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Literature summary for 1.4.3.16 extracted from

  • Bifulco, D.; Pollegioni, L.; Tessaro, D.; Servi, S.; Molla, G.
    A thermostable L-aspartate oxidase: a new tool for biotechnological applications (2013), Appl. Microbiol. Biotechnol., 97, 7285-7295.
    View publication on PubMed

Application

Application Comment Organism
biotechnology StLASPO represents an appropriate biocatalyst for the resolution of racemic solutions of D,L-aspartate and a well-suited protein scaffold to evolve a L-amino acid oxidase activity by protein engineering Sulfurisphaera tokodaii

Cloned(Commentary)

Cloned (Comment) Organism
expressed in Escherichia coli BL21(DE3) cells Sulfurisphaera tokodaii
expression in Escherichia coli in the active form as holoenzyme Sulfurisphaera tokodaii

Inhibitors

Inhibitors Comment Organism Structure
D-Aspartate weak inhibition; weak inhibition Sulfurisphaera tokodaii
oxaloacetate weak inhibition; weak inhibition Sulfurisphaera tokodaii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
0.33
-
O2 pH 8, 37°C Sulfurisphaera tokodaii
1.3
-
L-aspartate apparent value, at pH 8.0 and 37°C Sulfurisphaera tokodaii
13.3
-
L-aspartate pH 8, 37°C Sulfurisphaera tokodaii
18.1
-
L-asparagine apparent value, at pH 8.0 and 37°C Sulfurisphaera tokodaii

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
51000
-
gel filtration Sulfurisphaera tokodaii
53647
-
1 * 53647, calculated from amino acid sequence Sulfurisphaera tokodaii
536467
-
x * 536467, calculated from sequence Sulfurisphaera tokodaii

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
L-asparagine + H2O + O2 Sulfurisphaera tokodaii
-
4-amino-2,4-dioxobutanoate + NH3 + H2O2
-
?
L-aspartate + H2O + O2 Sulfurisphaera tokodaii the enzyme oxidizes L-aspartate to iminosuccinate, which is then non-enzymatically hydrolyzed to oxaloacetate oxaloacetate + NH3 + H2O2
-
?
additional information Sulfurisphaera tokodaii the enzyme is absolutely stereoselective, since no activity is detected on D-aspartate. The enzyme shows no activity with L-phenylalanine, L-glutamate, glycine, L-proline, and L-alanine ?
-
?

Organism

Organism UniProt Comment Textmining
Sulfurisphaera tokodaii KC333624 synthetic construct
-
Sulfurisphaera tokodaii Q972D2
-
-

Purification (Commentary)

Purification (Comment) Organism
-
Sulfurisphaera tokodaii
HiTrap nickel chelating affinity column chromatography, and Superdex 200 gel filtration Sulfurisphaera tokodaii

Storage Stability

Storage Stability Organism
4°, purified enzyme at pH 7.5, several months, nploss of activity Sulfurisphaera tokodaii
4°C, pH 7.5, in the dark, stable for months Sulfurisphaera tokodaii

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
L-asparagine + H2O + O2
-
Sulfurisphaera tokodaii 4-amino-2,4-dioxobutanoate + NH3 + H2O2
-
?
L-asparagine + O2 Vmax/Km is 63fold lower compared to L-aspartate Sulfurisphaera tokodaii 4-amino-2-imino-4-oxobutanoate + H2O2
-
?
L-aspartate + H2O + O2 the enzyme oxidizes L-aspartate to iminosuccinate, which is then non-enzymatically hydrolyzed to oxaloacetate Sulfurisphaera tokodaii oxaloacetate + NH3 + H2O2
-
?
L-aspartate + O2 the enzyme is absolutely stereoselective, since no activity is detected on D-aspartate Sulfurisphaera tokodaii iminosuccinate + H2O2
-
?
additional information the enzyme does not show activity on L-phenylalanine (50–100 mM), L-glutamate (50–100 mM), glycine (50–100 mM), L-proline (50–100 mM) and L-alanine (50–100 mM) Sulfurisphaera tokodaii ?
-
?
additional information the enzyme is absolutely stereoselective, since no activity is detected on D-aspartate. The enzyme shows no activity with L-phenylalanine, L-glutamate, glycine, L-proline, and L-alanine Sulfurisphaera tokodaii ?
-
?

Subunits

Subunits Comment Organism
? x * 536467, calculated from sequence Sulfurisphaera tokodaii
monomer 1 * 53647, calculated from amino acid sequence Sulfurisphaera tokodaii

Synonyms

Synonyms Comment Organism
LASPO
-
Sulfurisphaera tokodaii
StLASPO
-
Sulfurisphaera tokodaii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
assay at Sulfurisphaera tokodaii
60 80
-
Sulfurisphaera tokodaii

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
40 95 40°C: about 50% of maximal activity, 95°C: about 60% of maximal activity Sulfurisphaera tokodaii

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
37
-
more than 70% of the initial activity is recovered after 60 min at 37°C Sulfurisphaera tokodaii
80
-
stable up to Sulfurisphaera tokodaii
80
-
the enzyme is fully stable up to 80°C (400 min). When the temperature is increased to above 85°C, a sharp decrease in enzyme stability is apparent and the activity is lost in about 100 min at 100°C Sulfurisphaera tokodaii
83
-
Tm-value determinedv by fluoresecence at 340 nm Sulfurisphaera tokodaii
86
-
Tm-value determinedv by fluoresecence at 525 nm Sulfurisphaera tokodaii

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.05
-
L-aspartate pH 8, 37°C Sulfurisphaera tokodaii
1.05
-
L-aspartate apparent value, at pH 8.0 and 37°C Sulfurisphaera tokodaii

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
10
-
-
Sulfurisphaera tokodaii

pH Range

pH Minimum pH Maximum Comment Organism
8.8 12 pH 8.8: about 50% of maximal activity, pH 12.0: about 70% of maximal activity Sulfurisphaera tokodaii

pH Stability

pH Stability pH Stability Maximum Comment Organism
7 10 60 min, stable Sulfurisphaera tokodaii
7 10 no significant change in activity is observed up to 400 min of incubation at pH 7.5. More than 70% of the initial activity is recovered after 60 min at 37°C. Below pH 8.0 and above pH 10.0, a significant time-dependent inactivation is apparent Sulfurisphaera tokodaii

Cofactor

Cofactor Comment Organism Structure
FAD FAD-containing flavoprotein, tight binding of the FAD cofactor Sulfurisphaera tokodaii
FAD contains 1 FAD per protein monomer Sulfurisphaera tokodaii