Screening And Characterization Of Keratinase Production Microorganism And Preliminary Study In Solid-State Fermentation

Keratin is a kind of protein with compact structure,which is widely distributed in nature.It is difficult to degrade by common proteinase because of the massive amount of intermolecular disulfide bonds.Keratinase derives from microorganisms,it can specially cleave disulfide bonds and turn keratin into peptides as well as amino acids.Analysis shows that million tons of feather waste could be turned into products by taking advantages of keratinase.What else,also protect the environment.Bacillus subtilis KS12,capable to generate keratinase,was isolated from soil samples.Keratinase enzyme activity was obtained through liquid fermentation?46.89 U/mL?.Research on enzymatic properties showed,the extracellular keratinase was stable in a broad range of pH?7.5-9.0?,with optimum at pH 7.5,its enzyme activity was obviously inhibited under acid condition.Optimum temperature for keratinse was 37?,it was not resistant to high temperature,and 50% of keratinase activity was lost after incubated at 60? for 1 hour.The metal ions Ca²⁺ and Mg²⁺?5 mol/L?could enhance keratinase activity.However,study showed that heavy metal ions would generally restrain enzyme activity,especially Cu2+.5% Tween 80 expressed a positive effect on keratinase activity,nevertheless,SDS and H₂O₂ presented apparent inhibition on keratinase.It is believed that organic solvents might be poisonous to keratinase,because none of them show any positive promotion on keratinase activity.In order to further understand the capability of Bacillus subtilis KS12 in fermentation,optimal condition of feather meal and bran mixed solid state fermentation was determined through single factor experiments.The results showed that the best conditions of fermentation were 70% feather meal and 30% bran dosage,55% initial moisture of solid medium,density of Ca?OH?2 and glucose were 0.2% and 3% respectively,10% inoculum dosage of Bacillus subtilis KS12,fermented at 37? for 3 days.The highest keratinase activity?356.34�7.21 U/g?was detected at 72 hours of fermentation,meanwhile,soluble protein content was 60.46�1.69 mg/g.Results of shallow tray fermentation experiment showed,after 36 hours of fermentation,keratinase activity reached 542.46�15.47 U/g,soluble protein content was 45.41�2.27 mg/g.Compared with unfermented medium,simulative vitro digestibility result of fermentation products was improved by 28.1%.