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Literature summary extracted from

  • Kirmair, L.; Seiler, D.L.; Skerra, A.
    Stability engineering of the Geobacillus stearothermophilus alcohol dehydrogenase and application for the synthesis of a polyamide 12 precursor (2015), Appl. Microbiol. Biotechnol., 99, 10501-10513.
    View publication on PubMed

Application

EC Number Application Comment Organism
1.1.1.1 synthesis semi-preparative biocatalysis at 60°C using the stabilized mutant C257L, employing butyraldehyde for in situ cofactor regeneration with only catalytic amounts of NAD+, yields up to 23% conversion of omega-hydroxy lauric acid methyl ester to omega-oxo lauric acid methyl ester after 30 min Geobacillus stearothermophilus

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.1.1.1 expression in Escherichia coli Geobacillus stearothermophilus

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.1 C257L mutation introduced to improve stability under oxidzing conditions. Mutant exhibits prolonged stability and an elevated inactivation temperature Geobacillus stearothermophilus

Inhibitors

EC Number Inhibitors Comment Organism Structure
1.1.1.1 Butyraldehyde
-
 Geobacillus stearothermophilus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.1.1.1 0.066
-
 12-oxolauric acid methyl ester wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 0.072
-
 NADH wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 0.086
-
 12-hydroxylauric acid methyl ester wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 0.1
-
 12-hydroxylauric acid methyl ester mutant C257L, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 0.108
-
 Butyraldehyde wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 0.239
-
 NAD+ wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 0.364
-
 acetaldehyde wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 0.611
-
 n-butanol wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 0.91
-
 ethanol wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 1.5
-
 ethanol mutant C257L, pH 8.0, 60°C Geobacillus stearothermophilus

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.1 Geobacillus stearothermophilus P42328
-
-

Storage Stability

EC Number Storage Stability Organism
1.1.1.1 15°C, 50 mM sodium phosphate buffer, pH 8.0, half-life of wild-type 7 h, half-life of mutant C257L 17 h Geobacillus stearothermophilus
1.1.1.1 4°C, 50 mM sodium phosphate buffer, pH 7.0, 10 mM 2-mercaptoethanol, 10% glycerol, both wild-type and mutant C357L stable for several months Geobacillus stearothermophilus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.1 12-hydroxylauric acid methyl ester + NAD+
-
 Geobacillus stearothermophilus 12-oxolauric acid methyl ester + NADH + H+ product is a key intermediate for biobased polyamide 12 production ?
1.1.1.1 12-oxolauric acid methyl ester + NADH + H+
-
 Geobacillus stearothermophilus 12-hydroxylauric acid methyl ester + NAD+
-
 ?
1.1.1.1 acetaldehyde + NADH + H+
-
 Geobacillus stearothermophilus ethanol + NAD+
-
 r
1.1.1.1 butyraldehyde + NAD+
-
 Geobacillus stearothermophilus n-butanol + NADH + H+
-
 r
1.1.1.1 ethanol + NAD+
-
 Geobacillus stearothermophilus acetaldehyde + NADH + H+
-
 r
1.1.1.1 n-butanol + NADH + H+
-
 Geobacillus stearothermophilus butyraldehyde + NAD+
-
 r

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.1.1.1 62.6
-
 wild-type, thermal denaturation midpoint Geobacillus stearothermophilus
1.1.1.1 65.3
-
 mutant C257L, thermal denaturation midpoint Geobacillus stearothermophilus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.1.1.1 27.6
-
 12-hydroxylauric acid methyl ester wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 34.2
-
 NAD+ wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 34.3
-
 12-hydroxylauric acid methyl ester mutant C257L, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 195
-
 n-butanol wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 305
-
 ethanol wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 308
-
 12-oxolauric acid methyl ester wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 326
-
 NADH wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 365
-
 ethanol mutant C257L, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 681
-
 acetaldehyde wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 931
-
 Butyraldehyde wild-type, pH 8.0, 60°C Geobacillus stearothermophilus

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.1 NADH
-
 Geobacillus stearothermophilus

Ki Value [mM]

EC Number Ki Value [mM] Ki Value maximum [mM] Inhibitor Comment Organism Structure
1.1.1.1 2.1
-
 Butyraldehyde wild-type, pH 8.0, 60°C Geobacillus stearothermophilus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.1.1.1 100
-
 NAD+ wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 240
-
 ethanol mutant C257L, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 320
-
 n-butanol wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 320
-
 12-hydroxylauric acid methyl ester wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 340
-
 12-hydroxylauric acid methyl ester mutant C257L, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 340
-
 ethanol wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 1900
-
 acetaldehyde wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 4500
-
 NADH wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 4700
-
 12-oxolauric acid methyl ester wild-type, pH 8.0, 60°C Geobacillus stearothermophilus
1.1.1.1 8600
-
 Butyraldehyde wild-type, pH 8.0, 60°C Geobacillus stearothermophilus