Isolation, purification and characterisation of a novel lysozyme from a rumen ciliate Entodinium caudatum
The rate of breakdown of a range of rumen bacteria by rumen protozoa in whole rumen fluid ranged from 11.5%/h for Butyrivibrio fibrisolvens SH13 to 2.7%/h for Eubacterium ruminantium 2388. When the rates of breakdown of the bacteria were measured using hen's egg white lysozyme, an N-acetylmuramidase, they ranged from 10.1%/h to 1.1%/h. There was correlation between levels of activity in lysozyme and rumen fluid, with R2=0.52 (P>0.01). The rates of breakdown of the rumen bacteria using mutanolysin, also an N-acetylmuramidase were also measured. They ranged from 16.0 to 1.2%/h, and the R2 value was 0.81. When the same bacteria were incubated with an N-acetylglucosaminidase, the rates of breakdown ranged from 3.9 to 0.27%/h, and there was no correlation with the activity in rumen fluid. This implied that the principal bacteriolytic activity in rumen fluid is similar to lysozyme (EC 126.96.36.199). Protozoal lysozyme was partially purified from Entodinium caudatum using a combination of cation exchange and gel filtration chromatography. The partially-purified enzyme resembled other lysozymes in that it was a basic protein which degraded Micrococcus lysodeikticus cell walls and had a high isoelectric point of pI9. It displayed optimal activity at pH 6.5, ionic strength of 0.05 M and at a temperature of 40 °C. It had an apparent affinity constant (Kapp) of 388 mg M. lysodeikticus lysed/I. It had an apparent molecular mass of 14 kDa, from SDS-PAGE, and its N-terminal amino acid sequence slightly resembled that of a distinct class of lysozymes found in Streptomyces species, the fungus Chalaropsis and the protozoan parasite Entamoeba histolytica. Thus the major type of bacteriolytic activity in rumen fluid was found to be a lysozyme-like enzyme which was partially purified and characterised. Further characterisation of this enzyme could provide important information that would be useful in developing a means of preventing wasteful breakdown of bacterial protein in the rumen.