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Literature summary for 5.1.3.11 extracted from
- Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis (2015), J. Mol. Catal. B, 120, 158-164 .
No PubMed abstract available
Application
| Application | Comment | Organism |
|---|---|---|
| synthesis | synthesis of lactulose, a non-digestible disaccharide widely used in food and pharmaceutical industries. Thermostability enhancement of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus by site-directed mutagenesis. Compared to the wild-type enzyme the mutant enzyme E161D/N365P shows approximately 4fold increase in the t1/2 value at 80°C and a 1.3fold increase in catalytic efficiency for lactulose production | Caldicellulosiruptor saccharolyticus |
Cloned(Commentary)
| Cloned (Comment) | Organism |
|---|---|
| expression in Escherichia coli BL21 (DE3) | Caldicellulosiruptor saccharolyticus |
| recombinant expression of His-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) | Caldicellulosiruptor saccharolyticus |
Protein Variants
| Protein Variants | Comment | Organism |
|---|---|---|
| E161D | site-directed mutagenesis, the mutant shows increased thermostability compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
| E161D/N365P | compared to the wild-type enzyme the mutant enzyme shows approximately 4fold increase in the t1/2 value at 80°C and a 1.3fold increase in catalytic efficiency for lactulose production. Its reaction temperature for maximum activity increases from 80°C to 87.5°C, and half denaturating guanidine-HCl concentration increased from 3.16 M to 3.55 M | Caldicellulosiruptor saccharolyticus |
| E161D/N365P | site-directed mutagenesis, the mutant's half-life is approximately 4fold higher than that of the wild-type enzyme. The reaction temperature for maximum activity increased from 80°C to 87.5°C, and catalytic efficiency (kcat/Km) for lactulose production is increased 29%. The mutant E161D/N365P is more stable against chemical denaturation and shows also a broader pH profile than the wild-type. The most thermostable mutant E161D/N365P displays a 1.12 and 1.32fold increase in isomerization activity and epimerization activity, respectively | Caldicellulosiruptor saccharolyticus |
| E161D/S180P/S351G | compared to the wild-type enzyme the mutant enzyme shows approximately 3.3fold increase in the t1/2 value at 80°C. Catalytic efficiency (kcat/Km) of mutant E161D/S180P/S351G decreases to approximately 56% of the wild type enzyme with a 1.6fold increase in apparent Km value | Caldicellulosiruptor saccharolyticus |
| E161D/S180P/S351G | site-directed mutagenesis, the mutant shows a 3.3fold increase in half-life compared to wild-type, the mutant has both decreased isomerization activity and epimerization activity compared to the wild-type | Caldicellulosiruptor saccharolyticus |
| E161D/S180P/S351G/N365P | site-directed mutagenesis, the mutant shows reduced thermostability compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
| E94G | site-directed mutagenesis, the mutant shows increased thermostability compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
| E94G/E161D | site-directed mutagenesis, the mutant shows a similar half-life compared to wild-type | Caldicellulosiruptor saccharolyticus |
| N365P | site-directed mutagenesis, the mutant shows increased thermostability compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
| S180P | site-directed mutagenesis, the mutant shows increased thermostability compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
| S304G | site-directed mutagenesis, the mutant shows reduced thermostability compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
| S351G | site-directed mutagenesis, the mutant shows increased thermostability compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
| S99P | site-directed mutagenesis, the mutant shows highly reduced thermostability compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
| T110G | site-directed mutagenesis, the mutant shows reduced thermostability compared to the wild-type enzyme | Caldicellulosiruptor saccharolyticus |
KM Value [mM]
| KM Value [mM] | KM Value Maximum [mM] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| additional information | - |
additional information | Michaelis-Menten kinetics | Caldicellulosiruptor saccharolyticus | |
| 417.9 | - |
lactose | pH 7.5, 80°C, mutant enzyme E161D/N365P | Caldicellulosiruptor saccharolyticus |  |
| 417.9 | - |
lactose | recombinant mutant E161D/N365P, pH 7.5, 80°C | Caldicellulosiruptor saccharolyticus | |
| 581.37 | - |
lactose | pH 7.5, 80°C, wild-type enzyme | Caldicellulosiruptor saccharolyticus | |
| 581.37 | - |
lactose | recombinant wild-type enzyme, pH 7.5, 80°C | Caldicellulosiruptor saccharolyticus | |
| 932.78 | - |
lactose | pH 7.5, 80°C, mutant enzyme E161D/S180P/S351G | Caldicellulosiruptor saccharolyticus | |
| 932.78 | - |
lactose | recombinant mutant E161D/S180P/S351G, pH 7.5, 80°C | Caldicellulosiruptor saccharolyticus | |
Natural Substrates/ Products (Substrates)
| Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| lactose | Caldicellulosiruptor saccharolyticus | - |
lactulose | - |
r | |
Organic Solvent Stability
| Organic Solvent | Comment | Organism |
|---|---|---|
| guanidine-HCl | half denaturating guanidine-HCl concentration: 3.16 M (wild-type enzyme), 2.97 M (mutant enzyme E161D/S180P/S351G), 3.55 M (mutant enzyme E161D/N365P) | Caldicellulosiruptor saccharolyticus |
Organism
| Organism | UniProt | Comment | Textmining |
|---|---|---|---|
| Caldicellulosiruptor saccharolyticus | A4XGA6 | - |
- |
Purification (Commentary)
| Purification (Comment) | Organism |
|---|---|
| recombinant His-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and gel filtration | Caldicellulosiruptor saccharolyticus |
Specific Activity [micromol/min/mg]
| Specific Activity Minimum [µmol/min/mg] | Specific Activity Maximum [µmol/min/mg] | Comment | Organism |
|---|---|---|---|
| 6.89 | - |
purified recombinant mutant E161D/S180P/S351G, pH 7.5, 80°C, substrate lactose | Caldicellulosiruptor saccharolyticus |
| 10.77 | - |
purified recombinant wild-type enzyme, pH 7.5, 80°C, substrate lactose | Caldicellulosiruptor saccharolyticus |
| 12.06 | - |
purified recombinant mutant E161D/N365P, pH 7.5, 80°C, substrate lactose | Caldicellulosiruptor saccharolyticus |
Substrates and Products (Substrate)
| Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
|---|---|---|---|---|---|---|
| lactose | - |
Caldicellulosiruptor saccharolyticus | epilactose | - |
? | |
| lactose | - |
Caldicellulosiruptor saccharolyticus | lactulose | - |
? | |
| lactose | - |
Caldicellulosiruptor saccharolyticus | lactulose | - |
r | |
Subunits
| Subunits | Comment | Organism |
|---|---|---|
| More | three-dimensional structure analysis | Caldicellulosiruptor saccharolyticus |
Synonyms
| Synonyms | Comment | Organism |
|---|---|---|
| cellobiose 2-epimerase | - |
Caldicellulosiruptor saccharolyticus |
| CsCE | - |
Caldicellulosiruptor saccharolyticus |
Temperature Optimum [°C]
| Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 80 | - |
assay at | Caldicellulosiruptor saccharolyticus |
| 80 | - |
wild-type enzyme | Caldicellulosiruptor saccharolyticus |
| 85 | - |
mutant E161D/S180P/S351G | Caldicellulosiruptor saccharolyticus |
| 87.5 | - |
mutant E161D/N365P | Caldicellulosiruptor saccharolyticus |
Temperature Stability [°C]
| Temperature Stability Minimum [°C] | Temperature Stability Maximum [°C] | Comment | Organism |
|---|---|---|---|
| 80 | - |
purified recombinant enzymes, wild-type enzyme CsCE has a half-life of thermal inactivation (t1/2) of approximately 140 min, while the half-lives of mutants E161D/S180P/S351G and E161D/N365P increase to 462.7 min and 554.0 min, respectively | Caldicellulosiruptor saccharolyticus |
| 80 | - |
t1/2: 141.3 min (wild-type enzyme), 462.7 min (mutant enzyme E161D/S180P/S351G), 554.0 min (mutant enzyme E161D/N365P) | Caldicellulosiruptor saccharolyticus |
| 87.5 | - |
the most thermostable mutant E161D/N365P displays an approximate 20% increase in activity at 87.5°C compared with that at 80°C | Caldicellulosiruptor saccharolyticus |
| 95 | - |
recombinant wild-type enzyme loses more than 80% activity at 95°C, while mutants E161D/S180P/S351G and E161D/N365P retain more than 50% and 70% activity, respectively | Caldicellulosiruptor saccharolyticus |
Turnover Number [1/s]
| Turnover Number Minimum [1/s] | Turnover Number Maximum [1/s] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 72.44 | - |
lactose | pH 7.5, 80°C, mutant enzyme E161D/S180P/S351G | Caldicellulosiruptor saccharolyticus | |
| 72.44 | - |
lactose | recombinant mutant E161D/S180P/S351G, pH 7.5, 80°C | Caldicellulosiruptor saccharolyticus | |
| 75.57 | - |
lactose | pH 7.5, 80°C, mutant enzyme E161D/N365P | Caldicellulosiruptor saccharolyticus | |
| 75.57 | - |
lactose | recombinant mutant E161D/N365P, pH 7.5, 80°C | Caldicellulosiruptor saccharolyticus | |
| 81.55 | - |
lactose | pH 7.5, 80°C, wild-type enzyme | Caldicellulosiruptor saccharolyticus | |
| 81.55 | - |
lactose | recombinant wild-type enzyme, pH 7.5, 80°C | Caldicellulosiruptor saccharolyticus | |
pH Optimum
| pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
|---|---|---|---|
| 7.5 | - |
wild-type and mutant enzymes | Caldicellulosiruptor saccharolyticus |
| 7.5 | - |
wild-type enzyme, mutant enzyme E161D/S180P/S351G, mutant enzyme E161D/N365P | Caldicellulosiruptor saccharolyticus |
pH Range
| pH Minimum | pH Maximum | Comment | Organism |
|---|---|---|---|
| 6 | 8.5 | activity range, the mutant E161D/N365P showed a broader pH/activity profile than wild type enzyme, especially at basic side | Caldicellulosiruptor saccharolyticus |
| 6 | 8.5 | the mutant E161D/N365P shows a broader pH/activity profile than wild type enzyme, especially at basic side. At pH 8.5, the activity decreases about 16% and 5% for wild-type enzyme and mutant E161D/N365P, respectively. At pH 6.0, about 44%, 40% and 55% of the maximum activity is obtained for wild-type enzyme, mutant enzyme E161D/S180P/S351G and mutant enzyme E161D/N365P, respectively | Caldicellulosiruptor saccharolyticus |
General Information
| General Information | Comment | Organism |
|---|---|---|
| additional information | three-dimensional structure, structure modeling | Caldicellulosiruptor saccharolyticus |
| physiological function | cellobiose 2-epimerase from the thermophile Caldicellulosiruptor saccharolyticus (CsCE) catalyzes the isomerization of lactose into lactulose, a non-digestible disaccharide widely used in food and pharmaceutical industries | Caldicellulosiruptor saccharolyticus |
kcat/KM [mM/s]
| kcat/KM Value [1/mMs-1] | kcat/KM Value Maximum [1/mMs-1] | Substrate | Comment | Organism | Structure |
|---|---|---|---|---|---|
| 0.078 | - |
lactose | pH 7.5, 80°C, mutant enzyme E161D/S180P/S351G | Caldicellulosiruptor saccharolyticus | |
| 0.078 | - |
lactose | recombinant mutant E161D/S180P/S351G, pH 7.5, 80°C | Caldicellulosiruptor saccharolyticus | |
| 0.14 | - |
lactose | pH 7.5, 80°C, wild-type enzyme | Caldicellulosiruptor saccharolyticus | |
| 0.14 | - |
lactose | recombinant wild-type enzyme, pH 7.5, 80°C | Caldicellulosiruptor saccharolyticus | |
| 0.181 | - |
lactose | pH 7.5, 80°C, mutant enzyme E161D/N365P | Caldicellulosiruptor saccharolyticus | |
| 0.181 | - |
lactose | recombinant mutant E161D/N365P, pH 7.5, 80°C | Caldicellulosiruptor saccharolyticus | |
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