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

• HNL was purified from Prunus amygdalus turcomanica seeds for the first time.
• PatHNL was determined to be homotetramer in native form.
• Immobilized HNLs was prepared for the enantiopure cyanohydrin synthesis.
• Immobilized HNLs achieved the synthesis of cyanohydrins with a higher yield and enantiopurity.

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 °C for all HNL preparations. For mandelonitrile cleavage, the apparent Km – Vmax values were 0.38 mM – 197.0 U mg protein−1 for the free HNL, 1.30 mM – 26.0 U mg 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 Km – Vmax 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 – 0.54 U 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 immobilized HNL preparations were better than the free HNL in the synthesis of abovementioned cyanohydrins except 2-chloromandelonitrile with higher yields and enantiopurities.

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