title: Development of an ultrahigh-temperature process for the enzymatic hydrolysis of lactose. III. Utilization of two thermostable beta-glycosidases in a continuous ultrafiltration membrane reactor and galacto-oligosaccharide formation under steady-state conditions.
abstract: 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.
authors.affiliation: Division of Biochemical Engineering, Institute of Food Technology, Universität für Bodenkultur Wien (BOKU), Muthgasse 18, A-1190 Vienna, Austria.
authors.forename: Inge
authors.initials: I
authors.lastname: Petzelbauer
authors.forename: Barbara
authors.initials: B
authors.lastname: Splechtna
authors.forename: Bernd
authors.initials: B
authors.lastname: Nidetzky
mesh_terms: D019149:Bioreactors; D004795:Enzyme Stability; D005959:Glucosidases / Q000378:metabolism*; D006358:Hot Temperature; D006868:Hydrolysis; D007700:Kinetics; D007785:Lactose / Q000378:metabolism*; D009844:Oligosaccharides / Q000378:metabolism*; D013696:Temperature; D014462:Ultrafiltration / Q000295:instrumentation
chemical_list: D009844:Oligosaccharides; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase; D007785:Lactose