title: Isolation, purification, and properties of Penicillium charlesii alkaline protease.
abstract: A serine protease with a pH optimum from 7 to 9 and activity over the range of pH 3 to 10 was isolated and purified from culture filtrates of Penicillium charlesii 16 days after inoculation. The enzyme was purified by the following sequence of procedures: (i) gel permeation chromatography through Sephacryl S-200, (ii) DEAE-Sepharose anion-exchange chromatography, and (iii) fast protein liquid chromatography (FPLC) over Superose 12. Anion-exchange chromatography separated the protease activity into a major activity (protease PII, 82%) and two minor activities (proteases PI and PIII, 10 and 8%, respectively, of the total activity). Protease PII has a molecular mass of 44 kilodaltons. Purified preparations of this enzyme are susceptible to autodegradation. FPLC of heat-treated PII gave one major species (PIIa), whereas untreated enzyme resulted in three species (PIIb, PIIc, and PIId). PIIb and PIIc also catalyzed the hydrolysis of protein (hide powder azure). PIIb and PIIc were in the molecular mass range of 10 to 20 kilodaltons. Protease PII is completely inhibited by phenylmethylsulfonyl fluoride (PMSF). The protease has primary substrate specificity for phenylalanyl or arginyl amino acyl residues attached to amines. The enzyme has amidase, but no esterase activity toward similar synthetic substrates such as occurs with trypsinlike microbial serine proteases. The addition of PMSF (final concentration, 10(-4) M) to 1- and 2-day-old cultures of P. charlesii inhibited the production of extracellular peptidophosphogalactomannan (pPGM) by 41 and 34%, respectively, and inhibited the alkaline protease activity by 85%. These results suggest that the production and release of pPGM may be affected by alkaline protease.
authors: Department of Microbiology and Cell Science, University of Florida, Gainesville 32611.
authors: C A
authors: CA
authors: Abbas
authors: S
authors: S
authors: Groves
authors: J E
authors: JE
authors: Gander
mesh_terms: D002851:Chromatography, High Pressure Liquid; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D010407:Penicillium / Q000201:enzymology*; D012697:Serine Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D015842:Serine Proteinase Inhibitors; D013379:Substrate Specificity
chemical_list: D015842:Serine Proteinase Inhibitors; D012697:Serine Endopeptidases