Hydroxynitrile Lyase of Wild Apricot (Prunus armeniaca L.): Purification, Characterization and Application in Synthesis of Enantiopure Mandelonitrile

Abstract

Hydroxynitrile lyases (HNLs) are increasingly finding application in synthesis of enantiomerically pure cyanohydrins. Cyanohydrins are important intermediates in the production of pharmaceuticals and agrochemicals. In the present studies seeds of wild apricot (Prunus armeniaca L.) have emerged as potential source of hydroxynitrile lyase. The HNL of wild apricot (ParsHNL) was purified 8.1 fold and 18.2 % yield with a specific activity of 141 units mg−1 protein. The SDS-PAGE of the enzyme revealed that it consists of subunits of 40 and 37 kDa. However, the molecular weight of holoenzyme was assessed to be 360 kDa. The enzyme showed maximum activity in 0.1 M sodium-citrate buffer having pH 4.75 at 25 °C. Thermostability studies revealed that this HNL showed activity up to 70 °C temperature and quite stable up to 50 °C. Activation energy of ParsHNL was calculated to be 37.83 kJ mol−1. This enzyme has K m of 3.76 mM, V max of 188.4 µmol mg−1 min−1 and k cat of 1130.4 s−1 using mandelonitrile as substrate while for reverse reaction using benzaldehyde as substrate it showed K m of 16.1 mM, V max of 7.21 µmol mg−1 min−1 and k cat of 43.3 s−1. Synthesis of mandelonitrile was carried out using ParsHNL and finally 8.88 mmole (1.184 g) of mandelonitrile was recovered which corresponded to 89 % molar conversion with 96 % ee for R-mandelonitrile. The yield of mandelonitrile was 411 µmol mg−1h−1. These results indicated that ParsHNL has very high potential for synthesis of cyanohydrins and can be used for the production of enantiopure cyanohydrins.