Continuous production of lactulose by immobilized thermostable β-glycosidase from Pyrococcus furiosus

A continuous enzymatic process for the production of the prebiotic disaccharide lactulose through transgalactosylation was developed using free and immobilized β-glycosidase from Pyrococcus furiosus. The hyperthermostable β-glycosidase (CelB) was immobilized onto an anion-exchange resin (Amberlite IRA-93) or onto Eupergit® C with immobilization yields of 72% and 83%, respectively. The immobilized biocatalysts demonstrated specific activities of 920 nkat g−1 dry carrier and 1500 nkat g−1 dry carrier at 75 °C with p-nitrophenyl-β-d-galactopyranoside as substrate. Continuous biotransformations in packed-bed reactors using carrier bound CelB and in an enzyme membrane reactor using free CelB were carried out at 75 °C. Maximum lactulose yields of 43% related to the initial lactose concentration were reached with the carrier bound CelB preparations. The corresponding productivities were 52 g lactulose l−1 h−1 (Amberlite IRA-93) and 15 g lactulose l−1 h−1 (Eupergit® C), respectively. The free enzyme tested in an enzyme membrane reactor showed a product yield of 41% and a productivity of 12 g lactulose l−1 h−1 in the first day. While both carrier bound CelB preparations were 100% stable for at least 14 days, the half-life of the free CelB in the enzyme membrane reactor was only about 1.5 days.