Thermolysin-like protease from Bacillus thermoproteolyticus

Enzyme Description

Species

Bacillus stearothermophilus ↗

Extremophile

Yes thermophilic organism

EC Number

3.4.24.4 ↗

Sequence

Length: 319 amino acids

VAGASTVGVGRGVLGDQKYINTTYSSYYGYYYLQDNTRGSGIFTYDGRNRTVLPGSLWTDGDNQFTASYDAAAVDAHYYAGVVYDYYKNVHGRLSYDGSNAAIRSTVHYGRGYNNAFWNGSQMVYGDGDGQTFLPFSGGIDVVGHELTHAVTDYTAGLVYQNESGAINEAMSDIFGTLVEFYANRNPDWEIGEDIYTPGVAGDALRSMSDPAKYGDPDHYSKRYTGTQDNGGVHTNSGIINKAAYLLSQGGVHYGVSVNGIGRDKMGKIFYRALVYYLTPTSNFSQLRAACVQAAADLYGSTSQEVNSVKQAFNAVGVY

Johanna Mansfeld et al. (2007) — The stability of engineered thermostable neutral proteases from Bacillus stearothermophilus in organic solvents and detergents

Biotechnology and Bioengineering · doi:10.1002/bit.21292 ↗ · Stability - C50 Stability - Incubation

69 measurements ▼

Database ID

UniProt: P06874 ↗

Sequence Annotation

Inferred - from protein name

Protein Source

Recombinant, host bacterium Escherichia coli

Experimental Data (69 measurements)

69 measurements — page 4 of 4

Mutation	Property	Assay	Solvent	Solvent Volume	Aqueous Reference	WT Reference	Measured Value	Units	Solution	pH	Temperature	Substrate(s)	Product(s)	Cofactor(s)	Shaking	Comments
A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L	Stability - Incubation	Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase	Dimethylformamide (DMF)	90%	100.0	—	0.0	%	Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2	Incubation: 7.5, Assay: 7.5	Incubation: 37°C, Assay: 37°C	0.5% (w/v) Casein	free amino acids , Peptides	—	—	Non-incubated control (in %), assay in aqueous phase \| Clear about diluted conditions in assay ("diluted to a final concentration of the water-miscible organic solvent of <0.5% (v/v).")/For mutants, had to shift the residue numbering of 3 for all mutants except A4T and G8C compared to article numbering to be consistent, suggests an indel in the proteins sequence between protein used in the article and UniProt, but could not find the article protein sequence
G8C/N63C/C291L	Stability - Incubation	Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase	Dimethylformamide (DMF)	60%	100.0	—	13.0	%	Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2	Incubation: 7.5, Assay: 7.5	Incubation: 37°C, Assay: 37°C	0.5% (w/v) Casein	free amino acids , Peptides	—	—	Non-incubated control (in %), assay in aqueous phase \| Clear about diluted conditions in assay ("diluted to a final concentration of the water-miscible organic solvent of <0.5% (v/v).")/For mutants, had to shift the residue numbering of 3 for all mutants except A4T and G8C compared to article numbering to be consistent, suggests an indel in the proteins sequence between protein used in the article and UniProt, but could not find the article protein sequence
G8C/N63C/C291L	Stability - Incubation	Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase	Dimethylformamide (DMF)	80%	100.0	—	0.0	%	Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2	Incubation: 7.5, Assay: 7.5	Incubation: 37°C, Assay: 37°C	0.5% (w/v) Casein	free amino acids , Peptides	—	—	Non-incubated control (in %), assay in aqueous phase \| Clear about diluted conditions in assay ("diluted to a final concentration of the water-miscible organic solvent of <0.5% (v/v).")/For mutants, had to shift the residue numbering of 3 for all mutants except A4T and G8C compared to article numbering to be consistent, suggests an indel in the proteins sequence between protein used in the article and UniProt, but could not find the article protein sequence
G8C/N63C/C291L	Stability - Incubation	Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase	Dimethylformamide (DMF)	90%	100.0	—	0.0	%	Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2	Incubation: 7.5, Assay: 7.5	Incubation: 37°C, Assay: 37°C	0.5% (w/v) Casein	free amino acids , Peptides	—	—	Non-incubated control (in %), assay in aqueous phase \| Clear about diluted conditions in assay ("diluted to a final concentration of the water-miscible organic solvent of <0.5% (v/v).")/For mutants, had to shift the residue numbering of 3 for all mutants except A4T and G8C compared to article numbering to be consistent, suggests an indel in the proteins sequence between protein used in the article and UniProt, but could not find the article protein sequence
A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L	Stability - Incubation	Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase	Dimethylformamide (DMF)	10%	100.0	—	98.0	%	Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2	Incubation: 7.5, Assay: 7.5	Incubation: 37°C, Assay: 37°C	0.5% (w/v) Casein	free amino acids , Peptides	—	—	Non-incubated control (in %), assay in aqueous phase \| Clear about diluted conditions in assay ("diluted to a final concentration of the water-miscible organic solvent of <0.5% (v/v).")/For mutants, had to shift the residue numbering of 3 for all mutants except A4T and G8C compared to article numbering to be consistent, suggests an indel in the proteins sequence between protein used in the article and UniProt, but could not find the article protein sequence
A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L	Stability - Incubation	Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase	Dimethylformamide (DMF)	20%	100.0	—	97.0	%	Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2	Incubation: 7.5, Assay: 7.5	Incubation: 37°C, Assay: 37°C	0.5% (w/v) Casein	free amino acids , Peptides	—	—	Non-incubated control (in %), assay in aqueous phase \| Clear about diluted conditions in assay ("diluted to a final concentration of the water-miscible organic solvent of <0.5% (v/v).")/For mutants, had to shift the residue numbering of 3 for all mutants except A4T and G8C compared to article numbering to be consistent, suggests an indel in the proteins sequence between protein used in the article and UniProt, but could not find the article protein sequence
A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L	Stability - Incubation	Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase	Dimethylformamide (DMF)	40%	100.0	—	83.0	%	Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2	Incubation: 7.5, Assay: 7.5	Incubation: 37°C, Assay: 37°C	0.5% (w/v) Casein	free amino acids , Peptides	—	—	Non-incubated control (in %), assay in aqueous phase \| Clear about diluted conditions in assay ("diluted to a final concentration of the water-miscible organic solvent of <0.5% (v/v).")/For mutants, had to shift the residue numbering of 3 for all mutants except A4T and G8C compared to article numbering to be consistent, suggests an indel in the proteins sequence between protein used in the article and UniProt, but could not find the article protein sequence
A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L	Stability - Incubation	Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase	Dimethylformamide (DMF)	60%	100.0	—	5.0	%	Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2	Incubation: 7.5, Assay: 7.5	Incubation: 37°C, Assay: 37°C	0.5% (w/v) Casein	free amino acids , Peptides	—	—	Non-incubated control (in %), assay in aqueous phase \| Clear about diluted conditions in assay ("diluted to a final concentration of the water-miscible organic solvent of <0.5% (v/v).")/For mutants, had to shift the residue numbering of 3 for all mutants except A4T and G8C compared to article numbering to be consistent, suggests an indel in the proteins sequence between protein used in the article and UniProt, but could not find the article protein sequence
A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L	Stability - Incubation	Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase	Dimethylformamide (DMF)	80%	100.0	—	0.0	%	Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2	Incubation: 7.5, Assay: 7.5	Incubation: 37°C, Assay: 37°C	0.5% (w/v) Casein	free amino acids , Peptides	—	—	Non-incubated control (in %), assay in aqueous phase \| Clear about diluted conditions in assay ("diluted to a final concentration of the water-miscible organic solvent of <0.5% (v/v).")/For mutants, had to shift the residue numbering of 3 for all mutants except A4T and G8C compared to article numbering to be consistent, suggests an indel in the proteins sequence between protein used in the article and UniProt, but could not find the article protein sequence

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

80%

100.0

—

1.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Non-incubated control (in %), assay in aqueous phase | Clear about diluted conditions in assay ("diluted to a final concentration of the water-miscible organic solvent of <0.5% (v/v).")/For mutants, had to shift the residue numbering of 3 for all mutants except A4T and G8C compared to article numbering to be consistent, suggests an indel in the proteins sequence between protein used in the article and UniProt, but could not find the article protein sequence

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

90%

100.0

—

14.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

80%

100.0

—

22.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

60%

100.0

—

45.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

40%

100.0

—

76.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

20%

100.0

—

85.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

10%

100.0

—

91.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

90%

100.0

—

0.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

60%

100.0

—

3.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

40%

100.0

—

21.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

20%

100.0

—

66.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

10%

100.0

—

92.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - C50

Volume of organic solvent for which half of the activity, measured by absorbance spectrophotmetry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase, is lost after incubation at 37°C for 48 hours

Dimethylformamide (DMF)

—

45.8

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

Not applicable control (in % (v/v)) | For mutants, had to shift the residue numbering of 3 for all mutants except A4T and G8C compared to article numbering to be consistent, suggests an indel in the proteins sequence between protein used in the article and UniProt, but could not find the article protein sequence

C291L

Stability - C50

Isopropanol

—

44.5

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - C50

1,4-Dioxane

—

32.1

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - C50

Dimethyl Sulfoxide (DMSO)

—

32.4

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - C50

Acetonitrile

—

30.6

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - C50

Acetone

—

26.0

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - C50

Dimethylformamide (DMF)

—

29.8

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - C50

Methanol

—

56.6

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - C50

Methanol

—

86.4

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - C50

Isopropanol

—

65.5

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - C50

1,4-Dioxane

—

47.9

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - C50

Acetonitrile

—

51.6

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - C50

Acetone

—

50.1

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - C50

Methanol

—

87.1

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - C50

Isopropanol

—

65.9

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - C50

1,4-Dioxane

—

48.3

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - C50

Dimethyl Sulfoxide (DMSO)

—

49.1

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - C50

Acetonitrile

—

52.4

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - C50

Dimethyl Sulfoxide (DMSO)

—

48.7

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - C50

Dimethylformamide (DMF)

—

46.8

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - C50

Acetone

—

50.9

% (v/v)

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

80%

100.0

—

18.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

90%

100.0

—

25.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

80%

100.0

—

76.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

60%

100.0

—

86.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

10%

100.0

—

91.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

20%

100.0

—

86.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

40%

100.0

—

78.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

60%

100.0

—

43.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

40%

100.0

—

100.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

90%

100.0

—

16.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

10%

100.0

—

99.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

20%

100.0

—

94.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

40%

100.0

—

98.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

60%

100.0

—

93.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

80%

100.0

—

65.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

90%

100.0

—

40.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

10%

100.0

—

97.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Methanol

20%

100.0

—

96.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

40%

100.0

—

70.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

10%

100.0

—

100.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

20%

100.0

—

74.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

40%

100.0

—

16.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

60%

100.0

—

10.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

80%

100.0

—

1.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

90%

100.0

—

0.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

10%

100.0

—

97.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

20%

100.0

—

99.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

90%

100.0

—

0.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

60%

100.0

—

13.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

80%

100.0

—

0.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

G8C/N63C/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

90%

100.0

—

0.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

10%

100.0

—

98.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

20%

100.0

—

97.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

40%

100.0

—

83.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

60%

100.0

—

5.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L

Stability - Incubation

Activity after incubation (48 hours at 37°C), measured by absorbance spectrophotometry (free aromatic residues absorbance measurement, 275 nm) in aqueous phase

Dimethylformamide (DMF)

80%

100.0

—

0.0

Incubation: 50 mM Tris-HCl, 5 mM CaCl2, Assay: 50 mM Tris-HCl, 5 mM CaCl2

Incubation: 7.5, Assay: 7.5

Incubation: 37°C, Assay: 37°C

0.5% (w/v) Casein

free amino acids, Peptides

—

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Mutations in this dataset (4)

WT A4T/G8C/T59A/G61A/N63C/T66F/S68P/A72P/C291L G8C/N63C/C291L C291L

Visualization : Stability — Incubation

One bar per measurement. Colour = solvent, shade = solvent volume. — — — Reference value. Hover for details.

Mutation Effect

Mutation impact on enzyme stability and function in the presence of organic solvent: comparison of wild-type and mutant values in identical conditions.

Visualization : Stability — C₅₀

One bar per measurement. Colour = solvent, shade = temperature. Hover for details.

Structure

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Thermolysin-like protease from Bacillus thermoproteolyticus

Enzyme Description

Sequence

Experimental Data (69 measurements)

Visualization : Stability — Incubation

Mutation Effect

Visualization : Stability — C50

Structure

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Visualization : Stability — C₅₀