Rhizopus arrhizus lipase

Acyl-CoAs ([1-¹⁴ C]-labeled an unlabeled) used for enzymatic assays were synthesized according to the method developed by Sánchez et al. [47 (link)]. [1-¹⁴C]-fatty acids and triolein [carboxyl-¹⁴C] were purchased from Perkin Elmer or American Radiochemicals. Labeled diacylglycerol was derived through partial lipase treatment of triolein [carboxyl-¹⁴C] with Rhizopus arrhizus lipase (Sigma-Aldrich). The lipase products were separated on TLC plates (silica gel 60; Sigma-Aldrich). The DAG was eluted from the silica with methanol: chloroform (2:1, v/v), extracted with chloroform by Bligh and Dyer method (1959). Non-radioactive fatty acids and diacylglycerol were ordered from Larodan or Sigma-Aldrich. Internal standard of heptadecanoic acid methyl ester, coenzyme A and TAG standard for thin-layer chromatography were purchased also from Sigma-Aldrich.

Neutral lipids were separated by TLC as described above. TAG fractions were eluted from the silica gel with a small volume of chloroform/ methanol (1:2, v/v), adding 0.9% NaCl and centrifuging the mixture in a glass tube. The chloroform phase (TAGs) was transferred to a clean glass tube and dried under N₂. The fatty acid composition of the sn-2 position of TAGs was determined from sn-2 monoacylglycerols (MAG) generated by lipase digestion of the TAGs. The positional analysis of TAG was conducted using Rhizopus arrhizus lipase (Sigma, St. Louis, MO) as described by Bafor et al. [23 (link)]. Half to one mg of TAG was resuspended in 1 mL of diethyl ether and 0.8 mL of buffer containing 0.1 M-Tris/HCl, pH 7.7, 5 mM CaCl₂ and 1,200 units of Rhizopus arrhizus lipase. The mixture was incubated at room temperature for 2–3 h with vigorous shaking. After removal of the diethyl ether with a stream of N₂, the lipids were extracted as described by Bligh & Dyer [19 (link)]. Lipase hydrolysis products (from the CHCl₃ layer) were separated by TLC on silica gel G60 plates with hexane/diethyl ether/acetic acid (70:140:3, v/v/v) [21 (link), 24 (link)]. For fatty acid analysis, lipid fractions of MAG and TAG species were scraped from the TLC plate into glass tubes and FAMEs were prepared and analyzed by GC.

The H1244 transformants bearing CzDGAT1A and CzDGTT5 were each grown in liquid SD/-ura medium containing 2% (w/v) galactose for 18 h at 30 °C, and then harvested for microsome preparation using a French pressure cell (Spectronics Instruments, NY, USA). The detailed procedures were described by Liu et al. [40 ]. The resulting microsomal membrane pellets were resuspended in microsome storage buffer (50 mM Tris–HCl, pH 7.5, 10% glycerol) to give a protein concentration of 10 µg µL⁻¹ for immediate use or stored at − 80 °C.
The in vitro DGAT assay was conducted according to our previously described procedures [40 ]. The acyl-CoAs tested included palmitoyl-CoA (C16:0-CoA), hexadecenoyl-CoA (C16:1-CoA), stearoyl-CoA (C18:0-CoA), oleoyl-CoA (C18:1-CoA), linoleoyl-CoA (C18:2-CoA), α-linolenoyl-CoA (C18:3n3-CoA), γ-linolenoyl-CoA (C18:3n6-CoA), arachidonyl-CoA (C20:4-CoA), eicosapentaenoyl-CoA (C20:5-CoA), and docosahexaenoyl-CoA (C22:6-CoA). The DAGs tested were C18:1/C16:0-, C16:0/C18:1-, and C18:1/C18:1-DAGs. C16:0/C18:1- and C18:1/C18:1-DAGs were purchased from Larodan Fine Chemicals (Malmo, Sweden), whereas C18:1/C16:0-DAG was prepared by partial digestion of C18:1/C16:0/C18:1-TAG (Larodan Fine Chemicals) with Rhizopus arrhizus lipase (Sigma-Aldrich, MO, USA) and recovery of DAG.