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Literature summary for 5.1.3.11 extracted from

  • Shen, Q.; Zhang, Y.; Yang, R.; Pan, S.; Dong, J.; Fan, Y.; Han, L.
    Enhancement of isomerization activity and lactulose production of cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (2016), Food Chem., 207, 60-67 .
    View publication on PubMed
Search for more literature for 5.1.3.11

Application

Application Comment Organism
synthesis industrial application of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase (CsCE) for lactulose synthesis is limited by low enzyme activity and formation of epilactose as by-product, the mutant G4-C5, i.e. R5M/I52V/A12S/K328I/F231L, shows improved enzyme activity without compromised thermostability. The yield of lactulose catalyzed by mutant G4-C5 increases to approximately 76% with no obvious epilactose detected, indicating that mutant G4-C5 is more suitable for lactulose production than the wild-type enzyme. The mutant does not produc epilactose as side-product from lactose, in contrast to the wild-type enzyme. Although epilactose is also a bifidus factor, formation of epilactose can complicate lactulose purification Caldicellulosiruptor saccharolyticus
synthesis industrial application of Caldicellulosiruptor saccharolyticus cellobiose 2-epimerase for lactulose synthesis is limited by low enzyme activity and formation of epilactose as by-product. The yield of lactulose catalyzed by mutant R5M/A12S/I52V/F231L/K328I increases to approximately 76% with no epilactose detected, indicating that the mutant is more suitable for lactulose production than the wild-type enzyme Caldicellulosiruptor saccharolyticus

Cloned(Commentary)

Cloned (Comment) Organism
a random mutant library is prepared via error-prone PCR, identification of mutant R5M/I52V/A12S/K328I/F231L with emhanced enzyme activity and improved performance, recombinant expression of wild-type enzyme and mutants in Escherichia coli strain BL21(DE3) Caldicellulosiruptor saccharolyticus
wild-type and mutant enzymes without His-tag are produced in Escherichia coli BL21 (DE3) Caldicellulosiruptor saccharolyticus

Protein Variants

Protein Variants Comment Organism
A12S random mutagenesis, the mutant shows increased activity compared to the wild-type Caldicellulosiruptor saccharolyticus
A12S t1/2 at 80°C increases approximately 20 min compared with that of the wild-type enzyme Caldicellulosiruptor saccharolyticus
A12S/K328I random mutagenesis, the mutant shows increased activity compared to the wild-type Caldicellulosiruptor saccharolyticus
A12S/K328I t1/2 at 80°C decreases approximately 8 min compared with that of the wild-type enzyme Caldicellulosiruptor saccharolyticus
R5M/A12S/F231L/K328I random mutagenesis, the mutant shows increased activity compared to the wild-type Caldicellulosiruptor saccharolyticus
R5M/A12S/F231L/K328I t1/2 at 80°C decreases approximately 24 min compared with that of the wild-type enzyme Caldicellulosiruptor saccharolyticus
R5M/A12S/I52V/F231L/K328I t1/2 at 80°C increases approximately 18 min compared with that of the wild-type enzyme. The mutant enzyme retains higher activity than the wild-type enzyme at low temperatures. The mutant enzyme shows a broader pH-activity profile than the wild-type enzyme. The yields of lactulose and epilactose catalyzed from lactose by the wild-type enzyme reaches around 57% and 16%, respectively, after 4 h. The concentration of lactulose catalyzed by mutant enzyme R5M/A12S/I52V/F231L/K328I rapidly increases from 0 min to 90 min and then increases gradually to the steady-state level with a yield of lactulose up to approximately 76%. No obvious epilactose is detected at any time Caldicellulosiruptor saccharolyticus
R5M/I52V/A12S/K328I/F231L random mutagnesis, the mutant shows 2.8 and 3.0fold increases in specific activity and kcat/Km for lactulose production, respectively, without compromising thermostability. The yield of lactulose catalyzed by mutant G4-C5 increases to approximately 76% with no obvious epilactose detected, indicating that mutant G4-C5 is more suitable for lactulose production than 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
358.24
-
 lactose pH 7.5, 80°C, mutant enzyme R5M/A12S/I52V/F231L/K328I Caldicellulosiruptor saccharolyticus
358.24
-
 lactose pH 7.5, 80°C, recombinant mutant R5M/A12S/I52V/F231L/K328I Caldicellulosiruptor saccharolyticus
402.12
-
 lactose pH 7.5, 80°C, mutant enzyme R5M/A12S/F231L/K328I Caldicellulosiruptor saccharolyticus
402.12
-
 lactose pH 7.5, 80°C, recombinant mutant R5M/A12S/F231L/K328I Caldicellulosiruptor saccharolyticus
475.43
-
 lactose pH 7.5, 80°C, mutant enzyme A12S/K328I Caldicellulosiruptor saccharolyticus
475.43
-
 lactose pH 7.5, 80°C, recombinant mutant A12S/K328I Caldicellulosiruptor saccharolyticus
537.12
-
 lactose pH 7.5, 80°C, mutant enzyme A12S Caldicellulosiruptor saccharolyticus
537.12
-
 lactose pH 7.5, 80°C, recombinant mutant A12S Caldicellulosiruptor saccharolyticus
613.54
-
 lactose pH 7.5, 80°C, wild-type enzyme Caldicellulosiruptor saccharolyticus
613.54
-
 lactose pH 7.5, 80°C, recombinant wild-type enzyme Caldicellulosiruptor saccharolyticus

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
cellobiose Caldicellulosiruptor saccharolyticus
-
 4-O-beta-D-glucopyranosyl-D-mannose
-
 r

Organism

Organism UniProt Comment Textmining
Caldicellulosiruptor saccharolyticus A4XGA6
-
-

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
10.77
-
 substrate: lactose, pH 7.5, 80°C, wild-type enzyme Caldicellulosiruptor saccharolyticus
13.68
-
 substrate: lactose, pH 7.5, 80°C, mutant enzyme A12S Caldicellulosiruptor saccharolyticus
17.45
-
 substrate: lactose, pH 7.5, 80°C, mutant enzyme A12S/K328I Caldicellulosiruptor saccharolyticus
19.6
-
 substrate: lactose, pH 7.5, 80°C, mutant enzyme R5M/A12S/F231L/K328I Caldicellulosiruptor saccharolyticus
30.08
-
 substrate: lactose, pH 7.5, 80°C, mutant enzyme R5M/A12S/I52V/F231L/K328I Caldicellulosiruptor saccharolyticus

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 lactose the yields of lactulose and epilactose catalyzed by the wild-type enzyme reaches around 57% and 16%, respectively, after 4 h Caldicellulosiruptor saccharolyticus lactulose + epilactose
-
 r
cellobiose
-
 Caldicellulosiruptor saccharolyticus 4-O-beta-D-glucopyranosyl-D-mannose
-
 r
lactose the yields of lactulose and epilactose catalyzed by the wild-type enzyme reaches around 57% and 16%, respectively, after 4 h. The concentration of lactulose catalyzed by mutant enzyme R5M/A12S/I52V/F231L/K328I rapidly increases from 0 min to 90 min and then increases gradually to the steady-state level with a yield of lactulose up to approximately 76%. No obvious epilactose is detected at any time Caldicellulosiruptor saccharolyticus epilactose
-
 ?
lactose the yields of lactulose and epilactose catalyzed by the wild-type enzyme reaches around 57% and 16%, respectively, after 4 h. The concentration of lactulose catalyzed by mutant enzyme R5M/A12S/I52V/F231L/K328I rapidly increases from 0 min to 90 min and then increases gradually to the steady-state level with a yield of lactulose up to approximately 76%. No obvious epilactose is detected at any time Caldicellulosiruptor saccharolyticus lactulose
-
 ?
additional information the concentration of lactulose catalyzed by mutant G4-C5 rapidly increases from 0 min to 90 min and then increased gradually to the steady-state level with a yield of lactulose up to approximately 76%. No obvious epilactose is detected at any time intervals with mutant G4-C5 (R5M/I52V/A12S/K328I/F231L) Caldicellulosiruptor saccharolyticus ?
-
 ?

Subunits

Subunits Comment Organism
More enzyme three-dimensional structure, overview 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
-
-
 Caldicellulosiruptor saccharolyticus
80
-
 assay at Caldicellulosiruptor saccharolyticus

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
80
-
 t1/2: 141 min (wild-type enzym), 161.2 min (mutant enzyme A12S), 132.5 (mutant enzyme A12S/K328I), 116.9 min (mutant enzyme R5M/A12S/F231L/K328I), 159.4 min (mutant enzyme R5M/A12S/I52V/F231L/K328I) Caldicellulosiruptor saccharolyticus

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
43.26
-
 lactose pH 7.5, 80°C, recombinant wild-type enzyme Caldicellulosiruptor saccharolyticus
43.26
-
 lactose pH 7.5, 80°C, wild-type enzyme Caldicellulosiruptor saccharolyticus
47.5
-
 lactose pH 7.5, 80°C, mutant enzyme A12S Caldicellulosiruptor saccharolyticus
47.5
-
 lactose pH 7.5, 80°C, recombinant mutant A12S Caldicellulosiruptor saccharolyticus
51.77
-
 lactose pH 7.5, 80°C, mutant enzyme A12S/K328I Caldicellulosiruptor saccharolyticus
51.77
-
 lactose pH 7.5, 80°C, recombinant mutant A12S/K328I Caldicellulosiruptor saccharolyticus
55.83
-
 lactose pH 7.5, 80°C, mutant enzyme R5M/A12S/F231L/K328I Caldicellulosiruptor saccharolyticus
55.83
-
 lactose pH 7.5, 80°C, recombinant mutant R5M/A12S/F231L/K328I Caldicellulosiruptor saccharolyticus
75.27
-
 lactose pH 7.5, 80°C, mutant enzyme R5M/A12S/I52V/F231L/K328I Caldicellulosiruptor saccharolyticus
75.27
-
 lactose pH 7.5, 80°C, recombinant mutant R5M/A12S/I52V/F231L/K328I Caldicellulosiruptor saccharolyticus

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7.5
-
-
 Caldicellulosiruptor saccharolyticus
7.5
-
 assay at Caldicellulosiruptor saccharolyticus

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
0.0705
-
 lactose pH 7.5, 80°C, wild-type enzyme Caldicellulosiruptor saccharolyticus
0.0705
-
 lactose pH 7.5, 80°C, recombinant wild-type enzyme Caldicellulosiruptor saccharolyticus
0.0884
-
 lactose pH 7.5, 80°C, mutant enzyme A12S Caldicellulosiruptor saccharolyticus
0.0884
-
 lactose pH 7.5, 80°C, recombinant mutant A12S Caldicellulosiruptor saccharolyticus
0.1089
-
 lactose pH 7.5, 80°C, recombinant mutant A12S/K328I Caldicellulosiruptor saccharolyticus
0.109
-
 lactose pH 7.5, 80°C, mutant enzyme A12S/K328I Caldicellulosiruptor saccharolyticus
0.1389
-
 lactose pH 7.5, 80°C, recombinant mutant R5M/A12S/F231L/K328I Caldicellulosiruptor saccharolyticus
0.139
-
 lactose pH 7.5, 80°C, mutant enzyme R5M/A12S/F231L/K328I Caldicellulosiruptor saccharolyticus
0.21
-
 lactose pH 7.5, 80°C, mutant enzyme R5M/A12S/I52V/F231L/K328I Caldicellulosiruptor saccharolyticus
0.2101
-
 lactose pH 7.5, 80°C, recombinant mutant R5M/A12S/I52V/F231L/K328I Caldicellulosiruptor saccharolyticus