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Appl. Environ. Microbiol., Nov 1995, 4004-4011, Vol 61, No. 11
Copyright © 1995, American Society for Microbiology

Genetic and biochemical characterization of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H lysin

A Vasala, M Valkkila, J Caldentey and T Alatossava
Department of Genetics, University of Oulu, Finland.

LL-H, a virulent phage of Lactobacillus delbrueckii subsp. lactis, produces a peptidoglycan-degrading enzyme, Mur, that is effective on L. delbrueckii, Lactobacillus acidophilus, Lactobacillus helveticus, and Pediococcus damnosus cell walls. In this study, the LL-H gene mur was cloned into Escherichia coli, its nucleotide sequence was determined, and the enzyme produced in E. coli was purified and biochemically characterized. Mur was purified 112-fold by means of ammonium sulfate precipitation and cation-exchange chromatography. The cell wall- hydrolyzing activity was found to be associated with a 34-kDa protein. The C-terminal domain of Mur is not essential for catalytic activity since it can be removed without destroying the lytic activity. The N- terminal sequence of the purified lysin was identical to that deduced from the nucleotide sequence, but the first methionine is absent from the mature protein. The N-terminal part of this 297-amino-acid protein had homology with several Chalaropsis-type lysozymes. Reduction of purified and Mur-digested L. delbrueckii cell wall material with labeled NaB3H4 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 around 5.0. The LL-H lysin Mur is stable at temperatures below 60 degrees C.

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