Centa

Bla specific activity (A_Bla) was determined photometrically as described elsewhere (Bebrone et al. 2001 (link)). CENTA (Sigma-Aldrich/Merck, Germany) was used as substrate and the change in absorption was measured at 405 nm (ε 6400 L mol⁻¹ cm⁻¹) over 1 min. The activity test consisted of 920 μL KPi buffer (100 mmol L⁻¹, pH 7.5), 70 μL CENTA (5 mmol L⁻¹), and 10 μL sample solution.
The influence of the different tags and their varying molecular masses on the activity of Bla is described by the initial catalytic activity $\delta {\overline{A}}_{\mathit{initial}}=\frac{k_{taggedBla}}{k_{untaggedBla}}$ with k_Enzyme = (A_Enzyme · M_Enzyme).
PGA specific activity (A_PGA) was determined photometrically with NIPAB (abcr GmbH, Germany) as substrate measuring the change in absorption at 405 nm (ε 9090 L mol⁻¹ cm⁻¹) over 1 min. The activity test consisted of 800 μL KPi buffer (100 mmol L⁻¹, pH 7.5), 100 μL NIPAB (5 mmol L⁻¹), and 100 μL sample solution.
RjFDH specific activity (A_RjFDH) was determined photometrically via NAD⁺ reduction (Sigma-Aldrich/Merck, Germany) measuring the change in absorption at 340 nm (ε 6300 L mol⁻¹ cm⁻¹) over 1 min. The activity test consisted of 775 μL KPi buffer (100 mmol L⁻¹, pH 7.5), 100 μL potassium formate (1 mol L⁻¹), 25 μL NAD⁺ (100 mmol L⁻¹), and 100 μL sample solution.