Purification and immobilization of hydroxynitrile lyase from different fruits seeds

In this study, we aimed to purify and characterize hydroxynitrile lyase (HNL) from the seeds of three wild type and three cultivated type of Prunus fruits which have not been used as HNL sources up to now, to immobilize onto Eupergit CM by covalently or cross-linked to obtain cross-linked HNL aggregates (HNL-CLEA), and to investigate the application of free and immobilized HNLs for the kinetic resolution of (R/S)-mandelonitrile and the synthesis of (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxy mandelonitrile and (R)-2-hydroxy-4-phenylbutyronitrile. Thermal and storage stabilities of all HNL preparations were also determined and the reusabilities of immobilized HNLs in batch type reactor were investigated. Purified HNLs were consist of four identical subunits and the molecular weight of each one is 25 kDa. Of free HNLs the highest Vmax value was determined as 197.2 U/mg prot. for wild type almond and the highest Vmax values were determined as 126.2 U/g Eup CM and 48 U/g CLEA for cultivated apricot HNL-Eupergit CM and cultivated apricot HNL-CLEA, respectively. Immobilized HNL preparations showed better thermal stabilities at 50°C and storage stabilities at 5°C than those of corresponding free HNLs. The highest reusability was obtained with the cultivated plum HNL-Eupergit CM and the productivity of 20 mg sample was determined as 4x103 U/g Eup CM when the remaining activity was 50% of its initial activity. Using free HNLs of different types, the highest (R)-mandelonitrile, (R)-2-chloromandelonitrile, (R)-3,4-dihydroxy mandelonitrile, R)-2-hydroxy-4-phenylbutyronitrile yields were determined as 91, 66, 95, 88% , respectively and the highest enantiomeric excess (ee%) values were obtained as 94, 47, 99, 94%, respectively. Better results were obtained with immobilized HNLs, especially in the case of HNL-CLEAs for the synthesis of above mentioned enantiopure cyanohydrins.