Complexity of the Native Cell Wall Proteinase of Lactococcus lactis subsp. cremoris HP and Purification of the Enzyme

Abstract

The native cell wall-associated proteinase P 1 from Lactococcus lactis subsp. cremoris HP (basonym: Streptococcus cremoris HP) is a high molecular weight component of complex structure. Two electrophoretically distinguishable, proteolytically active components (1 and 2) were released from this organism when milk-grown cells were resuspended in a calcium-free buffer at pH 6.5 and 25 °C. These components may disintegrate to give several catalytically active derivatives of distinguishable, lower molecular weight. It appeared that component 2 (molecular weight about 126.000) is a more stable derivative of component 1 (molecular weight about 133.000). The smallest proteolytically active derivative identified, has a molecular weight of about 60.000. Proteinase P 1 can be purified rapidly and with acceptable activity yields (45 %), albeit mainly as component 2, using the affinity adsorbent carbobenzoxy-D-phenylalanine-triethylene-tetramine-sepharose. A similar result, with respect to the component isolated and the efficiency of the procedure, was obtained by means of co-precipitation of the enzyme during dialysis followed by Sepharose CL-6B gel filtration. The enzyme is a serine proteinase with a pH optimum around 6.4. Ca 2+ (and Mn 2+ ) ions confer thermostability on the enzyme. Bivalent cations (possibly Ca 2+ ) appear to have an essential structural function in as far as they maintain the catalytic(ally) (most efficient) conformation. A working hypothesis is presented to account for the observations and to describe both the stabilization of the proteinase in a complex structure and the integration of this complex within the peripheral cell wall network.