β-xylosidase from Selenomonas ruminantium: Immobilization, stabilization, and application for xylooligosaccharide hydrolysis

Abstract

The tetrameric β-xylosidase from Selenomonas ruminantium is very stable in alkaline pH allowing it to easily immobilize by multipoint covalent attachments on highly activated glyoxyl agarose gels. Initial immobilization resulted only in slight stabilization in relation to the free enzyme, since involvement of all subunits was not achieved. Coating the catalyst with aldehyde-dextran or polyethylenimine, fully stabilized the quaternary structure of the enzyme rendering much more stabilization to the biocatalyst. The catalyst coated with polyethylenimine of molecular weight 1300 is the most stable one exhibiting an interesting half-life of more than 10 days at pH 5.0 and 50 °C, being, therefore, 240-fold more stable than free enzyme. Optimum activity was observed in the pH range 4.0–6.0 and at 55 °C. The catalyst retained its side activity against p-nitrophenyl α-l-arabinofuranoside and it was inhibited by xylose and glucose. Kinetic parameters with p-nitrophenyl β-d-xylopyranoside as substrate were V_max 0.20 μmol.min⁻¹ mg prot.⁻¹, K_m 0.45 mM, K_cat 0.82 s⁻¹, and K_cat/K_m 1.82 s⁻¹ mM⁻¹. Xylose release was observed from the hydrolysis of xylooligosaccharides with a decrease in the rate of xylose release by increasing substrate chain-length. Due to the high thermostability and the complete stability after five reuse cycles, the applicability of this biocatalyst in biotechnological processes, such as for the degradation of lignocellulosic biomass, is highly increased.

Keywords:

• β-xylosidase
• Selenomonas ruminantium
• enzyme immobilization
• enzyme stabilization
• xylooligosaccharide hydrolysis

Declaration of interest

The authors declare not having any conflict of interest.

Part of this work was sponsored by the Spanish Ministry of Science and Innovation (Project BIO-2012-36861). C.R.F.T. gratefully acknowledges to CAPES/Ministry of Education, Brazil, through the Program Science Without Borders for the postdoctoral scholarship [Grant 3134-13-0].