Purification and characterization of proteases from Bacillus amyloliquefaciens isolated from traditional soybean fermentation starter.
Bacillus amyloliquefaciens FSE-68 isolated from meju, a Korean soybean fermentation starter, was identified on the basis of biophysical tests and 16S rRNA gene sequence. A neutral metalloprotease (NPR68) and an alkaline serine-protease (APR68) were purified by ammonium sulfate precipitation and cation exchange chromatography and identified on the basis of their activities at different pH values and the selective protease inhibitors. The molecular weights of NPR68 and APR68 measured with ESI-MS were 32743 (+/- 0.8) and 27443 (+/- 0.5) Da, respectively. Against oxidized insulin chains, the NPR68 has a cleavage preference at the site where leucine is located as a P1' residue followed by phenylalanine, and the APR68 has broad specificity and favors leucine at the P1 site. These results indicate that the proteases are natural variants of subtilisin and bacillolysin.
Cho
Seong-Jun
SJ
Graduate School of Biotechnology, Korea University, Seoul 136-701, Korea.
Oh
Sung-Hoon
SH
Pridmore
R David
RD
Juillerat
Marcel A
MA
Lee
Cherl-Ho
CH
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D001407:Bacillus / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins; D002412:Cations; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004795:Enzyme Stability; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases / Q000737:chemistry / Q000378:metabolism; D045726:Metalloproteases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D010802:Phylogeny; D011480:Protease Inhibitors / Q000494:pharmacology; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013025:Glycine max; D013379:Substrate Specificity; D020860:Subtilisin / Q000737:chemistry / Q000378:metabolism
D001426:Bacterial Proteins; D002412:Cations; D011480:Protease Inhibitors; D010450:Endopeptidases; D045726:Metalloproteases; D012697:Serine Endopeptidases; D020860:Subtilisin; D008666:Metalloendopeptidases; C071298:bacillolysin; D000645:Ammonium Sulfate