Isolation, Identification Of Feather Keratin Degrading Bacteria And Cloning, Expression Of Keratinase Gene

Four feather-degrading strain bacteria, isolated and screened from soil and sludge of long-time deposit of waste feather and feather processing plants, were classified as Stenotrophomonas sp, Microbacterium sp, Bacillus sp and Chryseobacterium sp. Stenotrophomonas strain showed the strongest feather-drgrading ability among the isolated microorganisms. Phylogenetic analysis, cloning and expression of keratinase gene were studied of the strain.The 16S rDAN gene sequences for Stenotrophomonas strain was amplified and sequenced. The results showed that the complete 16S rDAN gene sequence was 1509bp. A phylogenetic tree was constructed by comparing the 16S rDAN sequence of the strain (GenBank number: FJ765513) with other relaive bacteria species in the GenBank database. In the phylogenetic tree, the studied strain, Stenotrophomonas maltophilia strain 1.22 constitute a branch with the similarity value 99.8%. According to its morphological, physiological and biochemical characteristics, and phylogenetic analysis, the microorganism was identified as Stenitrophomonas maltophilia strain YHYJ-1.The gene kerD encoding keratinase was cloned by PCR. Sequences analysis reveals that the sequences with the length of 1905bp encoding a polypeptide of 634 amino acid residues. (G+C)% was 68.9%, and with a signal pepetide. The alignment result of kerD protein indicates that it belongs to PA superfamily with the homology to serine protease. Via blast, the deduced amino acid sequence of this keratinase showed 28-35% identities to the previously reported keratinase gene, which shared high homology with the deduced keratinase sequence from S. maltophilia R551-3. The gene kerD is a novely kind of keratianse firstly reported from S.maltophilia. Rrecombinant plasmid(pET-kerD) was constructed and transformed into E.coli BL21 to express in order to verify the function of the gene kerD. A preliminary analysis of keratinase in E.coli BL21 showed that this keratinse was highly expressed in E.coli BL21 with the endocellular enzyme activity (10.5U/mL) induced after 4 hour, which proved this kerD had the activity of keratinase. Its optimal reaction temperature and pH were 50℃and 7.8, respectively.Several kinds of feather-degrading microorganisms were isolated in this study,which provided experimental materials for feather degrading.It was first reported that keratinase gene was cloned from S.maltophilia. The result we got laid solid base for the further research on keratinase from S. maltophilia YHYJ-1 and provided valuable figures for research on the relationship between structure and function of keratinase and helped to clarify the catalytic mechanisim of keratinase from the molecular level.