Isolation And Identification Of Feather-degradation Strains And Study On The Enzyme-producing Conditions

The growing poultry industry has brought a large amount of feather waste,which is usually buried or burned,resulting in environmental pollution and waste of protein resources.Traditional feather utilization,such as high temperature and high pressure hydrolysis,will consume a lot of energy,which is neither economical nor environmentally friendly.Microbial degradation of keratin,as a natural and efficient way of keratin degradation,has attracted great attention due to its energy saving and environmental protection,and has a broad application prospect.In this paper,44 strains were isolated from soil samples collected from a bamboo-forest scattered chicken farm in Hunan Province and feather powder as sole carbon and nitrogen source.One strain,named WYM39,had the strongest degradation ability of feathers,and could completely degrade 1 g of intact feathers contained in 100 mL liquid feather medium within 4 days.In addition,there is a strain WYM40,which can degrade feathers and decompose cellulose.Because it can be compared with WYM39 which isolated from the same environment,it is also studied in a further step.Combined with physiological and biochemical characteristics,morphological observation and 16s rDNA sequence analysis,it was preliminarily identified that both WYM39 and WYM40 belong to Streptomyces.The optimum enzyme production conditions and enzymatic properties of the strain WYM39 were studied,the results showed that the optimum fermentation temperature was30℃,the optimum fermentation time was 96 h,the optimum initial pH was 7.0,the optimum initial NaCl concentration was 0%.The optimum reaction temperature was 60℃,the optimum reaction pH was 8.0,and 89%of the enzyme activity could be preserved after 30min water bath at 60℃.The enzyme activity of crude enzyme solution could be inhibited when the concentration of Ca²⁺、Mg²⁺and K⁺reached 5 mmol/m L.The enzyme activity of crude enzyme could be increased several times by adding10μL/m Lβ-mercaptoethanol and10μg/mL dithiothreitol to the reaction system.The enzyme activity of crude enzyme solution could be inhibited by PMSF,indicating that it belonged to serine protease.At the same time,both surfactant SDS and organic solvent isopropanol（which can be used as solvent for detergents）can promote the enzyme activity of the crude enzyme solution,which indicates that the keratinase has the potential to be used in detergent industry.In addition,the enzyme production conditions and enzymatic properties of strain WYM40 were studied,the results showed that the enzyme activity in crude enzyme solution was lower than that of strain WYM39,but the enzyme production conditions and enzymatic properties were similar to strain WYM39.Amycolatopsis sp.BJA-103 is a strain isolated from rhizosphere soil of valeriana,which can efficiently degrade feather keratin.In this study,the crude enzyme solution of strain BJA-103 was separated and purified by ammonium sulfate gradient salting out and DEAE-cellulose column chromatography.One of the keratinases which named BJA-103-199was successfully obtained.The amino acid sequence of the whole enzyme was determined by comparing the LC-MS detection results with the theoretical sequence.The enzymatic properties of purified keratinase BJA-103-199 were studied.The results showed that the optimum reaction conditions were determined as follows:60℃,pH 7,and 54%of the enzyme activity could be preserved after 30 min water bath at 60℃,and the activity was inhibited by metal ion(Ca²⁺,Mg²⁺,K⁺,5-500μmol)and PMSF（1 mmol/mL）.β-mercaptoethanol（10μL/mL）and DTT（10μg/m L）can increase the enzyme activity by about twenty times.The purified keratinase can degrade many substrates,including soluble azo-casein,casein,albumin bovine V,bovine hemoglobin,and insoluble keratin azure,feather powder.