Purification of Bacteroides amylophilus protease.
A protease was released by Bacteroides amylophilus cells in late stationary phase, approximately 12 hr after maximum cell density was reached. The protease was concentrated by adsorption on diethylaminoethyl (DEAE)-Sephadex and was purified 532-fold by DEAE-Sephadex chromatography, by G-200-Sephadex gel filtration, and by isoelectric focusing. The purified protease was active between pH 4.5 and 12.0 with optima at pH 6.0 and 11.5. Evidence against there being a single protease was given by the differential inhibition of esterase and protease activities by some inhibitors. There was some evidence that only a single protease was present as the ratio of protease activity at various pH values did not alter significantly during purification or when the purified protease was partially heat-inactivated or treated with two specific trypsin-type protease inhibitors: N-alpha-tosyl-l-lysylchloromethyl ketone or phenylmethane-sulfonyl fluoride. Two forms of the same protease were found by acrylamide gel electrophoresis. Gel filtration confirmed the presence of protease in 30,000 and 60,000 molecular-weight forms. Treatment with 1 mm ethylenediaminetetraacetic acid or with 4 m urea failed to convert the 60,000-molecular-weight to the 30,000-molecular-weight species.
D000179:Acrylates; D000681:Amylases / Q000378:metabolism; D001439:Bacteroides / Q000201:enzymology* / Q000254:growth & development; D002474:Cell-Free System; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003470:Culture Media; D004492:Edetic Acid / Q000494:pharmacology; D004589:Electrophoresis, Disc; D004950:Esterases / Q000378:metabolism; D005782:Gels; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007475:Ion Exchange Resins; D007525:Isoelectric Focusing; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000302:isolation & purification* / Q000378:metabolism; D011480:Protease Inhibitors; D013696:Temperature; D014508:Urea / Q000494:pharmacology
D000179:Acrylates; D003470:Culture Media; D005782:Gels; D007475:Ion Exchange Resins; D011480:Protease Inhibitors; D014508:Urea; D004492:Edetic Acid; D004950:Esterases; D000681:Amylases; D010447:Peptide Hydrolases