Effect of Glycosylation on the Biocatalytic Properties of Hydroxynitrile Lyase from the Passion Fruit, Passiflora edulis: A Comparison of Natural and Recombinant Enzymes.

Abstract

A hydroxynitrile lyase from the passion fruit Passiflora edulis (PeHNL) was isolated from the leaves and showed high stability in biphasic co-organic solvent systems for cyanohydrin synthesis. Cyanohydrins are important building blocks for the production of fine chemicals and pharmaceuticals. Thus, to enhance production yields of PeHNL for industrial applications, we cloned and expressed recombinant PeHNL in Escherichia coli BL21(DE3) and Pichia pastoris GS115 cells without a signal peptide sequence. The aim of this study is to determine the effect of N-glycosylation on enzyme stability and catalytic properties in microbial expression systems. PeHNL from leaves (PeHNL-N) and that expressed in P. pastoris (PeHNL-P) were glycosylated, whereas that expressed in E. coli (PeHNL-E) was not. The enzymes PeHNL-N and PeHNL-P showed much better thermostability, pH stability, and organic solvent tolerance than the deglycosylated enzyme PeHNL-E and the deglycosylated mutant N105Q from P. pastoris (PeHNL-P-N105Q). The glycosylated PeHNL-P also efficiently performed transcyanation of (R)-mandelonitrile with a 98 % enantiomeric excess in a biphasic system with diisopropyl ether. These data demonstrate the efficacy of these methods for improving enzyme expression and stability for industrial application through N-glycosylation.