Alcohol acyl transferase activity assay

Escherichia coli strain C43 (DE3) containing either no plasmid (negative control), pET21a::AAT16, pET21a::AAT16-S99G, or pET21a::AAT16-L178F were grown at 37 °C in LB overnight. Cultures were then inoculated to 1% in 250 mL of TB +/− ampicillin (100 μg/mL) and grown at 37 °C and 250 rpm until an OD₆₀₀ of 1.0 was reached. At this point gene expression was induced with the addition of 0.4 mM IPTG and cultures were further incubated another 16 h at 18 °C. Following this, E. coli cells were harvested through centrifugation (4,000 rpm for 40 min), resuspended in extraction buffer (50 mM Tris HCl pH 8.5, 10% (v/v) glycerol, and 1 mM DTT; Complete™ protease inhibitor tablet-EDTA-free, Roche, Basel, Switzerland), and sonicated on ice until clear. Samples were then centrifuged (4,000 rpm for 20 min) to pellet cell debris, and the crude cell lysates were concentrated using Amicon 10 kDa MWCO centrifugal filters. Protein concentration was determined using the Bradford assay (Bradford, 1976).

Reactions were carried out in a total volume of 500 μL containing: 20 μL crude cell lysate of E. coli strain C43 (DE3) expressing either AAT16, AAT16-S99G, or AAT16-L178F enzyme, buffer (50 mM Tris HCl pH 8.5, 10% (v/v) glycerol, and one mM DTT), 20 mM butanol, and 0.5 mM octanoyl-CoA. Both substrates were present at saturating concentrations. Reactions were incubated at 30 °C and 250 rpm for 30 min before being halted with the addition of 100 μL of 10% (w/v) SDS. Each reaction was then extracted into 150 μL of hexane for future product analysis by GC-MS. All assays were carried out in triplicate.