Designing nano-biocatalyst with improved enzymatic activity and stability
In the current scenario, there is a need to develop clean, reliable, biocompatible and benign processes for the industrial manufacturing of chemicals. Notwithstanding all these advantages of enzymes, industrial application of enzymes is often hampered by a lack of long-term operational stability and difficult recovery and re-use of the enzyme. Hence, re-engineering of enzymes with high activities in the given environments is required for enzymatic catalysis in industrial biocatalytic processes. The redesign of enzyme can be achieved by chemical approaches including immobilization and chemical modification which represents a simple but effective route. In most of the conventional immobilization methods, the activity of enzymes is usually lower than its native counterpart which is mainly due to the hindered substrate accessing and/or unfavourable conformational transition of enzyme within the matrix. Hence, it is necessary to develop new method which can amplify catalytic activity along with the improvement in the enzyme properties. Recently, the construction of organic–inorganic hybrid material is a rapidly expanding field of material chemistry for its advanced designs with a specific structure and functionality. Organic–inorganic hybrid platform can be prepared simply by using metal ions as the inorganic component and the organic component at room temperature under aqueous conditions. The main advantages of using hybrid organic-inorganic matrix for enzyme immobilization are: (i). Organic-inorganic platform can help to form strong bonding with enzyme which can prevent the leaching of enzyme in reaction mixture. (ii) The presence of metallic counterpart in hybrid material can exhibit the allosteric effect on enzyme which can ultimately enhance the enzyme activity. (iii) Hybrid material can stabilize the conformational structure in various in-hospitalized conditions and chemical environment.
To enhance robustness, thermal stability, and extended the shelf-life time for industrial applications, in this research work, an organic-inorganic hybrid platform for enzyme immobilization has been developed via rapid single pot technique using biomineralization methodology. The allosteric effect (due to metal ion) and structural configuration of enzyme not only helped to enhance the enzyme activity but also improved the stability (mechanically robustness and thermally stable) due to protective shield. Further, the immobilized enzyme was characterized by powdered X-ray diffraction (XRD), Fourier transform infrared (FT-IR) and confocal scanning laser microscopy. The size and morphology were analysed by scanning electron microscopy (SEM). Also, the kinetic parameters (Vmax and KM) and thermal stability of free and immobilized enzyme were determined in terms of thermal deactivation constant (kd), half-life(t1/2) and deactivation energy (Ed). In addition, conformational changes occurring after immobilization were estimated by FT-IR data analysis tools. Lastly, reusability and storage stability of enzymes were studied to check its durability and industrial feasibility. This exploration of immobilization technology is anticipated to inspire further advancement in the novel design and functionality of the support matrix for a wide range of applications.