ABSTRACT
AIMS: To clone and sequence the pepX gene from Streptococcus thermophilus. METHODS AND RESULTS: Three pairs of primers were used in polymerase chain reactions using as template the total DNA from Strep. thermophilus ACA-DC 4 in order to amplify, clone and sequence the pepX gene. Sequence analysis revealed an open reading frame of 2268 nucleotides encoding a protein of 755 amino acids. The calculated molecular mass of 85 632 Da agreed well with the apparent molecular mass of 80 000 Da previously determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and gel filtration for the monomeric form of the purified enzyme. CONCLUSIONS: The pepX gene from Strep. thermophilus ACA-DC 4 was cloned and sequenced. The PepX protein showed significant sequence similarity with PepX enzymes from other lactic acid bacteria and contained a motif which was almost identical with the active site motif of the serine-dependent PepX family. SIGNIFICANCE AND IMPACT OF THE STUDY: There are economic and technological incentives for accelerating and controlling the process of cheese ripening. To achieve this, starters may be modified by introducing appropriate genes from other food-grade bacteria. New or additional peptidase activities may alter or improve the proteolytic properties of lactic acid bacteria.
Subject(s)
Cheese/microbiology , Dipeptidyl-Peptidases and Tripeptidyl-Peptidases/genetics , Streptococcus/enzymology , Streptococcus/genetics , Cloning, Molecular , DNA Primers , DNA, Bacterial/analysis , Molecular Sequence Data , Sequence Analysis, DNA , Sequence Homology, Amino Acid , Streptococcus/classificationABSTRACT
Lactobacillus delbrueckii subsp. lactis ACA-DC 178, which was isolated from Greek Kasseri cheese, produces a cell-wall-bound proteinase. The proteinase was removed from the cell envelope by washing the cells with a Ca2+-free buffer. The crude proteinase extract shows its highest activity at pH 6.0 and 40 degrees C. It is inhibited by phenylmethylsulfonyl fluoride, showing that the enzyme is a serine-type proteinase. Considering the substrate specificity, the enzyme is similar to the lactococcal PI-type proteinases, since it hydrolyzes beta-casein mainly and alpha- and kappa-caseins to a much lesser extent. The cell-wall-bound proteinase from L. delbrueckii subsp. lactis ACA-DC 178 liberates four main peptides from beta-casein, which have been identified.
Subject(s)
Caseins/metabolism , Endopeptidases/metabolism , Lactobacillus/enzymology , Amino Acid Sequence , Caseins/chemistry , Cell Wall/enzymology , Cheese/microbiology , Endopeptidases/isolation & purification , Hydrogen-Ion Concentration , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Substrate Specificity , TemperatureABSTRACT
An intracellular X-prolyl-dipeptidyl aminopeptidase from Streptococcus thermophilus ACA-DC 4, isolated from traditional Greek yoghurt, was purified by anion exchange and gel filtration chromatography. A single band of molecular weight of about 80,000 appeared in SDS-PAGE; by gel filtration it was shown that the native enzyme was dimeric. The peptidase showed optimum activity on glycyl-prolyl 4-nitroanilide at pH 7.0 and at 50 degrees C, with K(m) = 3.1 mM and Vmax = 3500 U mg-1; over 50 degrees C the enzyme activity declined rapidly. It was inactivated by PMSF; sulfhydryl group reagents and metal chelators had little effect on enzyme activity.
