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Literature summary for 5.1.3.11 extracted from

  • Ito, S.; Hamada, S.; Ito, H.; Matsui, H.; Ozawa, T.; Taguchi, H.; Ito, S.
    Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus (2009), Biotechnol. Lett., 31, 1065-1071.
    View publication on PubMed
Search for more literature for 5.1.3.11

Cloned(Commentary)

Cloned (Comment) Organism
recombinant wild type and mutants are expressed in BL21(DE3) Escherichia coli cells Ruminococcus albus

Protein Variants

Protein Variants Comment Organism
E246A production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
E246Q production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
E308A production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
E308D production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
E308Q production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
F114A production by site-directed mutagenesis, mutant has a very low specific activity toward cellobiose and lactose, residue protrudes into the active-site cleft in the model core barrel structure Ruminococcus albus
H243A production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
H243K production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
H243R production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
H374A production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
H374K production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
H374R production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
M106T mutant is active Ruminococcus albus
M179T mutant is active Ruminococcus albus
M212T mutant is active Ruminococcus albus
M251T mutant is inactive Ruminococcus albus
M270T mutant is active Ruminococcus albus
M378T mutant is inactive Ruminococcus albus
M380T mutant is active Ruminococcus albus
M62T mutant is active Ruminococcus albus
M95T mutant is active Ruminococcus albus
M96T mutant is active Ruminococcus albus
R377A production by site-directed mutagenesis, mutant retains 43% of the activity toward cellobiose and 23% activity toward lactose, compaired to the wild type Ruminococcus albus
R377H production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
R52A production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
R52H production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
R52K production by site-directed mutagenesis, complete loss of activity towards cellobiose and lactose Ruminococcus albus
W249F mutant completely loses acitivity toward cellobiose and lactose Ruminococcus albus
W303F 11% and 1.9% activity toward cellobiose and lactose, compared to the wild type, contributes to catalysis, residue protrudes into the active-site cleft in the model core barrel structure Ruminococcus albus
W304F mutant completely loses acitivity toward cellobiose and lactose Ruminococcus albus
W55F mutant is active, the specific activities toward cellobiose and lactose of the mutant retains more than 80% of those of the wild type enzyme although their Km values are increase greatly Ruminococcus albus

Inhibitors

Inhibitors Comment Organism Structure
N-bromosuccinimide
-
 Ruminococcus albus

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
13.8
-
 cellobiose wild type enzyme, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
18
-
 cellobiose M270T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
20
-
 cellobiose M212T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
33
-
 lactose wild type, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
34
-
 cellobiose M380T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
37
-
 cellobiose R377A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
54
-
 cellobiose M62T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
56
-
 cellobiose F114A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
56
-
 cellobiose M95T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
72
-
 lactose W55F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
73
-
 lactose R377A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
84
-
 cellobiose M378T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
95
-
 lactose F114A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
108
-
 cellobiose W303F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
121
-
 cellobiose M96T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
127
-
 cellobiose W55F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
128
-
 cellobiose M106T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
145
-
 lactose W303F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
146
-
 cellobiose M179T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
43700
-
 approximate molecular weight for wild type and mutant proteins, determined by SDS-PAGE Ruminococcus albus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
cellobiose Ruminococcus albus cellobiose 2-epimerase catalyzes a hydroxyl stereoisomerism at C-2 of the reducing terminal glucose moiety 4-O-beta-D-glucopyranosyl-D-mannose
-
 r
lactose Ruminococcus albus
-
 4-O-beta-D-galactopyranosyl-D-mannose epilactose r

Organism

Organism UniProt Comment Textmining
Ruminococcus albus P0DKY4
-
-

Purification (Commentary)

Purification (Comment) Organism
purification of wild-type and mutant proteins by successive chromatography on columns of Q-Sepharose FF and hydroxyapatite Ruminococcus albus

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
0.44
-
 F114A mutant, kinetic value for cellobiose Ruminococcus albus
0.53
-
 W303F mutant, kinetic value for cellobiose Ruminococcus albus
1.15
-
 F114A mutant, kinetic value for cellobiose Ruminococcus albus
1.4
-
 M378T mutant, kinetic value for cellobiose Ruminococcus albus
5.6
-
 M106T mutant, kinetic value for cellobiose Ruminococcus albus
5.99
-
 W303F mutant, kinetic value for cellobiose Ruminococcus albus
6.3
-
 M179T mutant, kinetic value for cellobiose Ruminococcus albus
6.35
-
 M212T mutant, kinetic value for cellobiose Ruminococcus albus
6.6
-
 R377A mutant, kinetic value for cellobiose Ruminococcus albus
8.73
-
 M96T mutant, kinetic value for cellobiose Ruminococcus albus
9
-
 M270T mutant, kinetic value for cellobiose Ruminococcus albus
10.9
-
 M62T mutant, kinetic value for cellobiose Ruminococcus albus
12.1
-
 R377A mutant, kinetic value for cellobiose Ruminococcus albus
12.3
-
 M95T mutant, kinetic value for cellobiose Ruminococcus albus
22.7
-
 W55F mutant, kinetic value for cellobiose Ruminococcus albus
25.8
-
 M380T mutant, kinetic value for cellobiose Ruminococcus albus
28.4
-
 wild type enzyme, kinetic value for lactose Ruminococcus albus
42
-
 W55F mutant, kinetic value for cellobiose Ruminococcus albus
51
-
 wild type enzyme, kinetic value for cellobiose Ruminococcus albus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
cellobiose cellobiose 2-epimerase catalyzes a hydroxyl stereoisomerism at C-2 of the reducing terminal glucose moiety Ruminococcus albus 4-O-beta-D-glucopyranosyl-D-mannose
-
 r
lactose
-
 Ruminococcus albus 4-O-beta-D-galactopyranosyl-D-mannose epilactose r

Synonyms

Synonyms Comment Organism
cellobiose 2-epimerase
-
 Ruminococcus albus

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
 assay at Ruminococcus albus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.79
-
 lactose F114A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
4.04
-
 lactose W303F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
4.64
-
 cellobiose F114A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
5.16
-
 cellobiose M378T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
14.4
-
 cellobiose M270T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
20.9
-
 lactose R377A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
26.6
-
 cellobiose R377A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
29.7
-
 cellobiose M106T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
30.3
-
 cellobiose M62T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
31.5
-
 cellobiose M212T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
33.3
-
 cellobiose M95T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
36.9
-
 cellobiose M179T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
40
-
 cellobiose M380T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
43.8
-
 cellobiose M96T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
46.4
-
 cellobiose W303F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
52.1
-
 lactose wild type, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
63.3
-
 cellobiose wild type enzyme, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
78.3
-
 lactose W55F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
215
-
 cellobiose W55F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus

pH Range

pH Minimum pH Maximum Comment Organism
5 5.5 no activity Ruminococcus albus
6 9.5 activity Ruminococcus albus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.019
-
 lactose F114A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
0.028
-
 lactose W303F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
0.06
-
 cellobiose M378T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
0.08
-
 cellobiose F114A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
0.23
-
 cellobiose M106T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
0.25
-
 cellobiose M179T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
0.285
-
 lactose R377A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
0.37
-
 cellobiose M96T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
0.43
-
 cellobiose W303F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
0.58
-
 cellobiose M62T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
0.61
-
 cellobiose M95T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
0.72
-
 cellobiose R377A mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
0.8
-
 cellobiose M270T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
1.08
-
 lactose W55F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
1.17
-
 cellobiose M380T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
1.57
-
 cellobiose M212T mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
1.58
-
 lactose wild type, 100 mM sodium phosphate, pH 7.5, 20 mM lactose, 30°C Ruminococcus albus
1.69
-
 cellobiose W55F mutant, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus
4.62
-
 cellobiose wild type enzyme, 100 mM sodium phosphate, pH 7.5, 20 mM cellobiose, 30°C Ruminococcus albus