Construction Of KerT/Wb600 Keratinase Unhairing System And Investigation Of Its Mechanism

With the increasingly strict environmental requirements of the country,the development and application of cleaning agents and cleaning technology in leather production have attracted extensive attention.Unhairing is important in leather production while it is one of the most serious pollution processes.The unhairing process mainly requires the removing of hair,and the alkali sulfide unhairing method is widely used in tanneries.Although the traditional unhairing method has the advantages of low cost,safe and high efficiency,it causes a lot of pollution in the unhairing process.Due to the requirement of clean production,the biotechnology has attracted attention in the tanning process.In the unhairing process,if the traditional alkali-sulfide,method can be partially or completely replaced by enzyme method,the unhairing process can be clean and green.In this research,the KerT/Wb600 protease obtained by genetic modification is used in the unhairing process,which has high degradability of keratin and low degradability of collagen.Firstly,the enzymatic properties of the KerT/Wb600 protease are studied to explore the optimal conditions of the enzyme,and the hydrolysis ability of KerT/Wb600protease on keratin,collagen and glycoprotein was studied,which provided a reference for the application of the KerT/Wb600 protease in the unhairing process.The unhairing ability of KerT/Wb600 protease is explored by observing the unhairing effect on goatskins,In addition,the amylase was screened,and the combined enzyme unhairing effect of amylase and KerT/Wb600 protease is studied and the co-action mechanism of the two enzymes is investig.Scanning electron microscopy(SEM)and ultra-depth of field microscopy were used to investigate the co-action mechanism of the two enzymes.In the unhairing application,goatskin and cowhide are selected as raw materials,and enzyme-painting and enzyme-bathing unhairing are both used.Moreover,two tanning methods including aldehyde-vegetable combined tanning and chrome-vegetable combined tanning are carried out to explore the influence of enzyme unhairing on tanning.Afterwards,the cow hair waste generated in the unhairing process is used for the preparation of carbon materials,and the carbon is further used to the preliminary dye adsorption application.In the analysis of properties of KerT/Wb600 protease,the optimum activity of the KerT/Wb600 protease is determined at 40?and p H=10.5.It should be considered when design the enzymatic unhairing process.By studying the influence of different concentrations of Ca²⁺on protease activity,it is found that the concentration of Ca²⁺used in conventional liming process will not inhibit the activity of KerT/Wb600 protease.Compared with the conventional 2709 alkaline protease,the collagenase ability of KerT/Wb600 is significantly reduced,which decrease the damage of collagen fiber during unhairing process.In addition,in order to construct a co-action unhairing system with KerT/Wb600 and glycosidase,the unhairing effect of medium-temperature amylase is studied and its synergistic effect with KerT/Wb600 protease is investigated by performing water-bathing unhairing of goatskin in beaker.The results show that KerT/Wb600protease mainly hydrolyzes the keratin in the hair root,while medium-temperature amylase could remove the whole hair follicle from the skin.On the basis of the research on the enzymatic properties of KerT/Wb600 protease,the painting-unhairing of goatskin with KerT/Wb600 protease is carried out,and the synergism of the KerT/Wb600 protease and amylase is explored.The results show that the hair of goatskin can be completely removed by using KerT/Wb600 protease alone(the concentration of enzyme was 320 U/m L)or combined with amylase(the concentration of the two enzymes was 240 U/m L).By observing the physicochemical properties of crust leather which are tanned by aldehyde-vegetable tanning and chromium-vegetable tanning with enzyme unhairing,the tensile strength of aldehyde-vegetable leather is effectively improved owing to enzyme unhairing.The chrome-vegetable leather has high shrinkage temperature and good tear strength.It is due to enzymatic unhairing promotes the binding of tanning agent and functional groups of collagen fiber.The enzyme-bathing unhairing of cowhides is perfermed in leather drum.The results show that KerT/Wb600 protease unhairing can be used on its own and in conjunction with medium temperature amylas to remove hair,and the usage of sulfide is reduced.When the enzyme activities of KerT/Wb600 protease and amylase are 80 U/m L separately with 1%sodium sulfide,high-quality leather with high tensile strength and tear strength can be obtained.Finally,the collected hair waste is used for the preparation of carbon materials,and they are used for dye adsorption.The results show that porous carbon materials with high specific surface area can be obtained by carbonization and activation of cow hair,and it has a certain application prospect in the field of dye adsorption.