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Literature summary for 2.4.1.7 extracted from

  • Cerdobbel, A.; De Winter, K.; Aerts, D.; Kuipers, R.; Joosten, H.; Soetaert, W.; Desmet, T.
    Increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis (2011), Protein Eng. Des. Sel., 24, 829-834.
    View publication on PubMed
Search for more literature for 2.4.1.7

Cloned(Commentary)

Cloned (Comment) Organism
expression of His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Bifidobacterium adolescentis

Protein Variants

Protein Variants Comment Organism
A498H site-directed mutagenesis, the mutant shows reduced activity and thermostability compared to the wild-type enzyme Bifidobacterium adolescentis
D445P site-directed mutagenesis, the mutant shows slightly decreased activity and increased thermostability compared to the wild-type enzyme Bifidobacterium adolescentis
D445P/D446G site-directed mutagenesis, the mutant shows reduced activity and increased thermostability compared to the wild-type enzyme Bifidobacterium adolescentis
D445P/D446P site-directed mutagenesis, the mutant shows reduced activity and unaltered thermostability compared to the wild-type enzyme Bifidobacterium adolescentis
D445P/D446T site-directed mutagenesis, the mutant shows reduced activity and increased thermostability compared to the wild-type enzyme Bifidobacterium adolescentis
D446G site-directed mutagenesis, the mutant shows similar activity and thermostability as the wild-type enzyme Bifidobacterium adolescentis
D446P site-directed mutagenesis, the mutant shows slightly increased activity and the same thermostability compared to the wild-type enzyme Bifidobacterium adolescentis
D446T site-directed mutagenesis, the mutant shows reduced activity and slightly reduced thermostability compared to the wild-type enzyme Bifidobacterium adolescentis
L306H site-directed mutagenesis, the mutant shows similar activity and thermostability as the wild-type enzyme Bifidobacterium adolescentis
additional information increasing the thermostability of sucrose phosphorylase by a combination of sequence- and structure-based mutagenesis, substitution of the most flexible residues with amino acids that occur more frequently at the corresponding positions in related sequences, and substitutions to promote electrostatic interactions Bifidobacterium adolescentis
N325D/V473H site-directed mutagenesis, the mutant shows reduced activity and thermostability compared to the wild-type enzyme Bifidobacterium adolescentis
N414D site-directed mutagenesis, the mutant shows reduced activity and thermostability compared to the wild-type enzyme Bifidobacterium adolescentis
Q331E site-directed mutagenesis, the mutant shows reduced activity and increased thermostability compared to the wild-type enzyme Bifidobacterium adolescentis
Q460E/E485H site-directed mutagenesis, the mutant shows reduced activity and increased thermostability compared to the wild-type enzyme Bifidobacterium adolescentis
R393N site-directed mutagenesis, the mutant shows reduced activity and increased thermostability compared to the wild-type enzyme Bifidobacterium adolescentis

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
6.5
-
 sucrose pH 7.0, 60°C, recombinant wild-type enzyme Bifidobacterium adolescentis

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
sucrose + phosphate Bifidobacterium adolescentis
-
 D-fructose + alpha-D-glucose 1-phosphate
-
 ?

Organism

Organism UniProt Comment Textmining
Bifidobacterium adolescentis
-
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography Bifidobacterium adolescentis

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
213
-
 purified recombinant wild-type enzyme, pH 7.0, 37°C Bifidobacterium adolescentis

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
additional information assay method with production of alpha-D-glucose-1-phosphate is coupled to the reduction of NAD+ in the presence of phosphoglucomutase and glucose-6-phosphate dehydrogenase Bifidobacterium adolescentis ?
-
 ?
sucrose + phosphate
-
 Bifidobacterium adolescentis D-fructose + alpha-D-glucose 1-phosphate
-
 ?

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
 assay at Bifidobacterium adolescentis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
60
-
 purified wild-type enzyme, pH 7.0, 24 h, 30% activity remaining, purified mutants show 15.3-42.9% remaining activity, overview Bifidobacterium adolescentis

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
201
-
 sucrose pH 7.0, 60°C, recombinant wild-type enzyme Bifidobacterium adolescentis

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
 assay at Bifidobacterium adolescentis

General Information

General Information Comment Organism
evolution the enzyme belongs to glycoside hydrolase family GH 13 and follows the typical doubledisplacement mechanism of retaining glycosidases Bifidobacterium adolescentis