Isolation And Identification Of An Efficient Feather-degrading Strain Streptomyces Albidoflavus Fea-10 And Heterologous Expression Of Its Keratinase Gene

Keratinase is a kind of protease,which could degrade recalcitrant keratin?such as feather,hair,wool et al?,widely exist in nature.Keratinase always can be classified as serine protease and metallo protease.Since keratin can not be degraded by normal protease,and it has a veraty of number,there are millions of tons of keratinous wastes produced every year.But these keratinous waste are not be piled up in the nature,the keratinolytic microbial produce keratinase to degrade this waste and release the pressure on the environment.At present,it was reported that bacteria,fungi and actinomycetes could produce keratinase.Some Archaea strains were also reported could be keratinase producer.Keratinolytic microbial can use the low-cost keratinous wastes as cluture substration to generate keratinase.It make keratinase has low production cost,and keratinase have many applications in various industrial,so it has high value in use.A feather degrading strain Fea-10 was isolated from a chicken farm soil.Fea-10 was preliminary identified as Streptomyces albidoflavus.It had high-efficiency feather-degrading activity,and could degrade intact feather.By analysis the N-terminal sequence of some known keratinase in the genome of Fea-10,two possible keratinase genes gm2886 and gm2888 were found.They all had singal,pro-peptide and mature region.Ligated these two genes to pET15b-SUMO and transformed into BL21?DE3?,respectively.There was no protein expression.Replaced the vector and host to pET28 a and Rossta?DE3?,only gm2888 could obtain inclusion body of inactive;Then ligated the proenzyme sequence without singal of these two gene to pET22 b,only 2886 could get a soluble recombination protein,but it didn't have keratinase activity.Considered the Streptomyces expression system was more suitable for keratinase from Streptomyces,and the extracellular environment of Streptomyces could help proenzyme successfully fold to actived mature keratinase.Use the Streptomyces-E.coli shuttle integrated expression plasmid pSET152 as vector,Streptomyces pactum ACT12 as host.Construct the vector of gm2886 with native promoter and erm promoter,respectively.Construct the vector of gm2888 with erm promoter.Transformed into ACT12 by conjugational transfer.Used milk tablet to first screen the zygote,indicated zygote with larger degradation circle into TSBY fermentation,collected supernatant and added ammonium sulfate to sediment protein.Only gm2886 with erm promoter could get recombination protein.The keratinase was purified by nickel affinity chromatography and charactered its enzymatic properties.The optimal temperature and pH of recombinant protein for proteolysis of casein was 50? and pH 10.0,respectively.Metal ion Ca²⁺,Mg²⁺,K⁺ could inhibite the activity of recombinant protein,and PMSF also inhibited the activity of recombinant protein,so it was serine protease.Recombinant protein degraded soluble substrate such as BSA,casein,azo-casein and hemoglobin,as well as insoluble substrate such as feather and azure keratin.