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Literature summary extracted from

  • Martinez-Martinez, I.; Navarro-Fernandez, J.; Garcia-Carmona, F.; Sanchez-Ferrer, A.
    Implication of a mutation in the flavin binding site on the specific activity and substrate specificity of glycine oxidase from Bacillus subtilis produced by directed evolution (2008), J. Biotechnol., 133, 1-8.
    View publication on PubMed

Cloned(Commentary)

EC Number Cloned (Comment) Organism
1.4.3.19 expressed in Escherichia coli Rosetta (DE3) cells Bacillus subtilis

Protein Variants

EC Number Protein Variants Comment Organism
1.4.3.19 I15V the mutant exhibits a 7fold higher specific activity compared to the wild type enzyme Bacillus subtilis

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.4.3.19 0.4
-
 glycine mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 0.5
-
 N-ethylglycine mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 0.51
-
 sarcosine mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 0.6
-
 glycine-ethyl-ester mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 0.6
-
 glycine wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 0.6
-
 sarcosine wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 1.8
-
 glycine-ethyl-ester wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 2.8
-
 N-ethylglycine wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 7.9
-
 D-proline mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 8.9
-
 D-proline wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 217
-
 D-alanine mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 290
-
 D-pipecolate mutant enzyme I15V, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 315
-
 D-alanine wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis
1.4.3.19 2000
-
 D-pipecolate wild type enzyme, in 0.4 mM 4-aminoantipyrine, 7 mM phenol, 4.4 U/ml horseradish peroxidase, 0.0002 mM FAD, and 75 mM sodium diphosphate (pH 8.5) at 37°C Bacillus subtilis

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.4.3.19 40500
-
 4 * 40500, gel filtration Bacillus subtilis
1.4.3.19 43000
-
 4 * 43000, SDS-PAGE Bacillus subtilis
1.4.3.19 162000
-
 gel filtration Bacillus subtilis
1.4.3.19 172000
-
 SDS-PAGE Bacillus subtilis

Organism

EC Number Organism UniProt Comment Textmining
1.4.3.19 Bacillus subtilis O31616
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.4.3.19 100 kDa ultrafiltration, HisTrap FF Ni2+-chelating affinity column chromatography, and phenyl-Sepharose FF hydrophobic column chromatography Bacillus subtilis

Specific Activity [micromol/min/mg]

EC Number Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.4.3.19 0.9
-
 mutant enzyme I15V, from crude extract Bacillus subtilis
1.4.3.19 8.6
-
 mutant enzyme I15V, after 9.3fold purification Bacillus subtilis

Storage Stability

EC Number Storage Stability Organism
1.4.3.19 -80°C, 75 mM sodium diphosphate buffer pH 8.5 containing 10% glycerol, several months, no loss of activity Bacillus subtilis

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.4.3.19 D-alanine + H2O + O2
-
 Bacillus subtilis pyruvate + NH3 + H2O2
-
 ?
1.4.3.19 D-methionine + H2O + O2
-
 Bacillus subtilis ?
-
 ?
1.4.3.19 D-phenylalanine + H2O + O2
-
 Bacillus subtilis phenylpyruvate + NH3 + H2O2
-
 ?
1.4.3.19 D-pipecolate + H2O + O2
-
 Bacillus subtilis ?
-
 ?
1.4.3.19 D-proline + H2O + O2
-
 Bacillus subtilis ?
-
 ?
1.4.3.19 glycine + H2O + O2
-
 Bacillus subtilis glyoxylate + NH3 + H2O2
-
 ?
1.4.3.19 glycine-ethyl-ester + H2O + O2
-
 Bacillus subtilis ?
-
 ?
1.4.3.19 N-ethylglycine + H2O + O2
-
 Bacillus subtilis glyoxylate + ethylamine + H2O2
-
 ?
1.4.3.19 sarcosine + H2O + O2
-
 Bacillus subtilis glyoxylate + methylamine + H2O2
-
 ?

Subunits

EC Number Subunits Comment Organism
1.4.3.19 homotetramer 4 * 43000, SDS-PAGE Bacillus subtilis
1.4.3.19 homotetramer 4 * 40500, gel filtration Bacillus subtilis

Synonyms

EC Number Synonyms Comment Organism
1.4.3.19 GOX
-
 Bacillus subtilis

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.4.3.19 60 80 at 60°C, the I15V mutant shows a similar profile than the wild type enzyme but with lower residual activity after 20 min, at 65°C the mutant goes through activation (160%) during the first 15 min, the activity then falls with a time-dependent trend and reaches a half-life value similar to the wild type enzyme, at higher temperatures (70-80°C) the activity decreases rapidly in both cases, but with half-life value higher for I15V mutant than for wild type enzyme Bacillus subtilis

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
1.4.3.19 6.5 9.5 the incubation of both enzymes for 1 h shows highest stability between pH 6.5 and pH 9.5, the mutant enzyme is also stable at pH below pH 6.5 retaining about 70% of its initial activity, this stability at a lower pH is in contrast to the sharp decrease in pH-stability showed by wild type GOX which retains only about 37% of the initial activity Bacillus subtilis