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Applied and Environmental Microbiology, November 2000, p. 4772-4778, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Streptococcus thermophilus Cell
Wall-Anchored Proteinase: Release, Purification, and Biochemical and
Genetic Characterization
María Dolores
Fernandez-Espla,
Peggy
Garault,
Véronique
Monnet, and
Françoise
Rul*
Unité de Recherche de Biochimie et
Structure des Protéines, Institut National de la Recherche
Agronomique, 78352 Jouy-en-Josas Cedex, France
Received 12 May 2000/Accepted 4 August 2000
Streptococcus thermophilus CNRZ 385 expresses a cell
envelope proteinase (PrtS), which is characterized in the present work, both at the biochemical and genetic levels. Since PrtS is resistant to
most classical methods of extraction from the cell envelopes, we
developed a three-step process based on loosening of the cell wall by
cultivation of the cells in the presence of glycine (20 mM), mechanical
disruption (with alumina powder), and enzymatic treatment (lysozyme).
The pure enzyme is a serine proteinase highly activated by
Ca2+ ions. Its activity was optimal at 37°C and pH 7.5 with acetyl-Ala-Ala-Pro-Phe-paranitroanilide as substrate. The study of
the hydrolysis of the chromogenic and casein substrates indicated that
PrtS presented an intermediate specificity between the most divergent
types of cell envelope proteinases from lactococci, known as the PI and
PIII types. This result was confirmed by the sequence determination of
the regions involved in substrate specificity, which were a mix between
those of PI and PIII types, and also had unique residues. Sequence
analysis of the PrtS encoding gene revealed that PrtS is a member of
the subtilase family. It is a multidomain protein which is maturated and tightly anchored to the cell wall via a mechanism involving an
LPXTG motif. PrtS bears similarities to cell envelope proteinases from
pyogenic streptococci (C5a peptidase and cell surface proteinase) and
lactic acid bacteria (PrtP, PrtH, and PrtB). The highest homologies were found with streptococcal proteinases which lack, as PrtS, one
domain (the B domain) present in cell envelope proteinases from all
other lactic acid bacteria.
*
Corresponding author. Mailing address: Unité de
Recherche de Biochimie et Structure des Protéines, Institut
National de la Recherche Agronomique, 78352 Jouy-en-Josas Cedex,
France. Phone: (33) 1 34 65 21 49. Fax: (33) 1 34 65 21 63. E-mail:
rul{at}jouy.inra.fr.

Present address: Instituto del Frío (CSIC), Departamento de
Cienca y Tecnología de Productos Lácteos, Ciudad
Universitaria,
28040 Madrid,
Spain.
Applied and Environmental Microbiology, November 2000, p. 4772-4778, Vol. 66, No. 11
0099-2240/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
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