GENETIC AND BIOCHEMICAL-CHARACTERIZATION OF THE LACTOBACILLUS-DELBRUECKII SUBSP LACTIS BACTERIOPHAGE-LL-H LYSIN

LL-H, a virulent phage of lactobacillus delbrueckii subsp, lactis, pro duces a peptidoglycan-degrading enzyme, Mur, that is effective on L. d elbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus, and P ediococcus damnosus cell walls, In this study, the LL-H gene mur was c loned into Escherichia coli, its nucleotide sequence was determined, a nd the enzyme produced in E. coli was purified and biochemically chara cterized, Mur was purified 112-fold by means of ammonium sulfate preci pitation and cation-exchange chromatography. The cell wall-hydrolyzing activity was found to be associated with a 34-kDa protein, The C-term inal domain of Mur is not essential for catalytic activity since it ca n be removed without destroying the lytic activity, The N-terminal seq uence of the purified lysin was identical to that deduced from the nuc leotide sequence, but the first methionine is absent from the mature p rotein, The N-terminal part of this 297 amino-acid protein had homolog y with several Chalaropsis-type lysozymes. Reduction of purified and M ur-digested L. delbrueckii cell wall material with labeled (NaBH)-H-3, indicated that the enzyme is a muramidase. The temperature optimum of purified Mur is between 30 and 40 degrees C, and the pH optimum is ar ound 5.0, The LL-H lysin Mur is stable at temperatures below 60 degree s C.