普洛药业股份有限公司

● In-house library of amino transferases were screened for the target reaction. ● The best hit gave the desired product enantiomer but with low yield (30% for 10 mM substrate & 100 mM alanine). ● Mutagenesis in the enzyme’s substrate pocket did not improve the activity. ● Random mutagenesis with ep-PCR in combination of RapidFire 460 HT mass spectrometry system resulted in a variant with 5 times higher activity

● In-house nitrilases were screened against the target reaction. Two were found with moderate activity for the hydrolysis of terminal nitrile group. ● The structure of the enzymes were obtained via homologous modeling to help identify substrate pockets. ● The residues in the pocket were subjected to mutagenesis and screening (shown at right). Seventeen variants with 4-11 times improved activity was obtained. ● A variant showed. ● Process was developed to scale up the reaction in 40 kg scale, affording 3 kg product with 99% ee and 73% yield (substrate was recycled via racemization).

● Screening of in-house lipases and esterases to find an active enzyme for the kinetic resolution of a racemic alcohol via esterification. ● The reaction was transferred to chemical process team for scale-up and integration with downstream derivatization.