Immobilization and stabilization of d-hydantoinase from Vigna angularis and its use in the production of N-carbamoyl-d-phenylglycine. Improvement of the reaction yield by allowing chemical racemization of the substrate

• Published in: Process biochemistry (1991) Vol. 95; pp. 251 - 259
• Main Authors: Becaro, Aline Aparecida, Mendes, Adriano Aguiar, Adriano, Wellington Sabino, Lopes, Laiane Antunes, Vanzolini, Kenia Lourenço, Fernandez-Lafuente, Roberto, Tardioli, Paulo Waldir, Cass, Quezia Bezerra, Giordano, Raquel de Lima Camargo
• Format: Journal Article
• Language: English
• Published: Barking Elsevier Ltd 01.08.2020; Elsevier BV
• Subjects: Beads; Borohydrides; d,l-phenylhydantoin; D-hydantoinase; Enzymatic activity; Enzyme activity; Enzymes; Glyoxyl agarose; Hydantoinase; Immobilization; Magnesium; N-carbamoyl-d-phenylglycine; Phenylglycine; Racemization; Reduction; Stability; Substrates; Vigna angularis; Zinc; Glyoxyl agarose; d-hydantoinase; d,l-phenylhydantoin; N-carbamoyl-d-phenylglycine
• ISSN: 1359-5113; 1873-3298
• DOI: 10.1016/j.procbio.2020.02.017

[Display omitted] •D-hydantoinase immobilized on glyoxyl agarose presented ca.60 % recovered activity.•Presence of Zn+2 in the active site was necessary to maintain the catalytic activity.•Immobilized d-hydantoinase was 86-fold more stable than the free enzyme.•d,l-phenylhydantoin was efficiently hydrolyzed to N-carbamoyl-d-phenylglycine.•High enantioselectivity (>99 %) was achieved under substrate racemization conditions. This work reports the immobilization of a multimeric d-hydantoinase (DHTase) from Vigna angularis (E.C. 3.5.2.2.) on agarose beads activated with glyoxyl groups aiming to improve its stability via multipoint covalent attachment. The final reduction with sodium borohydride resulted in a drop in enzyme activity that could be decreased by adding Zn2+ or Mg2+. The optimal preparation with high activity (58 % recovered activity) and stability (around 86-fold more stable than the free enzyme) was obtained by DHTase immobilization on glyoxyl agarose for 24 h at 25 °C and pH 10.05, and a borohydride reduction step in the presence of 10 mM Zn2+ (DHTase-Glx). The enzyme was almost fully immobilized on glyoxyl agarose (19.8 mg/g of support) when offering 20 mg/g. This immobilized biocatalyst was used to catalyze the hydrolysis of d,l-phenylhydantoin under substrate racemization conditions, which produced 99 % of N-carbamoyl-d-phenylglycine after 9 h reaction.