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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 <jats:italic>Chamberlinius hualienensis<\/jats:italic> was shown to have the highest specific activity for (<jats:italic>R<\/jats:italic>)-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 <jats:italic>Parafontaria tonominea<\/jats:italic> was expressed in <jats:italic>Escherichia coli<\/jats:italic> SHuffle T7 and showed high specific activity for (<jats:italic>R<\/jats:italic>)-mandelonitrile synthesis and stability at a range of pHs and temperatures. The stability of millipede HNLs is likely due to disulfide bond(s). The <jats:italic>E. coli<\/jats:italic> cells expressing HNL produced (<jats:italic>R<\/jats:italic>)-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.<\/jats:p>","DOI":"10.1038\/s41598-018-20190-x","type":"journal-article","created":{"date-parts":[[2018,2,8]],"date-time":"2018-02-08T15:47:39Z","timestamp":1518104859000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":20,"title":["Hydroxynitrile lyases from cyanogenic millipedes: molecular cloning, heterologous expression, and whole-cell biocatalysis for the production of (R)-mandelonitrile"],"prefix":"10.1038","volume":"8","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-7533-430X","authenticated-orcid":false,"given":"Takuya","family":"Yamaguchi","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aem","family":"Nuylert","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6047-4676","authenticated-orcid":false,"given":"Atsutoshi","family":"Ina","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tsutomu","family":"Tanabe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3645-3952","authenticated-orcid":false,"given":"Yasuhisa","family":"Asano","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2018,2,14]]},"reference":[{"key":"20190_CR1","doi-asserted-by":"publisher","first-page":"1457","DOI":"10.1016\/j.phytochem.2008.02.019","volume":"69","author":"M Zagrobelny","year":"2008","unstructured":"Zagrobelny, M., Bak, S. & M\u00f8ller, B. 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