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Literature summary for 3.1.1.75 extracted from

  • Tan, L.T.; Hiraishi, T.; Sudesh, K.; Maeda, M.
    Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285 (2013), Appl. Microbiol. Biotechnol., 97, 4859-4871.
    View publication on PubMed

Cloned(Commentary)

Cloned (Comment) Organism
gene phaZRpiT1, DNA and amino acid sequence determination and analysis of wild-type and mutant enzymes, recombinant expression of soluble His-tagged wild-type and mutant enzymes in Escherichia coli Ralstonia pickettii

Protein Variants

Protein Variants Comment Organism
A66V random mutagenesis, the mutant enzymes shows increased activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
A66V/N251S/T290A random mutagenesis, the mutant enzymes shows altered activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
A66V/N285D/G310G random mutagenesis, the mutant enzymes shows altered activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
A66V/T145S/N285D random mutagenesis, the mutant enzymes shows altered activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
G108G/Q125L/R230R/K260M/T284S/G310G random mutagenesis, the mutant enzymes shows altered activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
additional information mutated phaZRpiT1 genes generated by error-prone PCR are fused to the oprI gene from Pseudomonas aeruginosa to display them as anchored fusion proteins on Escherichia coli cell surface, screening for activity with 4-nitrophenyl butyrate. The wholecell catalysis is a promising and suitable tool to screen for PHB depolymerases with enhanced catalytic activity Ralstonia pickettii
N285D site-directed mutagenesis, the mutant enzymes shows increased activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
N285D/G310G random mutagenesis, the mutant enzymes shows altered activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
N285Y random mutagenesis, the mutant enzymes shows increased activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
S92S/G180D/I292F random mutagenesis, the mutant enzymes shows altered activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
S92S/T145S/N285Y random mutagenesis, the mutant enzymes shows altered activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
T145S/S246T/T252T/S257T random mutagenesis, the mutant enzymes shows altered activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
T252T/S257T/N285D random mutagenesis, the mutant enzymes shows altered activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
T37A/C51C/N285Y random mutagenesis, the mutant enzymes shows altered activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii
T37A/N285D random mutagenesis, the mutant enzymes shows altered activity with 4-nitrophenyl butyrate as compared to the wild-type enzyme Ralstonia pickettii

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
additional information kinetics of random mutants, overview Ralstonia pickettii
0.05
-
4-nitrophenyl hexanoate recombinant mutant A66V/N251S/T290A, pH 7.0, 30°C Ralstonia pickettii
0.05
-
4-nitrophenyl pentanoate recombinant mutant A66V/N251S/T290A, pH 7.0, 30°C Ralstonia pickettii
0.05
-
4-nitrophenyl hexanoate recombinant mutant G108G/Q125L/R230R/K260M/T284S/G310G, pH 7.0, 30°C Ralstonia pickettii
0.06
-
4-nitrophenyl hexanoate recombinant mutant A66V/T145S/N285D, pH 7.0, 30°C Ralstonia pickettii
0.06
-
4-nitrophenyl hexanoate recombinant mutant S92S/G180D/I292F, pH 7.0, 30°C Ralstonia pickettii
0.06
-
4-nitrophenyl hexanoate recombinant mutant T145S/S246T/T252T/S257T, pH 7.0, 30°C Ralstonia pickettii
0.07
-
4-nitrophenyl hexanoate recombinant mutant A66V/N285D/G310G, pH 7.0, 30°C Ralstonia pickettii
0.09
-
4-nitrophenyl hexanoate recombinant mutant A66V, pH 7.0, 30°C Ralstonia pickettii
0.09
-
4-nitrophenyl pentanoate recombinant mutant A66V, pH 7.0, 30°C Ralstonia pickettii
0.09
-
4-nitrophenyl pentanoate recombinant mutant S92S/G180D/I292F, pH 7.0, 30°C Ralstonia pickettii
0.09
-
4-nitrophenyl pentanoate recombinant mutant T145S/S246T/T252T/S257T, pH 7.0, 30°C Ralstonia pickettii
0.09
-
4-nitrophenyl pentanoate recombinant wild-type enzyme, pH 7.0, 30°C Ralstonia pickettii
0.1
-
4-nitrophenyl pentanoate recombinant mutant A66V/N285D/G310G, pH 7.0, 30°C Ralstonia pickettii
0.1
-
4-nitrophenyl hexanoate recombinant wild-type enzyme, pH 7.0, 30°C Ralstonia pickettii
0.16
-
4-nitrophenyl pentanoate recombinant mutant A66V/T145S/N285D, pH 7.0, 30°C Ralstonia pickettii
0.16
-
4-nitrophenyl butyrate recombinant mutant S92S/G180D/I292F, pH 7.0, 30°C Ralstonia pickettii
0.16
-
4-nitrophenyl butyrate recombinant mutant T145S/S246T/T252T/S257T, pH 7.0, 30°C Ralstonia pickettii
0.19
-
4-nitrophenyl butyrate recombinant mutant G108G/Q125L/R230R/K260M/T284S/G310G, pH 7.0, 30°C Ralstonia pickettii
0.23
-
4-nitrophenyl butyrate recombinant mutant A66V, pH 7.0, 30°C Ralstonia pickettii
0.23
-
4-nitrophenyl butyrate recombinant mutant A66V/N251S/T290A, pH 7.0, 30°C Ralstonia pickettii
0.24
-
4-nitrophenyl acetate recombinant mutant A66V/N285D/G310G, pH 7.0, 30°C Ralstonia pickettii
0.24
-
4-nitrophenyl pentanoate recombinant mutant G108G/Q125L/R230R/K260M/T284S/G310G, pH 7.0, 30°C Ralstonia pickettii
0.26
-
4-nitrophenyl acetate recombinant mutant A66V, pH 7.0, 30°C Ralstonia pickettii
0.27
-
4-nitrophenyl propionate recombinant wild-type enzyme, pH 7.0, 30°C Ralstonia pickettii
0.28
-
4-nitrophenyl acetate recombinant mutant G108G/Q125L/R230R/K260M/T284S/G310G, pH 7.0, 30°C Ralstonia pickettii
0.29
-
4-nitrophenyl hexanoate recombinant mutant T37A/N285Y, pH 7.0, 30°C Ralstonia pickettii
0.3
-
4-nitrophenyl hexanoate recombinant mutant S92S/T145S/N285Y, pH 7.0, 30°C Ralstonia pickettii
0.31
-
4-nitrophenyl propionate recombinant mutant A66V/N251S/T290A, pH 7.0, 30°C Ralstonia pickettii
0.32
-
4-nitrophenyl hexanoate recombinant mutant N285Y, pH 7.0, 30°C Ralstonia pickettii
0.37
-
4-nitrophenyl butyrate recombinant mutant A66V/N285D/G310G, pH 7.0, 30°C Ralstonia pickettii
0.38
-
4-nitrophenyl propionate recombinant mutant S92S/G180D/I292F, pH 7.0, 30°C Ralstonia pickettii
0.38
-
4-nitrophenyl propionate recombinant mutant T145S/S246T/T252T/S257T, pH 7.0, 30°C Ralstonia pickettii
0.41
-
4-nitrophenyl acetate recombinant mutant A66V/N251S/T290A, pH 7.0, 30°C Ralstonia pickettii
0.41
-
4-nitrophenyl hexanoate recombinant mutant T252T/S257T/N285D, pH 7.0, 30°C Ralstonia pickettii
0.43
-
4-nitrophenyl hexanoate recombinant mutant T37A/N285D, pH 7.0, 30°C Ralstonia pickettii
0.45
-
4-nitrophenyl hexanoate recombinant mutant N285D, pH 7.0, 30°C Ralstonia pickettii
0.46
-
4-nitrophenyl propionate recombinant mutant A66V, pH 7.0, 30°C Ralstonia pickettii
0.46
-
4-nitrophenyl butyrate recombinant mutant A66V/T145S/N285D, pH 7.0, 30°C Ralstonia pickettii
0.46
-
4-nitrophenyl pentanoate recombinant mutant T37A/N285Y, pH 7.0, 30°C Ralstonia pickettii
0.51
-
4-nitrophenyl butyrate recombinant mutant T252T/S257T/N285D, pH 7.0, 30°C Ralstonia pickettii
0.53
-
4-nitrophenyl propionate recombinant mutant G108G/Q125L/R230R/K260M/T284S/G310G, pH 7.0, 30°C Ralstonia pickettii
0.54
-
4-nitrophenyl propionate recombinant mutant T37A/N285Y, pH 7.0, 30°C Ralstonia pickettii
0.56
-
4-nitrophenyl acetate recombinant mutant S92S/G180D/I292F, pH 7.0, 30°C Ralstonia pickettii
0.56
-
4-nitrophenyl acetate recombinant mutant T145S/S246T/T252T/S257T, pH 7.0, 30°C Ralstonia pickettii
0.56
-
4-nitrophenyl butyrate recombinant wild-type enzyme, pH 7.0, 30°C Ralstonia pickettii
0.58
-
4-nitrophenyl pentanoate recombinant mutant S92S/T145S/N285Y, pH 7.0, 30°C Ralstonia pickettii
0.59
-
4-nitrophenyl pentanoate recombinant mutant N285Y, pH 7.0, 30°C Ralstonia pickettii
0.63
-
4-nitrophenyl butyrate recombinant mutant N285D, pH 7.0, 30°C Ralstonia pickettii
0.63
-
4-nitrophenyl butyrate recombinant mutant T37A/N285Y, pH 7.0, 30°C Ralstonia pickettii
0.66
-
4-nitrophenyl acetate recombinant mutant T37A/N285D, pH 7.0, 30°C Ralstonia pickettii
0.66
-
4-nitrophenyl pentanoate recombinant mutant T37A/N285D, pH 7.0, 30°C Ralstonia pickettii
0.67
-
4-nitrophenyl butyrate recombinant mutant T37A/N285D, pH 7.0, 30°C Ralstonia pickettii
0.67
-
4-nitrophenyl acetate recombinant mutant T37A/N285Y, pH 7.0, 30°C Ralstonia pickettii
0.68
-
4-nitrophenyl propionate recombinant mutant N285D, pH 7.0, 30°C Ralstonia pickettii
0.71
-
4-nitrophenyl pentanoate recombinant mutant T252T/S257T/N285D, pH 7.0, 30°C Ralstonia pickettii
0.73
-
4-nitrophenyl acetate recombinant wild-type enzyme, pH 7.0, 30°C Ralstonia pickettii
0.78
-
4-nitrophenyl butyrate recombinant mutant N285Y, pH 7.0, 30°C Ralstonia pickettii
0.82
-
4-nitrophenyl acetate recombinant mutant A66V/T145S/N285D, pH 7.0, 30°C Ralstonia pickettii
0.89
-
4-nitrophenyl pentanoate recombinant mutant N285D, pH 7.0, 30°C Ralstonia pickettii
1.01
-
4-nitrophenyl propionate recombinant mutant T252T/S257T/N285D, pH 7.0, 30°C Ralstonia pickettii
1.07
-
4-nitrophenyl propionate recombinant mutant A66V/N285D/G310G, pH 7.0, 30°C Ralstonia pickettii
1.17
-
4-nitrophenyl propionate recombinant mutant A66V/T145S/N285D, pH 7.0, 30°C Ralstonia pickettii
1.25
-
4-nitrophenyl butyrate recombinant mutant S92S/T145S/N285Y, pH 7.0, 30°C Ralstonia pickettii
1.28
-
4-nitrophenyl propionate recombinant mutant N285Y, pH 7.0, 30°C Ralstonia pickettii
1.29
-
4-nitrophenyl acetate recombinant mutant N285D, pH 7.0, 30°C Ralstonia pickettii
1.29
-
4-nitrophenyl propionate recombinant mutant T37A/N285D, pH 7.0, 30°C Ralstonia pickettii
1.35
-
4-nitrophenyl acetate recombinant mutant N285Y, pH 7.0, 30°C Ralstonia pickettii
1.96
-
4-nitrophenyl acetate recombinant mutant S92S/T145S/N285Y, pH 7.0, 30°C Ralstonia pickettii
2.43
-
4-nitrophenyl acetate recombinant mutant T252T/S257T/N285D, pH 7.0, 30°C Ralstonia pickettii
2.7
-
4-nitrophenyl propionate recombinant mutant S92S/T145S/N285Y, pH 7.0, 30°C Ralstonia pickettii

Metals/Ions

Metals/Ions Comment Organism Structure
Ca2+ activates Ralstonia pickettii

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
poly((R)-3-hydroxybutyrate) + H2O Ralstonia pickettii
-
?
-
?

Organism

Organism UniProt Comment Textmining
Ralstonia pickettii P12625 gene phaZRpiT1
-
Ralstonia pickettii T1 P12625 gene phaZRpiT1
-

Purification (Commentary)

Purification (Comment) Organism
recombinant soluble His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography Ralstonia pickettii

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-nitrophenyl acetate + H2O
-
Ralstonia pickettii 4-nitrophenol + acetate
-
?
4-nitrophenyl acetate + H2O
-
Ralstonia pickettii T1 4-nitrophenol + acetate
-
?
4-nitrophenyl butyrate + H2O
-
Ralstonia pickettii 4-nitrophenol + butyrate
-
?
4-nitrophenyl butyrate + H2O
-
Ralstonia pickettii T1 4-nitrophenol + butyrate
-
?
4-nitrophenyl hexanoate + H2O
-
Ralstonia pickettii 4-nitrophenol + hexanoate
-
?
4-nitrophenyl hexanoate + H2O
-
Ralstonia pickettii T1 4-nitrophenol + hexanoate
-
?
4-nitrophenyl pentanoate + H2O
-
Ralstonia pickettii 4-nitrophenol + pentanoate
-
?
4-nitrophenyl pentanoate + H2O
-
Ralstonia pickettii T1 4-nitrophenol + pentanoate
-
?
4-nitrophenyl propionate + H2O
-
Ralstonia pickettii 4-nitrophenol + propionate
-
?
4-nitrophenyl propionate + H2O
-
Ralstonia pickettii T1 4-nitrophenol + propionate
-
?
poly((R)-3-hydroxybutyrate) + H2O
-
Ralstonia pickettii ?
-
?

Synonyms

Synonyms Comment Organism
PhaZRpiT1
-
Ralstonia pickettii
PHB depolymerase
-
Ralstonia pickettii
poly[(R)-3-hydroxybutyrate] depolymerase
-
Ralstonia pickettii

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
30
-
esterase assay at Ralstonia pickettii
37
-
poly((R)-3-hydroxybutyrate)-degrading activity assay at Ralstonia pickettii

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
7
-
esterase assay at Ralstonia pickettii
7.5
-
poly((R)-3-hydroxybutyrate)-degrading activity assay at Ralstonia pickettii

General Information

General Information Comment Organism
additional information the enzyme has three functional domains to effectively degrade solid poly((R)-3-hydroxybutyrate) materials, and its catalytic domain catalyzes the ester bond cleavage of the substrate Ralstonia pickettii