Entrapment of surface-displayed BhCA in alginate beads: an integrated approach to enhance enzyme stability and reusability

Enzyme immobilisation has emerged as an effective strategy to enhance the operational stability, reusability, and catalytic performance of biocatalysts for industrial applications. However, conventional immobilisation methods often suffer from limitations such as enzyme leakage, reduced activity, and poor mass transfer efficiency. In the present study, we report a novel approach of increasing the reusability of enzymes by combining surface display technique with entrapment in calcium alginate matrix. This immobilisation approach was optimised using carbonic anhydrase (CA) as a model enzyme, where whole cells displaying CA on the surface were entrapped in Ca-alginate beads, resulting in the formation of CA biocatalytic beads. Various parameters for immobilisation in Ca-alginate beads were systematically optimised, followed by their characterisation. This approach resulted in a substantial enhancement in thermostability of CA with a T1/2 of 8 h at 70 °C, as well as improved resistance to some of the major CA inhibitors. An exceptional improvement in reusability has been observed, maintaining 100% of their initial activity after 20 consecutive catalytic cycles. The method also led to improvement in the shelf life of the immobilised enzyme, retaining 92% of activity after 35 days of storage at 4 °C. A lab-scale bioreactor was also developed for the continuous mineralisation of CO2 into CaCO3 using CA biocatalytic beads, highlighting their strong potential for efficient CO2 capture under steady-state operation. This immobilisation strategy proposes an efficient route for immobilising various enzymes providing enhancement in enzyme performance, longevity and cost effectiveness for application in industrial bioprocesses.