Hydroxynitrile lyases from cyanogenic millipedes: molecular cloning, heterologous expression, and whole-cell biocatalysis for the production of (R)-mandelonitrile

Takuya Yamaguchi,Atsutoshi Ina,Yasuhisa Asano,Aem Nuylert,Tsutomu Tanabe

doi:10.1038/s41598-018-20190-x

Takuya Yamaguchi, Atsutoshi Ina + Show 3 more

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https://doi.org/10.1038/s41598-018-20190-x

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Abstract

Hydroxynitrile lyases (HNLs), which are key enzymes in cyanogenesis, catalyze the cleavage of cyanohydrins into carbonyl compounds and hydrogen cyanide. Since HNLs also catalyze the reverse reaction, they are used industrially for the asymmetric synthesis of cyanohydrins, which are valuable building blocks of pharmaceuticals and fine chemicals. HNLs have been isolated from cyanogenic plants and bacteria. Recently, an HNL from the cyanogenic millipede Chamberlinius hualienensis was shown to have the highest specific activity for (R)-mandelonitrile synthesis, along with high stability and enantioselectivity. However, no HNLs have been isolated from other cyanogenic millipedes. We identified and characterized HNLs from 10 cyanogenic millipedes in the Paradoxosomatidae and Xystodesmidae. Sequence analyses showed that HNLs are conserved among cyanogenic millipedes and likely evolved from one ancestral gene. The HNL from Parafontaria tonominea was expressed in Escherichia coli SHuffle T7 and showed high specific activity for (R)-mandelonitrile synthesis and stability at a range of pHs and temperatures. The stability of millipede HNLs is likely due to disulfide bond(s). The E. coli cells expressing HNL produced (R)-mandelonitrile with 97.6% enantiomeric excess without organic solvents. These results demonstrate that cyanogenic millipedes are a valuable source of HNLs with high specific activity and stability.

Highlights

Hydroxynitrile lyases (HNLs), which are key enzymes in cyanogenesis, catalyze the cleavage of cyanohydrins into carbonyl compounds and hydrogen cyanide
All of these eleven cDNAs encoding HNL were heterologously expressed in insect cells, five cDNAs encoding HNL were able to express in Escherichia coli, and transgenic E. coli cells expressing a cDNA encoding HNL were used as whole-cell catalysts to synthesize (R)-mandelonitrile
NttHNL was R-selective HNL and the specific activity of purified NttHNL for (R)-mandelonitrile synthesis was 4702 U/mg, lower than that of ChuaHNL (7420 U/mg)[18], but three times higher than that of PaHNL (1450 U/mg)[19], which is used industrially. These results suggested that cyanogenic millipedes are a good source of HNLs with high specific activity for cyanohydrin synthesis

Summary

Introduction

Hydroxynitrile lyases (HNLs), which are key enzymes in cyanogenesis, catalyze the cleavage of cyanohydrins into carbonyl compounds and hydrogen cyanide. An HNL from the cyanogenic millipede Chamberlinius hualienensis was shown to have the highest specific activity for (R)-mandelonitrile synthesis, along with high stability and enantioselectivity. The HNL from Parafontaria tonominea was expressed in Escherichia coli SHuffle T7 and showed high specific activity for (R)-mandelonitrile synthesis and stability at a range of pHs and temperatures. The E. coli cells expressing HNL produced (R)-mandelonitrile with 97.6% enantiomeric excess without organic solvents These results demonstrate that cyanogenic millipedes are a valuable source of HNLs with high specific activity and stability. ChuaHNL showed the highest specific activity for (R)-mandelonitrile synthesis among all characterized HNLs, and high enantioselectivity and stability at a www.nature.com/scientificreports/. Range of temperatures and pHs. Other cyanogenic millipedes may contain HNLs with high specific activity and stability. All of these eleven cDNAs encoding HNL were heterologously expressed in insect cells, five cDNAs encoding HNL were able to express in Escherichia coli, and transgenic E. coli cells expressing a cDNA encoding HNL were used as whole-cell catalysts to synthesize (R)-mandelonitrile

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