Efficiency Assessment between Entrapment and Covalent Bond Immobilization of Mutant β-Xylosidase onto Chitosan Support

Abstract

The Y509E mutant of β-xylosidase from Geobacillus stearothermophilus (XynB2Y509E) (which also bears xylanase activity) has been immobilized in chitosan spheres through either entrapment or covalent bond formation methods. The maximum immobilization yield by entrapment was achieved by chitosan beads developed using a 2% chitosan solution after 1 h of maturation time in CFG buffer with ethanol. On the other hand, the highest value in covalent bond immobilization was observed when employing chitosan beads that were prepared from a 2% chitosan solution after 4 h of activation in 1% glutaraldehyde solution at pH 8. The activity expressed after immobilization by covalent bonding was 23% higher compared to the activity expressed following entrapment immobilization, with values of 122.3 and 99.4 IU.g−1, respectively. Kinetic data revealed that catalytic turnover values were decreased as compared to a free counterpart. Both biocatalysts showed increased thermal and pH stability, along with an improved storage capacity, as they retained 88% and 40% of their activity after being stored at 4 °C for two months. Moreover, XynB2Y509E immobilized by covalent binding also exhibited outstanding reusability, retaining 92% of activity after 10 cycles of reuse. In conclusion, our results suggest that the covalent bond method appears to be the best choice for XynB2Y509E immobilization.

Structural Characterization of β-Xylosidase XynB2 from Geobacillus stearothermophilus CECT43: A Member of the Glycoside Hydrolase Family GH52

“…The reaction was stopped by the addition of 1 M Na 2 CO 3 . An extinction coefficient of ∆ε = 18 mM −1 cm −1 was used for pNP [20,21]. Glycerol inhibition was tested to concentrations of up to 50% (v:v).…”

“…Protein Engineering has been explored on the GH52 scaffold, allowing the introduction/improvement of xylanase [18] or glycosynthase activities [19]. A mutant form of XynB2 from Geobacillus stearothermophilus CECT43 also proved useful as an immobilized biocatalyst, such as cross-linked enzyme aggregates or covalently immobilized enzymes, resulting in pH stability and thermostability improvement of the biocatalyst [20,21]. These results suggest that β-xylosidase might be a good candidate for the generation of cross-linked enzyme crystals [22], for which protein crystallization is a mandatory pre-requisite.…”

Phenol Removal from Wastewater Using Tyrosinase Enzyme Immobilized in Granular Activated Carbon and Activated Chitosan Beads

“…Figure4apresents the enzymatic activity results obtained using T-GAC, and Figure4billustrates the results of the enzymatic activity of the T-ACBs with different GA concentrations and chitosan beads without activation.The immobilizations in GAC and chitosan beads mainly occurred via physical adsorption, so enzyme desorption is more accessible in case of operational changes. The ACBs' support was subjected to a chemical activation process with glutaraldehyde, a cross-linking agent, which introduces aldehyde functional groups into the chitosan spheres that form covalent bonds with the amine (NH 2 ) and thiol (SH) functional groups of tyrosinase[34]. Some authors have observed that during the immobilization of enzymes in GAC, the percentage of immobilized enzymes usually stabilizes under less than 120 min of contact between the enzyme and the support[28][29][30].…”