Studies on protein engineering of a microbial elastase

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Alkalophilic Bacillus sp. Ya-B strain secretes alkaline elastase which has high activity in the high alkaline range. Bacillus subtilis transformed with a plasmid carrying the alkaline elastase structural gene alone does not result in the production and/or secretion of active elastase. B. subtilis transformed with a plasmid carrying the cloned elastase gene and its 5'-flanking region (about 800 bp), however, can secrete active elastase. We determined the whole nucleotide sequence of the 5'-flanking region and found an open reading frame encoding 49 amino acids. The gene named aleR seems to be necessary for the expression of elastase gene on B. subtilis. We postulate that AleR protein is an activating enzyme of pro-elastase.On the other hand, alkaline elastase is expected as a potential meat tenderizer. It is known that the pH of raw meat is 5-6. We tested the meat tenderizing activity of alkaline elastase at pH 6.0. The enzyme still shows substantial activities toward collagen and elastin with small effects to myofibril. The enzyme, therefore, is proven to be suitable for meat tenderizing use.Finally we studied the relationship between the structure and activity of elastase. Alkaline elastase has a sequence homology of 55% to subtilisin BPN' and subtilisin E. From the comparison of primary amino acid sequence, we introduced 4 acid amino deletion into subtilisin E, and observed a distinct alteration of subtilisin E to an elastase-like enzyme.