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Literature summary for 5.4.99.11 extracted from

  • Duan, X.; Cheng, S.; Ai, Y.; Wu, J.
    Enhancing the thermostability of Serratia plymuthica sucrose isomerase using B-factor-directed mutagenesis (2016), PLoS ONE, 11, e0149208 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

Cloned (Comment) Organism
gene smuA, synthetic gene, sequence comparisons, recombinant expression of codon-optimized wild-type and mutant enzymes in Escherichia coli strain BL21(DE3), subcloning in Escherichia coli strain JM109 Serratia plymuthica

Protein Variants

Protein Variants Comment Organism
E175N site-directed mutagenesis, the mutant shows increased activity and exhibits an identical pH optimum and a slightly increased optimal temperature (35°C) compared to wild-type enzyme (30°C) Serratia plymuthica
E175N/K576 site-directed mutagenesis, the mutant shows increased activity and exhibits an identical pH optimum and a slightly increased optimal temperature (35°C) compared to wild-type enzyme (30°C) Serratia plymuthica
G176D site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Serratia plymuthica
K174D site-directed mutagenesis, the mutant shows reduced activity compared to wild-type Serratia plymuthica
K576D site-directed mutagenesis, the mutant shows increased activity and exhibits an identical pH optimum and a slightly increased optimal temperature (35°C) compared to wild-type enzyme (30°C) Serratia plymuthica
additional information the half-lives of the enzyme mutants E175N, K576D and E175N/K576D are 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme at 45°C, respectively. The Km values for the E175N, K576D and E175N/K576D mutants decrease by 6.6%, 2.0% and 11.0%, respectively, and their kcat/Km values increase by 38.2%, 4.2% and 19.4%, respectively, compared with those of the wild-type enzyme. After optimizing the conditions for isomaltulose production at 45°C, the E175N, K576D and E175N/K576D mutants display slightly improved isomaltulose yields, compared with the wild-type enzyme. The catalytic efficiencies (kcat/Km values) of E175N, K576D and E175N/K576D are increased by 38.2%, 4.2% and 19.4%, respectively Serratia plymuthica
N577K site-directed mutagenesis, the mutant's activity is similar to the wild-type enzyme Serratia plymuthica
S575D site-directed mutagenesis, the mutant's activity is similar to the wild-type enzyme Serratia plymuthica

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
26.8
-
sucrose recombinant mutant E175N/K576D, pH 6.0, 30°C Serratia plymuthica
28.1
-
sucrose recombinant mutant E175N pH 6.0, 30°C Serratia plymuthica
29.5
-
sucrose recombinant mutant K576D, pH 6.0, 30°C Serratia plymuthica
30.1
-
sucrose recombinant wild-type enzyme, pH 6.0, 30°C Serratia plymuthica

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
sucrose Serratia plymuthica
-
6-O-alpha-D-Glucopyranosyl-D-fructofuranose
-
r
sucrose Serratia plymuthica AS9
-
6-O-alpha-D-Glucopyranosyl-D-fructofuranose
-
r

Organism

Organism UniProt Comment Textmining
Serratia plymuthica D0VX20
-
-
Serratia plymuthica AS9 D0VX20
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant wild-type enzyme and mutants E175N, K576D, and E175N/K576D from Escherichia coli strain BL21(DE3) by ammonium sulfate fractionation, dialysis, and cation exchange chromatography, to homogeneity Serratia plymuthica

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
957.5
-
purified recombinant wild-type enzyme, pH 6.0, 30°C, substrate sucrose Serratia plymuthica
1017.6
-
purified recombinant mutant E175N pH 6.0, 30°C, substrate sucrose Serratia plymuthica
1045.7
-
purified recombinant mutant K576D, pH 6.0, 30°C, substrate sucrose Serratia plymuthica
1218.9
-
purified recombinant mutant E175N/K576D, pH 6.0, 30°C, substrate sucrose Serratia plymuthica

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
sucrose
-
Serratia plymuthica 6-O-alpha-D-Glucopyranosyl-D-fructofuranose
-
r
sucrose
-
Serratia plymuthica AS9 6-O-alpha-D-Glucopyranosyl-D-fructofuranose
-
r

Subunits

Subunits Comment Organism
? x 67560, sequence calculation, x * 65000, recombinant enzyme, SDS-PAGE Serratia plymuthica

Synonyms

Synonyms Comment Organism
sucrose isomerase
-
Serratia plymuthica

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
recombinant wild-type enzyme Serratia plymuthica
35
-
recombinant enzyme mutants E175N, K576D, and E175N/K576D Serratia plymuthica

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
20 50 temperature profiles of wild-type and mutant enzymes, overview Serratia plymuthica

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
45
-
at 45°C and pH 6.0, the half-life of wild-type enzyme is 39.2 minutes. Whereas, the half-lives of mutants E175N, K576D, and E175N/K576D are 90.2 minutes, 69.8 minutes, and 300 minutes, respectively, which are 2.30, 1.78 and 7.65 times greater than that of the wild-type enzyme Serratia plymuthica

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
992.8
-
sucrose recombinant wild-type enzyme, pH 6.0, 30°C Serratia plymuthica
1014.3
-
sucrose recombinant mutant K576D, pH 6.0, 30°C Serratia plymuthica
1055.8
-
sucrose recombinant mutant E175N/K576D, pH 6.0, 30°C Serratia plymuthica
1280.3
-
sucrose recombinant mutant E175N pH 6.0, 30°C Serratia plymuthica

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6
-
recombinant wild-type and mutant enzymes Serratia plymuthica

pH Range

pH Minimum pH Maximum Comment Organism
4 9 pH profiles of wild-type and mutant enzymes, overview Serratia plymuthica

pI Value

Organism Comment pI Value Maximum pI Value
Serratia plymuthica sequence calculation
-
6.86

General Information

General Information Comment Organism
additional information proteins AS9 PalI and SmuA differ in only one amino acid, structure of the wild-type sucrose isomerases is obtained by single point mutation through pymol using the crystal structure of SmuA from Protaminobacter rubrum strain CBS 547.77 (PDB ID 3GBD). Homology models of structures of the mutant sucrose isomerases are constructed based on the crystal structure of SmuA, using the EMBL-EBI server Serratia plymuthica

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
33
-
sucrose recombinant wild-type enzyme, pH 6.0, 30°C Serratia plymuthica
34.4
-
sucrose recombinant mutant K576D, pH 6.0, 30°C Serratia plymuthica
39.4
-
sucrose recombinant mutant E175N/K576D, pH 6.0, 30°C Serratia plymuthica
45.6
-
sucrose recombinant mutant E175N pH 6.0, 30°C Serratia plymuthica