Design of robust immobilized Xys1[delta] xylanase biocatalysts: Process intensification for XOS production from lignocellulosic residues

Resumen

Xylo-oligosaccharides (XOS) have been described as prebiotics and display beneficial effects upon human health. The xylanase Xys1 , from Streptomyces halstedii JM8, was immobilized on glyoxyl-agarose beads by multipoint covalent attachment. The resulting immobilized Xys1 biocatalyst was 62-fold more thermo-stable than its soluble counterpart, and was used to catalyse the hydrolysis reaction of xylan for XOS production. A strategy based on surface coating with layers of polymers was developed to further increase the thermo-stability of this biocatalyst. The optimal modification consisted of surface coating with a bilayer formed by a layer of derived dextran polymer and a layer of polyethylenimine. The optimised biocatalyst was 550-fold more stable than the soluble Xys1 enzyme (at 70 ºC, pH 7). This optimised biocatalyst was used for production of xylo-oligosaccharides from soluble xylans of various sources in batch mode. Total reaction yields for beechwood, wheat and corncob xylan were 93% in 4 h, 44% in 5 h and 100% in 1 h, respectively. At these time points, xylan conversion yields to XOS were 65 % for beechwood xylan; 31% for wheat arabinoxylan; and 76% for corncob xylan. The optimised biocatalyst was reused for 15 reaction cycles of beechwood xylan hydrolysis without affecting its catalytic activity. To further increase the thermo-stability of the immobilized Xys1 biocatalyst, a strategy based on intensification of rigidification of the enzyme surface region involved in multipoint covalent attachment was developed. For this purpose, 1 4 additional lysine residues were introduced by substitution of native arginine residues to form 1 4 additional covalent bonds between enzyme and immobilization support. These covalent bonds, if formed, did not promote increased thermo-stabilizing effects, as they did not probably provide more intense rigidification of this enzyme surface area. Continuous flow reaction of corncob xylan hydrolysis for XOS production has been developed with an optimised packed-bed reactor (PBR). For this purpose, the xylanase Xys1 was immobilized by multipoint covalent immobilization on supports consisting of methacrylic polymer matrices previously functionalized with glyoxyl groups. The aim was to avoid enzyme leaching and to allow high flow rates since these supports display enhanced mechanical properties. The optimal support presented an area BET value of 69.5 m2/g and a narrow poresize distribution (80 nm pore size diameter). The resulting immobilized Xys1 biocatalyst onto this support displayed a maximum protein loading of 20 mg/g support, and high thermostability. A PBR was developed with this biocatalyst and used for continuous xylan hydrolysis at different flow rates. The specific volumetric productivity for XOS, 3,277 gXOS genzyme -1 h-1, was achieved at 10 mL/min flow rate with minimum production of xylose.