Study On The Properties And Tissue Location Of Hydroxynitrile Lyase From Amygdalus Pedunculatus Pall

Cyanohydrin is an important organic intermediate for the synthesis of fine chemicals and pharmaceuticals.Hydroxynitrile lyases?HNLs? is an important biological enzyme used to catalyze the asymmetric synthesis of cyanohydrin,because these enzymes exhibit high catalytic efficiency and are very economical.The crude extract from the Amygdalus pedunculata Pall seeds contains 20-30% protein and is a potential source of plant protein.In the present study,the purification route of HNL was established.enzymatic kinetics using mandelonitrile as substrate,the effects of temperature,pH,and metal ions on enzymatic activity,as well as the application of catalytic synthesis of cyanohydrin and nitroethanol was studied.In addition,the antibody of APHNL were prepared,and the distribution of APHNL during its growth and development was studied by western blot and immunohistochemical localization.The details are as follows:1.Study on purification technology route.The HNL from A.pedunculata Pall?APHNL? was purified 138 fold and 4.20%yield with a specific activity of 661 U/mg by crude extraction,ammonium sulfate salting out,anion exchange chromatography column,hydrophobic chromatography column and ultrafiltration centrifugation.SDS-PAGE result showed the enzyme to be present as a monomer and its molecular weight was near 61 kDa.2.Study on enzymatic properties.The study of temperature and pH on the stability of APHNL activity showed that APHNL is a heat-resistant protein with activity up to 80?,stable up to 60? and denaturation temperature as high as 91.4? and it can exist stably at pH 6.0-12.The study of oxalate ion on enzyme activity showed that the enzyme activity decreased with the increase of oxalate concentration.The study of metal ions on enzyme activity showed that Fe³⁺,Ba²⁺and Fe²⁺had almost no effect on enzyme activity.Mn⁴⁺,Mg²⁺,Cd²⁺,Al³⁺inhibited partial enzyme activity.Other metal ions showed slight inhibition.Ag⁺ and Hg²⁺ had obvious inhibition on the activity.APHNL has a Km of 0.5 mM,Vmax of 665.9?mol mg^-1 min^-1,Kcat of 676.5 s^-1 and Kcat/Km of 1353 s^-1 mM^-1 using mandelonitrile as substrate,which indicated that APHNL is a potential biological enzyme.3.Study on catalytic activity.Asymmetric cyanohydrin was synthesized by using benzaldehyde,m-phenoxybenzaldehyde and HCN as substrates and APHNL as catalyst.Molarconversionof?R?-mandelonitrileand?R?-2-Hydroxy-2-?3-phenoxy-phenyl?-acetonitrile were 90% and 98% with 94%and 93% ee,respectively.Nitroethanol was synthesized by using benzaldehyde,m-nitrobenzaldehyde,p-nitrobenzaldehyde and nitromethane as substrates and APHNL as catalyst.When benzaldehyde was used as a substrate,the product 1-phenyl-2-nitroethanol was formed,but it had no chirality and was an external racemate.The other two aldehydes and nitromethane did not react.The results showed that the APHNL had catalytic selectivity and was an excellent biocatalyst for the synthesis of cyanohydrin enantiomers.4.Study on tissue location.New Zealand rabbit was immunized by 95% electrophoretic pure APHNL to obtain positive serum which was purified by affinity purification to get polyclonal antibody.SDS-PAGE showed that the purity of the antibody was over 90% and the molecular weight was 55 kDa.The results of western blot and immunohistochemical studies showed that APHNL were mainly distributed in embryo and endosperm during fruit development,protoplast of seed coat surface cells,and protoplast and procambia of cotyledon during fruit nucleation stage,and protoplast of seed coat surface cells,and protoplast and procambia of cotyledon during fruit ripening stage,and showed an increasing trend with fruit ripening.