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

Abstract

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.

Mandelonitrile lyase MDL2-mediated regulation of seed amygdalin and oil accumulation of Prunus sibirica

“…HNLs is generally divided into two kinds of FAD containing MDLs and non-FAD containing HNLs based on avoprotein (FAD) content. Of note, almost HNLs have been identi ed with asymmetric synthesis of mandelonitrile in different families of cyanogenic plants [32][33][34][35][36][37][38][39][40], and MDLs have been reported only in black cherry and almond with speci c activity for the dissociation of mandelonitrile [31,[41][42][43][44], which raised a vital question of which MDL was contributed speci cally to decrease amygdalin accumulation of P. sibirica seeds. Here, we applied a combined analysis of genomewide annotation of mandelonitrile dissociation-associated MDL family members (Table 1) and the correlation with accumulative level of mandelonitrile and amygdalin and transcriptional amount of MDL family member in seeds across various germplasms or developmental periods of P. sibirica (Fig.…”

“…Almost FAD-independent HNLs in several plants (such as Arabidopsis, cassava, Japanese apricot, almond and peach) have been identi ed with asymmetric synthesis of mandelonitrile by the assay of overexpression, mutant, or catalytic activity [32][33][34][35][36][37][38][39][40]. To date, however, no MDL gene has been conducted on genetic transformation in plants to unravel its function.…”

Hydroxynitrile Lyase Discovery, Engineering, and Promiscuity towards Asymmetric Synthesis: Recent Progress

“…and ee than the WT towards hydrocyanation of 4methylthiobenzaldehyde [39] DtHNL Gibson assembly method D85A, S87A, Y101F, Y117F, Y161F and D85S-S87D resulted in inactive mutants, decreased the activity by at least 90 %., produced insoluble protein and prevented activity measurements [29] PeHNL SDM PeHNL-P-N105Q poor thermostability, pH stability, organic solvent tolerance, and enantioselectivity than the WT [40] PeHNL SDM PCR followed by cloning H8A, Y30F, E54A, E54Q, R56A, S66A, N101A and N101D PeHNL~107 reduced activity for cleavage and synthesis of (R)-MN than the WT increased activity for cleavage and synthesis of (R)-MN than the WT [41] PeHNL PCR followed by cloning PeHNL~107 improved stability, conv. and enantiselectivity in synthesis of nine (R)cyanohydrins [42] ChuaHNL-PpPDI SDM N99Q, N109Q, N123Q, N99Q-N109Q, N99Q-N123Q and N109Q-N123Q N123Q low protein production revealed that sugar chains are essential for the high-level production in His-ChuaHNL higher specific activity towards three of the eight aromatic aldehydes in their chiral cyanohydrin synthesis [43] ChuaHNL SDM R38A, Y40F, D56E, Y103F and K117R lost or decreased activity in cyanogenesis and cleavage of (R)-MN [44] PlamHNL SDM PlamHNL-N85Y higher enantioselectivity and conv. in (R)-2-Cl-MN synthesis [45] LuHNL SDM SDM of the catalytic residues elucidated a novel reaction mechanism for cyanohydrin decomposition involving the cyano-zinc complex and hydrogen-bonded interaction of the hydroxyl group of cyanohydrin with Glu323/Thr65 and H 2 O/Lys162 of LuHNL [46] GtHNL SDM (based on results of epPCR and SSM)…”

“…Nuylert, et al. reported two new HNLs from passion fruit leaves, Pe HNL‐Ny ( Passiflora edulis forma flavicarpa yellow passion fruit), and Pe HNL‐Np ( Passiflora edulis Sims purple passion fruit) [42] . Between the two, the former showed much better thermostability, pH stability, and organic solvent tolerance than the latter probably because it is glycosylated at Asn105, while the latter not.…”

A Comprehensive Overview of Novel Hydroxynitrile Lyases

“…When a 15-amino-acid-residue C-terminal truncated form of PeHNL was made and expressed in E. coli, it produced an enzyme that was not glycosylated. When non-glycosylated and C-terminally truncated mutant PeHNL107 is expressed in P. pastoris , it enhances stability and reusability to the same extent as glycosylated native PeHNL (Nuylert et al ., 2017b).…”