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.
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.
Okamoto
H
H
Shionogi Research Laboratories, Shionogi & Co. Ltd, Osaka, Japan.
Fujiwara
T
T
Nakamura
E
E
Katoh
T
T
Iwamoto
H
H
Tsuzuki
H
H
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D018698:Glutamic Acid / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010446:Peptide Fragments; D011480:Protease Inhibitors; D011993:Recombinant Fusion Proteins; D018698:Glutamic Acid; C108269:BSase protein, Bacillus subtilis; D012697:Serine Endopeptidases; D008666:Metalloendopeptidases