ABSTRACT
Effects of a thiol-dependent serine proteinase of Bacillus intermedius on peptide substrates and insulin B-chain were studied. The enzyme preferably splits peptide bonds formed by carboxyl groups of hydrophobic amino acids. Ca2+ increases the thermal stability of the proteinase significantly. The kinetic characteristics of hydrolysis of Z-Ala-Ala-Leu-pNA by this enzyme was determined as K(m) = 1.25 mM and kcat = 0.15 sec-1. The enzyme has high stability to DMFA and isopropanol, and is able to catalyze peptide bond synthesis.
Subject(s)
Bacillus/enzymology , Serine Endopeptidases/metabolism , Sulfhydryl Compounds/metabolism , Amino Acid Sequence , Calcium/metabolism , Catalysis , Molecular Sequence Data , Sequence Homology, Amino Acid , SolventsABSTRACT
The conditions for growth and serine proteinase biosynthesis were studied in the batch culture of Bacillus intermedius. The synthesis of the enzyme was found to be inhibited by glucose and by the amino acid mixture and activated by the addition of organic compounds-casein and gelatin-to the medium. Inorganic phosphate in the medium increased the enzyme yield. The enzyme activity was shown to be twofold higher upon the addition of corn extract (1.8%) in the medium than in the medium with meat peptone (1.7-2.2%) and inorganic phosphate (0.28-0.30 miligram).
Subject(s)
Bacillus/enzymology , Endopeptidases/biosynthesis , Amino Acids/pharmacology , Caseins/metabolism , Culture Media , Endopeptidases/metabolism , Gelatin/metabolism , Glucose/pharmacology , Hydrogen-Ion Concentration , Peptones/pharmacology , Phosphates/metabolism , Plant Extracts/pharmacology , Protease Inhibitors/pharmacology , Substrate Specificity , Zea mays/chemistryABSTRACT
Extracellular serine proteinase has been isolated from the cultural medium of Bacillus intermedius 3-19 using CM-cellulose chromatography and affinity chromatography on bacitracin-Sepharose. The specificity of the proteinase with a wide range of natural and synthetic substrates has been investigated. The greatest activity was observed with tripeptides containing C-terminal Leu or Phe. The enzyme was completely inhibited by diisopropylfluorophosphate and partly inhibited by thiol-specific reagents. It is concluded that B. intermedius proteinase is a thiol-dependent serine proteinase pertaining to the subtilisin group. The amino acid composition of the enzyme has been determined. The enzyme contains one to three 1/2 cysteine residues, one of which is supposedly a Cys residue. The N-terminal amino acid sequences of the protein is AQTVPYGIPQIKAPA-.
Subject(s)
Bacillus/enzymology , Serine Endopeptidases/metabolism , Amino Acid Sequence , Bacillus/physiology , Chromatography, Affinity , Chromatography, Ion Exchange , Molecular Sequence Data , Serine Endopeptidases/isolation & purification , Spores, Bacterial , Substrate SpecificityABSTRACT
Extracellular alkaline proteinase was isolated from the Bacillus intermedius culture medium. The enzyme was purified by ion-exchange chromatography 200-fold to apparent homogeneity and has a specific activity of 950 u./mg. The proteinase was maximally active at pH 10, 50 degrees C and is stable at pH 6.3-11.0. EDTA, o-phenanthroline or p-chloromercuribenzoate did not affect the enzyme activity, while phenylmethylsulfonyl fluoride inhibited it by 95-97%. It was concluded that the enzyme is subtilisin-like and belongs to the serine proteinase family of Bacilli.
