Biotechnology and Bioengineering, Vol.77, No.6, 619-631, 2002
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 (SsbetaGly) 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 70degreesC were determined, Eupergit C was best among the carriers in regard to retention of native-like activity and stability of SsbetaGly and CelB over the pH range 3.0-7.5. Its protein binding capacity of similar to0.003 (on a mass basis) was one-third times that of CPG, while immobilization yields were typically 80% in each case. Activation energies for lactose conversion by the immobilized enzymes at pH 5.5 were in the range 5060 kJ/mol. This is compared to values of similar to75 kJ/mol for the free enzymes. Immobilization expands the useful pH range for CelB and SsbetaGly by approximately 1.5 pH units toward pH 3.5 and pH 4.5, respectively. A packed-bed enzyme reactor was developed for the continuous conversion of lactose in different media, including whey and milk, and operated over extended reaction times of up to 14 days. The productivities of the Eupergit C-immobilized enzyme reactor were determined at dilution rates between 1 and 12 h(-1), and using 45 and 170 g/L initial lactose. Results of kinetic modeling for the same reactor, assuming plug flow and steady state, suggest the presence of mass-transfer limitation of the reaction rate under the conditions used. Formation of galactooligosaccharides in the continuous packed-bed reactor and in the batch reactor using free enzyme was closely similar in regard to yield and individual saccharide components produced.