Synthesis of 1-cyanocyclohexaneacetic acid in a partial-mixed recirculating bed reactor with resin-immobilized nitrilase.

Abstract

Immobilized enzyme bioreactors provide a controlled reaction environment and integrated reaction-separation processes for biocatalysis. In this study, a biocatalytic process based on resin-immobilized nitrilase and a partial-mixed recirculating bed bioreactor was developed for the synthesis of 1-cyanocyclohexaneacetic acid, a gabapentin intermediate. The resin catalyst prepared by immobilizing the regioselective nitrilase AcN-S on the activated amino resin LX-1000EPHA achieved 99.12% immobilization yield, 73.40 U/g specific activity, and 95.42% activity recovery when loaded with 10 mg/g crude enzyme. The resin catalyst (100 g/L) could convert 1 M (148.2 g/L) substrate 1-cyanocyclohexaneacetonitrile to 1-cyanocyclohexaneacetic acid within 18 h, achieving a conversion of 95.40%. At a substrate concentration of 0.5 M, > 85% conversion could still be achieved after 15 batches. In a partial-mixed recirculating bed reactor, the resin catalyst (100 g/L) could completely convert 500 mM substrate within 10 h, and achieve > 90% conversion after 20 batches, with residual activity of 93.23%. Resin activation and cross-linking treatment after immobilization were found to improve operational stability, reduce protein leakage, and ensure high immobilization yield and activity recovery. The reactor provided a low-shear environment and recirculating flow, which together improve catalyst reusability and reduce product inhibition. The constructed reaction system provides a solution for the efficient conversion of slightly soluble/insoluble substrates and the integration of reaction and separation.