Development of an ultra-high-temperature process for the enzymatic hydrolysis of lactose. I. The properties of two thermostable beta-glycosidases

Recombinant beta-glycosidases from hyperthermophilic Sulfolobus solfataricu s (Ss beta Gly) and Pyrococcus furiosus (CelB) have been characterized with regard to their potential use in lactose hydrolysis at about 70 degrees C or greater. Compared with Ss beta Gly, CelB is approximately 15 times more stable against irreversible denaturation by heat, its operational half-life time at 80 degrees C and pH 5.5 being 22 days. The stability of CelB but n ot that of Ss beta Gly is decreased 4-fold in the presence of 200 mM lactos e at 80 degrees C. CelB displays a broader pH/activity profile than Ss beta Gly, retaining at least 60% enzyme activity between pH 4 and 7. Both enzym es have a similar activation energy for lactose hydrolysis of approximately 75 kJ/mol (pH 5.5), and this is constant between 30 and 95 degrees C. D-Ga lactose is a weak competitive inhibitor against the release of D-glucose fr om lactose (K-i approximate to 0.3 M), and at 80 degrees C the ratio of K-i ,K-D-galactose to K-m,K-lactose is 2.5 and 4.0 for CelB and Ss beta Gly, re spectively. Ss beta Gly is activated up to 2-fold in the presence of D-gluc ose with respect to the maximum rate of glycosidic bond cleavage, measured with o-nitrophenyl beta-D-galactoside as the substrate. By contrast, CelB i s competitively inhibited by D-glucose and has a K-i of 76 mM. The transfer of the galactosyl group from lactose to accepters such as lactose or D-glu cose rather than water is significant for both enzymes and depends on the i nitial lactose concentration as well as the time-dependent substrate/produc t ratio during batchwise lactose conversion. It is approximately 1.8 times higher for Ss beta Gly, compared with CelB. Overall, CelB and Ss beta Gly s hare their catalytic properties with much less thermostable beta-glycosidas es and thus seem very suitable for lactose hydrolysis at greater than or eq ual to 70 degrees C. (C) 1999 John Wiley & Sons, Inc.