FIG. 2.

Effect of pH toward LpNAL activity. (A) pH profile for the Neu5Ac synthesis (▪) and hydrolysis (•) activity of LpNAL determined by HPLC. Assay conditions at 37°C were 100 mM mannosamine, 30 mM pyruvate, and 87 μg enzyme for synthetic assay and 10 mM Neu5Ac and 1.5 μg for hydrolytic assay. The buffers (20 mM) used were sodium acetate (pH 5 to 5.5), sodium phosphate (pH 6 to 8), Tris-HCl (pH 8.5 to 9), and glycine (pH 9.5 to 11.5). (B) pH stability profile in the synthetic direction. Samples were analyzed after 15 days of incubation at 37°C in the different pH media to determine residual activity under the standard reaction, using 100 mM mannosamine and 30 mM pyruvate as substrates. Buffers used were the same as above.