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 | 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 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 | Comment | Organism | Structure |
---|---|---|---|
Ca2+ | activates | Ralstonia pickettii |
Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|
poly((R)-3-hydroxybutyrate) + H2O | Ralstonia pickettii | - |
? | - |
? |
Organism | UniProt | Comment | Textmining |
---|---|---|---|
Ralstonia pickettii | P12625 | gene phaZRpiT1 | - |
Ralstonia pickettii T1 | P12625 | gene phaZRpiT1 | - |
Purification (Comment) | Organism |
---|---|
recombinant soluble His-tagged wild-type and mutant enzymes from Escherichia coli by nickel affinity chromatography | Ralstonia pickettii |
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 | Comment | Organism |
---|---|---|
PhaZRpiT1 | - |
Ralstonia pickettii |
PHB depolymerase | - |
Ralstonia pickettii |
poly[(R)-3-hydroxybutyrate] depolymerase | - |
Ralstonia pickettii |
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 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 | 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 |