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Genetic and biochemical characterization of the Lactobacillus delbrueckii subsp. lactis bacteriophage LL-H lysin
A Vasala1, M Välkkilä, J Caldentey
1Department of Genetics, University of Oulu, Finland.
Abstract:
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.
Insights
A novel bacteriophage lysin, Mur, effectively degrades cell walls of Lactobacillus species. This enzyme, a muramidase, shows optimal activity at pH 5.0 and is stable below 60°C.
Area of Science:
- Microbiology
- Enzymology
- Molecular Biology
Background:
- Lactobacillus delbrueckii subsp. lactis phage LL-H produces a peptidoglycan-degrading enzyme, Mur.
- Mur is effective against cell walls of L. delbrueckii, L. acidophilus, L. helveticus, and Pediococcus damnosus.
Purpose of the Study:
- Clone and sequence the LL-H mur gene in Escherichia coli.
- Purify and biochemically characterize the Mur enzyme produced in E. coli.
Main Methods:
- Gene cloning into E. coli
- Nucleotide sequencing
- Enzyme purification (ammonium sulfate precipitation, cation-exchange chromatography)
- Biochemical characterization (activity assays, N-terminal sequencing, homology analysis)
Main Results:
- The mur gene was cloned and sequenced.
- Mur was purified 112-fold to a 34-kDa protein.
- The enzyme is a muramidase with optimal activity at 30-40°C and pH 5.0.
- The C-terminal domain is non-essential for activity; the N-terminal region shows homology to Chalaropsis-type lysozymes.
- Mur is stable below 60°C.
Conclusions:
- The LL-H lysin Mur is a muramidase effective against various Lactobacillus and Pediococcus cell walls.
- Biochemical characterization provides insights into its catalytic mechanism and stability.
- The enzyme's properties suggest potential applications in biotechnology or probiotics.