NUCLEOTIDE-SEQUENCE AND EXPRESSION OF KERA, THE GENE ENCODING A KERATINOLYTIC PROTEASE OF BACILLUS-LICHENIFORMIS PWD-1

Bacillus licheniformis PWD-1 (ATCC 53757) secretes keratinase, a proteolytic enzyme which is active on whole feathers, By amino acid sequence similarity and phenylmethylsulfonyl fluoride inhibition, the keratinase was demonstrated to be a serine protease, The entire nucleotide sequence of the coding and flanking regions of the keratinase structure gene, kerA, was determined, A fixed oligonucleotide primer derived from the N-terminal sequence of the purified enzyme and a second random oligonucleotide primer were used in a procedure called PCR walking, which was developed to amplify and sequence the upstream and downstream regions of kerA, Another method, PCR screening, was conducted with a lambda phage vector with inserted PWD-1 genomic DNA fragments as templates and with the known sequences of the vector arms and the N-terminal sequence of the enzyme as primers, PCR amplification and sequence analysis of the lambda library completed the entire kerA sequence and established a set of gene deletions, The kerA gene shares a 97% sequence identity with the gene encoding subtilisin Carlsberg from B. licheniformis NCIMB 6816. The putative promoters, ribosome binding sites, and transcriptional terminators are also similar in these two bacteria, The deduced amino acid sequences indicate only three amino acid differences between the two mature proteases, Northern (RNA) analysis demonstrates that transcriptional regulation controls kerA expression on different growth media.