Enhanced transglucosylation/hydrolysis ratio of mutants of Pyrococcus furiosus β-glucosidase: Effects of donor concentration, water content, and temperature on activity and selectivity in hexanol

Research output: Contribution to journalArticlepeer-review

Abstract

The transglucosylation reaction catalyzed by wild-type β-glucosidase CelB from hyperthermophilic Pyrococcus furiosus and active site mutants (M424K, F426Y, M424K/F426Y) was studied. The conversion of pentyl-β-glucoside to hexyl-β-glucoside in hexanol was used as a model transglucosylation reaction. Hydrolysis to glucose was a side reaction. The activity (rates of hydrolysis and transglucosylation) and the selectivity (S value) were measured as a function of pentyl-β-glucoside concentration (5-240 mM), water content (1-100% v/v), and temperature (50-95°C). All mutants had lower activity than the wild-type enzyme, but they had higher selectivity, which means that they provided a higher ratio of transglucosylation product to hydrolysis product. The largest increase in S-value (2.6 fold) was obtained by the F426Y mutant, which resulted in increased hexyl-β-glucoside yield from 56% to 69%. In addition, the F426Y enzyme had higher selectivity over the wide range of temperatures tested. The activity of CelB wild-type and CelB F426Y increased as a function of water activity (aw), and complete activation by the water was obtained in a two-phase system with 20% water phase. In contrast to CelB wild-type, the F426Y mutant had transferase activity as low as aw = 0.29. Surprisingly, the S value increased with increasing water activity up to aw = 0.92. At still higher water content the S value decreased.

Original languageEnglish
Pages (from-to)656-665
Number of pages10
JournalBiotechnology and Bioengineering
Volume75
Issue number6
DOIs
Publication statusPublished - 2001 Dec 20

Subject classification (UKÄ)

  • Biocatalysis and Enzyme Technology

Free keywords

  • βglucosidase
  • Activity
  • Hexanol
  • Selectivity
  • Transglucosylation

Fingerprint

Dive into the research topics of 'Enhanced transglucosylation/hydrolysis ratio of mutants of Pyrococcus furiosus β-glucosidase: Effects of donor concentration, water content, and temperature on activity and selectivity in hexanol'. Together they form a unique fingerprint.

Cite this