Synergistic Decomposing Of Keratinous Fibers With Chemical-Enzyme System And The Structure Characterization And Performance Analysis Of The Products

Keratin has been widely concerned by researchers due to its abundant sources,low cost,and safety.At present,only some fine and soft wool and down feathers have been directly used in the textile industry.Large amounts of keratinous wastes like leftovers from the wool industry,waste wool fabrics,and animal and human hair are produced annually.They are directly discarded,buried,or burned,causing severe environmental pollution and waste of resources.Commonly used keratin degradation or dissolution methods include chemical,biological,enzymatic,and physical processes.Those methods have disadvantages like severe damage to the backbone structure of keratin chains and amino acids,high reagent costs,or low degradation efficiency.Therefore,it is essential to develop a green,environmentally friendly,efficient,and controllable keratin waste degradation method to"turn waste into treasure."This project takes wool and hair as the research object.Exploring the L-cysteine-protease synergetic system's dissolution and degradation efficiency,products,and action positions on keratin fibers,we reveal the L-cysteine-protease's dissolution or degradation rules and mechanism system on keratin fibers to achieve the goal of controllable separation of different keratin products.The specific research content and results are as follows:(1)Firstly,the mechanism of action of the L-cysteine solution on wool fibers is investigated.The effect of the pH condition of L-cysteine solution on the dissolution rate,dissolution position,and the products are examined and analyzed to reveal the reduction and dissolution regulation of L-cysteine on wool fibers.After wool fibers are treated with L-cysteine solution at varying pH conditions at room temperature,the weight loss rate of wool is examined to evaluate the dissolution efficiency.The morphology of the wool residues is observed to judge the dissolution position of wool fiber.The dissolved keratin's molecular weight,chemical structure,and properties are characterized by SDS-PAGE,FTIR,TG,and DSC,respectively.The results show that,when pH is 7-10,L-cysteine can penetrate into the fiber and the reduction of disulfide bond increased with pH,but there is no significant damage to the fiber;With the increase of pH(11-13.5),the wool dissolution rate increased significantly,and L-cysteine solution can effectively dissolve cortical keratin while retaining the scale structure;the typical bands corresponding to the keratin extracted at pH 11-12(dimer,type I and II keratin,keratin-associated protein(KAP)were retained completely,indicating that disulfide,hydrogen and salt bridge between keratin are broken.When pH?13,the corresponding typical keratin bands are missing,indicating that the keratin peptide bonds are further damaged.Based on this,L-cysteine solution with a pH of 12 replaces the traditional thiol reducing agent and urea system to extract keratin,with a yield of 56.03%,avoiding the use of urea and reducing the cost.(2)Based on elucidating the effect of L-cysteine on wool fiber,the degradation effect,degradation location,and degradation products of L-cysteine,8 M urea,protease Esperase and their different combinations on wool are further investigated to establish a green and efficient keratin fiber degradation system and clarify its degradation rules.The wool fibers are treated with three kinds of reagents and their different combinations at pH 10.15.Then the dissolution rate is evaluated by measuring the weight loss rate of wool,and the structure and performance of the corresponding products in the degraded residue and solution are characterized and analyzed.The morphology and structure of the residues are characterized using SEM,FTIR,X-ray diffraction(XRD),and amino acid composition analysis to judge the degradation position.The products in the hydrolysates are determined by SDS-PAGE and gel permeation chromatography(GPC).The results indicate that single L-cysteine and 8 M urea had no significant degradation effect on wool,and Esperase had a limited degradation effect on wool fiber,resulting in a 25.03%weight loss rate.When L-cysteine is mixed with either urea or Esperase,the weight-loss rates of wool significantly increase to 60.03%and 91.63%,respectively.Combining L-cysteine and 8 M urea could effectively dissolve keratin in the cortex and keep the scales intact.The main products in the solution were keratin and soluble polypeptide.The L-cysteine-Esperase system can effectively degrade keratin fibers,and the residues are cortical cells.The main products in the hydrolysate are free amino acids soluble peptides with a molecular weight of less than 1000 Da.(3)The cuticle is an essential component of wool fiber;it is difficult to dissolve or degrade during cortical keratin extraction.To explore the degradation effect of the L-cysteine-enzyme system on wool cuticles,firstly,the high-purity cuticle cells are isolated,and their structure and properties are characterized and analyzed in detail.Then,the degradation effect of Esperase and Ker BV in the absence or presence of L-cysteine on cuticle are investigated.Cuticle-FA and Cuticle-L+U are separated by the formic acid method and ultrasonic technique,respectively.The physicochemical structures and properties of Cuticle-FA and Cuticle-L+U are characterized by SEM,FTIR,Raman,Solid-state 13C NMR,and TG.Cuticle-FA and Cuticle-L+U are treated with Esperase or Ker Bv in the absence or presence of L-cysteine separately.Then the products in hydrolysates are detected by size exclusion chromatography(SEC)at 212 nm and 280 nm,while the cuticle residues are observed using SEM.Compared to raw wool,Cuticles contains a higher content of?-sheets and random coil structures(Cuticle-FA:80.08%;Cuticle-L+U:52.32%,wool)and better thermal stability;compared to Cuticle-L+U,Cuticle-FA showing higher content of disulfide bond and higher extractable rate.After being treated with single Esperase or Ker Bv,both cuticles remain intact;While in the presence of L-cysteine,both enzymes can degrade both flaky cuticles into smaller fragments and soluble polypeptide and amino acids.(4)The L-cysteine-enzyme degradation system is extended to the separation of the main keratin components of hair,and the hair cuticle removal method based on the L-cysteine-enzyme system is established,then the structure and properties of the cuticle and descaled hairs are characterized and analyzed.A cuticle separation method is established by comparing the degree of hair scale exfoliation by Esperase and Savinase,and cuticle and descaled hair H-E are isolated.Descaled hair H-El and H-SL are isolated using combined L-cysteine and Esperase or Savinase t.The physicochemical structures and properties of the cuticle and three descaled hair are characterized by electron microscopy,FTIR,solid-state 13C NMR,SDS-PAGE,TG,and DSC.The results showed that the pure hair scale can be separated by filtration,centrifugation,and re-suspension after 3-day treatment with Esperase,and the yield is about 3.5%.After introducing L-cysteine,both Esperase and Savinase can effectively remove hair scales within 4 h.The ¹³C CP/MAS NMR spectra show that the content of?-sheet and random structure in scaly keratin is higher(74%),while the relative content of?-helical structure in the keratin of hair and three descaled hair is higher(48%-61%).The extractable rate of the cuticle is only 26%,the corresponding SDS-PAGE show that the molecular weight of the extracted protein is about 10-20 k Da.In comparison,the extraction rate of descaled hair protein and original hair are both 60%.The molecular weight of KAP and keratin remains intact.(5)The separation of melanosomes from keratinous fibers also requires completely degrade keratin.Therefore,the reducing reagent-enzyme system is extended to melanin isolation to establish processing to extract keratin and melanosome.KAP and keratin are successively extracted from hair using the DTT-urea system,and the residues are further degraded by esperase to release the melanosomes.The physical and chemical structures and properties are characterized using SEM,FTIR,13 C NMR,and UV-vis spectroscopy.The results show that yield of melanosomes is 1.3%,and it has a rice-grained structure with the long and short axes of 904±171 nm and 341±75 nm,respectively.The melanosomes have typical indole and pyrrole and aromatic structures and good thermal stability below 150?.Both melanosome suspension and polyacrylamide-melanosomes composite film can effectively filter ultraviolet rays.