A Preblocking Strategy with Rapid Immobilization of Nitrile Hydratase and Precise Control of Hydrophobic Microenvironment for High Regioselective Amide Transformation

Abstract

The control over enzyme immobilization and its microenvironment at the molecular level is deemed crucial. In this study, a "preblocking" method was performed through the use of natural polymer agarose resins after activation by phenyl-, methyl-, and ethyl-vinyl sulfones combined with divinyl sulfones prior to the specific immobilization of a his-tag fused recombinant nitrile hydratase (ReNHase). This technique primarily allows for the establishment of a hydrophobic microenvironment to enhance the immobilization and mass transfer and avoid inactivation caused by traditional backfilling. The purified ReNHase showed specific activity and regioselectivity of adiponitrile in an aqueous solution as 28.43 U•mg −1 and 90.2%, respectively. Increasing the density of hydrophobic groups promotes the adsorption extent and immobilization yield of ReNHase. Optimized DVS:PVS 1:4 hydrophobic microenvironment could finish immobilization in 4h. Eminent expressed activity and regioselectivity of 94.6% and 93.9% was exhibited, respectively. The approach demonstrated exceptional selectivity in the biotransformation at the molecular level. Furthermore, the method was found to preserve enzymatic activity over seven cycles and exhibited significantly enhanced resilience to variations in the temperature and substrate concentration. The ReNHase@DVS:PVS 1:4 still retained 98% of the maximum activity, while ReNHase only retained 64% of activity at 55 °C. The ReNHase@DVS:PVS 1:4 still retained 55% of the maximum activity, while ReNHase was completely inactive at 200 mM adiponitrile. A packed-bed reactor was constructed, where the biotransformation of 5.64 g of 5-cyanovaleramide was carried out continuously for 48 h at a flow rate of 0.60 mL/min, achieving a peak space-time yield reported to be 0.93 mol•h −1 •L −1 . The "preblocking" technique developed in this study is anticipated to provide new methodology in the catalysis of hydrophobic substrates.