Purification and characterization of an endopeptidase from Lactococcus lactis subsp. cremoris Wg2.
An endopeptidase has been purified to homogeneity from a crude cell extract of Lactococcus lactis subsp. cremoris Wg2 by a procedure that includes diethyl-aminoethane-Sephacel chromatography, phenyl-Sepharose chromatography, hydroxylapatite chromatography, and fast protein liquid chromatography over an anion-exchange column and a hydrophobic-interaction column. Gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated a molecular mass of the purified enzyme of 70,000 Da. The endopeptidase can degrade several oligopeptides into various tetra-, tri-, and dipeptides. The endopeptidase has no aminopeptidase, carboxypeptidase, dipeptidase, or tripeptidase activity. It is optimally active at pH 6.0 to 6.5 and in the temperature range of 30 to 38 degrees C. The enzyme is inactivated by the chemical agents 1,10-phenanthroline, ethylenedinitrilotetraacetate, beta-mercaptoethanol, and phenylmethylsulfonyl fluoride and is inhibited by Cu2+ and Zn2+. The ethylenedinitrilotetraacetate- or 1,10-phenanthroline-treated enzyme can be reactivated by Co2+. Immunoblotting with specific antibodies raised against the purified endopeptidase indicated that the enzyme is also present in other Lactococcus spp., as well as in Lactobacillus spp. and Streptococcus salivarius subsp. thermophilus.
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Department of Microbiology, University of Groningen, Haren, The Netherlands.
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D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D002364:Caseins / Q000378:metabolism; D002845:Chromatography; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D013294:Lactococcus lactis / Q000201:enzymology*; D008670:Metals / Q000494:pharmacology; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D013696:Temperature
D000596:Amino Acids; D002364:Caseins; D008670:Metals; D010450:Endopeptidases