出版社：American Society for Biochemistry and Molecular Biology
摘要：In the present study, we propose a continuous assay for the screening of sn -2 lipases by using triacylglycerols (TAGs) from Aleurites fordii seed (tung oil) and a synthetic TAG containing the α-eleostearic acid at the sn -2 position and the oleic acid (OA) at the sn -1 and sn -3 positions [1,3- O -dioleoyl-2- O -α-eleostearoyl- sn -glycerol ( sn -OEO)]. Each TAG was coated into a microplate well, and the lipase activity was measured by optical density increase at 272 nm due to transition of α-eleostearic acid from the adsorbed to the soluble state. The sn -1,3-regioselective lipases human pancreatic lipase (HPL), LIP2 lipase from Yarrowia lipolytica (YLLIP2), and a known sn -2 lipase, Candida antarctica lipase A (CALA) were used to validate this method. TLC analysis of lipolysis products showed that the lipases tested were able to hydrolyze the sn -OEO and the tung oil TAGs, but only CALA hydrolyzed the sn -2 position. The ratio of initial velocities on sn -OEO and tung oil TAGs was used to estimate the sn -2 preference of lipases. CALA was the enzyme with the highest ratio (0.22 ± 0.015), whereas HPL and YLLIP2 showed much lower ratios (0.072 ± 0.026 and 0.038 ± 0.016, respectively). This continuous sn -2 lipase assay is compatible with a high sample throughput and thus can be applied to the screening of sn -2 lipases.
