Purification and partial characterization of an extracellular serine-proteinase of Streptomyces cyaneus isolated from Brazilian cerrado soil.
Streptomyces cyaneus, a micro-organism isolated from Brazilian cerrado soil, produces an extracellular proteinase (SCP), which was purified 22-fold to homogeneity from culture supernatant fluid, using a single aprotinin-agarose affinity chromatography step. It is produced at a level corresponding to approximately 15% of total protein, but its physiological function has yet to be determined. The molecular mass of this S. cyaneus proteinase was estimated to be 120 kDa by gel filtration high performance liquid chromatography, and it migrates by SDS-PAGE as a single band of 30 kDa. It was optimally active at 25 degrees C and pH 9.0, and was fully inhibited by the serine-proteinase inhibitors PMSF and TPCK. A Km value of 1. 86 x 10-5 mmol l-1, and Vmax of 2.0 x 10-2 mmol l-1 (Abs247 nm microg-1 min-1), were calculated for alpha-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate.
Petinate
S D
SD
Departamento de Microbiologia Geral, Universidade Federal do Rio de Janeiro, Brazil.
Branquinha
M H
MH
Coelho
R R
RR
Vermelho
A B
AB
Giovanni-De-Simone
S
S
D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D008970:Molecular Weight; D012697:Serine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D012988:Soil Microbiology; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature; D014105:Tosyl Compounds / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology
D015842:Serine Proteinase Inhibitors; D014105:Tosyl Compounds; D014108:Tosylphenylalanyl Chloromethyl Ketone; C003614:4-toluenesulfonyl fluoride; D012697:Serine Endopeptidases