CN103439500A - Visible tracking and detecting method for protease in leather treatment process - Google Patents
Visible tracking and detecting method for protease in leather treatment process Download PDFInfo
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Abstract
The invention discloses a visible tracking and detecting method for protease in a leather treatment process. The visible tracking and detecting method comprises the following steps of firstly, under an alkaline condition, marking protease through fluorescein isothiocyanate (FITC) to obtain FITC marker enzyme, then treating a skin block without furs with the marker enzyme under room temperature and shading conditions, and preparing a slice from the skin block treated by the marker enzyme to observe a distribution state of the enzyme in the skin under a fluorescence microscope. According to the visible tracking and detecting method, the permeability of the enzyme in the skin and the acting degree of the enzyme to the skin can be detected according to the strong fluorescence characteristic of the FITC, and a permeation mode of the enzyme in the skin can be obtained according to the difference of treatment time. The enzyme firstly permeates into the skin through pores and the peripheries of hair follicles and then gradually diffuses into a skin substrate to act on fibrous stroma proteins. According to the visible tracking and detecting method, a theoretical basis is supplied to the application of the enzyme to manufacturing of leather; enzyme with proper molecular weight is selected for treating collagen according to fiber structures of the collagen in different treatment stages of manufacturing of leather, and the controllable treatment of enzyme on the leather protein and the collagen is realized by analyzing the permeability and the acting degree of the enzyme in the collagens.
Description
Technical field
The present invention relates to the detection method of a kind of enzyme in materials with hide glue fibril, be specifically related to the visual tracking detection method of a kind of proteinase in leather is processed.
Background technology
Along with being on the rise of curriery pollution problem, the environment-friendly type process hides has caused process hides researcher's extensive concern with the research of material.Wherein, enzyme preparation is as a kind of material of efficient, environmental protection; applicating history in process hides for a long time, mainly utilizes hydrolytic enzyme to remove the non-collagen tissues such as protein in skin, fat, is infiltration and the combination of material in the later stage operation (the Swarna V.Kanth for preparing; R.Venba; B.Madhan; N.K.Chandrababu; S.Sadulla.Cleaner tanning practices for tannery pollution abatement:Role of enzymes in eco-friendly vegetable tanning.J.Clean Prod., 2009,17:507 – 515; Puvanakrishnan R.Ecofriendly lime and Sulfide free enzymatic dehairing of skins and hides using a bacterial alkaline protease.Chemosphere., 2008,70:1015 – 1024).
Animal Skin is as a kind of insoluble special substrate; to protect the integrality of hide fibre structure in whole process hides process; the influence factor that enzyme is processed skin is a lot; except the impact of enzyme action condition, to it, the infiltration in skin and hydrolysis fiber interstitial thereof have very important effect to enzyme molecular weight.Such as seepage velocity and the degree of enzyme in skin of different sized molecules amounts is variant, and then cause its difference of hydrolysis to skin; Can select the enzyme that is applicable to molecular weight by compound synergy according to the special construction of the different operation materials with hide glue fibril of process hides, reach better treatment effect.Therefore relate to the research of molecular weight scheduling theory and will the application in leather provide the theoretical direction effect to enzyme.
In process hides, use in the research of enzyme preparation, in leather enzyme processing procedure, the penetration degree of enzyme in skin changes along with the variation of time, if the enzyme infiltration state in skin that has a kind of method can detect the different molecular weight size reaches interfibrillar substance effect degree in skin, will the applied research in process hides provide very broad theory support to enzyme.The earliest, someone transfers to the proteinase of different levels in the enzymatic depilation pigskin on casein, enzyme reacts with casein and produces white dot, length of penetration and quantity (Yang Y.Z.Test of enzyme in dehaired pelt.China Leather by the known enzyme of spot, 2002,31:20 – 22), still, deactivated proteinase in processing for enzyme, this method is difficult to detect its perviousness.
Summary of the invention
The object of the present invention is to provide the visual tracking detection method of a kind of proteinase in leather is processed, the method is applicable to various proteinase, and the different time that can process at skin is observed infiltration and the distribution of enzyme in skin, can be used for penetration mode and the seepage velocity of enzyme molecule in skin of comparison different molecular weight simultaneously.
For achieving the above object, the technical solution used in the present invention comprises the following steps:
1) take the damping fluid that the pH value is 7.0～9.0 is solvent, prepares respectively enzyme solutions and fluorescein isothiocynate solution;
2) to dripping fluorescein isothiocynate solution in enzyme solutions, mix, in the reaction system that makes to obtain, the mass ratio of enzyme and fluorescein isothiocynate is 100:(0.5～5.0), then in 4 ℃ of lower lucifuges, react 10h, obtain reactant liquor;
3) reactant liquor is packed into molecular interception scope is less than in the bag filter of enzyme molecular weight, then the damping fluid concussion dialysis that to put into the pH value be 7.0～9.0 is not until there is free fluorescein isothiocynate in the lye that dialysis obtains, stop dialysis, now in bag filter, residue is FITC marker enzyme solution;
4) damping fluid that is 7.0～9.0 by the pH value after the taking-up of the FITC marker enzyme solution in bag filter is diluted, obtaining concentration is the FITC marker enzyme solution after the 0.4-1mg/mL dilution; To the dilution after FITC marker enzyme solution in put into cut the hair skin bit, in the water-bath oscillator, in the room temperature lucifuge, shake, after finishing, concussion takes out the skin bit of processing, the skin bit surface of then processing with distilled water flushing, to remove free fluorescein isothiocynate, obtains the skin bit after the FITC marker enzyme is processed; Skin bit freezing-microtome freezing microtome section after the FITC marker enzyme is processed, and section is locked on microslide in order in the distribution of fluorescence microscopy Microscopic observation enzyme in skin.
Damping fluid in described step 1), step 3) and step 4) is phosphate buffer or glycocoll-sodium hydrate buffer solution.
Enzyme in described step 1) is 2709 alkali proteases, Genecor TanG proteinase, Genecor SoakL proteinase or Genecor LimeG proteinase.
The concentration of the enzyme solutions in described step 1) is 2mg/mL, and the concentration of fluorescein isothiocynate solution is 1mg/mL.
In described step 3), when dialysis concussion changes at interval of 2h the damping fluid that 1 pH value is 7.0～9.0; While stopping dialysing, the lye that the concussion dialysis obtains is 0 in 480nm place absorbance.
The skin bit that in described step 4), every 10-30g cuts hair adds the FITC marker enzyme solution after 20-50mL dilution.
Described step 4) is cut the skin bit of hair and is made with skin by process hides.
In described step 4), process hides is ox-hide or the sheepskin after pre-immersion fleshing with skin.
In described step 4), the time of lucifuge concussion is 12h-24h.
The mixed liquor of the phosphate buffer that the mounting medium that in described step 4), section is used during sealing is glycerine and pH value=7.4, and in mixed liquor, the volume ratio of the phosphate buffer of glycerine and pH value=7.4 is 9:1.
Compared with prior art, beneficial effect of the present invention is:
The present invention utilizes the isothiocyano in fluorescein isothiocynate (FITC) molecule to react the amino key of formation sulphur carbon with the amino in the zymoprotein molecule under alkali condition, even and enzyme loses activity, because its structure does not change, therefore, no matter be that the activated enzyme of tool or deactivated enzyme all can pass through the FITC mark.Because FITC has strong fluorescence, and FITC labelled protein enzyme shows yellow green under fluorescent microscope; Therefore, utilize the proteinase of FITC mark no matter be that tool is activated, or deactivated, can both process infiltration and the distribution of different time enzyme in skin at fluorescence microscopy Microscopic observation skin, can be used for penetration mode and the seepage velocity of comparison different molecular weight enzyme molecule in skin simultaneously; Through evidence, according to the penetration mode of the known enzyme of the difference in processing time in skin, be: at first the enzyme molecule by penetrating in skin around pore and hair follicle, then is spread in scytoblastema matter the effect of being hydrolyzed of interfibrillar substance albumen gradually.Therefore, the present invention can utilize FITC labelled protein enzyme to process skin bit and can realize that the infiltration in skin reaches the visual tracking detection to the skin effect degree to enzyme.
The accompanying drawing explanation
The profile fluorescent microscope photo that Fig. 1 is ox-hide after FITC mark Genecor TanG enzyme processing ox-hide of the present invention; Wherein, a is the ox-hide profile fluorescent microscope photo that in embodiment 1, FITC mark Genecor TanG enzyme is processed 12h, and b is the ox-hide profile fluorescent microscope photo that in embodiment 2, FITC mark Genecor TanG enzyme is processed 24h.
Embodiment
Embodiment 1:
1) take the phosphate buffer that the pH value is 8.0 is solvent, prepares respectively the Genecor TanG protein enzyme solution of 2mg/mL and the FITC solution of 1mg/mL;
2) slowly drip the FITC solution of 0.2mL and shake up in the Genecor TanG protein enzyme solution of 10mL, in the reaction system that makes to obtain, the mass ratio of Genecor TanG proteinase and FITC is 100:1, then, in 4 ℃ of lower lucifuge reaction 10h, obtains reactant liquor;
3) reactant liquor is packed into molecular interception scope is less than in the bag filter of Genecor TanG protease molecule amount, then the phosphate buffer concussion dialysis that to put into the pH value be 8.0, change at interval of 2h the phosphate buffer that 1 pH value is 8.0 in the earthquake dialysis procedure, until the lye that dialysis obtains is 0 in 480nm place absorbance, show not have free FITC in the lye that obtains of dialysis, stop dialysis, now in bag filter, residue is FITC mark Genecor TanG enzyme solutions;
4) phosphate buffer that is 8.0 by the pH value after the taking-up of the FITC mark Genecor TanG enzyme solutions in bag filter is diluted to 45mL, obtaining concentration is the FITC mark Genecor TanG enzyme solutions after the 0.44mg/mL dilution; To the dilution after FITC mark Genecor TanG enzyme solutions in put into 22g cut the hair the ox-hide piece, shake 12h in the room temperature lucifuge in the water-bath oscillator, then finish concussion and take out the ox-hide piece of processing, the ox-hide piece surface of processing with distilled water flushing again, to remove free FITC, obtains the ox-hide piece after FITC mark Genecor TanG enzyme is processed; Ox-hide piece freezing-microtome freezing microtome section after FITC mark Genecor TanG enzyme is processed, then with the mixed liquor of the phosphate buffer of glycerine and pH value=7.4 as mounting medium, section is locked on microslide in order in the distribution of fluorescence microscopy Microscopic observation enzyme in ox-hide; Wherein, in mixed liquor, the volume ratio of the phosphate buffer of glycerine and pH value=7.4 is 9:1.
Embodiment 2:
1) take the phosphate buffer that the pH value is 8.0 is solvent, prepares respectively the Genecor TanG protein enzyme solution of 2mg/mL and the FITC solution of 1mg/mL;
2) slowly drip the FITC solution of 0.2mL and shake up in the Genecor TanG protein enzyme solution of 10mL, in the reaction system that makes to obtain, the mass ratio of Genecor TanG proteinase and FITC is 100:1, then, in 4 ℃ of lower lucifuge reaction 10h, obtains reactant liquor;
3) reactant liquor is packed into molecular interception scope is less than in the bag filter of Genecor TanG protease molecule amount, then the phosphate buffer concussion dialysis that to put into the pH value be 8.0, change at interval of 2h the phosphate buffer that 1 pH value is 8.0 in the earthquake dialysis procedure, until the lye that dialysis obtains is 0 in 480nm place absorbance, show not have free FITC in the lye that obtains of dialysis, stop dialysis, now in bag filter, residue is FITC mark Genecor TanG enzyme solutions;
4) phosphate buffer that is 8.0 by the pH value after the taking-up of the FITC mark Genecor TanG enzyme solutions in bag filter is diluted to 45mL, obtaining concentration is the FITC mark Genecor TanG enzyme solutions after the 0.44mg/mL dilution; To the dilution after FITC mark Genecor TanG enzyme solutions in put into 22g cut the hair the ox-hide piece, shake 24h in the room temperature lucifuge in the water-bath oscillator, then finish concussion and take out the ox-hide piece of processing, the ox-hide piece surface of processing with distilled water flushing again, to remove free FITC, obtains the ox-hide piece after FITC mark Genecor TanG enzyme is processed; Ox-hide piece freezing-microtome freezing microtome section after FITC mark Genecor TanG enzyme is processed, then with the mixed liquor of the phosphate buffer of glycerine and pH value=7.4 as mounting medium, section is locked on microslide in order in the distribution of fluorescence microscopy Microscopic observation enzyme in ox-hide; Wherein, in mixed liquor, the volume ratio of the phosphate buffer of glycerine and pH value=7.4 is 9:1.
In Fig. 1 a-b, the part of dotted arrow indication is yellow green, and this yellow green is partly the distribution of FITC mark Genecor TanG enzyme in ox-hide; Wherein, Fig. 1 a is the ox-hide profile fluorescence photo that in embodiment 1, FITC mark Genecor TanG enzyme is processed 12h, can find out that Genecor TanG enzyme mainly concentrates on around pore and hair follicle, illustrate in the initial stage Genecor TanG proteinase master of enzyme processing penetrates into kraft fibers by pore.Fig. 1 b is the skin profile fluorescence photo that in embodiment 2, FITC mark Genecor TanG enzyme is processed 24h, can find out Genecor TanG enzyme diffusion between the hide fiber beyond pore.After processing 12h and 24h by contrast Genecor TanG enzyme, the distribution of Genecor TanG enzyme in ox-hide is known: at first Genecor TanG enzyme molecule by penetrating in skin around pore and hair follicle, then is spread in scytoblastema matter the effect of being hydrolyzed of interfibrillar substance albumen gradually.
Embodiment 3:
1) take glycocoll-sodium hydrate buffer solution that the pH value is 9.0 is solvent, prepares respectively the Genecor LimeG protein enzyme solution of 2mg/mL and the FITC solution of 1mg/mL;
2) slowly drip the FITC solution of 0.5mL and shake up in the Genecor LimeG protein enzyme solution of 10mL, in the reaction system that makes to obtain, the mass ratio of Genecor LimeG proteinase and FITC is 100:2.5, in 4 ℃ of lower lucifuge reaction 10h, obtain reactant liquor again;
3) reactant liquor liquid is packed into molecular interception scope is less than in the bag filter of Genecor LimeG protease molecule amount, then put into the pH value and be 9.0 glycocoll-sodium hydrate buffer solution concussion dialysis, change at interval of 2h glycocoll-sodium hydrate buffer solution that 1 pH value is 9.0 in the earthquake dialysis procedure, until the lye that dialysis obtains is 0 in 480nm place absorbance, show not have free FITC in the lye that obtains of dialysis, stop dialysis, now in bag filter, residue is FITC mark Genecor LimeG enzyme solutions;
4) glycocoll-sodium hydrate buffer solution that is 9.0 by the pH value after the taking-up of the FITC mark Genecor LimeG enzyme solutions in bag filter is diluted to 30mL, obtaining concentration is the FITC mark Genecor LimeG enzyme solutions after the 0.6mg/mL dilution; FITC mark Genecor LimeG enzyme after dilution is put into the sheepskin piece that 10g cuts hair in molten, shake 24h in the room temperature lucifuge in the water-bath oscillator, then finish concussion and take out the sheepskin piece of processing, the sheepskin piece surface of processing with distilled water flushing again, to remove free FITC, obtains the sheepskin piece after FITC mark Genecor LimeG enzyme is processed; Sheepskin piece freezing-microtome freezing microtome section after FITC mark Genecor LimeG enzyme is processed, then with the mixed liquor of the phosphate buffer of glycerine and pH value=7.4 as mounting medium, section is locked on microslide in order in the distribution of fluorescence microscopy Microscopic observation enzyme in sheepskin; Wherein, in mixed liquor, the volume ratio of the phosphate buffer of glycerine and pH value=7.4 is 9:1.
Embodiment 4:
1) take the phosphate buffer that the pH value is 7.0 is solvent, prepares respectively the Genecor SoakL protein enzyme solution of 2mg/mL and the FITC solution of 1mg/mL;
2) slowly drip the FITC solution of 1.0mL and shake up in the Genecor SoakL protein enzyme solution of 10mL, in the reaction system that makes to obtain, the mass ratio of Genecor SoakL proteinase and FITC is 100:5, then, in 4 ℃ of lower lucifuge reaction 10h, obtains reactant liquor;
3) reactant liquor is packed into molecular interception scope is less than in the bag filter of Genecor SoakL protease molecule amount, then the phosphate buffer concussion dialysis that to put into the pH value be 7.0, change at interval of 2h the phosphate buffer that 1 pH value is 7.0 in the earthquake dialysis procedure, until the lye that dialysis obtains is 0 in 480nm place absorbance, show not have free FITC in the lye that obtains of dialysis, stop dialysis, now in bag filter, residue is FITC mark Genecor SoakL enzyme solutions;
4) phosphate buffer that is 7.0 by the pH value after the taking-up of the FITC mark Genecor SoakL enzyme solutions in bag filter is diluted to 50mL, obtain concentration and be FITC mark Genecor SoakL enzyme solutions after the 0.4mg/mL dilution and put into the ox-hide piece that 25g cuts hair in the FITC mark Genecor SoakL enzyme solutions after dilution, shake 12h in the room temperature lucifuge in the water-bath oscillator, then finish concussion and take out the ox-hide piece of processing, the ox-hide piece surface of processing with distilled water flushing again is to remove free FITC, obtain the ox-hide piece after FITC mark Genecor SoakL enzyme is processed, ox-hide piece freezing-microtome freezing microtome section after FITC mark Genecor SoakL enzyme is processed, then with the mixed liquor of the phosphate buffer of glycerine and pH value=7.4 as mounting medium, section is locked on microslide in order in the distribution of fluorescence microscopy Microscopic observation enzyme in ox-hide, wherein, in mixed liquor, the volume ratio of the phosphate buffer of glycerine and pH value=7.4 is 9:1.
Embodiment 5:
1) take the phosphate buffer that the pH value is 8.0 is solvent, prepares respectively 2709 alkaline protease solutions of 2mg/mL and the FITC solution of 1mg/mL;
2) slowly drip the FITC solution of 0.1mL and shake up in 2709 alkaline protease solutions of 10mL, in the reaction system that makes to obtain, the mass ratio of 2709 alkali proteases and FITC is 100:0.5, then, in 4 ℃ of lower lucifuge reaction 10h, obtains reactant liquor;
3) reactant liquor is packed into molecular interception scope is less than in the bag filter of 2709 basic protein enzyme molecular weights, then the phosphate buffer concussion dialysis that to put into the pH value be 8.0, change at interval of 2h the phosphate buffer that 1 pH value is 8.0 in the earthquake dialysis procedure, until the lye that dialysis obtains is 0 in 480nm place absorbance, show not have free FITC in the lye that obtains of dialysis, stop dialysis, now in bag filter, residue is the alkaline enzyme solutions of FITC mark 2709;
4) phosphate buffer that is 8.0 by the alkaline enzyme solutions of FITC mark 2709 in bag filter by the pH value is diluted to 20mL, and obtaining concentration is the alkaline enzyme solutions of FITC mark 2709 after the 1mg/mL dilution; To the dilution after the alkaline enzyme solutions of FITC mark 2709 in put into 10g cut the hair the ox-hide piece, shake 18h in the room temperature lucifuge in the water-bath oscillator, then finish concussion and take out the ox-hide piece of processing, the ox-hide piece surface of processing with distilled water flushing again, to remove free FITC, obtains the ox-hide piece after the alkaline enzyme of FITC mark 2709 is processed; Ox-hide piece freezing-microtome freezing microtome section after the alkaline enzyme of FITC mark 2709 is processed, then with the mixed liquor of the phosphate buffer of glycerine and pH value=7.4 as mounting medium, section is locked on microslide in order in the distribution of fluorescence microscopy Microscopic observation enzyme in ox-hide; Wherein, in mixed liquor, the volume ratio of the phosphate buffer of glycerine and pH value=7.4 is 9:1.
Embodiment 6:
1) take glycocoll-sodium hydrate buffer solution that the pH value is 9.0 is solvent, prepares respectively the Genecor LimeG protein enzyme solution of 2mg/mL and the FITC solution of 1mg/mL;
2) slowly drip the FITC solution of 0.5mL and shake up in the Genecor LimeG protein enzyme solution of 10mL, in the reaction system that makes to obtain, the mass ratio of Genecor LimeG proteinase and FITC is 100:2.5, in 4 ℃ of lower lucifuge reaction 10h, obtain reactant liquor again;
3) reactant liquor liquid is packed into molecular interception scope is less than in the bag filter of Genecor LimeG protease molecule amount, then put into the pH value and be 9.0 glycocoll-sodium hydrate buffer solution concussion dialysis, change at interval of 2h glycocoll-sodium hydrate buffer solution that 1 pH value is 9.0 in the earthquake dialysis procedure, until the lye that dialysis obtains is 0 in 480nm place absorbance, show not have free FITC in the lye that obtains of dialysis, stop dialysis, now in bag filter, residue is FITC mark Genecor LimeG enzyme solutions;
4) glycocoll-sodium hydrate buffer solution that is 9.0 by the pH value after the taking-up of the FITC mark Genecor LimeG enzyme solutions in bag filter is diluted to 30mL, obtaining concentration is the FITC mark Genecor LimeG enzyme solutions after the 0.6mg/mL dilution; FITC mark Genecor LimeG enzyme after dilution is put into the sheepskin piece that 30g cuts hair in molten, shake 24h in the room temperature lucifuge in the water-bath oscillator, then finish concussion and take out the sheepskin piece of processing, the sheepskin piece surface of processing with distilled water flushing again, to remove free FITC, obtains the sheepskin piece after FITC mark Genecor LimeG enzyme is processed; Sheepskin piece freezing-microtome freezing microtome section after FITC mark Genecor LimeG enzyme is processed, then with the mixed liquor of the phosphate buffer of glycerine and pH value=7.4 as mounting medium, section is locked on microslide in order in the distribution of fluorescence microscopy Microscopic observation enzyme in sheepskin; Wherein, in mixed liquor, the volume ratio of the phosphate buffer of glycerine and pH value=7.4 is 9:1.
In above-described embodiment 1,2 for the Genecor TanG proteinase of preparing Genecor TanG protein enzyme solution purchased from outstanding can company of section, in embodiment 3,6, the Genecor LimeG proteinase of preparation Genecor LimeG protein enzyme solution is purchased from company of outstanding person's energy section, in embodiment 4, the Genecor SoakL proteinase of preparation Genecor SoakL protein enzyme solution is purchased from company of outstanding person's energy section, and in embodiment 5,2709 alkali proteases of preparation 2709 alkaline protease solutions are purchased from the magnificent prosperous and powerful Bioisystech Co., Ltd in east, Beijing.
The ox-hide piece of cutting hair in embodiment of the present invention 1-2,4-5 step 4) is to select the ox-hide after pre-immersion fleshing to make; The sheepskin piece of cutting hair in embodiment 3,6 step 4) is to select the sheepskin after pre-immersion fleshing to make.In addition, the molecular interception scope of the bag filter adopted in step 3) of the present invention can be chosen according to the molecular weight of enzyme, and molecule is held back and should be less than enzyme molecular weight, guarantees that the enzyme molecule can not see through.
The present invention is according to process hides different disposal stage materials with hide glue fibril structure, can select the enzyme of suitable molecular weight to process collagen, because selected enzyme is crossed through the FITC mark, therefore, can be under fluorescent microscope by enzyme analysis the perviousness in collagen and effect degree, reaching enzyme provides the purpose of theoretical foundation to the controlled processing of lime-preserved egg bletilla collagen.In molecular biosciences, fluorescein, usually used as a kind of probe tracking Study on Protein or enzyme of visual research, has been applied to (Rita A in a lot of research; Carla S; Raul M; Margarida C; Antonio M.Cunha; Jos é Carlos Rodriguez-Cabello; ArturCavaco-Paulo.Proteolytic Enzyme Engineering:A Tool for Wool.Biomacromolecules., 2009,10,1655 – 1661).Fluorescein isothiocynate (FITC) can react with the amino on zymoprotein because of the reactive group on its molecule, therefore through being commonly used for the mark of protein and enzyme.FITC has hyperfluorescenceZeng Yongminggaoyingguang, when excitation wavelength is 490-495nm, at fluorescent microscope, can observe yellowish green material, or, under UV-irradiation, (Celine H can detect by an unaided eye; Dudal J.L.; Salima P.; Olivier G.; Emmanuel P.Fluorescein isothiocyanate-labeled human plasma fibronectin in extracellular matrix remodeling.Anal.Biochem., 2008,372:62 – 71).
Claims (10)
1. the visual tracking detection method of a proteinase in leather is processed, is characterized in that, comprises the following steps:
1) take the damping fluid that the pH value is 7.0～9.0 is solvent, prepares respectively enzyme solutions and fluorescein isothiocynate solution;
2) to dripping fluorescein isothiocynate solution in enzyme solutions, mix, in the reaction system that makes to obtain, the mass ratio of enzyme and fluorescein isothiocynate is 100:(0.5～5.0), then in 4 ℃ of lower lucifuges, react 10h, obtain reactant liquor;
3) reactant liquor is packed into molecular interception scope is less than in the bag filter of enzyme molecular weight, then the damping fluid concussion dialysis that to put into the pH value be 7.0～9.0 is not until there is free fluorescein isothiocynate in the lye that dialysis obtains, stop dialysis, now in bag filter, residue is FITC marker enzyme solution;
4) damping fluid that is 7.0～9.0 by the pH value after the taking-up of the FITC marker enzyme solution in bag filter is diluted, obtaining concentration is the FITC marker enzyme solution after the 0.4-1mg/mL dilution; To the dilution after FITC marker enzyme solution in put into cut the hair skin bit, in the water-bath oscillator, in the room temperature lucifuge, shake, after finishing, concussion takes out the skin bit of processing, the skin bit surface of then processing with distilled water flushing, to remove free fluorescein isothiocynate, obtains the skin bit after the FITC marker enzyme is processed; Skin bit freezing-microtome freezing microtome section after the FITC marker enzyme is processed, and section is locked on microslide in order in the distribution of fluorescence microscopy Microscopic observation enzyme in skin.
2. the visual tracking detection method of proteinase according to claim 1 in leather is processed, it is characterized in that: the damping fluid in described step 1), step 3) and step 4) is phosphate buffer or glycocoll-sodium hydrate buffer solution.
3. the visual tracking detection method of proteinase according to claim 1 in leather is processed, it is characterized in that: the enzyme in described step 1) is 2709 alkali proteases, Genecor TanG proteinase, Genecor SoakL proteinase or Genecor LimeG proteinase.
4. the visual tracking detection method in leather is processed according to the described proteinase of claim 1 or 3, it is characterized in that: the concentration of the enzyme solutions in described step 1) is 2mg/mL, the concentration of fluorescein isothiocynate solution is 1mg/mL.
5. the visual tracking detection method of proteinase according to claim 1 in leather is processed is characterized in that: in described step 3), change at interval of 2h the damping fluid that 1 pH value is 7.0～9.0 during the concussion dialysis; While stopping dialysing, the lye that the concussion dialysis obtains is 0 in 480nm place absorbance.
6. the visual tracking detection method of proteinase according to claim 1 in leather is processed is characterized in that: the skin bit that in described step 4), every 10-30g cuts hair adds the FITC marker enzyme solution after the 20-50mL dilution.
7. the visual tracking detection method in leather is processed according to the described proteinase of claim 1 or 6, it is characterized in that: described step 4) is cut the skin bit of hair and is made with skin by process hides.
8. the visual tracking detection method in leather is processed according to claim 7 or described proteinase is characterized in that: in described step 4) process hides with skin ox-hide or the sheepskin after for the fleshing that soaks in advance.
9. the visual tracking detection method of proteinase according to claim 7 in leather is processed, it is characterized in that: in described step 4), the time of lucifuge concussion is 12h-24h.
10. the visual tracking detection method of proteinase according to claim 1 in leather is processed, it is characterized in that: the mixed liquor of the phosphate buffer that the mounting medium that in described step 4), section is used during sealing is glycerine and pH value=7.4, and in mixed liquor, the volume ratio of the phosphate buffer of glycerine and pH value=7.4 is 9:1.
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