Hydrolysis of lactose by hyperthermophilic beta-glycosidases from the archaea Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) was carried out at 70 degrees C in a continuous stirred-tank reactor (CSTR) coupled to a 10-kDa cross-flow ultrafiltration module to recycle the enzyme. Recirculation rates of > or =1 min(-1), reaction of proteins with reducing sugars, and enzyme adsorption onto the membrane are major "operational" factors of enzyme inactivation in the CSTR. They cause the half-life times of SsbetaGly and CelB to be reduced two- and eight-fold, respectively, the average value for both enzymes now being approximately 5 to 7 days. Using lactose at initial concentrations of 45 and 170 g/L, the CSTR was operated at a constant conversion level of approximately 80% for more than 2 weeks without the occurrence of microbial contamination. The productivities for the SsbetaGly-catalyzed conversion of lactose were determined at different dilution rates and initial substrate concentrations, and exceed by a factor of < or =2 those observed with CelB under otherwise identical conditions. This difference reflects the approximately eight-fold stronger product inhibition of CelB by D-glucose. While the maximum total galacto-oligosaccharide production (90-100 mM) at 170 g/L lactose in the CSTR was not different from that in the batch reactor (CelB) or was greater by approximately 25% (SsbetaGly), continuous and batchwise reactions with both enzymes differed markedly with regard to relative proportions of the individual saccharide components present at 80% substrate conversion. The CSTR yielded an up to four-fold greater ratio of disaccharides to trisaccharides concomitant with a 5- to 30-fold larger relative proportion of beta-D-Galp-(1-->3)-D-Glc in the product mixture. The results show that apart from continuous hydrolysis of lactose at 70 degrees C, a CSTR charged with SsbetaGly or CelB and operated at steady-state conditions could be a useful reaction system for the production of galacto-oligosaccharides in which composition is narrower and more easily programmable, in terms of the individual components contained, as compared to the batchwise reaction.

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Recombinant beta-glycosidases from hyperthermophilic Sulfolobus solfataricus (SsbetaGly) and Pyrococcus furiosus (CelB) have been characterized with regard to their potential use in lactose hydrolysis at about 70 degrees C or greater. Compared with SsbetaGly, CelB is approximately 15 times more stable against irreversible denaturation by heat, its operational half-life time at 80 degrees C and pH 5.5 being 22 days. The stability of CelB but not that of SsbetaGly is decreased 4-fold in the presence of 200 mM lactose at 80 degrees C. CelB displays a broader pH/activity profile than SsbetaGly, retaining at least 60% enzyme activity between pH 4 and 7. Both enzymes have a similar activation energy for lactose hydrolysis of approximately 75 kJ/mol (pH 5.5), and this is constant between 30 and 95 degrees C. D-Galactose is a weak competitive inhibitor against the release of D-glucose from lactose (Ki approximately 0.3 M), and at 80 degrees C the ratio of Ki, D-galactose to Km,lactose is 2.5 and 4.0 for CelB and SsbetaGly, respectively. SsbetaGly is activated up to 2-fold in the presence of D-glucose with respect to the maximum rate of glycosidic bond cleavage, measured with o-nitrophenyl beta-D-galactoside as the substrate. By contrast, CelB is competitively inhibited by D-glucose and has a Ki of 76 mM. The transfer of the galactosyl group from lactose to acceptors such as lactose or D-glucose rather than water is significant for both enzymes and depends on the initial lactose concentration as well as the time-dependent substrate/product ratio during batchwise lactose conversion. It is approximately 1.8 times higher for SsbetaGly, compared with CelB. Overall, CelB and SsbetaGly share their catalytic properties with much less thermostable beta-glycosidases and thus seem very suitable for lactose hydrolysis at >/=70 degrees C.