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Literature summary for 3.1.1.74 extracted from

  • Koschorreck, K.; Liu, D.; Kazenwadel, C.; Schmid, R.D.; Hauer, B.
    Heterologous expression, characterization and site-directed mutagenesis of cutinase CUTAB1 from Alternaria brassicicola (2010), Appl. Microbiol. Biotechnol., 87, 991-997.
    View publication on PubMed
Search for more literature for 3.1.1.74

Cloned(Commentary)

Cloned (Comment) Organism
PCR product digested with EcoRI and NotI, and ligated in corresponding sites of the linearized pPICZalphaA vector. Transformation of Escherichia coli DH5alpha cells. Wild-type and mutants heterologously expressed in Pichia pastoris X-33 cells under the control of the methanol-inducible AOX1 promoter Alternaria brassicicola

Protein Variants

Protein Variants Comment Organism
A84F mutation in the small helical flap, significantly increases the activity towards longer chain substrates like 4-nitrophenyl palmitate Alternaria brassicicola
I183A mutation in the hydrophobic binding loop, drastically reduces the overall activity Alternaria brassicicola
L181A mutation in the hydrophobic binding loop, drastically reduces the overall activity Alternaria brassicicola
L80A mutation in the hydrophobic binding loop, drastically reduces the overall activity Alternaria brassicicola

Molecular Weight [Da]

Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
20000
-
 1 * 24000, SDS-PAGE, glycosylated CUTAB1. 1 * 20000, SDS-PAGE, non-glycosylated CUTAB1 Alternaria brassicicola
24000
-
 1 * 24000, SDS-PAGE, glycosylated CUTAB1. 1 * 20000, SDS-PAGE, non-glycosylated CUTAB1 Alternaria brassicicola

Organism

Organism UniProt Comment Textmining
Alternaria brassicicola P41744 DSM 62008
-

Posttranslational Modification

Posttranslational Modification Comment Organism
glycoprotein the glycosylated form of CUTAB1 has a MW of 24000 Da. The non-glycosylated form of CUTAB1 has a MW of 20000 Da Alternaria brassicicola

Purification (Commentary)

Purification (Comment) Organism
wild-type and mutants purified by ultrafiltration, wild-type further purified by hydrophobic interaction chromatography and ammonium sulfate precipitation, 1.6fold with a yield of 70% Alternaria brassicicola

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
2.77
-
 carrier bound CUTAB1 with tributyrin as substrate, in 40 ml of 50 mM potassium phosphate buffer, pH 5.5 containing 25% ethanol, at 8°C, for 40 H Alternaria brassicicola
10
-
 crude extract, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
17
-
 1.6fold purified enzyme, with 4-nitrophenyl palmitate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
17
-
 wild-type, with 4-nitrophenyl palmitate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
30
-
 wild-type, with tripalmitin as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
41
-
 wild-type, with methyl acetate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
47
-
 wild-type, with tristearin as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
66
-
 mutant A84F, with tripalmitin as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
77
-
 mutant A84F, with tristearin as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
125
-
 wild-type, with ethyl caprylate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
125
-
 wild-type, with methyl caproate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
129
-
 wild-type, with methyl caprylate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
145
-
 wild-type, with methyl myristate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
157
-
 wild-type, with methyl propionate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
161
-
 wild-type, with methyl decanoate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
188
-
 mutant A84F, with 4-nitrophenyl palmitate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
197
-
 wild-type, with methyl laurate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
257
-
 wild-type, with methyl butyrate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
281
-
 wild-type, with ethyl butyrate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
454
-
 wild-type, with triolein as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
586
-
 mutant A84F, with triolein as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
948
-
 mutant A84F, with 4-nitrophenyl butyrate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
1057
-
 wild-type, with 4-nitrophenyl butyrate as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
1286
-
 wild-type, with tricaprylin as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
2019
-
 mutant A84F, with tricaprylin as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
3302
-
 wild-type, with tributyrin as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola
4394
-
 mutant A84F, with tributyrin as substrate, in 0.1 M Tris-HCl buffer, pH 8.0 with 0.5% (v/v) Triton X-100 and 0.1% (w/v) gum arabic Alternaria brassicicola

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
4-nitrophenyl butyrate + H2O
-
 Alternaria brassicicola 4-nitrophenol + butyrate
-
 ?
4-nitrophenyl palmitate + H2O
-
 Alternaria brassicicola 4-nitrophenol + palmitate
-
 ?
ethyl butyrate + H2O
-
 Alternaria brassicicola butyric acid + ethanol
-
 ?
ethyl caprylate + H2O
-
 Alternaria brassicicola caprylic acid + ethanol
-
 ?
methyl acetate + H2O
-
 Alternaria brassicicola acetic acid + methanol
-
 ?
methyl butyrate + H2O
-
 Alternaria brassicicola butyric acid + methanol
-
 ?
methyl caproate + H2O
-
 Alternaria brassicicola caproic acid + methanol
-
 ?
methyl caprylate + H2O
-
 Alternaria brassicicola caprylic acid + methanol
-
 ?
methyl decanoate + H2O
-
 Alternaria brassicicola decanoic acid + methanol
-
 ?
methyl laurate + H2O
-
 Alternaria brassicicola lauric acid + methanol
-
 ?
methyl myristate + H2O
-
 Alternaria brassicicola myristic acid + methanol
-
 ?
methyl propionate + H2O
-
 Alternaria brassicicola propionic acid + methanol
-
 ?
tributyrin + H2O
-
 Alternaria brassicicola ?
-
 ?
tricaprylin + H2O
-
 Alternaria brassicicola ?
-
 ?
triolein + H2O
-
 Alternaria brassicicola ?
-
 ?
tripalmitin + H2O
-
 Alternaria brassicicola ?
-
 ?
tristearin + H2O
-
 Alternaria brassicicola ?
-
 ?

Subunits

Subunits Comment Organism
monomer 1 * 24000, SDS-PAGE, glycosylated CUTAB1. 1 * 20000, SDS-PAGE, non-glycosylated CUTAB1 Alternaria brassicicola

Synonyms

Synonyms Comment Organism
CUTAB1
-
 Alternaria brassicicola
cutinase
-
 Alternaria brassicicola

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
40
-
-
 Alternaria brassicicola

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
40
-
 retains 84% of its initial activity at 40°C and pH 8.0 for 5 days Alternaria brassicicola

pH Range

pH Minimum pH Maximum Comment Organism
7 9 at both, room temperature and 40°C Alternaria brassicicola