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Literature summary extracted from

  • Ortiz-Soto, M.E.; Rivera, M.; Rudino-Pinera, E.; Olvera, C.; Lopez-Munguia, A.
    Selected mutations in Bacillus subtilis levansucrase semi-conserved regions affecting its biochemical properties (2008), Protein Eng. Des. Sel., 21, 589-595.
    View publication on PubMed

Activating Compound

EC Number Activating Compound Comment Organism Structure
2.4.1.10 levan 1.4fold increase in Vmax of wild-type in the presence of 15 g/l of levan is observed Bacillus subtilis
2.4.1.10 levan 3.7fold increase in Vmax of S164A in the presence of 15 g/l of levan is observed Bacillus subtilis
2.4.1.10 additional information levan has no effect on Y429N, a mutant that has lost the fructan-synthesizing activity Bacillus subtilis

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.1.10 expression in Escherichia coli Bacillus subtilis

Crystallization (Commentary)

EC Number Crystallization (Comment) Organism
2.4.1.10 mutant enzyme S164A, 12 days of micro-dialysis of the purified protein (8 g/l) against deionized water, PDB accession code 2VDT Bacillus subtilis

Protein Variants

EC Number Protein Variants Comment Organism
2.4.1.10 A344P site directed mutagenesis, same behavior like the wild-type Bacillus subtilis
2.4.1.10 F414W site directed mutagenesis, same behavior like the wild-type Bacillus subtilis
2.4.1.10 G361F site-directed mutagenesis, less stable than the wild-type, synthesizes mainly oligosaccharides, still catalyzes the synthesis of low amounts of polymer, pH-optimum 6, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis
2.4.1.10 H243L site-directed mutagenesis, less stable than the wild-type, pH-optimum 6, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis
2.4.1.10 I341V site-directed mutagenesis, pH-optimum 6 Bacillus subtilis
2.4.1.10 R360K site-directed mutagenesis, pH-optimum 6, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis
2.4.1.10 R360S site-directed mutagenesis, pH-optimum 6, decrease in activity, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis
2.4.1.10 R433A site-directed mutagenesis, synthesizes only oligosaccharides, pH-optimum 6-7, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis
2.4.1.10 S164A site-directed mutagenesis, S164A is catalytically important, as it maintains the nucleophile in an appropriate position regarding the sucrose molecule. S164A results in a 12fold more stable and less hydrolytic enzyme than the wild-type, with a half-life of 628.0 (+51.0) min, pH-optimum 6, decrease in activity, slightly higher affinity for sucrose Bacillus subtilis
2.4.1.10 S164K inactive Bacillus subtilis
2.4.1.10 Y429 site-directed mutagenesis, Y429 plays an indirect but important role in catalysis and acceptor specificity, as this is a key residue coordinating the sucrose position in the levansucrase binding pocket through a complex water network Bacillus subtilis
2.4.1.10 Y429A site-directed mutagenesis Bacillus subtilis
2.4.1.10 Y429N site-directed mutagenesis, synthesizes only oligosaccharides, pH-optimum 5-6, decrease in activity, affinity for sucrose is reduced, shift of reaction specificity (hydrolysis/transfer) Bacillus subtilis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.4.1.10 additional information
-
 additional information mutant enzyme S164K is inactive Bacillus subtilis
2.4.1.10 additional information
-
 additional information mutants A344P and F414W have the same behavior like the wild-type Bacillus subtilis
2.4.1.10 2.5
-
 sucrose mutant enzyme S164A, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
2.4.1.10 7.4
-
 sucrose mutant enzyme I341V, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
2.4.1.10 8
-
 sucrose wild-type, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
2.4.1.10 10.5
-
 sucrose mutant enzyme H243L, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
2.4.1.10 29.3
-
 sucrose mutant enzyme R433A, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
2.4.1.10 30
-
 sucrose mutant enzyme R369K, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
2.4.1.10 154
-
 sucrose mutant enzyme R360S, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
2.4.1.10 297.3
-
 sucrose mutant enzyme G361F, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis
2.4.1.10 319.4
-
 sucrose mutant enzyme Y429N, 0.5 U/ml of enzyme activity at 37°C and pH 6.0 Bacillus subtilis

Organism

EC Number Organism UniProt Comment Textmining
2.4.1.10 Bacillus subtilis P05655 expression in Escherichia coli
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.1.10 ion exchange chromatography Bacillus subtilis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.1.10 additional information mutant enzymes Y429N and R433A no longer produce levan but exclusively oligosaccharides Bacillus subtilis ?
-
 ?
2.4.1.10 sucrose + D-maltose
-
 Bacillus subtilis ?
-
 ?
2.4.1.10 sucrose + D-xylose
-
 Bacillus subtilis beta-D-fructofuranosyl-beta-D-xylopyranoside + D-glucose
-
 ?
2.4.1.10 sucrose + levan mutant enzymes H243L and S164A synthesize either high or low levan molecular weight Bacillus subtilis ?
-
 ?

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
2.4.1.10 additional information
-
 mutants A344P and F414W have the same behavior like the wild-type Bacillus subtilis
2.4.1.10 40
-
 t1/2: 16.0 min mutant enzym R433A, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
2.4.1.10 40
-
 t1/2: 17.21 min mutant enzym R360S, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
2.4.1.10 40
-
 t1/2: 31.4 min mutant enzym I341V, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
2.4.1.10 40
-
 t1/2: 42.11 min mutant enzym R360K, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
2.4.1.10 40
-
 t1/2: 47.04 min mutant enzym Y429N, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
2.4.1.10 40
-
 t1/2: 52.0 min wild-type, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
2.4.1.10 40
-
 t1/2: 628.0 min mutant enzym S164A, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
2.4.1.10 40
-
 t1/2: 7.9 min mutant enzym H243L, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis
2.4.1.10 40
-
 t1/2: less than 1min mutant enzym G361F, stability is determined following the loss of activity after incubation of 1 mg/ml of protein Bacillus subtilis

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.4.1.10 additional information
-
 additional information mutants A344P and F414W have the same behavior like the wild-type Bacillus subtilis
2.4.1.10 6.3
-
 sucrose mutant enzyme Y429N Bacillus subtilis
2.4.1.10 6.4
-
 sucrose mutant enzyme S164A Bacillus subtilis
2.4.1.10 13.5
-
 sucrose mutant enzyme R360S Bacillus subtilis
2.4.1.10 57.4
-
 sucrose mutant enzyme G361F Bacillus subtilis
2.4.1.10 87.5
-
 sucrose mutant enzyme R433A Bacillus subtilis
2.4.1.10 141.5
-
 sucrose mutant enzyme H243L Bacillus subtilis
2.4.1.10 164.6
-
 sucrose wild-type enzyme Bacillus subtilis
2.4.1.10 166.3
-
 sucrose mutant enzyme I341V Bacillus subtilis
2.4.1.10 170.2
-
 sucrose mutant enzyme R369K Bacillus subtilis