title: Purification and characterization of serine proteinase 2 from Bacillus intermedius 3-19.
abstract: A proteinase secreted in the late stationary phase was isolated from the culture fluid of Bacillus intermedius 3-19 by ion-exchange chromatography on CM-cellulose followed by FPLC on a Mono S column. The enzyme was completely inhibited by the serine proteinase inhibitors diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride. The maximum proteolytic activity against the synthetic chromogenic substrate Z-Ala-Ala-Leu-pNA was observed at pH 9.0. The molecular weight of the enzyme is 28 kD and its isoelectric point is 9.2. We have also determined pH- and thermostability and Km and kcat of this proteinase. The enzyme has been classified as a thiol-dependent serine proteinase. N-Terminal amino acid sequence (10 residues) and amino acid composition of the protein were also determined. By the mode of hydrolysis of peptide bonds in the oxidized B-chain of insulin, this enzyme is similar to the thiol-dependent serine proteinase 1 from B. intermedius 3-19 secreted during vegetative growth.
authors.affiliation: Department of Microbiology, Kazan State University, Kazan 420008, Russia. Nelly.Balaban@ksu.ru
authors.forename: N P
authors.initials: NP
authors.lastname: Balaban
authors.forename: A M
authors.initials: AM
authors.lastname: Mardanova
authors.forename: M R
authors.initials: MR
authors.lastname: Sharipova
authors.forename: L A
authors.initials: LA
authors.lastname: Gabdrakhmanova
authors.forename: E A
authors.initials: EA
authors.lastname: Sokolova
authors.forename: G N
authors.initials: GN
authors.lastname: Rudenskaya
authors.forename: I B
authors.initials: IB
authors.lastname: Leshchinskaya
mesh_terms: D000595:Amino Acid Sequence; D001407:Bacillus / Q000201:enzymology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007531:Isoflurophate / Q000494:pharmacology; D008969:Molecular Sequence Data; D010664:Phenylmethylsulfonyl Fluoride / Q000494:pharmacology; D011972:Receptor, Insulin / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry* / Q000302:isolation & purification* / Q000378:metabolism; D020860:Subtilisin / Q000737:chemistry / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007531:Isoflurophate; D010664:Phenylmethylsulfonyl Fluoride; D011972:Receptor, Insulin; D012697:Serine Endopeptidases; D020860:Subtilisin