Physiological And Biochemical Mechanism Of Keratin Degradation From Bacterial Stenotrophomonas Maltophilia DHHJ

The keratin waste is a renewable resource,but it has not yet gotten a high value-added applications.A large number of feather wastes have caused pollution in some places.Simple,feasible,economic,effective feather degradation processes are studied to improve the utilization and value of keratin wastes.In this way,we not only protect the environment but also make full use of waste resources.Microbial degradation of feathers is a hot topic with mild conditions,low energy consumption and pollution.At home and abroad the research of its mechanism is seldom.In this work,a novel kerationlytic bacterium,Stenotrophomonas maltophilia DHHJ,which isolated and preserved in our laboratory,was studied for the physiological and biochemical mechanisms of feather degradation including the morphological changes of bacteria and feathers,enzymolysis and purification of keratinase produced by bacterium,physical and chemical properties and mass spectrometry.In the fermentation test,feather is found to be the best one of the inducer feather,casein,and soybean.At the same time,keratinase is also an extracellular enzyme in the trials of extracellular enzymes and intracellular enzymes,while the enzyme of bacterial basal expression plays a certain role in feather degradation.In the feather degradation by S.maltophilia DHHJ,the feathers are basically digested and the large dents existed on the bacterium surface.And also the keratinase was detected in the fermentation broth.All these results indicate that the bacterium endocytosis and the secreted keratinase promote the degradation of feathers.The intracellular enzymes assist in degrading feathers.According to our laboratory conditions,ammonium sulphate precipitation followed by DEAE chromatography is used to purify the keratinase from crude enzyme.For ammonium sulphate precipitation,ammonium sulphate with different concentrations was added to culture filtrate.Considering the enzyme activity,the precipitate resulting from ammonium sulphate concentration between 0%-20%was suitable for the next purification.DEAE chromatography using a step-by-step elution was perfonned equilibrated with 0.05mol/L Tris-HCl?pH 7.8?buffer.Elution was perfonned with the same buffer containing different NaCl concentrations at a flow rate of 2 mL/min.The result shows that the eluent have a higher specific activity in a final concentration of 1 mol/L NaCl Tris-HCl.After two steps in purifition of crude enzyme,the fraction obtained from DEAE chromatography shows 7.02-fold purification,13.17%yield,and 116.28U/mg specific activity.The purified enzyme was analyzed by sodium dodecylsufate polyacrylamide gel electrophoresis?SDS-PAGE?.The results of SDS-PAGE indicated that the purified keratinase is a monomeric enzyme with a molecular mass of 19 kDa.The keratinase was purified to illustrate its physical and chemical characteristics.Keratinase is sensitive to temperature,and it is stable to conserve in the refrigerator at 4?.The loss rate of enzyme activity is 22.2%at 4?,while the keratinase shows no activity at 25? after 4 days.The keratinase activity was stimulated obviously by 10 mmoi/L Ca²⁺,Ba²⁺,organic solvents glycerol and ethanol?concentration between 0-20%?and enhanced slightly by 10 mmol/L Na+.However,the keratinase activity was inhibited by 10 mmol/L Zn²⁺,Cd²⁺,surfactant Tween 80 and SDS.In addition,the inhibition of pattern of ZnCl2 on keratinase activity belongs to competitive one.Compared to feathers,keratinase has a better affinity for casein.The NanoLC-ESI-MS/MS analysis report of keratinase after salting-out indicates that superoxide dismutase,D-alanyl-D-alanine carboxypeptidase,catalase,CKI/VRK protein kinase and binding protein may play a certain part in the process of microbial feather degradation.