Candida rugosa lipase covalently immobilized on facilely-synthesized carbon nitride nanosheets as a novel biocatalyst

The immobilization of lipase on solid supports provides a significant improvement to the stability and reusability of lipase. During immobilization, the restricted surface area and inferior separation capacity of matrix materials are crucial for obtaining high-quality immobilized lipase. Carbon nitride nanosheets (C₃N₄-NS) as a type of two-dimensional nanomaterial have attracted various attentions for their prominent 2D planar nanostructure, characteristic surface area, thermostability and biocompatibility. Herein, we report a rational design and fabrication of immobilized Candida rugosa lipase based on carbon nitride nanosheets (C₃N₄-NS) as the matrix. The synthetic C₃N₄-NS are characterized by transmission electron microscopy, Brunauer-Emmett-Teller gas sorptometry measurement, X-ray powder diffraction, Fourier transform infrared spectroscopy and thermogravimetric analysis. These results show that C₃N₄-NS possess an as-expected two-dimensional nanostructure with a large surface area of 74.374 m² g^-1. In addition, we chose glutaraldehyde-assisted covalent attachment to combine C₃N₄-NS and Candida rugosa lipase (CRL) via amino groups at the margins of C₃N₄-NS. The as-constructed immobilized lipase (C₃N₄-NS@CRL) exhibits satisfactory enzyme-loading (44.76 mg g^-1), pH-flexibility, thermostability (after 180 min at 50 °C, 67% of the initial activity remained) and recyclability (after 10 runs, 72% of the initial activity remained). When compared with the free CRL, all experimental data indicate that C₃N₄-NS@CRL exhibited improved stability and enhanced practicability. To our knowledge, this is the first report of the application of carbon nitride nanosheets to enzyme immobilization.