Cloning and expression of hydroxynitrile lyase gene from Eriobotrya japonica in Pichia pastoris(ENZYMOLOGY, PROTEIN ENGINEERING, AND ENZYME TECHNOLOGY)

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 K_m 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℃ 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.