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 solfataricus (SsbetaGly) 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 SsbetaGly, 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 not that of SsbetaGly is decreased 4-fold in the presence of 200 mM lactose at 80 degrees C. CelB displays a broader pH/activity profile than SsbetaGly, retaining at least 60% enzyme activity between pH 4 and 7. Both enzymes 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-Galactose is a weak competitive inhibitor against the release of D-glucose from lactose (Ki approximately 0.3 M), and at 80 degrees C the ratio of Ki, D-galactose to Km,lactose is 2.5 and 4.0 for CelB and SsbetaGly, respectively. SsbetaGly is activated up to 2-fold in the presence of D-glucose with respect to the maximum rate of glycosidic bond cleavage, measured with o-nitrophenyl beta-D-galactoside as the substrate. By contrast, CelB is competitively inhibited by D-glucose and has a Ki of 76 mM. The transfer of the galactosyl group from lactose to acceptors such as lactose or D-glucose rather than water is significant for both enzymes and depends on the initial lactose concentration as well as the time-dependent substrate/product ratio during batchwise lactose conversion. It is approximately 1.8 times higher for SsbetaGly, compared with CelB. Overall, CelB and SsbetaGly share their catalytic properties with much less thermostable beta-glycosidases and thus seem very suitable for lactose hydrolysis at >/=70 degrees C.
Petzelbauer
I
I
Division of Biochemical Engineering, Institute of Food Technology, Universität für Bodenkultur Wien (BOKU), Muthgasse 18, A-1190 Wien, Austria.
Nidetzky
B
B
Haltrich
D
D
Kulbe
K D
KD
D002863:Chromogenic Compounds / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D005947:Glucose / Q000494:pharmacology; D005959:Glucosidases / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007785:Lactose / Q000378:metabolism* / Q000494:pharmacology; D009598:Nitrophenylgalactosides / Q000494:pharmacology; D016944:Sulfolobus / Q000201:enzymology*; D013696:Temperature; D013997:Time Factors
D002863:Chromogenic Compounds; C055012:2-nitrophenylgalactoside; D009598:Nitrophenylgalactosides; D005959:Glucosidases; C104851:Sulfolobus solfataricus beta-glycosidase; D005947:Glucose; D007785:Lactose