Purification and characterization of serine proteinase of the Glu,Asp-specific enzyme family from Thermoactinomyces species

Biochemistry (Mosc). 1997 Feb;62(2):171-5.

Abstract

Enzyme catalyzing hydrolysis of a substrate of Glu,Asp-specific proteinases (Z-Glu-pNA) and cleaving bond Glu13-Ala14 in the oxidized insulin B chain was purified to homogeneity from the culture medium of Thermoactinomyces species using hydrophobic chromatography on phenyl-Sepharose CL 4B as the key purification step. The molecular weight of the proteinase is 23 kD. The enzyme is completely inhibited by diisopropyl fluorophosphate and is stable at pH 5-11. The pH optimum for the hydrolysis of azocasein as substrate is 8.5. The temperature optimum for proteolytic activity is 55 degrees C. The N-terminal sequence of the proteinase is: Ser-Val-Leu-Gly-Thr-Asp-Glu-Arg-Thr-Arg-Val-Thr-Asn-Thr-Thr-Thr-Tyr-Pro- Tyr- Trp-.

Publication types

  • Comparative Study

MeSH terms

  • Amino Acid Sequence
  • Binding Sites
  • Enzyme Stability
  • Hydrogen-Ion Concentration
  • Insulin / chemistry
  • Insulin / metabolism
  • Micromonosporaceae / enzymology*
  • Micromonosporaceae / genetics
  • Molecular Sequence Data
  • Molecular Weight
  • Sequence Homology, Amino Acid
  • Serine Endopeptidases / genetics
  • Serine Endopeptidases / isolation & purification*
  • Serine Endopeptidases / metabolism
  • Substrate Specificity
  • Temperature

Substances

  • Insulin
  • Serine Endopeptidases