Lysin activity - summary
Salt (0.3-0.4M) extracts wall associated lysin (WAL) from lysate low speed pellet.
Chlorella cells are resistant to nonionic, anionic and zwitterionic detergents, but sensitive to cationic detergents.
Assay lysin by chlorophyll release from Chlorella cell suspensions in the presence of Triton X-100. Assay suspensions contain ~0.3 to 1OD (665nm) of chlorophyll (1 OD of chlorophyll = 7*10exp7 cells)
Lysate supernatants contain several glycosidases (Alpha-D-NAcglucosamine> alpha-D-glucose> beta-D-galactose). The first two elute separately by ion exchange. None of these exhibit synergism with WAL lysin.
WAL lysin requires:
***Moderate salt concentration
***Calcium ion (~5mM), Strontium ion is equally effective
***Roughly neutral pH (6-8.5) (Ksp Ca(OH)2 = 7.2*10exp(-15))
WAL lysin is insensitive to:
***Oxidizing agents
***PMSF
***Alkylating agents
Antibody to PBCV inhibits WAL lysin as do EGTA, DEPC and proteases (proteinase K, trypsin)
WAL lysin acts on all three exsymbiotic algae, but not on C169
WAL lysin sensitivity: PBi> NC64a> SAG
Washing (NC64a) cells with 1M LiCl increases WAL lysin sensitivity 2-3 fold (presume this holds for all exsymbiotic Chlorella)
WAL lysin activity greater at 37C than at RT. Activity lost above 50C.
Cloned A215L has weak lysin activity as does commercial (crude) alginate lyase.
Commercial "cell wall degrading" enzymes including mixtures show (in general) no lysin activity.
Salt treatment of PBCV1 virus releases lysin active against PBi cells.
WAL lysin attaches to sulfated ion exchange columns. It elutes at several salt concentrations (usually above 0.4M); salt concentration of elution is erratic.
Lysin containing fractions contain multiple proteins (on SDS gels) including proteins roughly the size of PBCV1 capsid protein.
No obvious band in these fractions corresponds to A215L.
In no case has mixing fractions shown synergism.