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Literature summary for 1.10.3.2 extracted from

  • Tonin, F.; Rosini, E.; Piubelli, L.; Sanchez-Amat, A.; Pollegioni, L.
    Different recombinant forms of polyphenol oxidase A, a laccase from Marinomonas mediterranea (2016), Protein Expr. Purif., 123, 60-69 .
    View publication on PubMed
Search for more literature for 1.10.3.2

Cloned(Commentary)

Cloned (Comment) Organism
gene ppoA, the full-length and truncated enzyme is overexpressed in Escherichia coli strain Rosetta 2(DE3) and BL21(DE3) with and without a C-terminal His6-tag, method optimization, overview Marinomonas mediterranea

Protein Variants

Protein Variants Comment Organism
additional information elimination of the N-terminal sequence in mutant rMmPPOA-637 decreases the specific activity 15fold, which is partially restored in the presence of 1 M NaCl, and alters the secondary and tertiary structures and the pH dependence of optimal stability Marinomonas mediterranea

Inhibitors

Inhibitors Comment Organism Structure
additional information the recombinant His-tagged MmPPOA-695 shows a strong resistance to NaCl, DMSO, and Tween-80 Marinomonas mediterranea

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics Marinomonas mediterranea
0.011
-
 catechol native wild-type enzyme, pH 5.0, 25°C Marinomonas mediterranea
0.018
-
 catechol recombinant His-tagged enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
0.022
-
 catechol recombinant wild-type enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
0.172
-
 2,6-dimethoxyphenol recombinant wild-type enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
0.293
-
 2,6-dimethoxyphenol recombinant His-tagged enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
1.163
-
 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) recombinant His-tagged enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
1.263
-
 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) recombinant wild-type enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
1.413
-
 2,6-dimethoxyphenol recombinant mutant enzyme MmPPOA-637, pH 5.0, 25°C Marinomonas mediterranea
1.478
-
 catechol recombinant mutant enzyme MmPPOA-637, pH 5.0, 25°C Marinomonas mediterranea
1.84
-
 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) native wild-type enzyme, pH 5.0, 25°C Marinomonas mediterranea
1.98
-
 2,6-dimethoxyphenol native wild-type enzyme, pH 5.0, 25°C Marinomonas mediterranea
3.295
-
 2,6-dimethoxyphenol recombinant His-tagged mutant enzyme MmPPOA-637, pH 5.0, 25°C Marinomonas mediterranea

Localization

Localization Comment Organism GeneOntology No. Textmining
membrane membrane-bound. Adding nonionic detergents (Triton X100 and Tween-20) does not solubilize the enzyme from the cell membrane. The highest solubilization of MmPPOA is achieved by adding 1% SDS to the lysis buffer Marinomonas mediterranea 16020
-

Metals/Ions

Metals/Ions Comment Organism Structure
Cu2+ a multi-copper laccase Marinomonas mediterranea
NaCl native wild-type MmPPOA and recombinant wild-type MmPPOA-695-His (both the crude extract and the pure enzyme preparations) retain substantial part of the enzymatic activity in the presence of 1 M NaCl, while recombinant mutant His-tagged MmPPOA-637 activity shows a 90% decrease at 100 mM NaCl and about 20% increase at 1 M NaCl Marinomonas mediterranea

Organism

Organism UniProt Comment Textmining
Marinomonas mediterranea Q9KJB8
-
-
Marinomonas mediterranea MMB-1 Q9KJB8
-
-

Purification (Commentary)

Purification (Comment) Organism
recombinant His6-tagged wild-type and truncated mutant enzymes from Escherichia coli strain Rosetta 2(DE3) and BL21(DE3) by nickel affinity chromatography, dialysis, ultrafiltration, gel filtration, and again dialysis. The wild-type enzyme is purified by 16fold, the mutant enzyme by 3fold Marinomonas mediterranea

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
1.34
-
 purified recombinant enzyme, substrate 2,6-dimethoxyphenol, pH 5.0, 25°C Marinomonas mediterranea

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2 catechol + O2
-
 Marinomonas mediterranea 2 1,2-benzoquinone + 2 H2O
-
 ?
2 catechol + O2
-
 Marinomonas mediterranea MMB-1 2 1,2-benzoquinone + 2 H2O
-
 ?
2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) + O2
-
 Marinomonas mediterranea ?
-
 ?
2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) + O2
-
 Marinomonas mediterranea MMB-1 ?
-
 ?
2,6-dimethoxyphenol + O2
-
 Marinomonas mediterranea ?
-
 ?
2,6-dimethoxyphenol + O2
-
 Marinomonas mediterranea MMB-1 ?
-
 ?
additional information the enzyme shows broad substrate specificity. The recombinant His-tagged MmPPOA-695 shows kinetic properties on catechol higher than for known laccases. Laccase belongs to the group of blue, multicopper oxidases which couple the four-electron reduction of dioxygen to water with the oxidation of a broad range of substrates (including phenols, polyphenols, arylamines, anilines, hydroxyindols, and thiols) Marinomonas mediterranea ?
-
 ?
additional information the enzyme shows broad substrate specificity. The recombinant His-tagged MmPPOA-695 shows kinetic properties on catechol higher than for known laccases. Laccase belongs to the group of blue, multicopper oxidases which couple the four-electron reduction of dioxygen to water with the oxidation of a broad range of substrates (including phenols, polyphenols, arylamines, anilines, hydroxyindols, and thiols) Marinomonas mediterranea MMB-1 ?
-
 ?

Subunits

Subunits Comment Organism
? x * 70000, recombinant His-tagged MmPPOA-637 mutant, SDS-PAGE Marinomonas mediterranea

Synonyms

Synonyms Comment Organism
laccase
-
 Marinomonas mediterranea
MmPPO laccase
-
 Marinomonas mediterranea
MmPPOA
-
 Marinomonas mediterranea
More cf. EC 1.14.18.1 Marinomonas mediterranea
polyphenol oxidase A
-
 Marinomonas mediterranea
PpoA
-
 Marinomonas mediterranea

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
25
-
 assay at Marinomonas mediterranea

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
additional information
-
 the recombinant His-tagged MmPPOA-695 shows a very high thermal stabilit Marinomonas mediterranea
90
-
 above, high thermal stability of purified wild-type rMmPPOA-695-His and mutant rMmPPOA-637-His enzymes Marinomonas mediterranea
100
-
 inactivation through protein unfolding. The enzyme is refolded at 25°C but rests catalytically inactive Marinomonas mediterranea

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
0.084
-
 catechol recombinant mutant enzyme MmPPOA-637, pH 5.0, 25°C Marinomonas mediterranea
0.1
-
 2,6-dimethoxyphenol recombinant mutant enzyme MmPPOA-637, pH 5.0, 25°C Marinomonas mediterranea
0.21
-
 2,6-dimethoxyphenol recombinant His-tagged mutant enzyme MmPPOA-637, pH 5.0, 25°C Marinomonas mediterranea
0.92
-
 2,6-dimethoxyphenol recombinant wild-type enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
1.53
-
 catechol recombinant wild-type enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
1.74
-
 2,6-dimethoxyphenol recombinant His-tagged enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
1.98
-
 2,6-dimethoxyphenol native wild-type enzyme, pH 5.0, 25°C Marinomonas mediterranea
2
-
 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) recombinant His-tagged enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
2.18
-
 catechol recombinant His-tagged enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
2.5
-
 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) recombinant wild-type enzyme MmPPOA-695, pH 5.0, 25°C Marinomonas mediterranea
4.2
-
 catechol native wild-type enzyme, pH 5.0, 25°C Marinomonas mediterranea
4.5
-
 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) native wild-type enzyme, pH 5.0, 25°C Marinomonas mediterranea

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
3 9 recombinant wild-type enzyme, broad optimum Marinomonas mediterranea
4
-
 recombinant mutant enzyme MmPPOA-637 Marinomonas mediterranea

pH Range

pH Minimum pH Maximum Comment Organism
3 8 recombinant mutant enzyme MmPPOA-637, inactivation at pH 8.0 and above Marinomonas mediterranea
3 9 recombinant wild-type enzyme, active range and broad optimum Marinomonas mediterranea

General Information

General Information Comment Organism
malfunction elimination of the N-terminal sequence decreases the specific activity 15fold, which is partially restored in the presence of 1 M NaCl, and alters the secondary and tertiary structures and the pH dependence of optimal stability Marinomonas mediterranea
additional information the enzyme has a high redox potential Marinomonas mediterranea