Optimization of an innovative hollow-fiber process to produce lactose-reduced skim milk.

The research field for applications of lactose hydrolysis has been investigated for several decades. Lactose intolerance, improvement for technical processing of solutions containing lactose, and utilization of lactose in whey are the main topics for development of biotechnological processes. We report here the optimization of a hollow-fiber membrane reactor process for enzymatic lactose hydrolysis.

Hydrolysis of lactose by beta-glycosidase CelB from hyperthermophilic archaeon Pyrococcus furiosus: comparison of hollow-fiber membrane and packed-bed immobilized enzyme reactors for continuous processing of ultrahigh temperature-treated skim milk.

Recombinant beta-glycosidase CelB from the hyperthermophilic archaeon Pyrococcusfuriosus was produced through expression of the plasmid-encoded gene in Escherichia coli. Bioreactor cultivations of E. coli in the presence of the inductor isopropyl-1-thio-beta-D-galactoside (0.

Development of an ultrahigh-temperature process for the enzymatic hydrolysis of lactose. IV. Immobilization of two thermostable beta-glycosidases and optimization of a packed-bed reactor for lactose conversion.

Recombinant hyperthermostable beta-glycosidases from the archaea Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus furiosus (CelB) were covalently attached onto the insoluble carriers chitosan, controlled pore glass (CPG), and Eupergit C. For each enzyme/carrier pair, the protein-binding capacity, the immobilization yield, the pH profiles for activity and stability, the activity/temperature profile, and the kinetic constants for lactose hydrolysis at 70 degrees C were determined. Eupergit C was best among the carriers in regard to retention of native-like activity and stability of Ss beta Gly and CelB over the pH range 3.

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.

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.

Galactosyl transfer catalyzed by thermostable beta-glycosidases from Sulfolobus solfataricus and Pyrococcus furiosus: kinetic studies of the reactions of galactosylated enzyme intermediates with a range of nucleophiles.

The transfer of a galactosyl group from an enzyme to a number of neutral primary alcohols, phenol and azide has been studied during the reactions at 80 degrees C of thermostable beta-glycosidases from Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus furiosus (CelB) with 2-nitrophenyl beta-D-galactopyranoside or lactose (4-O-beta-D-galactopyranosyl D-glucopyranose) as substrates. The rate constant ratios, k(Nu)/k(water), for partitioning of the galactosylated enzyme intermediates between reaction with nucleophiles (k(Nu), M(-1) s(-1)) and water (k(water), s(-1)) have been determined from the difference in the initial velocities of the formation of 2-nitrophenol or D-glucose, and D-galactose. The results show that hydrophobic bonding interactions contribute approximately 8 kJ mol(-1) to the stabilization of the transition state for the reaction of galactosylated enzyme intermediates of Ss beta Gly and CelB with 1-butanol, compared to the transition state for the enzymatic reaction with methanol.