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

• Published in: Chembiochem : a European journal of chemical biology 2018-02, Vol.19 (4), p.312-316
• Main Authors: Lanfranchi, Elisa, Grill, Birgit, Raghoebar, Zainab, Van Pelt, Sander, Sheldon, Roger A., Steiner, Kerstin, Glieder, Anton, Winkler, Margit
• Format: Article
• Language: English
• Subjects: Aldehyde-Lyases - biosynthesis; Aldehyde-Lyases - chemistry; Aldehyde-Lyases - metabolism; Biocatalysis; Biocatalysts; Chemical synthesis; crosslinked enzyme aggregates; Crosslinking; Enantiomers; Enzymes; Enzymes, Immobilized - biosynthesis; Enzymes, Immobilized - chemistry; Enzymes, Immobilized - metabolism; Ferns; Ferns - enzymology; Ferns - microbiology; hydroxynitrile lyase; Immobilization; Komagataella phaffii; Mandelonitrile; Nitriles - chemistry; Nitriles - metabolism; Pichia - enzymology; Protein Aggregates; Stereoisomerism
• ISSN: 1439-4227; 1439-7633
• DOI: 10.1002/cbic.201700419

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. A million units per liter: The small hydroxynitrile lyase (HNL) from D. tyermannii can be produced in pro‐ and eukaryotic hosts in exceptionally high amounts. DtHNL constitutes up to 80 % of the soluble protein in P. pastoris. Immobilization as crosslinked enzyme aggregates (CLEAs) improves biocatalyst stability and allows DtHNL to be used at low pH.