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.
Highlights
Protease, like alcalase, is notable for its high biocatalytic activity and extensive adaptability in various fields (Jin et al, 2010; Zhang et al, 2018)
1.0 g hollow mesoporous silica spheres (HMSS)-NH2 was directly adding in a 25 mL ethyl alcohol which containing 1.5 mmol of different metal ions (CaCl2, ZnCl2, FeCl3, CuCl2), the mixture was stirred under a nitrogen atmosphere for 48 h at room temperature, the final products were filtered washed with acetone and ethyl alcohol, and air-dried for 24 h
A novel alcalase microarray with excellent biocatalytic performance was successfully constructed based on metal ion modified amino-functionalized HMSS
Summary
Like alcalase, is notable for its high biocatalytic activity and extensive adaptability in various fields (Jin et al, 2010; Zhang et al, 2018). There are four main methods for enzyme immobilization, such as physical adsorption, covalent binding, cross-linking of enzymes and entrapment (Xiao et al, 2018) Those conventional immobilization methods (e.g., physical adsorption, covalent binding, etc.) would lead to the losses of enzyme biocatalytic activity inevitably due to enzyme leakage and/or partial denaturation (Zhou and Hartmann, 2013). In this context, an efficient immobilization method should be developed which balance among catalytic activity, operational stability and economic applicability
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