Improving the Properties of Bacterial R‐Selective Hydroxynitrile Lyases for Industrial Applications

Abstract

Hydroxynitrile lyases (HNLs) catalyse the reversible cleavage of cyanohydrins to carbonyl compounds and HCN. The recent discovery of bacterial HNLs with a cupin fold gave rise to a new promising class of these enzymes. They are interesting candidates for the synthesis of cyanohydrins on an industrial scale owing to their high expression levels in Escherichia coli. The activity and enantioselectivity of the manganese‐dependent HNL from Granulicella tundricola (GtHNL) were significantly improved by site‐saturation mutagenesis of active site amino acids. The combination of beneficial mutations resulted in a variant with 490‐fold higher specific activity in comparison to the wild‐type enzyme. More importantly, GtHNL‐A40H/V42T/Q110H is a highly competitive alternative for the synthesis of chiral cyanohydrins, such as 2‐chlorobenzaldehyde cyanohydrin, (R)‐2‐hydroxy‐4‐phenylbutyronitrile, and (R)‐2‐hydroxy‐4‐phenyl‐3‐butene nitrile, which serve as intermediates for the synthesis of pharmaceuticals.

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

“…The specific activity of the improved mutant was 490‐fold higher than that of the wild‐type enzyme. Furthermore, high conversion and enantiomeric excess of different cyanohydrins of interest were obtained …”

“…Cyanohydrin synthesis : Syntheses of ( R )‐mandelonitrile and ( R )hydroxypivaldehyde cyanohydrin were performed in a biphasic system and analyzed as described in detail by Wiedner et al . Briefly, a solution of HCN (2 m ) in MTBE was prepared by dropwise addition of 30 % HCl to a dispersion of aqueous NaCN in MTBE.…”

“…Samples were acetylated prior to GC analyses. Standards and retention times of each compound were reported previously . For comparability, the aqueous phase contained approximately 0.2 mg Dt HNL1 in the different forms for ( R )‐mandelonitrile synthesis.…”

(R)‐Selective Nitroaldol Reaction Catalyzed by Metal‐Dependent Bacterial Hydroxynitrile Lyases

“…[11][12][13] As reported previously,t he proteins from Acidobacterium capsulatum ATCC 51196 (AcHNL) and Granulicella tundricula (GtHNL) as well as their variants containing the amino acid exchanges A40H,A 40R, V42T,a nd Q110H and combinations thereof were highly expressed as soluble proteins in E. coli reachingy ields > 50 %o ft otal soluble protein. [14] The ability of these enzymestoc atalyze the synthesis of b-nitro alcohols was examined by using atwo-phase system consisting of benzaldehyde and nitromethane dissolved in methyl tert-butyl ether (MTBE)a st he organic phase, and an aqueous phase comprising cell-free lysates or purified enzymesi np hosphate buffer at pH 6( Ta ble S1 in the Supporting Information and Table 1). As it was shown before that the HNL reactions catalyzed by GtHNL and AcHNL are manganese dependent,a ll enzymes were grownw ith manganese presentint he expression medium.…”

“…For HNL activity,w ep reviously reported the highest improvement for the triple variantsA 40H/V42T/ Q110H of both enzymes,w hich were superior to any of the single variants. [14] Interestingly,adifferent effect was observed for the Henry reaction:w hereas amino acid exchange at positions V42 and Q110 improved the activity of GtHNL to some extenta nd had no positive effect for AcHNL (Table S1), the triple variants showed more significant improvement, but asingle amino acid exchange at position A40 to histidine or arginine resulted in superior variants: AcHNL-A40H reached 75 % conversion after 4h and 99.3 %o ft he (R)e nantiomer of 2nitro-1-phenylethanol was formed ( Table 1). Almostn od ifference was observed whether cleared lysate or purified enzyme was applied( compare Tables 1a nd S1).…”