Purification and characterization of a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051; application to the recovery of bioactive peptides from fusion proteins by sequence-specific digestion |
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Authors: | H Okamoto T Fujiwara E Nakamura T Katoh H Iwamoto H Tsuzuki |
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Institution: | (1) Shionogi Research Laboratories, Shionogi & Co. Ltd. 5-12-4, Sagisu, Fukushima-ku, Osaka 553, Japan Tel.: (06) 458 5861, Fax: (06) 458 0987, JP |
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Abstract: | Screening cultures of nonpathogenic microorganisms led us to a glutamic-acid-specific endopeptidase from Bacillus subtilis ATCC 6051, which we purified and named BSase. The nucleotide sequence encoding BSase, with a molecular mass of 23 894 Da,
completely agreed with that of the mpr gene, which had been reported by Rufo Jr. and Sloma et al. to encode a metalloprotease J Bacteriol (1990) 172:1019–1023
and 1024–1029 respectively]. However, enzymatic characterization revealed it to have the catalytic triad of a serine protease
and not the consensus sequence of a metalloprotease, and it was inhibited by diisopropylfluorophosphate. We therefore consider
BSase (mpr) to be a serine protease. In the alignment of the acidic-amino-acid-specific proteases, the proteases from bacilli have a
highly conserved histidine residue, which is most important in the histidine triad in the proteases from streptomycetes. Furthermore,
Ca2+ was necessary for its activity and stability. BSase cleaved the C-terminal glutamic acid with high specificity and was very
stable over a wide pH range. On the basis of these properties, we tried to retrieve a bioactive peptide from a fusion protein
by sequence-specific digestion, and succeeded in obtaining the bioactive peptide. BSase was found to be very useful as a tool
for selective cleavage.
Received: 24 December 1996 / Received revision: 3 February 1997 / Accepted: 22 February 1997 |
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