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

✍ Scribed by Inge Petzelbauer; Bernd Nidetzky; Dietmar Haltrich; Klaus D. Kulbe

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 220 KB
• Volume: 64
• Category: Article
• ISSN: 0006-3592
• DOI: 10.1002/(sici)1097-0290(19990805)64:3<322::aid-bit8>3.0.co;2-9

✦ Synopsis

Recombinant ␤-glycosidases from hyperthermophilic Sulfolobus solfataricus (Ss␤Gly) and Pyrococcus furiosus (CelB) have been characterized with regard to their potential use in lactose hydrolysis at about 70°C or greater. Compared with Ss␤Gly, CelB is approximately 15 times more stable against irreversible denaturation by heat, its operational half-life time at 80°C and pH 5.5 being 22 days. The stability of CelB but not that of Ss␤Gly is decreased 4-fold in the presence of 200 mM lactose at 80°C. CelB displays a broader pH/activity profile than Ss␤Gly, 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°C. D- Galactose is a weak competitive inhibitor against the release of D-glucose from lactose (K i ≈ 0.3 M), and at 80°C the ratio of K i, D-galactose to K m,lactose is 2.5 and 4.0 for CelB and Ss␤Gly, respectively. Ss␤Gly 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 ␤-D-galactoside as the substrate. By contrast, CelB is competitively inhibited by D-glucose and has a K i 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 Ss␤Gly, compared with CelB. Overall, CelB and Ss␤Gly share their catalytic properties with much less thermostable ␤-glycosidases and thus seem very suitable for lactose hydrolysis at Ն70°C.