Enhancement of Alkaline Protease Activity and Stability via Covalent Immobilization onto Hollow Core-Mesoporous Shell Silica Nanospheres

Abstract

The stability and reusability of soluble enzymes are of major concerns, which limit their industrial applications. Herein, alkaline protease from Bacillus sp. NPST-AK15 was immobilized onto hollow core-mesoporous shell silica (HCMSS) nanospheres. Subsequently, the properties of immobilized proteases were evaluated. Non-, ethane- and amino-functionalized HCMSS nanospheres were synthesized and characterized. NPST-AK15 was immobilized onto the synthesized nano-supports by physical and covalent immobilization approaches. However, protease immobilization by covalent attachment onto the activated HCMSS–NH2 nanospheres showed highest immobilization yield (75.6%) and loading capacity (88.1 μg protein/mg carrier) and was applied in the further studies. In comparison to free enzyme, the covalently immobilized protease exhibited a slight shift in the optimal pH from 10.5 to 11.0, respectively. The optimum temperature for catalytic activity of both free and immobilized enzyme was seen at 60 °C. However, while the free enzyme was completely inactivated when treated at 60 °C for 1 h the immobilized enzyme still retained 63.6% of its initial activity. The immobilized protease showed higher Vmax, kcat and kcat/Km, than soluble enzyme by 1.6-, 1.6- and 2.4-fold, respectively. In addition, the immobilized protease affinity to the substrate increased by about 1.5-fold. Furthermore, the enzyme stability in various organic solvents was significantly enhanced upon immobilization. Interestingly, the immobilized enzyme exhibited much higher stability in several commercial detergents including OMO, Tide, Ariel, Bonux and Xra by up to 5.2-fold. Finally, the immobilized protease maintained significant catalytic efficiency for twelve consecutive reaction cycles. These results suggest the effectiveness of the developed nanobiocatalyst as a candidate for detergent formulation and peptide synthesis in non-aqueous media.

Bacterial and Fungal Proteolytic Enzymes: Production, Catalysis and Potential Applications

“…For peptidase application, the protein structural stability is a crucial biochemical property favoring its feasibility for commercial use. Enzymes are susceptible to denaturation by different physicochemical agents, including deamination, proteolysis, oxidation, inhibition, and structural changes due to pH and temperature [82][83][84][85][86].…”

Recent Advances in Enzyme-Nanostructure Biocatalysts with Enhanced Activity

“…Representative work on each aspect will be discussed here. Alkaline protease Hollow silica nanosphere 2.40 [78] Catalysts 2020, 10, 338 4 of 15…”

How to Lengthen the Long-Term Stability of Enzyme Membranes: Trends and Strategies

“…Fouling due to macromolecules would occur at the outer surface of the porous materials as described above; within the porous part, fouling can be avoided. On the basis of these ideas, porous carbon [94], microporous polymers [95,96], mesoporous zirconium [97], and mesoporous silica [98][99][100][101][102] have been developed. In particular, mesoporous silica has been given attention because it has designable pore size that can be utilized in implanting anchor molecules for covalent bonding.…”

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