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

Elisa Lanfranchi, Birgit Grill, Zainab Raghoebar, Sander Van Pelt, Roger A Sheldon, Kerstin Steiner, Anton Glieder, Margit Winkler

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Hydroxynitrile lyase from the white rabbit's foot fern Davallia tyermannii (DtHNL) catalyzes the enantioselective synthesis of α-cyanohydrins, which are key building blocks for pharmaceutical and agrochemical industries. An efficient and competitive process necessitates the availability and robustness of the biocatalyst. Herein, the recombinant production of DtHNL1 in Komagataella phaffii, yielding approximately 900 000 U L-1 , is described. DtHNL1 constitutes approximately 80 % of the total protein content. The crude enzyme was immobilized. Crosslinked enzyme aggregates (CLEAs) resulted in significant enhancement of the biocatalyst stability under acidic conditions (activity retained after 168 h at pH 2.4). The DtHNL1-CLEA was employed for (R)-mandelonitrile synthesis (99 % conversion, 98 % enantiomeric excess) in a biphasic system, and evaluated for the synthesis of (R)-hydroxypivaldehyde cyanohydrin under reaction conditions that immediately inactivated non-immobilized DtHNL1. The results show the DtHNL1-CLEA to be a stable biocatalyst for the synthesis of enantiomerically pure cyanohydrins under acidic conditions.

Original languageEnglish
Article number10.1002/cbic.201700419
Pages (from-to)312-316
JournalChemBioChem
Volume19
Issue number4
Early online date13 Nov 2017
DOIs
Publication statusPublished - 2018

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mandelonitrile lyase
Immobilization
Enzymes
Ferns
Agrochemicals
Immobilized Enzymes
Drug Industry
Foot
Rabbits

Fields of Expertise

  • Human- & Biotechnology

Cite this

Production of Hydroxynitrile Lyase from Davallia tyermannii (DtHNL) in Komagataella phaffii and Its Immobilization as a CLEA to Generate a Robust Biocatalyst. / Lanfranchi, Elisa; Grill, Birgit; Raghoebar, Zainab; Van Pelt, Sander; Sheldon, Roger A; Steiner, Kerstin; Glieder, Anton; Winkler, Margit.

In: ChemBioChem, Vol. 19, No. 4, 10.1002/cbic.201700419, 2018, p. 312-316.

Research output: Contribution to journalArticleResearchpeer-review

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abstract = "Hydroxynitrile lyase from the white rabbit's foot fern Davallia tyermannii (DtHNL) catalyzes the enantioselective synthesis of α-cyanohydrins, which are key building blocks for pharmaceutical and agrochemical industries. An efficient and competitive process necessitates the availability and robustness of the biocatalyst. Herein, the recombinant production of DtHNL1 in Komagataella phaffii, yielding approximately 900 000 U L-1 , is described. DtHNL1 constitutes approximately 80 {\%} of the total protein content. The crude enzyme was immobilized. Crosslinked enzyme aggregates (CLEAs) resulted in significant enhancement of the biocatalyst stability under acidic conditions (activity retained after 168 h at pH 2.4). The DtHNL1-CLEA was employed for (R)-mandelonitrile synthesis (99 {\%} conversion, 98 {\%} enantiomeric excess) in a biphasic system, and evaluated for the synthesis of (R)-hydroxypivaldehyde cyanohydrin under reaction conditions that immediately inactivated non-immobilized DtHNL1. The results show the DtHNL1-CLEA to be a stable biocatalyst for the synthesis of enantiomerically pure cyanohydrins under acidic conditions.",
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AU - Grill, Birgit

AU - Raghoebar, Zainab

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AU - Sheldon, Roger A

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AU - Glieder, Anton

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N2 - Hydroxynitrile lyase from the white rabbit's foot fern Davallia tyermannii (DtHNL) catalyzes the enantioselective synthesis of α-cyanohydrins, which are key building blocks for pharmaceutical and agrochemical industries. An efficient and competitive process necessitates the availability and robustness of the biocatalyst. Herein, the recombinant production of DtHNL1 in Komagataella phaffii, yielding approximately 900 000 U L-1 , is described. DtHNL1 constitutes approximately 80 % of the total protein content. The crude enzyme was immobilized. Crosslinked enzyme aggregates (CLEAs) resulted in significant enhancement of the biocatalyst stability under acidic conditions (activity retained after 168 h at pH 2.4). The DtHNL1-CLEA was employed for (R)-mandelonitrile synthesis (99 % conversion, 98 % enantiomeric excess) in a biphasic system, and evaluated for the synthesis of (R)-hydroxypivaldehyde cyanohydrin under reaction conditions that immediately inactivated non-immobilized DtHNL1. The results show the DtHNL1-CLEA to be a stable biocatalyst for the synthesis of enantiomerically pure cyanohydrins under acidic conditions.

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