A new and efficient colorimetric high-throughput screening method for triacylglycerol lipase directed evolution

A new and efficient method was developed for the first time using the targeted substrate, triacylglycerol, for the rapid screening of a large library of mutants for lipase directed evolution. The high-throughput method applies calcium chloride to convert the fatty acid produced from hydrolysis reaction to hydrochloric acid which is easy to release H+ detected by pH indicator. This new method exhibits significant advantages compared with the conventional method of applying a substituent of the actual targeted substrate, p-nitrophenyl palmitate as a high-throughput screening substrate. The results determined by the new method correlate well with those obtained from alkali titration and HPLC analysis. By applying the new method, a significantly enhanced mutant enzyme was obtained with lipase activity increased more than five-fold compared to wild type after only two cycles of directed evolution. In addition, the esterification activity of the mutant was also improved and the regioselectivity of the lipase was not changed.

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► The new high-throughput method applies calcium chloride to convert the fatty acid produced from hydrolysis of triacylglycerol reaction to hydrochloric acid which is easily detected by pH indicator.
► The new high-throughput screening method was successfully implemented in the directed evolution of RML and a significantly enhanced mutant enzyme was obtained.
► This new method exhibits significant advantages compared with the conventional method of applying a substituent of the actual targeted substrate, p-nitrophenyl palmitate as a high-throughput screening substrate.