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. 2020 Jun 10:8:565.
doi: 10.3389/fbioe.2020.00565. eCollection 2020.

Alcalase Microarray Base on Metal Ion Modified Hollow Mesoporous Silica Spheres as a Sustainable and Efficient Catalysis Platform for Proteolysis

Qi Zeng  1 , Qi Li  1 , Di Sun  1 , Mingming Zheng  1
Affiliations

Alcalase Microarray Base on Metal Ion Modified Hollow Mesoporous Silica Spheres as a Sustainable and Efficient Catalysis Platform for Proteolysis

Qi Zeng et al. Front Bioeng Biotechnol. .

Abstract

The industrial exploitation of protease is limited owing to its sensitivity to environmental factors and autolysis during biocatalytic processes. In the present study, the alcalase microarray (Bacillus licheniformis, alcalase@HMSS-NH2-Metal) based on different metal ions modified hollow mesoporous silica spheres (HMSS-NH2-Metal) was successfully developed via a facile approach. Among the alcalase@HMSS-NH2-Metal (Ca2+, Zn2+, Fe3+, Cu2+), the alcalase@HMSS-NH2-Fe3+ revealed the best immobilization efficiency and enzymatic properties. This tailor-made nanocomposite immobilized alcalase on a surface-bound network of amino-metal complex bearing protein-modifiable sites via metal-protein affinity. The coordination interaction between metal ion and alcalase advantageously changed the secondary structure of enzyme, thus significantly enhanced the bioactivities and thermostability of alcalase. The as-prepared alcalase@HMSS-NH2-Fe3+ exhibited excellent loading capacity (227.8 ± 23.7 mg/g) and proteolytic activity. Compared to free form, the amidase activity of alcalase microarray increased by 5.3-fold, the apparent kinetic constant Vmax/Km of alcalase@HMSS-NH2-Fe3+ (15.6 min-1) was 1.9-fold higher than that of free alcalase, and the biocatalysis efficiency increased by 2.1-fold for bovine serum albumin (BSA) digestion. Moreover, this particular immobilization strategy efficiently reduced the bioactivities losses of alcalase caused by enzyme leaking and autolysis during the catalytic process. The alcalase microarray still retained 70.7 ± 3.7% of the initial activity after 10 cycles of successive reuse. Overall, this study established a promising strategy to overcome disadvantages posed by free alcalase, which provided new expectations for the application of alcalase in sustainable and efficient proteolysis.

Keywords: alcalase immobilization; alcalase microarray; hollow mesoporous silica spheres (HMSS); metal ion modified nanocomposite; metal-protein affinity; proteolysis.

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Figures

Figure 1
Figure 1
SEM images of HMSS-NH2 with different magnification. (a): Mag = 3.0 kx, 10 μm, (b,c): Mag = 50 kx, 200 nm.
Figure 2
Figure 2
STEM images and Si, N, C, S and Fe element maps of HMSS-NH2 (1), HMSS-NH2-Fe3+ (2), and alcalase@HMSS-NH2-Fe3+ (3).
Figure 3
Figure 3
FT-IR spectra of HMSS-NH2, HMSS-NH2-Fe3+ and alcalase@HMSS-NH2-Fe3+.
Figure 4
Figure 4
Effect of pH value (A) and temperature (B) on the activity of free alcalase and alcalase@HMSS-NH2-Fe3+. (C) Thermal stability of the acalase@HMSS-NH2-Fe3+ and free alcalase.
Figure 5
Figure 5
Lineweaver-Burk double reciprocal plots of free alcalase and acalase@HMSS-NH2-Fe3+.
Figure 6
Figure 6
Fourier transform infrared (FT-IR) spectra of (A) free alcalase and (B) immobilized alcalase by Gaussian multi-peak fitting.
Figure 7
Figure 7
Hydrolysis degree of BSA with free alcalase, alcalase@HMSS-NH2 and alcalase@HMSS-NH2-Fe3+ as the biocatalyst. Hydrolysis conditions: biocatalysts: BSA =1:10 (w/w), 40°C, pH 7.5.
Figure 8
Figure 8
The reusability of acalase@HMSS-NH2-Fe3+ and acalase@HMSS-NH2. The reaction was incubated at 40°C, pH 7.5 for 5 min with 3 mL BAEE (10 mM) as substrate.
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