Immobilization of thermostable β-glucosidase variants on acrylic supports for biocatalytic processes in hot water

Two variants of the thermostable β-glucosidase TnBgl1A (wt and N221S/P342L) from Thermotoga neapolitana were immobilized on acrylic supports (Eupergit® C, Eupergit® C250L, and cryogel) and evaluated at conditions close to the boiling point of water. Thermo-gravimetric analysis showed the supports to be stable <250 °C. Both wt and N221S/P342L were covalently bound to oxirane-groups respectively via glutaraldehyde spacers, and for coupling reactions 26 Lys and 20 Ser/Thr were surface-located. Immobilized enzymes were active on all supports in the temperature range 80–95 °C, but the observed specific activity was low (≤19 U mg−1) using the cryogel. More than 91% of the initial activity was maintained after ten times recycling, and the same was recovered after 3 months storage at 4 °C for Eupergit® supports by simply incubating the preparation with bovine serum albumin. No storage loss was detectable on cryogels. The glutaraldehyde spacer improved activity on cryogels, but not on Eupergit® supports. Immobilization on Eupergit® C250L yielded the highest observed specific activity (254 U mg−1 for N221S/P342L) in a procedure including blocking of free oxirane-groups by BSA. This biocatalyst was used for on-line hydrolysis of quercetin-glucosides in a yellow onion extract at 80 °C, proving the immobilized biocatalyst to be promising in on-line systems for extraction and hydrolysis using hot pressurized water.