Information on EC 3.2.1.B28 - Pyrococcus furiosus beta-glycosidase

Please wait a moment until all data is loaded. This message will disappear when all data is loaded.
Specify your search results
Select one or more organisms in this record:
Show additional data
Do not include text mining results
Include (text mining) results (more...)
Include results (AMENDA + additional results, but less precise; more...)


The expected taxonomic range for this enzyme is: Pyrococcus furiosus

EC NUMBER
COMMENTARY hide
3.2.1.B28
preliminary BRENDA-supplied EC number
RECOMMENDED NAME
GeneOntology No.
Pyrococcus furiosus beta-glycosidase
-
REACTION
REACTION DIAGRAM
COMMENTARY hide
ORGANISM
UNIPROT
LITERATURE
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
show the reaction diagram
-
-
-
-
SUBSTRATE
PRODUCT                       
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate) hide
LITERATURE
(Substrate)
COMMENTARY
(Product) hide
LITERATURE
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
2,4-dinitrophenyl beta-D-glucopyranoside + H2O
2,4-dinitrophenol + D-glucopyranose
show the reaction diagram
-
-
-
-
?
2-nitrophenyl beta-D-galactopyranoside + H2O
2-nitrophenol + beta-D-galactopyranose
show the reaction diagram
2-nitrophenyl beta-D-galactopyranoside + H2O
2-nitrophenol + D-galactopyranose
show the reaction diagram
-
-
-
-
?
2-nitrophenyl beta-D-galactopyranoside + H2O
2-nitrophenol + D-galactopyranoside
show the reaction diagram
-
-
-
?
2-nitrophenyl beta-D-galactopyranoside + H2O
2-nitrophenol + D-galactose
show the reaction diagram
-
-
-
-
?
2-nitrophenyl beta-D-galactopyranoside-6-phosphate + H2O
2-nitrophenol + D-galactopyranoside-6-phosphate
show the reaction diagram
-
-
-
?
2-nitrophenyl beta-D-galactoside
2-nitrophenol + D-galactose
show the reaction diagram
-
-
-
-
r
2-nitrophenyl beta-D-glucopyranoside + H2O
2-nitrophenol + D-glucopyranose
show the reaction diagram
-
-
-
-
?
2-nitrophenyl beta-D-glucopyranoside + H2O
2-nitrophenol + D-glucopyranoside
show the reaction diagram
-
-
-
?
2-nitrophenyl beta-D-glucopyranoside + H2O
2-nitrophenol + D-glucose
show the reaction diagram
-
-
-
-
?
2-nitrophenyl beta-D-xylopyranoside + H2O
2-nitrophenol + D-xylopyranose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl alpha-L-arabinofuranoside + H2O
4-nitrophenol + L-arabinose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl alpha-L-arabinopyranoside + H2O
4-nitrophenol + L-arabinose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl beta-D-galactopyranoside + H2O
4-nitrophenol + beta-D-galactopyranose
show the reaction diagram
4-nitrophenyl beta-D-galactopyranoside + H2O
4-nitrophenol + beta-D-galactose
show the reaction diagram
-
catalytic efficiency (kcat/Km) for 4-nitrophenyl beta-D-galactopyranoside is 7.6% compared to catalytic catalytic efficiency (kcat/Km) for 4-nitrophenyl beta-D-glucopyranoside
-
-
?
4-nitrophenyl beta-D-galactopyranoside + H2O
4-nitrophenol + D-galactopyranose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl beta-D-galactopyranoside + H2O
4-nitrophenol + D-galactose
show the reaction diagram
4-nitrophenyl beta-D-galactoside
4-nitrophenol + beta-D-galactose
show the reaction diagram
-
CelB has a beta-galactosidase activity of 61% of the beta-glucosidase activity
-
-
?
4-nitrophenyl beta-D-galactoside + H2O
4-nitrophenol + D-galactose
show the reaction diagram
-
at 80C 61% of the activity with 4-nitrophenyl beta-D-glucoside
-
-
?
4-nitrophenyl beta-D-glucopyranoside + H2O
4-nitrophenol + beta-D-glucopyranose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl beta-D-glucopyranoside + H2O
4-nitrophenol + beta-D-glucose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl beta-D-glucopyranoside + H2O
4-nitrophenol + D-glucopyranose
show the reaction diagram
4-nitrophenyl beta-D-glucopyranoside + H2O
4-nitrophenol + D-glucose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl beta-D-glucoside
4-nitrophenol + beta-D-glucose
show the reaction diagram
-
CelB has a beta-galactosidase activity of 61% of the beta-glucosidase activity
-
-
?
4-nitrophenyl beta-D-glucoside + H2O
4-nitrophenol + beta-D-glucose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl beta-D-glucoside + H2O
4-nitrophenol + D-glucose
show the reaction diagram
-
-
-
-
?
4-nitrophenyl beta-D-mannopyranoside + H2O
4-nitrophenol + beta-D-mannopyranose
show the reaction diagram
4-nitrophenyl beta-D-mannopyranoside + H2O
4-nitrophenol + beta-D-mannose
show the reaction diagram
-
catalytic efficiency (kcat/Km) for 4-nitrophenyl beta-D-mannopyranoside is 0.7% compared to catalytic catalytic efficiency (kcat/Km) for 4-nitrophenyl beta-D-glucopyranoside
-
-
?
4-nitrophenyl beta-D-xylopyranoside + H2O
4-nitrophenol + beta-D-xylopyranose
show the reaction diagram
cellobiose + H2O
2 beta-D-glucose
show the reaction diagram
cellobiose + H2O
2 D-glucose
show the reaction diagram
-
-
-
-
?
cellobiose + H2O
?
show the reaction diagram
-
as active as 4-nitrophenyl-beta-D-glucopyranoside
-
-
?
cellobiose + H2O
D-glucose + D-glucose
show the reaction diagram
-
CelB is a little more active on the galactosides lactose and 4-nitrophenyl beta-D-galactopyranoside than on 4-nitrophenyl beta-D-glucopyranoside, whereas the efficiency of cellobiose hydrolysis is relatively low
-
-
?
daidzin + H2O
beta-D-glucose + daidzein
show the reaction diagram
-
-
-
-
?
diosmin + H2O
diosmetin + rutinose
show the reaction diagram
-
-
-
-
?
genistin + H2O
beta-D-glucose + genistein
show the reaction diagram
-
-
-
-
?
ginsenoside Rb1 + 4 H2O
protopanaxadiol aglycone + 4 D-glucopyranose
show the reaction diagram
-
-
-
-
?
ginsenoside Rb2 + 4 H2O
protopanaxadiol aglycone + 3 D-glucopyranose + L-arabinopyranose
show the reaction diagram
-
-
-
-
?
ginsenoside Rc + 4 H2O
protopanaxadiol aglycone + 3 D-glucopyranose + L-arabinopyranose
show the reaction diagram
-
-
-
-
?
ginsenoside Rd + 3 H2O
protopanaxadiol aglycone + 3 D-glucopyranose
show the reaction diagram
-
-
-
-
?
glycitin + H2O
beta-D-glucose + glycitein
show the reaction diagram
-
i.e. glycitein 7-O-beta-glucoside
-
-
?
hesperidin + H2O
hesperetin + rutinose
show the reaction diagram
-
-
-
-
?
isoquercitrin + H2O
quercetin + beta-D-glucose
show the reaction diagram
lactose + H2O
D-glucose + D-galactose
show the reaction diagram
lactose + N,N'-diacetylchitobiose
Gal-beta(1,4)-GlcNAc-beta(1,3)-Gal-beta(1,4)-Glc + H2O
show the reaction diagram
-
-
-
-
?
mannooligosaccharide + H2O
?
show the reaction diagram
hydrolysis of mannose from the reducing end
-
-
?
methyl beta-D-galactoside
methanol + D-galactose
show the reaction diagram
-
-
-
-
r
n glucose
gluco-oligosaccharides + (n-1) H2O
show the reaction diagram
-
-
study on the effect of pressure on the reaction equilibrium. Oligosaccharides are synthesized from glucose in an equilibrium reaction at pressures from 0.1 to 500 MPa. The enzyme remains active at 500 MPa. The equilibrium of the reaction is influenced by pressure and shifts towards the hydrolysis side, decreasing final oligosaccharide concentrations with increasing pressure. This pressure dependence of the final product concentration and the equilibrium constant can be described with a positive reaction volume of 2.4 mol/cm3
-
?
N-actelyglucosamine + lactose
Gal-beta(1,4)-GlcNAc + D-glucose
show the reaction diagram
-
-
transgalactosylation reaction
-
?
naringin + H2O
naringenin + rutinose
show the reaction diagram
-
-
-
-
?
narirutin + H2O
naringenin + neohesperidose
show the reaction diagram
-
-
-
-
?
neohesperidin + H2O
hesperetin + neohesperidose
show the reaction diagram
-
-
-
-
?
neoponcirin + H2O
isosakuranetin + rutinose
show the reaction diagram
-
-
-
-
?
pentyl beta-D-glucopyranoside + H2O
pentanol + beta-D-glucopyranose
show the reaction diagram
-
-
-
-
?
poncirin + H2O
isosakuranetin + neohesperidose
show the reaction diagram
-
-
-
-
?
quercitrin + H2O
quercetin + alpha-L-rhamnose
show the reaction diagram
rutin + H2O
quercetin + rutinose
show the reaction diagram
-
-
-
-
?
additional information
?
-
METALS and IONS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
NaCl
-
optimal concentration: 200 mM
additional information
-
divalent metal ions not required
INHIBITORS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
(5R,6R,7S,8S)-5-(hydroxymethyl)-2-phenyl-5,6,7,8-tetrahydroimidazol[1,2-a]pyridine-6,7,8-triol
1,3-dimethylimidazolium methylsulfate
-
the enzyme retains full catalytic efficiency for lactose hydrolysis at 80C in a 50% (v/v) solution of the ionic liquid 1,3-dimethylimidazoliummethylsulfate in sodium citrate buffer, pH 5.5. It is inactive but not irreversibly denatured at 70% (v/v) ionic liquid
1-methyl-1-butanol
-
10% (v/v), about% inhibition
2-propanol
-
10% (v/v), about% inhibition
acetone
-
10% (v/v), about% inhibition
D-galactose
-
measured with lactose as the substrate
D-glucose
DMSO
-
10% (v/v), about% inhibition
ethanol
-
10% (v/v), about 50% inhibition
formate
-
10% (v/v), about% inhibition
Isopropanol
-
10% (v/v), about% inhibition
methanol
-
10% (v/v), about% inhibition
additional information
-
not inhibited by EDTA
-
ACTIVATING COMPOUND
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
additional information
-
not activated by monovalent or divalent cations
-
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.12 - 42
2-nitrophenyl beta-D-galactopyranoside
16 - 38
2-nitrophenyl beta-D-galactopyranoside-6-phosphate
5.3
2-nitrophenyl beta-D-galactoside
-
release of 2-nitrophenol, pH 7.5, 80C
0.1 - 59
2-nitrophenyl beta-D-glucopyranoside
0.7 - 22
4-nitrophenyl beta-D-galactopyranoside
1.3 - 7.2
4-nitrophenyl beta-D-galactoside
0.02 - 13.9
4-nitrophenyl beta-D-glucopyranoside
2.6 - 49
4-nitrophenyl beta-D-glucoside
0.03 - 1.5
4-nitrophenyl beta-D-mannopyranoside
0.04 - 38
cellobiose
0.42
daidzin
-
pH 6.0, 95C
0.5
genistin
-
pH 6.0, 95C
0.33
glycitin
-
pH 6.0, 95C
1.6
hesperidin
-
pH 5.5, 95C
0.17
isoquercitrin
-
pH 5.0, 95C
0.01 - 220
lactose
861
methyl beta-D-galactoside
-
release of methanol, pH 7.5, 80C
1.7
naringin
-
pH 5.5, 95C
3.7
narirutin
-
pH 5.5, 95C
-
4.6
neohesperidin
-
pH 5.5, 95C
46
pentyl beta-D-glucopyranoside
-
pH 5.0, 80C, 6% water, 94% hexanol
3.095
quercitrin
-
pH 5.0, 95C
additional information
additional information
-
mutants enzymes R77Q and R77Q/N206D show 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
-
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.23 - 9600
2-nitrophenyl beta-D-galactopyranoside
26 - 171
2-nitrophenyl beta-D-galactopyranoside-6-phosphate
10000
2-nitrophenyl beta-D-galactoside
Pyrococcus furiosus
-
release of 2-nitrophenol, pH 7.5, 80C
0.22 - 3300
2-nitrophenyl beta-D-glucopyranoside
4.2 - 4863
4-nitrophenyl beta-D-galactopyranoside
0.05 - 2600
4-nitrophenyl beta-D-glucopyranoside
0.8 - 7.1
4-nitrophenyl beta-D-glucoside
0.11 - 65.9
4-nitrophenyl beta-D-mannopyranoside
0.1 - 670
cellobiose
1880
daidzin
Pyrococcus furiosus
-
pH 6.0, 95C
6050
genistin
Pyrococcus furiosus
-
pH 6.0, 95C
610
glycitin
Pyrococcus furiosus
-
pH 6.0, 95C
68.4
hesperidin
Pyrococcus furiosus
-
pH 5.5, 95C
53.3
isoquercitrin
Pyrococcus furiosus
-
pH 5.0, 95C
0.03 - 3800
lactose
8.3
methyl beta-D-galactoside
Pyrococcus furiosus
-
release of methanol, pH 7.5, 80C
27.7
naringin
Pyrococcus furiosus
-
pH 5.5, 95C
74.1
narirutin
Pyrococcus furiosus
-
pH 5.5, 95C
-
84.8
neohesperidin
Pyrococcus furiosus
-
pH 5.5, 95C
0.22
quercitrin
Pyrococcus furiosus
-
pH 5.0, 95C
additional information
additional information
Pyrococcus furiosus
-
mutants enzymes R77Q and R77Q/N206D show 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
-
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
1.89 - 1000
2-nitrophenyl beta-D-galactopyranoside
880
0.7 - 7.7
2-nitrophenyl beta-D-galactopyranoside-6-phosphate
210619
1900
2-nitrophenyl beta-D-galactoside
Pyrococcus furiosus
-
release of 2-nitrophenol, pH 7.5, 80C
7995
0.3 - 6480
2-nitrophenyl beta-D-glucopyranoside
1098
1.3 - 561
4-nitrophenyl beta-D-galactopyranoside
999
2.44 - 7337
4-nitrophenyl beta-D-glucopyranoside
510
1.3 - 49.8
4-nitrophenyl beta-D-mannopyranoside
2120
0.69 - 2400
cellobiose
82
42.1
hesperidin
Pyrococcus furiosus
-
pH 5.5, 95C
2513
0.18 - 20.4
lactose
114
0.01
methyl beta-D-galactoside
Pyrococcus furiosus
-
release of methanol, pH 7.5, 80C
45669
16.5
naringin
Pyrococcus furiosus
-
pH 5.5, 95C
2858
19.8
narirutin
Pyrococcus furiosus
-
pH 5.5, 95C
27254
18.5
neohesperidin
Pyrococcus furiosus
-
pH 5.5, 95C
9476
additional information
additional information
Pyrococcus furiosus
-
mutants enzymes R77Q and R77Q/N206D show 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
2
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
IMAGE
0.0000065 - 0.0098
(5R,6R,7S,8S)-5-(hydroxymethyl)-2-phenyl-5,6,7,8-tetrahydroimidazol[1,2-a]pyridine-6,7,8-triol
270
D-galactose
-
pH 5.5, 80C
43 - 300
D-glucose
SPECIFIC ACTIVITY [µmol/min/mg]
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
0.011
-
substrate: rutin, pH 5.5, 95C
0.081
-
substrate: neoponcirin, pH 5.5, 95C
0.085
-
substrate: diosmin, pH 5.5, 95C
0.091
-
substrate: poncirin, pH 5.5, 95C
0.102
-
substrate naringin, pH 5.5, 95C
0.103
-
substrate: neohesperidin, pH 5.5, 95C
0.153
-
substrate: narirutin, pH 5.5, 95C
0.172
-
substrate: hesperidin, pH 5.5, 95C
0.23
-
substrate: quercitrin, pH 5.0, 95C
0.28
-
mutant E372A, substrate 2-nitrophenyl beta-D-xylopyranoside, pH 3.0, 65C
0.55
-
substrate ginsenoside Rb2, pH 5.5, 95C
0.83
-
pH 5.0, 90C, addition of aerosol dioctyl sulfosuccinate
2.75
-
pH 5.0, 90C, enzyme immobilized on XAD-4
3.18
-
pH 5.0, 90C, enzyme immobilized on XAD-16
4.5
-
substrate 4-nitrophenyl alpha-L-arabinofuranoside, pH 5.5, 95C; substrate ginsenoside Rb1, pH 5.5, 95C
4.7
-
mutant E372A, substrate 2-nitrophenyl beta-D-glucopyranoside, pH 3.0, 65C
4.77
-
pH 5.0, 90C, enzyme immobilized on Celite
5.5
-
substrate ginsenoside Rd, pH 5.5, 95C
8.3
-
mutant E372A, substrate 2,4-dinitrophenyl beta-D-glucopyranoside, pH 3.0, 65C
13.04
-
pH 5.0, 90C, lyophilized enzyme
15
-
substrate 4-nitrophenyl alpha-L-arabinopyranoside, pH 5.5, 95C
66
-
wild-type, substrate cellobiose, pH 3.0, 65C
180
-
substrate: daidzin, pH 6.0, 95C
220
-
substrate: isoquercitrin, pH 5.0, 95C
291
-
wild-type, substrate 4-nitrophenyl beta-D-glucopyranoside, pH 3.0, 65C
320
-
wild-type, substrate 2,4-dinitrophenyl beta-D-glucopyranoside, pH 3.0, 65C
330
-
substrate: genistin, pH 6.0, 95C
529
-
substrate 2-nitrophenyl beta-D-galactopyranoside, pH 5.5, 95C
534
-
wild-type, substrate 2-nitrophenyl beta-D-glucopyranoside, pH 3.0, 65C
561
-
substrate 4-nitrophenyl beta-D-galactopyranoside, pH 5.5, 95C
828
-
substrate 2-nitrophenyl beta-D-glucopyranoside, pH 5.5, 95C
885
-
recombinant protein, 90C, pH 5.0
1049
-
wild-type, substrate 2-nitrophenyl beta-D-galactopyranoside, pH 3.0, 65C
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4
-
wild-type, substrate 2-nitrophenyl beta-D-galactopyranoside-6-phosphate
5 - 6
-
soluble enzyme and immobilized enzymes
pH RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.5 - 6
-
pH 4.5: about 85% of maximal activity, pH 6.0: about 60% of maximal activity
4.5 - 6.5
-
pH 4.5: about 50% of maximal activity, pH 6.5: about 60% of maximal activity, substrate: hesperidin
5 - 6
-
more than 80% of maximum activity
5 - 7
-
pH 5.0: 80% of maximal activity, pH 8.0: about 80% of maximal activity
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70
-
assay at
90
-
assay at
102 - 105
-
-
105
-
recombinant protein
additional information
-
CelB shows almost constant selcetivity for the glycon part of a donor substrate as the temperature increases. The specificity constant (Vmax/Km) ratio of phenyl-beta-glucoside and phenyl-beta-galactoside substrate decreases only slightly from 2.6 to 2.1 between 50 and 90C
TEMPERATURE RANGE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
70 - 110
-
70C: about 50% of maximal activity, 110C: about 45% of maximal activity
75 - 95
-
the activity increases from 75C to 95C, activity at 75C is about 40% of the activity at 95C, substrate: hesperidin
75 - 98
-
75C: about 50% of maximal activity, 98C: about 85% of maximal activity
80 - 110
-
80C: about 45% of maximal activity, 110C: about 65% of maximal activity
pI VALUE
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
4.4
-
isoelectric focusing
LOCALIZATION
ORGANISM
UNIPROT
COMMENTARY hide
GeneOntology No.
LITERATURE
SOURCE
MOLECULAR WEIGHT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
54580
-
x * 54580, calculated from sequence
54665
-
x * 54665, calculated from sequence
55500
-
x * 55500, SDS-PAGE, x * 55488, calculated
56326
-
4 * 56326, calculated from sequence
58000
-
4 * 58000, SDD-PAGE
230000
-
non-denaturing PAGE
SUBUNITS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
dodecamer
-
a monomeric form of the enzyme is constructed by removing the C-terminal region of the enzyme (the mutant lacks the C-terminal 23 residues and includes six substitutive mutations R170A, R220A, Y227F, F447S, R448V and E449K). The mutant enzyme forms a unique dodecameric structure consisting of two hexameric rings in the asymmetric unit of the crystal
monomer
-
a monomeric form of the enzyme is constructed by removing the C-terminal region of the enzyme (the mutant lacks the C-terminal 23 residues and includes six substitutive mutations R170A, R220A, Y227F, F447S, R448V and E449K). The mutant enzyme forms a unique dodecameric structure consisting of two hexameric rings in the asymmetric unit of the crystal
tetramer
Crystallization/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
3.3 A resolution structural model. CelB shows a homo-tetramer configuration, with subunits having a typical (betaalpha)8-barrel fold
-
hanging-drop vapour-diffusion method, a monomeric form of the enzyme is constructed by removing the C-terminal region (the mutant lacks the C-terminal 23 residues and includes six substitutive mutations R170A, R220A, Y227F, F447S, R448V and E449K) of the enzyme and its crystal structure is solved at a resolution of 2.8 A in space group
-
to 2.35 A resolution. There is one tetramer in the asymmetric unit and the dimeric molecule exhibits a structure that is stable towards sodium dodecyl sulfate even after boiling at 368 K. The elongation at the C-terminal end forms a hydrophobic patch at the dimer interface that might contribute to hyperthermostability
-
pH STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
10
-
75C, 90 min, stable
722214
TEMPERATURE STABILITY
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
75 - 80
-
the mutant enzyme that lacks the C-terminal 23 residues and includes six substitutive mutations R170A, R220A, Y227F, F447S, R448V and E449K is immediately inactivated between 75 and 80C
92
-
half-life: above 100 min
102
-
half-life: over 10 h
104
-
Tm-value
additional information
-
thermostability is not affected by the presence of Mg2+
GENERAL STABILITY
ORGANISM
UNIPROT
LITERATURE
chaotropes (I-, ClO4-, NO3- and Cs+) have a strong destabilising effect on the protein by perturbing hydrophobic interactions
-
denaturing effect of NaCl
-
half-life of free enzyme is 680 h in absence and 22 h in presence of 1 M glucose, half-life of cross-linked enzyme is 18 h in absence and 3 h in presence of 1 M glucose, half-life of cross-linked cells is 840 h in absence and 47 h in presnce of 1 M glucose, respectively
-
half-life times of the enzyme in hydrolysis of lactose is carried out at 70C in a continuous stirred-tank reactor coupled to a 10000 Da cross-flow ultrafiltration module (to recycle the enzyme) is approximately 5 to 7 days
-
the enzyme can be immobilized with little loss of enzyme activity and catalytic efficiency by covalent coupling of the proteins via the surface amino groups to insoluble carriers
-
ORGANIC SOLVENT
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
hexanol
-
enzyme catalyzes both hydrolysis and transglycosylation of glycosidc substrates. In hexanol/water two-phase systems, hydrolysis is by far the dominating reaction even though the total activity increases. In hexanol containing various amounts of water, the selectivity for the alcohol increases with increasing water activity. This counteracts the effect of higher water concentration and the transglycosylation/hydrolysis ratio increases with increasing water activity
Purification/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
Cloned/COMMENTARY
ORGANISM
UNIPROT
LITERATURE
expression in Escherichia coli BL21
-
expression in Escherichia coli, mutant enzymes Q150W and Q77R/Q150W
-
expression in Escherichia coli; overexpressed in Escherichia coli, resulting in high-level (up to 20% of total protein) production of the enzyme
-
expression in in Lactobacillus plantarum NC8 and Lactobacillus casei as hosts. Both lactobacilli harboring the pSIP409-celB vector produce active CelB in batch bioreactor cultivations while the specific CelB activity of the cell free extract is about 44% higher with Lactobacillus plantarum than with Lactobacillus casei using 4-nitrophenyl beta-galactoside as the substrate. A fedbatch bioreactor cultivation of Lactobacillus plantarum NC8 pSIP409-celB results in a specific CelB activity of 2500 nkat 4-nitrophenyl beta-D-galactopyranoside/mg protein after 28 h. A repeated dosage of the inducer (sakacin P inducing peptide) does not increase the enzyme expression further
-
expression of wild-type enzyme and mutant enzymes in Escherichia coli
-
expression using a baculovirus expression vector system in silkworm, Bombyx mori
-
recombinant mutant that lacks the C-terminal 23 residues and includes six substitutive mutations R170A, R220A, Y227F, F447S, R448V and E449Kis expressed in Escherichia coli BL21
-
EXPRESSION
ORGANISM
UNIPROT
LITERATURE
during growth of Pyrococcus furiosus on either cellobiose or laminarin, the activities of both beta-glucosidase and endoglucanase are increased at least fivefold compared with levels during growth on maltose or pyruvate, due to an enhanced transcription of both the celB gene and the lamA operon in the presence of these glucose-containing substrates
-
gene locus includes the celB gene, and a divergently orientated gene cluster, adhA-adhB-lamA, which codes for two alcohol dehydrogenases and an extracellular beta-1,3-endoglucanase. The in vivo and in vitro transcription initiation sites of both the celB gene and the lamA operon lie 25 nucleotides downstream of conserved TATA box motifs
-
ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
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 106C
E372D
-
200-fold reduction in specific activity
E372Q
-
1000-fold reduction in specific activity
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
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 106C
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 106C
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 106C
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 106C
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 106C
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 106C
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 106C
additional information
APPLICATION
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
degradation
-
encapsulation of CelB into silica microcapsules for degradation of biomass. The encapsulated enzyme is active at 80-100C, but diffusion of cellobiose into the silica microcapsules is a rate-limiting step
food industry
pharmacology
-
the hyperthermostable beta-glycosidase may be useful for food and pharmaceutical applications
synthesis
additional information
-
the enzyme converts flavanone glycoside to flavanone aglycone via a one-step reaction. It may be useful in the production of the flavanone aglycones naringenin and hesperetin from flavanone glycosides in citrus extracts