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 | 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 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 | 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 | UniProt | Comment | Textmining |
---|---|---|---|
Serratia plymuthica | D0VX20 | - |
- |
Serratia plymuthica AS9 | D0VX20 | - |
- |
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 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 | 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 | Comment | Organism |
---|---|---|
? | x 67560, sequence calculation, x * 65000, recombinant enzyme, SDS-PAGE | Serratia plymuthica |
Synonyms | Comment | Organism |
---|---|---|
sucrose isomerase | - |
Serratia plymuthica |
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 Minimum [°C] | Temperature Maximum [°C] | Comment | Organism |
---|---|---|---|
20 | 50 | temperature profiles of wild-type and mutant enzymes, overview | Serratia plymuthica |
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 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 Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|
6 | - |
recombinant wild-type and mutant enzymes | Serratia plymuthica |
pH Minimum | pH Maximum | Comment | Organism |
---|---|---|---|
4 | 9 | pH profiles of wild-type and mutant enzymes, overview | Serratia plymuthica |
Organism | Comment | pI Value Maximum | pI Value |
---|---|---|---|
Serratia plymuthica | sequence calculation | - |
6.86 |
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 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 |