Purification, immobilization and characterization of (R)-hydroxynitrile lyase from Prunus amygdalus turcomanica seeds and their applicability for synthesis of enantiopure cyanohydrins


ALAGÖZ D. , TÜKEL S. S. , YILDIRIM D.

JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC, cilt.101, ss.40-46, 2014 (SCI İndekslerine Giren Dergi) identifier identifier

  • Cilt numarası: 101
  • Basım Tarihi: 2014
  • Doi Numarası: 10.1016/j.molcatb.2013.12.019
  • Dergi Adı: JOURNAL OF MOLECULAR CATALYSIS B-ENZYMATIC
  • Sayfa Sayısı: ss.40-46

Özet

A hydroxynitrile lyase (HNL) was purified from wild almond seeds (Prunus amygdalus turcomanica Lincz.) for the first time. Native and subunit molecular masses of the HNL were determined as 100 and 25 kDa, respectively indicating that the enzyme is a homotetramer. The purified enzyme was immobilized onto Eupergit CM and Eupergit C 250 L supports and their lyase and carboligation (synthetic) activities were characterized in terms of optimal pH, temperature and kinetic parameters. While the optimal pH of the free HNL for the lyase activity was 6.0, it was 5.5 for both of the immobilized HNLs. Optimal temperature was determined as 25 degrees C for all HNL preparations. For mandelonitrile cleavage, the apparent K-m - V-max values were 0.38 mM - 197.0 U mg protein-1 for the free HNL, 1.30mM - 26.011mg protein(-1) for HNL immobilized onto Eupergit CM (HNL-Eup CM) and 0.95 mM - 17.5 U mg protein(-1) for HNL immobilized onto Eupergit C 250 L (HNL-Eup C 250 L), respectively. For the carboligation activity, the optimal pH was measured as 4.0 and optimal temperature was determined as 5 C for all of the HNL preparations. For mandelonitrile synthesis, the apparent K-m - V-max values were 14.0 mM -2.70 U mg protein(-1) for the free HNL, 41.0 mM - 0.49 U mg protein-1 for HNL-Eup CM and 38.0 mM - 0.54U mg protein(-1) for HNL-Eup C 250 L, respectively. All of the HNL preparations were employed for the synthesis of mandelonitrile, 2-chloromandelonitrile, 3,4-dihydroxymandelonitrile and 2-hydroxy-4-phenyl butyronitrile in a biphasic tert-butyl methyl ether-citrate buffer (pH 4.0) medium. The results showed that the iminobilized HNL preparations were better than the free HNL in the synthesis of abovementioned cyanohydrins except 2-chloromandelonitrile with higher yields and enantiopurities. (C) 2014 Elsevier B.V. All rights reserved.