6xHis-CBU0823 failed to produce detectable lactate in in vitro assays.

(A) Addition of 6xHis-oMLE produced significantly more pyruvate than 6xHis-CBU0823, and both produced significantly more than negative control and blank reactions. (B) Only 6xHis-oMLE yielded lactate amounts significantly above blank. Malic enzyme activity assays (3 mM malate, 5 mM NAD⁺ and 1 mM MnCl₂) were started with either 54 μg 6xHis-CBU0823, 26 μg 6xHis-oMLE, or equivalent volume of mock purification (negative control) or PBS (blank). Reactions were stopped after 20 minutes. Each reaction was analyzed by GC-MS and mean values compared (* p < 0.05, ** p < 0.01, *** p < 0.001). Error bars represent standard deviation from the mean from six independent replicates.