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Literature summary for 1.4.3.19 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
Search for more literature for 1.4.3.19

Cloned(Commentary)

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

Protein Variants

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

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
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
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
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
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
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
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
1.8
-
 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
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
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
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
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
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
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
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]

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

Organism

Organism UniProt Comment Textmining
Bacillus subtilis O31616
-
-

Purification (Commentary)

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

Specific Activity [micromol/min/mg]

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

Storage Stability

Storage Stability Organism
-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)

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

Subunits

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

Synonyms

Synonyms Comment Organism
GOX
-
 Bacillus subtilis

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
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

pH Stability pH Stability Maximum Comment Organism
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