Stabilization of Hydroxynitrile Lyases from Two Variants of Passion Fruit, Passiflora edulis Sims and Passiflora edulis Forma flavicarpa, by C-Terminal Truncation

Because the synthesis of chiral compounds generally requires a broad range of substrate specificity and stable enzymes, screening for better enzymes and/or improvement of enzyme properties through molecular approaches is necessary for sustainable industrial development. Herein, the discovery of unique hydroxynitrile lyases (HNLs) from two species of passion fruits, Passiflora edulis forma flavicarpa (yellow passion fruit, PeHNL-Ny) and Passiflora edulis Sims (purple passion fruit, PeHNL-Np), isolated and purified from passion fruit leaves is reported. These are the smallest HNLs (comprising 121 amino acids). Amino acid sequences of both enzymes are 99 % identical; there is a difference of one amino acid in a consensus sequence. PeHNL-Np has an Ala residue at position 107 and is nonglycosylated at Asn105. Because it was confirmed that natural and glycosylated PeHNL-Ny showed superior thermostability, pH stability, and organic tolerance to that of PeHNL-Np, it has been speculated that protein engineering around the only glycosylation site, Asn105, located at the C-terminal region of PeHNL-Ny, might contribute to the stabilization of PeHNL. Therefore, the focus is on improved stability of the nonglycosylated PeHNL by truncating its C-terminal region. The C-terminal-truncated PeHNLΔ₁₀₇ was obtained by truncating 15 amino acids from the C terminus followed by expression in Escherichia coli. PeHNLΔ₁₀₇ expressed in E. coli was not glycosylated, and showed improved thermostability, solvent stability, and reusability similar to that of the wild-type glycosylated form of PeHNL expressed in Pichia pastoris. These data reveal that the lack of the high-flexibility region at the C terminus of PeHNL might be a possible reason for improving the stability of PeHNL.