Stability and Specificity of the Cell Wall-Associated Proteinase from Lactococcus lactis subsp. cremoris H2 Released by Treatment with Lysozyme in the Presence of Calcium Ions

Abstract

The cell wall-associated proteinase from Lactococcus lactis subsp. crenwris H2 (isolate number 4409) was released from the cells by treatment with lysozyme, even in the presence of 50 mM calcium chloride. Cell lysis during lysozyme treatment was minimal. The proteinase activity released by lysozyme treatment fractionated on ion-exchange chromatography as three main forms, the molecular masses of which were determined by gel exclusion chromatography and polyacrylamide gel electrophoresis. Two of the enzyme forms released, 137 and 145 kDa, were the same as those released by incubation of cells in calcium-free phosphate buffer. In the * Corresponding author.

The physiology and biochemistry of the proteolytic system in lactic acid bacteria

“…The use of lysozyme to release the proteinase permits the inclusion of Ca 2÷ in the lysis mixture. The use of lysozyme in the presence of Ca 2÷ releases a more stable form of the proteinase which, at 180 kDa, is larger than any of the forms released by the Ca2÷-free release method, although smaller forms identical to those released by the conventional treatment are also present [55]. Since Ca 2÷ is known to stabilize the proteinase following release [46,54], the presence of the 180-kDa form of the enzyme may be due to the stabilizing effect of Ca 2 ÷ in the [ysis buffer so reducing the extent of further autoproteolytic shortening.…”

“…Following release, the proteinase is often found to exist in different molecular mass or antigenically distinct forms [41,50,52,54,55]. It is now clearly established that the different molecular mass forms from any one strain arise by autoproteolysis [54] and are thus artefacts of the release process rather than genetically distinct proteinase types.…”

“…An alternative means of releasing the enzyme from the cell is by lysozyme treatment [55]. Lysozyme treatment possibly effects release by partial digestion of the cell wall, weakening other interactions which are involved in cell wall binding.…”

“…Since the M r of the 180-kDa form is close to that predicted for the entire mature enzyme [32,33,41,42], this form must arise from cleavage at a site close to the membrane anchor. Cell lysis is minimal (less than 1%) using the lysozyme plus Ca 2÷ release procedure [55] so it is unlikely that the 180-kDa form includes the membrane anchor region itself.…”

“…When the proteinase is released using lysozyme in the presence of calcium, successive treatments continue to release further proteinase activity such that the total activity released may be several times greater than that determined originally on the whole cell suspension prior to release [55]. This may simply indicate that steric factors limit access of the casein substrate to surface-located active sites of the proteinase in situ or it may imply that some of the active proteinase molecules are masked in the intact cells and are releascd only when the wall structure is loosened by lyric enzymes.…”