, after frozen section, see
Examine and record the distribution situation of leather slice fluorescence intensity.
6. a kind of application method for the studying enzyme experimental provision of unidirectional mass transfer in the leather according to claim 5,
It is characterized in that, the leather sample is any part of Animal Skin.
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