Chemical and thermal stabilization of CotA laccase via a novel one-step expression and immobilization in muNS-Mi nanospheres

Abstract

A methodology that programs eukaryotic or bacterial cells to encapsulate proteins of any kind inside micro/nanospheres formed by muNS-Mi viral protein was developed in our laboratory. In the present study such “in cellulo” encapsulation technology is utilized for immobilizing a protein with an enzymatic activity of industrial interest, CotA laccase. The encapsulation facilitates its purification, resulting in a cost-effective, one-step way of producing immobilized enzymes for industrial use. In addition to the ability to be recycled without activity loss, the encapsulated protein showed an increased pH working range and high resistance to chemical inactivation. Also, its activity was almost unaffected after 30 min incubation at 90 °C and 15 min at the almost-boiling temperature of 95 °C. Furthermore, the encapsulated laccase was able to efficiently decolorate the recalcitrant dye RB19 at room temperature.

Lithographic Patterning of Nanoscale Arrays of the Oxidase Enzyme CotA: Effects on Activity and Stability

“…Even so, the CotA immobilized with this method still showed higher stability than previously reported immobilized CotA on glassy carbon electrodes (39% of initial activity after 7 days and 35% after 49 days) [ 48 ] or encapsulated in nanospheres (28% of initial activity over 1 day). [ 49 ]…”

“…Even so, the CotA immobilized with this method still showed higher stability than previously reported immobilized CotA on glassy carbon electrodes (39% of initial activity after 7 days and 35% after 49 days) [48] or encapsulated in nanospheres (28% of initial activity over 1 day). [49] The larger decline in activity of the enzymatic chips fabricated by photolithography compared to PPL could be due to the aforementioned higher portion of the non-covalently adsorbed enzyme, which gradually desorbs over repeated rounds of assays, resulting in an earlier fall in activity. This initial 40-day period is followed by a slower rate of decline similar to the PPLfabricated chips, which is likely due to the desorption of the alkylthiol SAM formed by AUT on Au, [45,50] combined with the eventual unfolding/degradation of the immobilized protein.…”

The potential of a novel enzyme-based surface plasmon resonance biosensor for direct detection of dopamine

“…In addition, the use of immobilized laccase as bio-recognition elements in SPR biosensors could be preferable, since not only enzyme immobilization is a useful technology to increase the thermal and chemical stability required for many medical and biotechnological applications such as biosensors but also, bacterial laccases are usually possess high stability under drastic conditions and our results in the laboratory showed that the laccase (CotA) from Bacillus sp. HR0 has high-level intrinsic stability [49][50][51] . Moreover, the conformational changes in immobilized enzyme enhance the laccase activity toward its phenolic substrates (such as dopamine) by shifting the optimal pH to acidic value.…”

Nanoparticle- and Microparticle-Based Vaccines against Orbiviruses of Veterinary Importance

“…Although the platform was originally developed in eukaryotic cells, wh of the muNS-MS range between 1 and 4 μm, it was also adapted to work in ba the spheres have a diameter of around 400 nm (nanospheres, muNS-N Therefore, this technology could also benefit from the capabilities of nanosiz optimize antigen uptake or prompt Th1 responses. In addition, the bacter easy to handle and cost-effective for protein production, adding vers methodology [100], and allows for the production of encapsulated protein for different uses [101]. Additionally, the IC-Tagging methodology was ada inside the endoplasmic reticulum (ER).…”

“…Therefore, this technology could also benefit from the capabilities of nanosize-particles to optimize antigen uptake or prompt Th1 responses. In addition, the bacterial version is easy to handle and cost-effective for protein production, adding versatility to the methodology [ 100 ], and allows for the production of encapsulated proteins or enzymes for different uses [ 101 ]. Additionally, the IC-Tagging methodology was adapted to work inside the endoplasmic reticulum (ER).…”