Immobilization of Geobacillus pallidus RAPc8 nitrile hydratase (NHase) reduces substrate inhibition and enhances thermostability

The nitrile hydratase (NHase) from the thermophilic strain Geobacillus pallidus RAPc8 was investigated for its potential application in the biocatalytic production of amides from nitriles. The recombinant NHase was immobilized to a range of insoluble matrices using various cross-linking agents. The immobilized preparation using Eupergit®C with 1-ethyl-3-(dimethylamino-propyl) carbodiimide (EDAC) cross-linking exhibited the highest immobilization efficiency (93%). The pH range and optimal temperature for activity were unchanged by immobilization but the thermostability of the Eupergit®C-NHase was improved compared with the soluble enzyme; at 60 °C the half-life of the immobilized NHase was 330 min as compared with 54.5 min for the soluble enzyme. Kinetic parameters Vmax (4.5 μmol mL−1 min−1), Km (17.3 mM) and kcat (3543.3 min−1) were obtained for the immobilized NHase at 50 °C, as compared with 48.8 μmol mL−1 min−1, 10.2 mM and 37777.1 min−1 respectively for the soluble enzyme. The operational stability was improved significantly by immobilization, with 85.7% of initial activity maintained after reuse for eight cycles. Most notably, the Eupergit®C-immobilized NHase showed substantially lower substrate inhibition (Ki = 194.7 mM) than the soluble enzyme (Ki = 101.0 mM). In the presence of various co-organic solvents, Eupergit®C-EDAC NHase showed statistically higher retention of activity than the non-immobilized control.