Hydroxynitrile　lyase　gene　(hnl)　from　Eriobotrya　japonica　was　successfully　amplified　using　the　method　of　SEFA　PCR　(Self-Formed　Adaptor　PCR).　The　complete　sequence　was　5.5kbp　in　length,　including　3100bp　of　the　upstream　promoter　region,　1659bp　of　the　coding　sequence,　three　introns　and　315bp　of　the　downstream　transcription　terminator.　The　phylogenetic　analysis　illustrated　that　the　obtained　hnl　exhibited　66-70%　identity　to　the　reported　isozymes　from　almond,　black　cherry　and　Japanese　apricot.　The　EjHNL　had　552　amino　acids　including　a　25　amino　acid-long　signal　peptide.　The　conserved　characteristic　structures　of　HNLs,　such　as　FAD-binding　motif,　N-glycosylation　sites　and　active　sites　were　observed.　The　coding　sequence　of　the　hnl　was　inserted　into　pPIC9K　vector　for　heterologous　expression　in　Pichia　pastoris.　The　HNL　activity　of　the　culture　supernatant　reached　15U/ml　after　96h　of　induction　by　methanol.　The　specific　activity　of　the　recombinant　HNL　was　about　197U/mg.　The　enantiomeric　excess　value　of　the　product　R-mandelonitrile　attained　98.6%　and　the　value　of　Km　of　the　recombinant　HNL　was　determined　to　be　0.47mM　based　on　the　kinetic　data.　The　optimum　temperature　and　pH　of　the　recombinant　HNL　were　40°C　and　6.0　respectively.　The　experimental　data　indicated　that　the　obtained　recombinant　HNL　showed　similar　catalytic　characteristics　with　the　natural　EjHNL.　The　expression　of　the　recombinant　HNL　in　P.　pastoris　could　present　another　available　biocatalyst　for　the　synthesis　of　R-selective　cyanohydrins.　©　2011　The　Society　for　Biotechnology,　Japan.