3.2.1.B28: Pyrococcus furiosus beta-glycosidase
This is an abbreviated version!
For detailed information about Pyrococcus furiosus beta-glycosidase, go to the full flat file.
Reaction
The enzyme produces mannose and glucose mainly from mannooligosaccharides and cellobiose by hydrolyzing them from the reducing end. It can not hydrolyze alpha-form substrates and bulk polymers like mannan and starch =
Synonyms
beta-glycosidase, Bgl, BGLPf, CelB, PFTG
ECTree
3.2.1.B28 Pyrococcus furiosus beta-glycosidaseAdvanced search results
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results (Engineering
Engineering on EC 3.2.1.B28 - Pyrococcus furiosus beta-glycosidase
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PROTEIN VARIANTS 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
A419T
159% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 109% with 4-nitrophenyl beta-D-galactoside, 53% residual activity after 1 h at 106°C
E386G
mutation in nucleophile residue E387, mutation completely abolishes activity under statndard conditions. The addition of 2 M sodium formate as an external nucleophile leads to the recovery of 8.40% activity with accumulation of oligosaccharides. At pH 3.0 and low concentrations of sodium formate buffer, the hyperthermophilic glycosynthase shows kcat values similar to those of the wild-type and 17fold higher than those observed at the usual reactivation conditions in 2 M sodium formate at pH 6.5
E417S
mutation in phosphate binding site, 5fold increase of the efficiency of hydrolyzing o-nitrophenol-beta-D-galactopyranoside-6-phosphate. Activity on nonphosphorylated sugars is largely reduced
E417S/M424K/F426Y
mutations in phosphate binding site, 3fold increase of the efficiency of hydrolyzing 2-nitrophenyl beta-D-galactopyranoside-6-phosphate. Activity on nonphosphorylated sugars is largely reduced
F426Y
I161V
110% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 37% with 4-nitrophenyl beta-D-galactoside, 84% residual activity after 1 h at 106°C
I67T
110% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 97% with 4-nitrophenyl beta-D-galactoside, 100% residual activity after 1 h at 106°C
K285R
155% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 103% with 4-nitrophenyl beta-D-galactoside, 58% residual activity after 1 h at 106°C
K70R
175% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 147% with 4-nitrophenyl beta-D-galactoside, 1% residual activity after 1 h at 106°C
M424K
M424K/F426Y
mutant has lower activity than the wild-type enzyme, but provides a higher ratio of transglucosylation product to hydrolysis products compared to wild-type enzyme
M424V
199% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 140% with 4-nitrophenyl beta-D-galactoside, 24% residual activity after 1 h at 106°C
N206N
about 300fold decrease in catalytic efficiency for 4-nitrophenyl-beta-D-glucospyranoside, about 50fold decrease in catalytic efficiency for 4-nitrophenyl-beta-D-galactoside
N415S
259% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 64% with 4-nitrophenyl beta-D-galactoside. 7.5fold increase in the ratio of 4-nitrophenyl beta-D-glucopyranoside/p-nitrophenyl beta-D-galactopyranoside hydrolysis, no decrease in thermostability
Q150W
the mutant enzyme has more affinity (Km) towards each substrate but shows lower turnover number (kcat) compared to the wild-type enzyme
Q77R/Q150W
the mutant enzyme has more affinity (Km) towards each substrate but shows lower turnover number (kcat) compared to the wild-type enzyme
R77Q
2000fold decrease in catalytic efficiency for 4-nitrophenyl-beta-D-glucospyranoside, 175fold decrease in catalytic efficiency for 4-nitrophenyl-beta-D-galactoside. The mutant enzyme shows a biphasic behavior for the hydrolysis of 4-nitrophenyl beta-D-mannopyranoside with separate kinetic parameters above and below 1 mM 4-nitrophenyl beta-D-mannopyranoside
R77Q/N206D
about 600fold decrease in catalytic efficiency for 4-nitrophenyl-beta-D-glucospyranoside, about 430fold decrease in catalytic efficiency for 4-nitrophenyl-beta-D-galactoside. The mutant enzyme shows a biphasic behavior for the hydrolysis of 4-nitrophenyl beta-D-mannopyranoside with separate kinetic parameters above and below 1 mM 4-nitrophenyl beta-D-mannopyranoside
T371A
148% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 149% with 4-nitrophenyl beta-D-galactoside, 14% residual activity after 1 h at 106°C
V211A
260% of wild-type activity with 4-nitrophenyl beta-D-glucoside, 151% with 4-nitrophenyl beta-D-galactoside, 1% residual activity after 1 h at 106°C
additional information
F426Y
mutant has lower activity than the wild-type enzyme, but provides a higher ratio of transglucosylation product to hydrolysis products compared to wild-type enzyme, 2.6fold increase. The mutant enzyme has higher selectivity over the wide range of temperatures tested
F426Y
mutation in phosphate binding site, results in an increased affinity for galactosides. Activity on nonphosphorylated sugars is largely reduced
M424K
mutant has lower activity than the wild-type enzyme, but provides a higher ratio of transglucosylation product to hydrolysis products compared to wild-type enzyme
M424K
mutation in phosphate binding site, shifting pH optimum from 5.0 to 6.0. Activity on nonphosphorylated sugars is largely reduced
additional information
generation of functional glycoside hydrolase hybrids by shuffling of the genes coding for Pyrococcus furiosus CelB and Sulfolobus solfataricus LacS. Generating of thermostable hybrid beta-glycosidases and isolating high-performance variants
additional information
a mutant lacks the C-terminal 23 residues and includes six substitutive mutations R170A, R220A, Y227F, F447S, R448V and E449K forms a unique dodecameric structure consisting of two hexameric rings in the asymmetric unit of the crystal. The lack of the C-terminal region does not affect the activity of the enzyme, but disrupts its oligomeric state and hyperthermostability
