Purification and characterization of a proteinase from the probiotic Lactobacillus rhamnosus OXY.
A proteinase produced by the human gastrointestinal isolate Lactobacillus rhamnosus strain OXY was identified and characterized. The prtR2 gene coding for proteinase activity was detected in the examined strain. The PCR primers used were constructed on the basis of the sequence of the prtR2 proteinase gene from Lactobacillus rhamnosus GG. The enzyme was purified by fast protein liquid chromatography (FPLC) using CM-Sepharose Fast Flow and Sephacryl S-300 columns. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the enzyme had a relatively low molecular mass of 60 kD. Protease activity was observed at a pH range from 6.5 to 7.5 with optimum k(cat)/K(m) values at pH 7.0 and 40°C. Maximum proteolytic activity (59 U mL(-1)) was achieved after 48 hr of cultivation. The activity of the enzyme was inhibited only by irreversible inhibitors specific for serine proteinases (PMSF and 3,4-dichloro-isocumarine), suggesting that the enzyme was a serine proteinase. Proteinase activity was increased by Ca(2+) and Mg(2+), and inhibited by Cu(2+), Zn(2+), Cd(2+), and Fe(2+).
Department of Biotechnology, Human Nutrition and Science of Food Commodities, University of Life Sciences, Lublin, Poland. awasko1@tlen.pl
Adam
A
Waśko
Marek
M
Kieliszek
Zdzisław
Z
Targoński
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification*; D002413:Cations, Divalent / Q000737:chemistry; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D041981:Gastrointestinal Tract / Q000382:microbiology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D052201:Lacticaseibacillus rhamnosus / Q000737:chemistry* / Q000201:enzymology / Q000302:isolation & purification; D008970:Molecular Weight; D010447:Peptide Hydrolases / Q000737:chemistry / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D015842:Serine Proteinase Inhibitors / Q000737:chemistry
D001426:Bacterial Proteins; D002413:Cations, Divalent; D017931:DNA Primers; D015842:Serine Proteinase Inhibitors; D010447:Peptide Hydrolases