In vitro 6xHis-oMLE activity assays for ME, LDH and MDH function, using the oMLE storage buffer as assay buffer.

(A) ME activity was observed for 6xHis-oMLE in a ME activity assay containing 10 mM malate, 1 mM NAD⁺ and 1 mM. Omission of MnCl₂ reduced activity. (B) MDH activity was observed for 6xHis-oMLE in an assay containing 5 mM malate and 0.5 mM NADH. Activity was greatly increased by the addition of 1 mM MnCl₂. (D) LDH activity was exhibited by 6xHis-oMLE in an LDH assay containing 5 mM pyruvate and 0.5 mM NADH. Activity was unchanged by the addition of 1 mM MnCl₂. All assays used 28.5 μg 6xHis-oMLE per well or the equivalent volume of 6xHis-mock purification negative control and were performed at 45°C in 100 mM HEPES/0.1 mMnCl₂ pH 6.0 buffer. Activity was measured using increase or decrease of light absorbance at 340 nm, correlating to NADH oxidation and NAD⁺ reduction respectively during enzyme activity. Error bars represent standard deviation around the mean from at least three independent experiments.

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