Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose: II. Oligosaccharide formation by two thermostable beta-glycosidases

During lactose conversion at 70 degrees C, when catalyzed by beta-glycosida ses from the archea Sulfolobus solfataricus (Ss beta Gly) and Pyrococcus fu riosus (CelB), galactosyl transfer to accepters other than water competes e fficiently with complete hydrolysis of substrate. This process leads to tra nsient formation of a range of new products, mainly disaccharides and trisa ccharides, and shows a marked dependence on initial substrate concentration and lactose conversion. Oligosaccharides have been analyzed quantitatively by using capillary electrophoresis and high performance anion-exchange chr omatography. At 270 g/L initial lactose, they accumulate at a maximum conce ntration of 86 g/L at 80% lactose conversion. With both enzymes, the molar ratio of trisaccharides to disaccharides is maximal at an early stage of re action and decreases directly proportional to increasing substrate conversi on. Overall, CelB produces about 6% more hydrolysis byproducts than Ss beta Gly. However, the product spectrum of Ss beta Gly is richer in trisacchari des, and this agrees with results obtained from the steady-state kinetics a nalyses of galactosyl transfer catalyzed by Ss beta Gly and CelB. The major transgalactosylation products of Ss beta Gly and CelB have been identified . They are beta-D-Galp-(1-->3)-Glc and beta-D-Galp-(1-->6)-Glc, and beta-D- Galp(1-->3)-lactose and beta-D-Galp(1-->6)-lactose, and their formation and degradation have been shown to be dependent upon lactose conversion. Both enzymes accumulate beta(1-->6)-linked glycosides, particularly allolactose, at a late stage of reaction. Because a high oligosaccharide concentration prevails until about 80% lactose conversion, thermostable beta-glycosidases are efficient for oligosaccharide production from lactose. Therefore, they prove to be stable and versatile catalysts for lactose utilization. (C) 20 00 John Wiley & Sons, Inc.