Immobilization of recombinant serine protease from Virgobacillus natechei FarDT on amino graphene-chitosan biocompatible nanohybrid for enhancing pH and thermal stability

The serine protease gene was heterologously expressed in Escherichia coli BL21 (DE3) using the PET 28a vector. The purified enzyme was immobilized on a nanohybrid of amino graphene and chitosan. The characterization of synthesized nanohybrids and immobilized enzymes was confirmed by Fourier transfor...

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Bibliographic Details
Published in:International journal of biological macromolecules 2024-11, Vol.279 (Pt 2), p.135254, Article 135254
Main Authors: Afrand, Mahboobeh, Sourinejad, Iman, Homaei, Ahmad, Hemmati, Roohullah
Format: Article
Language:English
Subjects:
Immobilization
Nanohybrid
Recombinant
Serine protease
Stability
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Summary:The serine protease gene was heterologously expressed in Escherichia coli BL21 (DE3) using the PET 28a vector. The purified enzyme was immobilized on a nanohybrid of amino graphene and chitosan. The characterization of synthesized nanohybrids and immobilized enzymes was confirmed by Fourier transform infrared (FTIR), X-ray diffraction (XRD), dynamic light scattering (DLS), and field emission scanning electron microscopy (FE-SEM). Immobilization increased the temperature optimum from 60 to 70 °C for both free and immobilized enzymes, while the optimal pH of the enzymes did not change post-immobilization (pH 8). The immobilized biocatalyst significantly enhanced thermal stability, as well as enzyme stability at significant pH ranges. After 30 days of storage, the immobilized enzymes exhibited approximately 83 % of their relative activity, while the free protease retained only 56 % of its initial activity. Stabilization also altered the kinetic parameters (increasing Km, decreasing Kcat/Km, and Vmax) and thermodynamic parameters (increasing enzyme half-life and activation energy). The study's outcomes represent a significant advancement in the realm of enzyme synthesis and its stabilization using several combined technologies, including enzyme production with recombinant DNA technology based on gene synthesis, and its stabilization using a hybrid substrate synthesized from nanomaterials. Based on these findings, the immobilized recombinant enzyme has high potential for industrial use as an efficient and stable biocatalyst.
ISSN:0141-8130
1879-0003
1879-0003
DOI:10.1016/j.ijbiomac.2024.135254