Partial purification and immobilization of a new (R)-hydroxynitrile lyase from seeds of Prunus pseudoarmeniaca

Hydroxynitrile lyase (HNL) from seeds of Prunus pseudoarmeniaca was partially purified by (NH4)2SO4 fractionation and covalently immobilized onto Eupergit C and Eupergit C 250 L. The percentages of bound protein per gram of Eupergit C and Eupergit C 250 L were about 81 and 98 of the initial amount of protein, respectively. Km and Vmax values were determined 2.23 mM and 0.54 U/mg prot. for the free HNL, 1.60 mM and 0.87 U/mg prot. for the immobilized HNL onto Eupergit C and 1.03 mM and 0.35 U/mg prot. for the immobilized HNL onto Eupergit C 250 L, respectively at optimized reaction conditions. The half lives (t1/2) and the thermal inactivation rate constants (ki) of free and immobilized HNLs were determined at 25 and 50 °C, immobilized HNLs displayed higher thermal stability. Carboligation activities of free and immobilized HNLs for (R)-mandelonitrile (R-MN) synthesis were also determined. Besides, reusabilities of immobilized HNLs for both lyase and carboligation activities were investigated by using batch type reactors.