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.
Highlights
Enzymes are widely used as alternative of chemical catalysts in various industries; and playing a significant role in development of ecofriendly technology, owing to their unique properties including high specificity, mild reaction conditions, low toxicity and selectivity [1,2]
Hollow core-mesoporous shell silica (HCMSS) nanospheres were synthesized by anionic surfactant through a soft-templating route assisted by ultrasonic waves
Negatively charged silica nuclei are reacted with anionic surfactant through a co-structure directing agent (3-aminopropyltrimethoxysilane (APMS)) to produce mesoporous silica nanospheres
Summary
Enzymes are widely used as alternative of chemical catalysts in various industries; and playing a significant role in development of ecofriendly technology, owing to their unique properties including high specificity, mild reaction conditions, low toxicity and selectivity [1,2]. Alkaline proteases, accounting alone for approximately 40% of the total global enzymes market, proved suitable for several industrial applications such as detergent, pharmaceutical, leather, dairy, silk, soy processing, brewery, meat tenderization, and waste management [5,6]. This is attributed mainly to significant activity and operational stability under harsh operational conditions, including high pH and temperature and in the presence of surfactants [4,7,8].
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