Fig. 4

Substrate specificity of WS and DGAT activities of Thraustochytrium roseum WS/DGAT proteins TrWSD4 and TrWSD5. Alcohol specificity of WS activity of TrWSD4 (a) and TrWSD5 (b) was determined by using the substrates of 30 μM palmitoyl-CoA and 100 μM linear alcohols of different chain lengths. Reactions were performed at 37 °C in mixtures including 25 mM sodium phosphate buffer (pH 7.4) and 1 mg/mL DTNB. The DGAT activity of TrWSD4 (c) and TrWSD5 (d) relative to WS activity of the corresponding recombinant enzyme is shown as percentage. The purified recombinant enzymes were used for WS and DGAT activity assays with the substrates of 15 μM palmitoyl-CoA together with 100 μM hexadecanol (WS activity) or 100 μM DAG (DGAT activity). Acyl-CoA specificity of DGAT activity of TrWSD4 (e) and TrWSD5 (f) was determined by using the substrates of DAG (100 μM) and acyl-CoAs with different chain lengths. Reactions were performed at 37 °C in mixtures including 25 mM sodium phosphate buffer (pH 7.4) and 1 mg/mL DTNB. Data represent mean values of three replicates ±S.D