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Literature summary 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

EC Number Application Comment Organism
1.4.3.16 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)

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

Inhibitors

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

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
1.4.3.16 0.33
-
 O2 pH 8, 37°C Sulfurisphaera tokodaii
1.4.3.16 1.3
-
 L-aspartate apparent value, at pH 8.0 and 37°C Sulfurisphaera tokodaii
1.4.3.16 13.3
-
 L-aspartate pH 8, 37°C Sulfurisphaera tokodaii
1.4.3.16 18.1
-
 L-asparagine apparent value, at pH 8.0 and 37°C Sulfurisphaera tokodaii

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
1.4.3.16 51000
-
 gel filtration Sulfurisphaera tokodaii
1.4.3.16 53647
-
 1 * 53647, calculated from amino acid sequence Sulfurisphaera tokodaii
1.4.3.16 536467
-
 x * 536467, calculated from sequence Sulfurisphaera tokodaii

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.4.3.16 L-asparagine + H2O + O2 Sulfurisphaera tokodaii
-
 4-amino-2,4-dioxobutanoate + NH3 + H2O2
-
 ?
1.4.3.16 L-aspartate + H2O + O2 Sulfurisphaera tokodaii the enzyme oxidizes L-aspartate to iminosuccinate, which is then non-enzymatically hydrolyzed to oxaloacetate oxaloacetate + NH3 + H2O2
-
 ?
1.4.3.16 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

EC Number Organism UniProt Comment Textmining
1.4.3.16 Sulfurisphaera tokodaii KC333624 synthetic construct
-
1.4.3.16 Sulfurisphaera tokodaii Q972D2
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
1.4.3.16
-
 Sulfurisphaera tokodaii
1.4.3.16 HiTrap nickel chelating affinity column chromatography, and Superdex 200 gel filtration Sulfurisphaera tokodaii

Storage Stability

EC Number Storage Stability Organism
1.4.3.16 4°, purified enzyme at pH 7.5, several months, nploss of activity Sulfurisphaera tokodaii
1.4.3.16 4°C, pH 7.5, in the dark, stable for months Sulfurisphaera tokodaii

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.4.3.16 L-asparagine + H2O + O2
-
 Sulfurisphaera tokodaii 4-amino-2,4-dioxobutanoate + NH3 + H2O2
-
 ?
1.4.3.16 L-asparagine + O2 Vmax/Km is 63fold lower compared to L-aspartate Sulfurisphaera tokodaii 4-amino-2-imino-4-oxobutanoate + H2O2
-
 ?
1.4.3.16 L-aspartate + H2O + O2 the enzyme oxidizes L-aspartate to iminosuccinate, which is then non-enzymatically hydrolyzed to oxaloacetate Sulfurisphaera tokodaii oxaloacetate + NH3 + H2O2
-
 ?
1.4.3.16 L-aspartate + O2 the enzyme is absolutely stereoselective, since no activity is detected on D-aspartate Sulfurisphaera tokodaii iminosuccinate + H2O2
-
 ?
1.4.3.16 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 ?
-
 ?
1.4.3.16 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

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

Synonyms

EC Number Synonyms Comment Organism
1.4.3.16 LASPO
-
 Sulfurisphaera tokodaii
1.4.3.16 StLASPO
-
 Sulfurisphaera tokodaii

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.4.3.16 37
-
 assay at Sulfurisphaera tokodaii
1.4.3.16 60 80
-
 Sulfurisphaera tokodaii

Temperature Range [°C]

EC Number Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
1.4.3.16 40 95 40°C: about 50% of maximal activity, 95°C: about 60% of maximal activity Sulfurisphaera tokodaii

Temperature Stability [°C]

EC Number Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
1.4.3.16 37
-
 more than 70% of the initial activity is recovered after 60 min at 37°C Sulfurisphaera tokodaii
1.4.3.16 80
-
 stable up to Sulfurisphaera tokodaii
1.4.3.16 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
1.4.3.16 83
-
 Tm-value determinedv by fluoresecence at 340 nm Sulfurisphaera tokodaii
1.4.3.16 86
-
 Tm-value determinedv by fluoresecence at 525 nm Sulfurisphaera tokodaii

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
1.4.3.16 1.05
-
 L-aspartate pH 8, 37°C Sulfurisphaera tokodaii
1.4.3.16 1.05
-
 L-aspartate apparent value, at pH 8.0 and 37°C Sulfurisphaera tokodaii

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.4.3.16 10
-
-
 Sulfurisphaera tokodaii

pH Range

EC Number pH Minimum pH Maximum Comment Organism
1.4.3.16 8.8 12 pH 8.8: about 50% of maximal activity, pH 12.0: about 70% of maximal activity Sulfurisphaera tokodaii

pH Stability

EC Number pH Stability pH Stability Maximum Comment Organism
1.4.3.16 7 10 60 min, stable Sulfurisphaera tokodaii
1.4.3.16 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

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