Chromatography of PBCV WAL lysin

Chromatography of PBCV WAL lysin)

Column is Pall Acrosep Q HyperD (quaternary ammonium). Five ml of sample 2, PBCV WAL lysin (see here) was applied. The column was eluted by step gradients of ammonium acetate (2ml each) of 0.05, 0.1, 0.2, 0.3, and 0.5M. Fractions 6-10 are 0.1M ammonium acetate.

10 microliter aliquots of column fractions were added to 100 microliters of cells suspended in MES plus 2mM CaCl2 and 0.5% Triton X-100. Suspensions were centrifuged 30" at 4500 RPM after incubating 30' at 37C.

See protein gels of these fractions

Note that WAL lysin elutes much earlier (0.1M ammonium acetate) than glycosidases of the soluble fractions

Tests for synergy

All samples contain 100 microliters of cells and 10 microliters of column fractions (mixtures contain 5 microliters each of two fractions). The combination of front and back fractions shows no synergy suggesting that the peak (7) is not the overlap of two activities. The sample on the far right contains the front fraction combined with fraction 23 of "soluble lysin" which contains both glucosidase and glucosaminidase, neither of which apparently synergizes with lysin.