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3.1.1.74: cutinase

This is an abbreviated version!
For detailed information about cutinase, go to the full flat file.

Word Map on EC 3.1.1.74

Reaction

cutin
+
H2O
= 2 cutin monomers

Synonyms

acidic cutinase, CcCUT1, CDEF1, CLE, Cut 5a, cut-2.KW3, Cut1, Cut11, Cut190, Cut2, Cut5a, CUTAB1, CutB, cuticle destructing factor 1, cutin esterase, cutin hydrolase, cutinase, cutinase 1, cutinase 2, cutinase-1, cutinase-like enzyme, cutinolytic polyesterase, CutL, CutL1, FspC, fungal cutinase, HIc, LC-cutinase, More, MYCTH_2110987, PET hydrolase, Tfu_0883, Thcut1, THCUT1 protein, Thc_Cut1, Thc_Cut2, TRIREDRAFT_60489

ECTree

     3 Hydrolases
         3.1 Acting on ester bonds
             3.1.1 Carboxylic-ester hydrolases
                3.1.1.74 cutinase

Engineering

Engineering on EC 3.1.1.74 - cutinase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
A84F
mutation in the small helical flap, significantly increases the activity towards longer chain substrates like 4-nitrophenyl palmitate
I183A
mutation in the hydrophobic binding loop, drastically reduces the overall activity
L181A
mutation in the hydrophobic binding loop, drastically reduces the overall activity
L80A
mutation in the hydrophobic binding loop, drastically reduces the overall activity
A102D/Q105R/G106E
pH-optima for activity and stability are identical to wild-type enzym. Improvement in Tm-value of 3.4°C. Increased half-life at 6°C relative to the wild-type enzyme of approximately 3fold
A102D/Q105R/G106E/N133A/S140P/E161T/A166P
large improvement of stability at 60°C
A102D/Q105R/G106E/N133A/S140P/E161T/A166P/K137E
large improvement of stability at 60°C
A102D/Q105R/G106E/Q98N/A99D/E109Q
thermodynamically most stable variant, improving on wild-type enzyme by 6.7 kJ/mol
A178P/V179P
loss of stability and activity
D30S
mutation increases the KD value for interaction with hydrophobin RolA
D30S/E31S/D142S/D171S
mutation D30S increases the KD value for interaction with hydrophobin RolA in comparison with mutant E31S/D142S/D171S
K174R/Y176F/A178E/D200R/G202E/D203E/D206R
mutant enzyme shows an increased kinetic stability
L26D/G28E/D30R/K67R
improvement in Tm-value of 0.7°C
N133A/S140P/E161T/A166P
proline mutations contribute to themostabilization by decreasing the entropy lost upon folding. Improvement in Tm of 1.7°C. Increased half-life at 6°C relative to the wild-type enzyme of approximately 2fold
Q110W/K114W
the mutant enzyme is retained in the endoplasmic reticulum whereas wild-type enzyme is secreted
R46P
the Tm-value is 3°C below that of wild-type enzyme
T84R/D86L/A99E/A100S
decrease in thermostability relative to the wild-type enzyme. Large losses in 4-nitrophenyl butyrate (about 70%) and poly(epsilon-caprolactone) (about 90%) activities
V150I/I136V
mutation do not provide any improvement in stability
A102D/Q105R/G106E
-
pH-optima for activity and stability are identical to wild-type enzym. Improvement in Tm-value of 3.4°C. Increased half-life at 6°C relative to the wild-type enzyme of approximately 3fold
-
L26D/G28E/D30R/K67R
-
improvement in Tm-value of 0.7°C
-
Q110W/K114W
-
the mutant enzyme is retained in the endoplasmic reticulum whereas wild-type enzyme is secreted
-
V150I/I136V
-
mutation do not provide any improvement in stability
-
H204N
L172K
site-directed mutagenesis, compared to the wild-type enzyme, the mutant exhibits higher enzymatic performance towards phenyl ester substrates of longer carbon chain length, yet its thermal stability is inversely affected
N177D
site-directed mutagenesis, the mutation aims to alter the surface electrostatics as well as to remove a potentially deamidation-prone asparagine residue. The mutant is more resilient to temperature increase with a 2.7fold increase in half-life at 50°C, accompanied by an increase in optimal temperature, as compared with wild-type enzyme, while the activity at 25°C is not compromised
N177D/L172K
site-directed mutagenesis, the double mutant shows enhanced activity towards phenyl ester substrates and enhanced thermal stability
F52W
site-directed mutagenesis, the mutant shows increased activity with 4-nitrophenyl palmitate by 4.86fold and altered substrate specificity toward substrates with longer chain lengths
L181F
site-directed mutagenesis, the mutant shows increased activity with 4-nitrophenyl palmitate by 4.86fold and altered substrate specificity toward substrates with longer chain lengths
F52W
-
site-directed mutagenesis, the mutant shows increased activity with 4-nitrophenyl palmitate by 4.86fold and altered substrate specificity toward substrates with longer chain lengths
-
L181F
-
site-directed mutagenesis, the mutant shows increased activity with 4-nitrophenyl palmitate by 4.86fold and altered substrate specificity toward substrates with longer chain lengths
-
A164E
-
amino acid substitution, 74% of wild-type activity
A164R
A185L
A185T
-
amino acid substitution, 142% of wild-type activity
A195S
A199C
A85F/G82A
-
optimal activity with triglyceride anolgues shifts towards slightly longer acyl ester chains
D111N
D134S
D33S
site-directed mutagenesis, the mutant shows 26% reduced activity in olive oil compared to the wild-type enzyme
E201K
G192Q
G26P
-
amino acid substitution, 68% of wild-type activity
G41A
-
amino acid substitution, 76% of wild-type activity
G82A
-
mutation has no influence on enzymatic properties
I183F
I204K
K140D
-
amino acid substitution, 39% of wild-type activity
K151M
-
amino acid substitution, 22% of wild-type activity
K151R
K168L
K65P
-
amino acid substitution, 99% of wild-type activity
L114C
-
comparative structural analysis of native enzyme and mutant enzymes
L114Y
L153A
-
amino acid substitution, 111% of wild-type activity
L153Q
L182A
L182W
L189A
activity with polyethylene terephthalate fibers is 78% of wild-type enzyme, activity with polyamide 6,6 fiber is 94% of wild-type activity
L189F
L81G/L182G
-
comparative structural analysis of native enzyme and mutant enzymes
N161D
N172K
N172K/R196E
N33S
74% of the activity of the wild-type enzyme
Q121L
-
comparative structural analysis of native enzyme and mutant enzymes
R156E
R156K
R156L
R156N
-
amino acid substitution, 89% of wild-type activity
R158L
-
amino acid substitution, 75% of wild-type activity
R17E/N172K
-
comparative structural analysis of native enzyme and mutant enzymes
R17S
-
amino acid substitution, 69% of wild-type activity
R196A
-
amino acid substitution, 75% of wild-type activity
R196E
R196K
R196L
R208A
S120A
S120C
-
comparative structural analysis of native enzyme and mutant enzymes
S129C
-
comparative structural analysis of native enzyme and mutant enzymes
S213C
-
comparative structural analysis of native enzyme and mutant enzymes
S57D
-
amino acid substitution, 61% of wild-type activity
S61D
-
amino acid substitution, 83% of wild-type activity
S82R
50% of the activity of the wild-type enzyme
S92C
-
comparative structural analysis of native enzyme and mutant enzymes
S92R
site-directed mutagenesis, the mutant shows 50% reduced activity in olive oil compared to the wild-type enzyme
T144C
T167L
T173K
T179C
T179E
-
amino acid substitution, 10% of wild-type activity
T179N
-
amino acid substitution, 119% of wild-type activity
T179Y
T18D
-
amino acid substitution, 65% of wild-type activity
T45D
-
amino acid substitution, 54% of wild-type activity
T80P
-
comparative structural analysis of native enzyme and mutant enzymes
V184A
Y119H
-
comparative structural analysis of native enzyme and mutant enzymes
L153Q
-
site-directed mutagenesis, the mutant shows transesterification activity similar to the wild-type enzyme
S54D
-
site-directed mutagenesis, the mutant shows reduced transesterification activity compared to the wild-type enzyme
T179C
-
site-directed mutagenesis, the mutant shows transesterification activity similar to the wild-type enzyme, T179C displays high stability in the presence of methanol with an activity loss of only 16% as compared to 90% loss of wild-type activity, the mutant is also more stable microencapsulated in reversed micelles of bis(2-ethylhexyl) sodium sulfosuccinate in isooctane
I36N/F70S
mutant engineered for cellulose acetate deacetylation, almost 2fold improvement in catalytic efficiency with both cellulose acetate and 4-nitrophenyl butanoate
I36S/F70A
mutant engineered for cellulose acetate deacetylation, 2fold improvement in catalytic efficiency with cellulose acetate and 4-nitrophenyl butanoate
H137L
site-directed mutageneis, the mutant exhibits a slightly increased Km value with the soluble substrate 4-nitrophenyl butyrate compared to the wild-type enzyme
H173L
36% of wild-type activity
S103A
S103T
S226P
mutant shows the highest activities toward tricaproin and tributyrin, the activity is greatly reduced toward tricaprylin
S226P/R228S
increase in PET degradation, improved activity and thermostability
S226P/R228S/S176A
inactive mutant enzyme
S226P
-
mutant shows the highest activities toward tricaproin and tributyrin, the activity is greatly reduced toward tricaprylin
-
S226P/R228S
-
increase in PET degradation, improved activity and thermostability
-
S226P/R228S/S176A
-
inactive mutant enzyme
-
cutinase-tryptophan,proline2
-
tryptophan tag, cutinase with varying length tryptophan tag (WP)2
cutinase-tryptophan,proline4
-
tryptophan tag, cutinase with varying length tryptophan tag (WP)4
S117A
site-directed mutagenesis, the mutation causes a 99% reduction in enzyme activity and also completely abolishes the elicitor activity of the protein
Y116A
site-directed mutagenesis, the mutation causes a 97% reduction in enzyme activity and also abolishes the elicitor activity of the protein
A68V/T253P
A30V
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows increased catalytic efficiency with polyethyleneterephthalate and higher kcat/KM values on soluble substrates compared to the wild-type enzyme
L183A
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows slightly increased catalytic efficiency with 4-nitrophenyl ester substrates compared to the wild-type enzyme
Q65E
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows slightly reduced catalytic efficiency with 4-nitrophenyl ester substrates compared to the wild-type enzyme
R187K
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows increased catalytic efficiency with 4-nitrophenyl ester substrates compared to the wild-type enzyme
R19S
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1
R19S/R29N/A30V
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows increased catalytic efficiency with 4-nitrophenyl ester substrates compared to the wild-type enzyme
R19SS
the mutant shows strongly increased PET hydrolysis activity compared to the wild-type enzyme
R29N
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows increased catalytic efficiency with polyethyleneterephthalate and higher kcat/KM values on soluble substrates compared to the wild-type enzyme
R29N/A30V
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows increased catalytic efficiency with polyethyleneterephthalate and higher kcat/KM values on soluble substrates compared to the wild-type enzyme
R29SS
the mutant shows strongly increased PET hydrolysis activity compared to the wild-type enzyme
L183A
-
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows slightly increased catalytic efficiency with 4-nitrophenyl ester substrates compared to the wild-type enzyme
-
Q65E
-
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows slightly reduced catalytic efficiency with 4-nitrophenyl ester substrates compared to the wild-type enzyme
-
R187K
-
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1, the mutant shows increased catalytic efficiency with 4-nitrophenyl ester substrates compared to the wild-type enzyme
-
R19S
-
site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1
-
I218A
-
engineering by site-directed mutagenesis modifying the active site, the mutant cutinase shows increased activity on polyester substrates. Mutation I218A creates space, activity on poly(ethylene terephthalate) is increased compared to the wild-type enzyme, with considerably higher hydrolysis efficiency
S130A
T101A/Q132A
-
engineering by site-directed mutagenesis modifying the active site, the mutant cutinase shows increased activity on polyester substrates. The double mutation Q132A/T101A both creates space and increases hydrophobicity. The activity of the double mutant on the soluble substrate p-nitrophenyl butyrate increased 2fold compared to wild-type cutinase, while on poly(ethylene terephthalate) the double mutant exhibits considerably higher hydrolysis efficiency
W86L
-
site-directed mutagenesis, the mutant exhibits an improvement in binding and catalytic efficiency of 1.4fold toward PET fiber compared with the wild-type enzyme
W86Y
-
site-directed mutagenesis, the mutant exhibits an improvement in binding and catalytic efficiency of 1.5fold toward PET fiber compared with the wild-type enzyme
W86L
-
site-directed mutagenesis, the mutant exhibits an improvement in binding and catalytic efficiency of 1.4fold toward PET fiber compared with the wild-type enzyme
-
W86Y
-
site-directed mutagenesis, the mutant exhibits an improvement in binding and catalytic efficiency of 1.5fold toward PET fiber compared with the wild-type enzyme
-
C275A/C292A
-
site-directed mutagenesis, the mutant lacks the disulfide bond formed by Cys275 and Cys292, resulting in increased instability
additional information