Enzymatic Preparation And Application Research Of Soluble Keratin

Keratin has potential application value in the field of biomedical materials due to its excellent biocompatibility and degradability.Because of the large number of disulfide bonds in natural keratin molecules,the solubility of keratin is poor.The preparation of soluble keratin with controllable molecular weight is very important for its biomedical application.At present,as the common methods,physical and chemical ways still have problems such as harsh conditions,large amount consumption of reagent and small keratin molecular fragments,which limit the performance and application of keratin-based materials.Enzymatic preparation of soluble keratin has mild conditions and a relatively controllable process,which is in line with the development direction of green manufacturing.China is a major producer for wool products,and a large amount of wool waste is produced during the production of wool fabrics.The purpose of this study is to prepare macromolecular soluble keratin by double enzyme method using wool as a substrate,and to prepare and characterize PHBV/keratin/AgNPs nanofibrous membranes by electrospinning and in situ reduction methods.The application research on the antibacterial properties and promoting animal wound healing of the above keratin-based materials was carried out.The main research contents of the paper are as follows:?1?The fermentation of keratinase:the composition and fermentation conditions of the medium were optimized to obtain the appropriate conditions for enzyme production,using the single factor and orthogonal experiments.The main components of the medium after optimized were peptone 14 g·L^-1,yeast extract 28 g·L^-1 and glycerol 30 g·L^-1.The incubation temperature was 37°C with an initial pH of 8.0.The enzyme activity in the flask was improved to 5926 U·m L^-1.The batch fermentation resulted in a maximum keratinase activity of 8949 U·m L^-1 at 36 h in a 7-L fermenter.Furthermore,the extracellular keratinase activity attained up to 16860 U·mL^-1 via fed-batch fermentation within 30 h.?2?Enzymatic preparation and crosslinking of keratin:In order to prepare macromolecular keratin and improve its mechanical properties,we hydrolyzed wool to obtain soluble keratin with relative molecular weight of about 25-40 kDa using keratinase.The hydrolysis rate of wool was 58%and the keratin yield was 44.7%.Then the soluble keratin was crosslinked by transglutaminase?TGase?,and the molecular weight of keratin was improved to 120 kDa.Four factors including the p H of keratin solution,the amount of TGase added,the reaction temperature and reaction time were optimized.The crosslinked keratin were characterized by SDS-PAGE,FTIR,CD and TGA,then compared with the soluble keratin hydrolyzed by acid method,alkali method and reducing method.The results shown that the molecular weight and thermal stability of cross-linked keratin?CK?prepared by double enzyme process were better than those prepared by acidic,alkali and reduction method.It was found that cross-lnked keratin can effectively promote cell proliferation and migration,which illustrated great biocompatibility.?3?Preparation and application of keratin biomaterials:the PHBV/CK nanofibrous mats were fabricated by the electrospinning of cross-linked keratin and hydroxybutyl-hydroxyl pentanoic acid copolyeter?PHBV?.The prepared nanofibrous mats demonstrated improved mechanical properties:the tensile strength of the PHBV/CK was improved to 3.1±0.2 MPa.The diameter of the nanofibers are 353±111 nm,and the fibers were randomly connected whose surface was smooth without beads.AgNPs were synthesized on the nanofibers via in situ bioreduction,using the above-mentioned keratinase as the reducing agent.It is demonstrated that the PHBV/CK/AgNPs nanofibrous mats possess favorable antibacterial properties on Escherichia coli and Staphylococcus aureus.The water contact angle of PHBV/CK/AgNPs was 46.6°and the water absorption was 399%,illustrating it is a hydrophilic material.The porous structure and hydrophilicity of the material are conducive to the exchange of nutrients and gases.The nanofiber membrane was used in the culture of L929cells and the cells showed good proliferation after cultivation for 24 h.Moreover,in vivo wound healing assessment,the PHBV/CK/AgNPs membrane displayed great wound healing ability:the wound healing rate was 93.7±1.8%on 14^th day,which was better than positive group.