Characterization of Cross-Linked Enzyme Aggregates of the Y509E Mutant of a Glycoside Hydrolase Family 52 β-xylosidase from G. stearothermophilus

Abstract

Cross-linked enzyme aggregates (CLEAs) of the Y509E mutant of glycoside hydrolase family 52 β-xylosidase from Geobacillus stearothermophilus with dual activity of β-xylosidase and xylanase (XynB2Y509E) were prepared. Ammonium sulfate was used as the precipitant agent, and glutaraldehyde as cross-linking agent. The optimum conditions were found to be 90% ammonium sulfate, 12.5 mM glutaraldehyde, 3 h of cross-linking reaction at 25 °C, and pH 8.5. Under these (most effective) conditions, XynB2Y509E-CLEAs retained 92.3% of their original β-xylosidase activity. Biochemical characterization of both crude and immobilized enzymes demonstrated that the maximum pH and temperature after immobilization remained unchanged (pH 6.5 and 65 °C). Moreover, an improvement in pH stability and thermostability was also found after immobilization. Analysis of kinetic parameters shows that the Km value of XynB2Y509E-CLEAs obtained was slightly higher than that of free XynB2Y509E (1.2 versus 0.9 mM). Interestingly, the xylanase activity developed by the mutation was also conserved after the immobilization process.

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

“…Site-directed mutagenesis of the xynB2 gene was developed using the QuikChange II kit (Stratagene, La Jolla, CA, USA); the plasmid with the mutation was named pJAVI100 [32]. The E. coli C43 strain harboring the plasmid pJAVI100 was cultivated in LB broth supplemented with 100 µg/mL ampicillin at 37 • C, 250 rpm.…”

“…The supernatant was collected and heat treated at 45 • C for 30 min and centrifuged again. The supernatant was dialyzed against 0.1 M citrate-phosphate-glycine buffer (CFG), pH 6.5 (29.41 g C 6 H 5 O 7 Na 2 .2H 2 O, 13.80 g NaH 2 PO 4 , and 7.51 g NH 2 CH 2 COOH in 1 L distilled water) at 4 • C overnight [32].…”

“…The reaction of immobilized XynB2 Y509E was initiated by the addition of 18 mg of beads with immobilized enzyme to 50 µL 2.2 mM substrate and 150 mM citrate-phosphate-glycine buffer at 50 • C. After 5 min, the reaction was stopped and color developed through the addition of 600 µL of 1M Na 2 CO 3 . Color intensity was read at 410 nm by using the extinction coefficient ∆ε = 18 mM −1 cm −1 [32] in an SP-830plus spectrophotometer (Metertech). One enzyme unit (IU) was defined as the amount of enzyme required to produce 1 µmol of pNP per minute, and specific activity was defined as units per mg weight of protein-containing spheres.…”

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.…”