Hydroxynitrile Lyase Isozymes from Prunus communis: Identification, Characterization and Synthetic Applications

Abstract

Biocatalysts originating from Badamu (Prunusc ommunis)h ave been applied to catalyze the asymmetric synthesis of (R)-4-methylsulfanylmandelonitrile,akey building block of thiamphenicol and florfenicol. Here,f our hydroxynitrile lyase (HNL) isozymes from Badamu were cloned and heterologously expressedi nPichia pastoris.T he biochemical properties and catalytic performances of these isozymes were comprehensively explored to evaluate their efficiency and selectivity in asymmetric synthesis.A mong then, PcHNL5 was identified with outstanding activity and enantioselectivity in asymmetric hydrocyanation. Under the optimized mild biphasicr eactionc onditions,s eventeen prochi-ral aromatic aldehydes were converted to valuable chiral cyanohydrins with good yields (up to 94%) and excellent optical purities (up to > 99.9% ee), which provide af acile access to numerous chiral amino alcohols,h ypoglycemic agents,a ngiotension converting enzyme (ACE)i nhibitors and b-blockers. This workt herefore underlines the importance of discovering the most potent biocatalyst among ag roup of isozymes for converting unnatural substrates into value-added products.

A High‐Throughput Screening Method for the Directed Evolution of Hydroxynitrile Lyase towards Cyanohydrin Synthesis

“…Therefore, ABAO can be directly used to quench the reaction and start the chromogenic assay. To investigate the accuracy of our HTS assays, the measured level of coloration and resultant substrate conversion were correlated to the GC analysis results for the hydrocyanation of aldehyde 1 (10 mM) by Pc HNL5 (in the form of a supernatant of yeast culture broth) [12] in an aqueous system (Figure 1). The assay demonstrated a high compatibility and sensitivity, resulting in detectable UV/Vis absorption variance at a conversion difference of only 1 %.…”

“…With optimized assay conditions in hand, the versatility of the ABAO assay methodology was investigated. The specific activities of wild‐type Pc HNL5 [12] toward a series of aldehydes (16 aromatic aldehydes ( α1 – α16 ), four heteroaromatic aldehydes ( α17 – α20 ), and eight aliphatic aldehydes ( α21 – α24 )) were determined using the modified ABAO assay method. To confirm the reliability of this assay methodology, the specific activities of HNL for hydrocyanation were simultaneously determined by GC.…”

“…The freedom of applying structurally diverse aldehydes prompted us to perform a substrate screening of several well‐known HNLs to validate the current HTS methodology. We tested the enzymatic hydrocyanation reactions of reported ( S )‐ Me HNL H103L [13] and its variant H103L/W128A, [14] ( R )‐ Pc HNL5 [12] and its variant L331A, [15] and industrially applied ( R )‐ Pa HNL5 variants N3I/I108M/A111G [6c] and V317A [16] . Four aldehydes, α2 , α4 , α12 , and α25 , were used as representative substrates (Table 1).…”

Production of Hydroxynitrile Lyase from Davallia tyermannii (DtHNL) in Komagataella phaffii and Its Immobilization as a CLEA to Generate a Robust Biocatalyst

“…The methylotrophic yeast K. phaffii (P. pastoris) was chosen as a host organism for Dt HNL1 production because of its capability to express recombinant proteins to high levels and its established molecular biology toolbox. Several HNLs have been successfully expressed from this host . The gene coding for Dt HNL1 was integrated into the K. phaffii CBS 7435 Mut S genome.…”

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

“…( Ps HNL), Prunus mume ( Pm HNL), Prunus armeniaca L. ( Pars HNL), Prunus communis ( Pc HNL), Linum usitatissimum ( Lu HNL), Phlebodium aureum ( Pha HNL), Arabidopsis thaliana ( At HNL), Eriobotrya japonica ( Ej HNL), Passiflora edulis forma flavicarpa ( Pe HNL), Davallia tyermannii ( Dt HNL), and Nandina domestica ( Nd HNL), and the S ‐selective ones derived from Hevea brasiliensis ( Hb HNL), Manihot esculenta ( Me HNL), Sorghum bicolor ( Sb HNL), and Baliospermum montanum ( Bm HNL) . Among the HNLs of plant origin, Sb HNL, Ej HNL, Pe HNL, Pa HNL, Pc HNL, and Pm HNL are glycosylated proteins, whereas, Lu HNL, Bm HNL, At HNL, Nd HNL, Hb HNL, Me HNL, and Pha HNL are not glycosylated. Furthermore, Ej HNL, Pa HNL Pc HNL, and Pm HNL contain flavin adenine dinucleotide (FAD), in contrast to Lu HNL, Pha HNL, At HNL, Pe HNL, Bm HNL, Hb HNL, Me HNL, Sb HNL, and Nd HNL which do not contain FAD.…”