Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2016 Jan 29;17(2):184.
doi: 10.3390/ijms17020184.

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

Abdelnasser Salah Shebl Ibrahim  1   2 , Ali A Al-Salamah  3 , Ahmed M El-Toni  4   5 , Khalid S Almaary  6 , Mohamed A El-Tayeb  7 , Yahya B Elbadawi  8 , Garabed Antranikian  9
Affiliations

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

Abdelnasser Salah Shebl Ibrahim et al. Int J Mol Sci. .

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 V(max), k(cat) and k(cat)/K(m), 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.

Keywords: alkaline protease; alkaliphiles; detergents; hollow core-mesoporous shell silica nanospheres; immobilization; nanotechnology.

PubMed Disclaimer

Figures

Figure 1
Figure 1
TEM images of amino-functionalized hollow core-mesoporous silica (HCMSS–NH2) nanospheres prepared by anionic surfactant.
Figure 2
Figure 2
(A) N2 adsorption-desorption isotherms and (B) pore size distribution of (a) non-functionalized, (b) amino-functionalized and (c) ethane-functionalized of hollow core-mesoporous shell silica nanospheres.
Figure 3
Figure 3
FT-IR spectra of (a) non-functionalized; (b) amino-functionalized and (c) ethane-functionalized hollow core-mesoporous shell silica nanospheres.
Figure 4
Figure 4
FTIR spectra of (a) Free NPST-AK15 protease enzyme (balck); (b) amino-functionalized hollow core-mesoporous silica (HCMSS–NH2) nanospheres (blue) and (c) NPST-AK15 protease immobilized onto amino-functionalized hollow core-mesoporous silica (HCMSS–NH2) nanospheres (red).
Figure 5
Figure 5
Loading efficiency of hollow core-mesoporous shell silica nanospheres for immobilization of NPST-AK15 protease.
Figure 6
Figure 6
Effect of pH on the activity of the free and immobilized NPST-AK15 protease. Enzyme activity was measured at 55 °C. The results represent the mean of three separate experiments, and error bars are indicated.
Figure 7
Figure 7
Effect of temperature on activity (A) and stability (B) of free and immobilized NPST-AK15 protease. Protease activity was measured under the standard assay conditions at various temperatures (30–75 °C) at pH 10. For thermal stability, the enzyme was pre-incubated at different temperatures for 2 h at pH 11.0, and the residual enzyme activities were estimated at regular intervals under standard assay conditions. The non-heated enzymes were taken as 100%. The results represent the mean of three separate experiments, and error bars are indicated.
Figure 7
Figure 7
Effect of temperature on activity (A) and stability (B) of free and immobilized NPST-AK15 protease. Protease activity was measured under the standard assay conditions at various temperatures (30–75 °C) at pH 10. For thermal stability, the enzyme was pre-incubated at different temperatures for 2 h at pH 11.0, and the residual enzyme activities were estimated at regular intervals under standard assay conditions. The non-heated enzymes were taken as 100%. The results represent the mean of three separate experiments, and error bars are indicated.
Figure 8
Figure 8
Estimation of kinetic parameters of the free and immobilized NPST-AK15 protease. The enzyme activity was measured at various casein concentrations (1.0–10.0 mg/mL) at pH 11 and 60 °C. The Km and Vmax values were determined using linearized Lineweaver-Burk plot. S: Substrate concentration; V: Protease specific activity.
Figure 9
Figure 9
Effect of organic solvents on stability of free and immobilized NPST-AK15 protease.
Figure 10
Figure 10
Effect of surfactants (A) and commercial detergents (B) on stability of free and immobilized NPST-AK15 protease stability. SDS: sodium dodecyl sulfate; CTAB: cetyltrimethylammonium bromide.
Figure 11
Figure 11
Reusability of NPST-AK15 protease immobilized within hollow core-mesoporous shell silica (HCMSS–NH2) nanospheres.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Jain D., Pancha I., Mishra S.K., Shrivastav A., Mishra S. Purification and characterization of haloalkaline thermoactive, solvent stable and SDS-induced protease from Bacillus sp.: A potential additive for laundry detergents. Bioresour. Technol. 2012;115:228–236. doi: 10.1016/j.biortech.2011.10.081. - DOI - PubMed
    1. Soozanipour A., Taheri-Kafrani A., Isfahani A.L. Covalent attachment of xylanase on functionalized magnetic nanoparticles and determination of its activity and stability. Chem. Eng. J. 2015;270:235–243. doi: 10.1016/j.cej.2015.02.032. - DOI
    1. Bouacem K., Bouanane-Darenfed A., Laribi-Habchi H., ben Elhoul M., Hmida-Sayari A., Hacene H., Ollivier B., Fardeau M.L., Jaouadi B., Bejar S. Biochemical characterization of a detergent stable serine alkalineprotease from Caldicoprobacter guelmensis. Int. J. Biol. Macromol. 2015;81:299–307. doi: 10.1016/j.ijbiomac.2015.08.011. - DOI - PubMed
    1. Ben Elhoul M., Jaouadi N.Z., Rekik H., Bejar W., Touioui S.B., Hmidi M., Badis A., Bejar S., Jaouadi B. A novel detergent-stable solvent-tolerant serine thiol alkaline protease from Streptomyces koyangensis TN650. Int. J. Biol. Macromol. 2015;79:871–882. doi: 10.1016/j.ijbiomac.2015.06.006. - DOI - PubMed
    1. Patil U., Chaudhari A. Purification and characterizationofsolvent-tolerant, thermostable, alkaline metalloprotease from alkalophilic Pseudomonas aeruginosa MTCC 7926. J. Chem. Technol. Biotechnol. 2009;84:1255–1262. doi: 10.1002/jctb.2169. - DOI

Publication types

LinkOut - more resources