Development of an ultrahigh‐temperature process for the enzymatic hydrolysis of lactose. III. Utilization of two thermostable β‐glycosidases in a continuous ultrafiltration membrane reactor and galacto‐oligosaccharide formation under steady‐state conditions

Hydrolysis of lactose by hyperthermophilic β‐glycosidases from the archaea Sulfolobus solfataricus (SsβGly) and Pyrococcus furiosus (CelB) was carried out at 70°C in a continuous stirred‐tank reactor (CSTR) coupled to a 10‐kDa cross‐flow ultrafiltration module to recycle the enzyme. Recirculation rates of ≥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 SsβGly and CelB to be reduced two‐ and eight‐fold, respectively, the average value for both enzymes now being ∼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 ∼80% for more than 2 weeks without the occurrence of microbial contamination. The productivities for the SsβGly‐catalyzed conversion of lactose were determined at different dilution rates and initial substrate concentrations, and exceed by a factor of ≤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 m M ) at 170 g/L lactose in the CSTR was not different from that in the batch reactor (CelB) or was greater by ∼25% (SsβGly), 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 β‐ D ‐Gal p ‐(1→3)‐ D ‐Glc in the product mixture. The results show that apart from continuous hydrolysis of lactose at 70°C, a CSTR charged with SsβGly 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. © 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 394–404, 2002; DOI 10.1002/bit.10106