Comparative Studies On The Activity Of Enzymes Related To Protein Catabolism In Rumen Bacteria And Protozoa Of Sheep

The aim of the study is to provide enzymology evidence related to the features of ruminal protein catabolism of sheep. Six 1-years old healthy Chinese merino sheep(Xinjiang Type) were choosed. These sheep weighing about 32±1.36 kg were installed with permanent fistula in the rumen. Rumen fluid was collected sequentially before feeding time(0h) and after feeding time1.5h, 3h, 6h, 9h and 12 h, which were repeated 3 times, when forage to concentrate ratio was 30:70.The determination of enzyme activity showed that the activities and specific activities of proteolytic enzyme, GPT, GOT and GDH all had a dynamic change showing an initial increase and then a decrease as the after feeding time passed by and were at a maximum after feeding 1.5h in ruminal bacteria and protozoa extracts of sheep. The dynamic changes of glutamate and NH4+content were similar. The specific activities of these four enzymes involved in protein metabolism in ruminal protozoa extracts were significantly higher than bacteria extracts at every feeding time(P<0.01). The average activity of proteolytic enzyme was significantly lower than bacteria(P<0.01) in each 1ml ruminal fluid bacteria and protozoa.The average activities of GDH and GPT of protozoa were higher than bacteria. The average activity of GOT was significantly higher than bacteria(P <0.01). The Km values showed, Km values for NAD in becteria and protozoa extracts were 2.6×10-7 M and 1.48×10-7 M, Km value of protozoa was smaller. Km values for glutamate in becteria and protozoa extracts were 8.41×10-6 M and 4.91×10-6 M, Km value of protozoa was smaller. Km values for NADH in becteria and protozoa extracts were 3.8×10-8M and 2.7×10-8 M,Km value of protozoa was smaller. Km values for 2-oxoglutarate in becteria and protozoa extracts were 1.16×10-6 M and 2.07×10-6 M, Km value of becteria was smaller. Km values for NH4+ in becteria and protozoa extracts were 2.97×10-5 M and 1.4×10-5 M, Km value of protozoa was smaller.Glutamate content of rumen protozoa extracts was significantly higher than bacteria(P<0.01).NH4+content of rumen protozoa extracts was significantly higher than bacteria(P<0.05). The results showed that the dynamic activity changes of proteolytic enzyme, GPT, GOT and GDH ofruminal bacteria and protozoa cells are similar as the changes of ruminal peptide ammonia and nitrogen concentration and the specific activities of these enzymes in protozoa cells are higher than bacteria. Protein catabolism of protozoa cells is more vigorous and amino acids deamination is more active. There is not only a mechanism re-using ammonia to synthesis amino acids, but also a transamination mechanism of glutamate in rumen protozoa cells.