Kinetic Analysis of Alginate Lyase Activity

Reactions in 200 µl volumes were set up in triplicate in a 96 well quartz plate. Alginate, polyG, and polyM substrates were in 20 mM Tris, 200 mM NaCl, pH 8 buffer. Substrate concentrations ranging from 4 mg/ml to 0.0625 mg/ml using two-fold serial dilutions were assayed. The quartz plate and substrate were equilibrated to 50°C, followed by the addition of the enzyme to obtain a final protein concentration of 0.25 µg/ml. Initial velocities were determined from each reaction over a ten minute run. To determine enzyme kinetics, the averaged initial velocities in milli-absorbance units (mAU) at 235 nm per minute versus the substrate concentrations were determined. As alginate is a polymer of variable length consisting of random combinations of mannuronic acid and guluronic acid residues, and as both have the same molecular weight (MW), substrate molarity was calculated using the MW of 176 g/mol for each monomer of uronic acid in the polymer (i.e. 194 g/mol monomer MW – 18 g/mol for the loss of H₂O during polymerization). Therefore, 4 mg/ml alginate is 22.7 mM monomer of uronic acid. Product concentrations were determined from the increase in absorbance at 235 nm using the extinction coefficient of 6150 M⁻¹ cm⁻¹[20] (link)–[22] (link). Velocity (V) at the tested substrate concentration was calculated as follows: V (mol/s)  =  (milliAU/min × min/60 sec × AU/1000milliAU × 1 cm)/(6150 M⁻¹ cm⁻¹) × (2 ×10⁻⁴ liters). Substrate molar concentrations and their associated velocity values were input in the Hyper32 program, http://homepage.ntlworld.com/john.easterby/hyper32.html[23] for calculating the maximal velocity, V_max, and the Michaelis-Menten constant, K_m, for each substrate using hyperbolic regression analysis. The turnover number, k_cat, for AlgMsp was calculated as follows: k_cat (s⁻¹)  =  V_max/E, where E is the mols of AlgMsp in the assay. Recombinant AlgMsp has a MW of 36,813 g/mol, therefore, at an enzyme concentration of 0.25 µg/ml, AlgMsp is equivalent to 6.79 nM, which is 1.36 pmol per 200 µl reaction.