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
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3.1.1.74
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fusarium
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solani
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pi
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lipase
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terephthalate
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p-nitrophenyl
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lipolytic
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thermobifida
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esterases
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insolens
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fusca
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humicola
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ideonella
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polybutylene
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sakaiensis
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industry
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polycaprolactone
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tributyrin
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degradation
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sulfosuccinate
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haematococca
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monilinia
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terephthalic
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petase
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synthesis
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nectria
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hydrophobins
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saccharomonospora
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biotechnology
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environmental protection
- 3.1.1.74
- fusarium
- solani
- pi
- lipase
- terephthalate
- p-nitrophenyl
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lipolytic
- thermobifida
- esterases
- insolens
- fusca
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humicola
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ideonella
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polybutylene
- sakaiensis
- industry
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polycaprolactone
- tributyrin
- degradation
- sulfosuccinate
- haematococca
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monilinia
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terephthalic
- petase
- synthesis
- nectria
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hydrophobins
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saccharomonospora
- biotechnology
- environmental protection
Reaction
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
Advanced search results
Engineering
Engineering on EC 3.1.1.74 - cutinase
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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
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
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
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
A102D/Q105R/G106E
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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
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Q110W/K114W
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the mutant enzyme is retained in the endoplasmic reticulum whereas wild-type enzyme is secreted
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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
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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
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L181F
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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
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A164R
A185L
A195S
A199C
A29S
A79G
A85F
A85F/G82A
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optimal activity with triglyceride anolgues shifts towards slightly longer acyl ester chains
A85W
D111N
D134S
D33S
site-directed mutagenesis, the mutant shows 26% reduced activity in olive oil compared to the wild-type enzyme
D83S
E201K
G192Q
G26A
I183F
I204K
I24S
K151R
K168L
L114Y
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
L81A
L81G/L182G
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comparative structural analysis of native enzyme and mutant enzymes
L99K
M98C
N161D
N172K
N172K/R196E
N84A
N84D
N84L
N84W
R156E
R156K
R156L
R17E
R17E/N172K
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comparative structural analysis of native enzyme and mutant enzymes
R17N
R196E
R196K
R196L
R208A
R78L
R78N
R88A
R96N
S120A
S42A
S54D
S54E
S54K
S54W
S92R
site-directed mutagenesis, the mutant shows 50% reduced activity in olive oil compared to the wild-type enzyme
T144C
T167L
T173K
T179C
T179Y
T18V
T19V
T45A
T45K
T50V
T80D
V184A
W69Y
Y38F
L153Q
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site-directed mutagenesis, the mutant shows transesterification activity similar to the wild-type enzyme
S54D
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site-directed mutagenesis, the mutant shows reduced transesterification activity compared to the wild-type enzyme
T179C
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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
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
Saccharomonospora viridis AHK190
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mutant shows the highest activities toward tricaproin and tributyrin, the activity is greatly reduced toward tricaprylin
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S226P/R228S
Saccharomonospora viridis AHK190
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increase in PET degradation, improved activity and thermostability
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cutinase-tryptophan,proline2
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tryptophan tag, cutinase with varying length tryptophan tag (WP)2
cutinase-tryptophan,proline4
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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
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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
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Q65E
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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
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R187K
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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
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R19S
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site-directed mutagenesis, mutation of isozyme Cut2 to the analogue residue of isozyme Cut1
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I218A
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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
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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
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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
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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
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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
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W86Y
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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
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C275A/C292A
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site-directed mutagenesis, the mutant lacks the disulfide bond formed by Cys275 and Cys292, resulting in increased instability
additional information
site-directed mutant, constructed, overexpressed, and purified
H204N
is catalytically inactive. Is not covalently modified by a 4fold excess of diethyl p-nitrophenyl phosphate, in contrast to the wild-type
A164R
site-directed mutagenesis, the mutant shows 59% reduced activity in olive oil compared to the wild-type enzyme
A185L
site-directed mutagenesis, the mutant shows unaltered activity in olive oil compared to the wild-type enzyme
A195S
site-directed mutagenesis, the mutant shows 62% reduced activity in olive oil compared to the wild-type enzyme
A199C
site-directed mutagenesis, the mutant shows no activity in olive oil
A29S
site-directed mutagenesis, the mutant shows 36% reduced activity in olive oil compared to the wild-type enzyme
A79G
site-directed mutagenesis, the mutant shows 50% reduced activity in olive oil compared to the wild-type enzyme
A85F
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optimal activity with triglyceride anolgues shifts towards slightly longer acyl ester chains
A85F
site-directed mutagenesis, the mutant shows 36% increased activity in olive oil compared to the wild-type enzyme
A85F
the mutant shows higher enzyme activity with hydrophobic, low-molecular-weight substrates in olive oil emulsions than the wild-type enzyme
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optimal activity with triglyceride anolgues shifts towards slightly longer acyl ester chains
A85W
site-directed mutagenesis, the mutant shows 9% increased activity in olive oil compared to the wild-type enzyme
A85W
the mutant shows higher enzyme activity with hydrophobic, low-molecular-weight substrates in olive oil emulsions than the wild-type enzyme
D111N
site-directed mutagenesis, the mutant shows 61% reduced activity in olive oil compared to the wild-type enzyme
D134S
site-directed mutagenesis, the mutant shows 63% reduced activity in olive oil compared to the wild-type enzyme
D83S
site-directed mutagenesis, the mutant shows 38% reduced activity in olive oil compared to the wild-type enzyme
E201K
site-directed mutagenesis, the mutant shows 46% reduced activity in olive oil compared to the wild-type enzyme
G192Q
site-directed mutagenesis, the mutant shows 56% reduced activity in olive oil compared to the wild-type enzyme
G26A
site-directed mutagenesis, the mutant shows 67% reduced activity in olive oil compared to the wild-type enzyme
I183F
site-directed mutagenesis, the mutant shows 75% reduced activity in olive oil compared to the wild-type enzyme
I204K
site-directed mutagenesis, the mutant shows 34% reduced activity in olive oil compared to the wild-type enzyme
I24S
site-directed mutagenesis, the mutant shows 96% reduced activity in olive oil compared to the wild-type enzyme
K151R
site-directed mutagenesis, the mutant shows 71% reduced activity in olive oil compared to the wild-type enzyme
K168L
site-directed mutagenesis, the mutant shows 17% reduced activity in olive oil compared to the wild-type enzyme
L114Y
site-directed mutagenesis, the mutant shows 80% reduced activity in olive oil compared to the wild-type enzyme
L153Q
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L153Q mutation also reduces the development of hydrophobic solvent accessible patches
shows the one- and two-fold higher ability to biodegrade aliphatic polyamide substrates. Activity with polyethylene terephthalate fibers is 5.3fold higher than wild-type enzyme, activity with polyamide 6,6 fiber is 119% of wild-type activity
L182A
site-directed mutagenesis, the mutant shows activity enhancement of 5fold toward high-molecular weight PET fibers compared to the wild-type enzyme
L182W
site-directed mutagenesis, the mutant shows 81% reduced activity in olive oil compared to the wild-type enzyme
L189F
site-directed mutagenesis, the mutant shows 9% increased activity in olive oil compared to the wild-type enzyme
L189F
the mutant shows higher enzyme activity with hydrophobic, low-molecular-weight substrates in olive oil emulsions than the wild-type enzyme
activity with polyethylene terephthalate fibers is 4fold higher than wild-type enzyme, activity with polyamide 6,6 fiber is 98% of wild-type activity
L81A
the mutant shows activity enhancement of 4fold toward high-molecular weight PET fibers compared to the wild-type enzyme
L99K
site-directed mutagenesis, the mutant shows 22% reduced activity in olive oil compared to the wild-type enzyme
M98C
site-directed mutagenesis, the mutant shows 65% reduced activity in olive oil compared to the wild-type enzyme
N161D
site-directed mutagenesis, the mutant shows 37% reduced activity in olive oil compared to the wild-type enzyme
N172K
site-directed mutagenesis, the mutant shows 55% reduced activity in olive oil compared to the wild-type enzyme
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comparative structural analysis of native enzyme and mutant enzymes
N84A
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26.5% of the activity of the wild-type enzyme with p-nitrophenylbutanoate as substrate
N84A
activity with polyethylene terephthalate fibers is 1.7fold higher than wild-type enzyme, activity with polyamide 6,6 fiber is 93% of wild-type activity
N84A
site-directed mutagenesis, the mutant shows 73.5% reduced activity in olive oil compared to the wild-type enzyme
N84A
the mutant shows activity enhancement of 1.7fold toward high-molecular weight PET fibers compared to the wild-type enzyme
N84D
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0.16% of the activity of the wild-type enzyme with p-nitrophenylbutanoate as substrate
N84D
site-directed mutagenesis, the mutant shows almost no activity in olive oil
N84L
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3.0% of the activity of the wild-type enzyme with p-nitrophenylbutanoate as substrate
N84L
site-directed mutagenesis, the mutant shows 97% reduced activity in olive oil compared to the wild-type enzyme
N84W
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0.11% of the activity of the wild-type enzyme with p-nitrophenylbutanoate as substrate
N84W
site-directed mutagenesis, the mutant shows almost no activity in olive oil
R156E
site-directed mutagenesis, the mutant shows 21% reduced activity in olive oil compared to the wild-type enzyme
R156K
site-directed mutagenesis, the mutant shows 15% increased activity in olive oil compared to the wild-type enzyme
R156L
site-directed mutagenesis, the mutant shows 29% reduced activity in olive oil compared to the wild-type enzyme
R17E
site-directed mutagenesis, the mutant shows 66% reduced activity in olive oil compared to the wild-type enzyme
R17N
site-directed mutagenesis, the mutant shows 69% reduced activity in olive oil compared to the wild-type enzyme
R196E
site-directed mutagenesis, the mutant shows 55% reduced activity in olive oil compared to the wild-type enzyme
R196K
site-directed mutagenesis, the mutant shows 62% reduced activity in olive oil compared to the wild-type enzyme
R196L
site-directed mutagenesis, the mutant shows 56% reduced activity in olive oil compared to the wild-type enzyme
R208A
site-directed mutagenesis, the mutant shows 36% reduced activity in olive oil compared to the wild-type enzyme
R78L
site-directed mutagenesis, the mutant shows 51% reduced activity in olive oil compared to the wild-type enzyme
R78N
site-directed mutagenesis, the mutant shows 66% reduced activity in olive oil compared to the wild-type enzyme
R88A
site-directed mutagenesis, the mutant shows 61% reduced activity in olive oil compared to the wild-type enzyme
R96N
site-directed mutagenesis, the mutant shows 43% reduced activity in olive oil compared to the wild-type enzyme
S120A
the mutant enzyme casries a 15 amino acid pro-peptide. The pro-peptide is affected by the presence of the micellar substrate
S120A
site-directed mutagenesis, the mutant shows no activity in olive oil
S42A
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0.22% of the activity of the wild-type enzyme with p-nitrophenylbutanoate as substrate
S42A
site-directed mutagenesis, the mutant shows almost no activity in olive oil
S54D
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S54D mutant of cutinase is significantly more resistant to sodium dioctyl sulfosuccinate denaturation than the wild type
S54E
site-directed mutagenesis, the mutant shows 66% reduced activity in olive oil compared to the wild-type enzyme
S54K
site-directed mutagenesis, the mutant shows unaltered activity in olive oil compared to the wild-type enzyme
S54W
site-directed mutagenesis, the mutant shows 11% reduced activity in olive oil compared to the wild-type enzyme
T144C
site-directed mutagenesis, the mutant shows 46% reduced activity in olive oil compared to the wild-type enzyme
T167L
site-directed mutagenesis, the mutant shows 46% reduced activity in olive oil compared to the wild-type enzyme
T173K
site-directed mutagenesis, the mutant shows 19% increased activity in olive oil compared to the wild-type enzyme
T173K
the mutant shows higher enzyme activity with hydrophobic, low-molecular-weight substrates in olive oil emulsions than the wild-type enzyme
T179C
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T179C mutation located close to the active centre and to disulfide bond Cys171-Cys178 introduced changes in the cutinase structure that are observed even in the cutinase region around the tryptophan residue. This mutation also reduces the development of hydrophobic solvent accessible patches
T179Y
site-directed mutagenesis, the mutant shows 31% increased activity in olive oil compared to the wild-type enzyme
T179Y
the mutant shows higher enzyme activity with hydrophobic, low-molecular-weight substrates in olive oil emulsions than the wild-type enzyme
T18V
site-directed mutagenesis, the mutant shows 10% reduced activity in olive oil compared to the wild-type enzyme
T19V
site-directed mutagenesis, the mutant shows 65% reduced activity in olive oil compared to the wild-type enzyme
T45A
site-directed mutagenesis, the mutant shows unaltered activity in olive oil compared to the wild-type enzyme
T45K
site-directed mutagenesis, the mutant shows 26% reduced activity in olive oil compared to the wild-type enzyme
T50V
site-directed mutagenesis, the mutant shows 75% reduced activity in olive oil compared to the wild-type enzyme
T80D
site-directed mutagenesis, the mutant shows 68% reduced activity in olive oil compared to the wild-type enzyme
activity with polyethylene terephthalate fibers is 2fold higher than wild-type enzyme, activity with polyamide 6,6 fiber is 98% of wild-type activity
V184A
the mutant shows activity enhancement of 2fold toward high-molecular weight PET fibers compared to the wild-type enzyme
W69Y
site-directed mutagenesis, the mutant shows 88% reduced activity in olive oil compared to the wild-type enzyme
Y38F
site-directed mutagenesis, the mutant shows 38% reduced activity in olive oil compared to the wild-type enzyme
S103A
site-directed mutageneis, the mutant exhibits a slightly increased Km value and a 2.3fold higher kcat with the soluble substrate 4-nitrophenyl butyrate compared to the wild-type enzyme
S103T
site-directed mutageneis, the mutant exhibits a slightly increased Km valuet with the soluble substrate 4-nitrophenyl butyrate compared to the wild-type enzyme
increase in both activity and thermostabililty, stable for 1 h below 60°C. Mutant is able to degrade various aliphatic and aliphatic coaromatic polyesters and hydrophilizes an amorphous PET film
A68V/T253P
Thermobifida alba AHK119
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increase in both activity and thermostabililty, stable for 1 h below 60°C. Mutant is able to degrade various aliphatic and aliphatic coaromatic polyesters and hydrophilizes an amorphous PET film
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S130A
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site-directed mutagenesis, catalytically inactive active site mutant, which is mostly located in the cytoplasm. Compared to the cells expressing the inactive cutinase mutant S130A, the cells expressing the truncated cutinase show increased membrane permeability and irregular morphology
CDEF1-deficient mutant (SALK-014093) that carries a T-DNA insertion in the coding region of CDEF1, shows no abnormal phenotypes, such as reduced fertility or reduced lateral root emergence
additional information
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CDEF1-deficient mutant (SALK-014093) that carries a T-DNA insertion in the coding region of CDEF1, shows no abnormal phenotypes, such as reduced fertility or reduced lateral root emergence
additional information
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because the organism has a low but significant FAE activity, it may be easier to introduce a high level of FAE activity in cutinases through point mutations
additional information
mutational analysis toward the thermostabilization of the enzyme. Mutants with increased thermal unfolding temperature and increase in the half-life of the enzyme activity at 60°C do not display improved rate or temperature optimum of enzyme activity. Surface salt bridge optimization produces enthalpic stabilization. Mutations to proline reduces the entropy loss upon folding. The lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the thermal unfolding temperature of the global structure
additional information
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mutational analysis toward the thermostabilization of the enzyme. Mutants with increased thermal unfolding temperature and increase in the half-life of the enzyme activity at 60°C do not display improved rate or temperature optimum of enzyme activity. Surface salt bridge optimization produces enthalpic stabilization. Mutations to proline reduces the entropy loss upon folding. The lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the thermal unfolding temperature of the global structure
additional information
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mutational analysis toward the thermostabilization of the enzyme. Mutants with increased thermal unfolding temperature and increase in the half-life of the enzyme activity at 60°C do not display improved rate or temperature optimum of enzyme activity. Surface salt bridge optimization produces enthalpic stabilization. Mutations to proline reduces the entropy loss upon folding. The lack of a correlative increase in the temperature optimum of catalytic activity with thermodynamic stability suggests that the active site is locally denatured at a temperature below the thermal unfolding temperature of the global structure
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additional information
the insertion mutant 49aILe shows 52% of the activity of the wild-type enzyme
additional information
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the insertion mutant 49aILe shows 52% of the activity of the wild-type enzyme
additional information
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a complete saturation mutagenesis approach to search cutinase for amino acids contributing to increased stability in the presence of the anionic surfactant. Mutants showing substitutions in the large hydrophobic crevice (S54D, S57D, S61D, K65P, R196A), that is thought to be the region more involved in the unfolding by anionics, will be very important to obtain an enzyme less sensitive to AOT
additional information
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stability of cutinase may be increased through mutations designed to avoid the transient formation of hydrophobic groups during protein movement. Because the organism has a low but significant ferulic acid esterase activity, it may be easier to introduce a high level of ferulic acid esterase activity in cutinases through point mutations
additional information
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cutinase is microencapsulated in reversed micelles of bis(2-ethylhexyl) sodium sulfosuccinate in isooctane for the production of alkyl esters, known as biodiesel, evaluation of the system stability using wild-type enzyme and three mutants, L153Q, T179C and S54D, method evaluation, overview. Loss of 45% of wild-type cutinase activity when incubated in the micellar system for 3 h, and an additional loss of 90% of the activity is observed in the presence of methanol after 10 min of incubation
additional information
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mutant myHiC, obtained by localised random mutagenesis, shows increased activity and decreased surfactanct sensitivity
additional information
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because the organism has a low but significant FAE activity, it may be easier to introduce a high level of FAE activity in cutinases through point mutations
additional information
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establishment of immobilized cutinase as a novel biocatalyst for the synthesis of functionalized acryclic esters by transesterification with transesterification of methyl acrylate with 6-mercapto-1-hexanol at a high molar ratio in a solvent free system as model reaction, overview
additional information
generation of codon-optimized enzyme for expression in Pichia pastoris
additional information
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generation of codon-optimized enzyme for expression in Pichia pastoris
additional information
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generation of codon-optimized enzyme for expression in Pichia pastoris
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additional information
exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for bis(benzoyloxyethyl)terephthalate and polyethyleneterephthalate. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on polyethyleneterephthalate films
additional information
exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for bis(benzoyloxyethyl)terephthalate and polyethyleneterephthalate. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on polyethyleneterephthalate films
additional information
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exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for bis(benzoyloxyethyl)terephthalate and polyethyleneterephthalate. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on polyethyleneterephthalate films
additional information
the cutinase id fused with binding modules from Hypocrea jecorina cellobiohydrolase I (CBM) and Alcaligenes faecalis polyhydroxyalkanoate depolymerase (PBM), respectively. The adsorption of the fusion enzymes to PET is increased, and PET hydrolysis activity of one of the fusions (Thc_Cut1 + CBM) is enhanced 3.8fold
additional information
the cutinase id fused with binding modules from Hypocrea jecorina cellobiohydrolase I (CBM) and Alcaligenes faecalis polyhydroxyalkanoate depolymerase (PBM), respectively. The adsorption of the fusion enzymes to PET is increased, and PET hydrolysis activity of one of the fusions (Thc_Cut1 + CBM) is enhanced 3.8fold
additional information
the cutinase is fused with binding modules from Hypocrea jecorina cellobiohydrolase I (CBM) and Alcaligenes faecalis polyhydroxyalkanoate depolymerase (PBM), respectively. The adsorption of the fusion enzymes to PET is increased, and PET hydrolysis activity of one of the fusions (Thc_Cut1 + CBM) is enhanced 3.8fold
additional information
the cutinase is fused with binding modules from Hypocrea jecorina cellobiohydrolase I (CBM) and Alcaligenes faecalis polyhydroxyalkanoate depolymerase (PBM), respectively. The adsorption of the fusion enzymes to PET is increased, and PET hydrolysis activity of one of the fusions (Thc_Cut1 + CBM) is enhanced 3.8fold
additional information
fusion of enzyme to the class II hydrophobins HFB4 and HFB7 or the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibit decreased kcat values on soluble substrates and strongly decreased the hydrophilicity of glass but cause only small changes in the hydrophobicity of polyethylene terephthalate. Upon fusion to HFB4 or HFB7, the hydrolysis of polyethylene terephthalate is enhanced over16fold over the level with the free enzyme. Fusion with the non-class II hydrophobin HFB9b does not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performs best when polyethylene terephthalate is preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion shows an increase in the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid
additional information
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fusion of enzyme to the class II hydrophobins HFB4 and HFB7 or the pseudo-class I hydrophobin HFB9b. The fusion enzymes exhibit decreased kcat values on soluble substrates and strongly decreased the hydrophilicity of glass but cause only small changes in the hydrophobicity of polyethylene terephthalate. Upon fusion to HFB4 or HFB7, the hydrolysis of polyethylene terephthalate is enhanced over16fold over the level with the free enzyme. Fusion with the non-class II hydrophobin HFB9b does not increase the rate of hydrolysis over that of the enzyme-hydrophobin mixture, but HFB9b performs best when polyethylene terephthalate is preincubated with the hydrophobins before enzyme treatment. The pattern of hydrolysis by the fusion shows an increase in the concentration of the product mono(2-hydroxyethyl) terephthalate relative to that of the main product, terephthalic acid
additional information
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exchange of the positively charged arginine (Arg19 and Arg29) located on the enzyme surface to the non-charged amino acids serine and asparagine strongly increased the hydrolysis activity for bis(benzoyloxyethyl)terephthalate and polyethyleneterephthalate. In contrast, exchange of the uncharged glutamine (Glu65) by the negatively charged glutamic acid lead to a complete loss of hydrolysis activity on polyethyleneterephthalate films
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additional information
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generation of a fusion protein, fusing cellobiohydrolase Is from Thermobifida fusca cellulase Cel6A (CBMCel6A) and Cellulomonas fimi cellulase CenA (CBMCenA), separately, to Thermobifida fusca cutinase. Both fusion proteins display catalytic properties and pH stabilities similar to those of Thermobifida fusca cutinase. Addition of pectinase enhances the cotton fiber binding activities of cutinase-CBMCel6A and cutinase-CBMCenA by 40%, and 45%, respectively. A dramatic increase of up to 3fold is observed in the amount of fatty acids released from cotton fiber by the combination of cutinase-CBM fusion proteins with pectinase
additional information
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generation of a fusion protein, fusing cellobiohydrolase Is from Thermobifida fusca cellulase Cel6A (CBMCel6A) and Cellulomonas fimi cellulase CenA (CBMCenA), separately, to Thermobifida fusca cutinase. Both fusion proteins display catalytic properties and pH stabilities similar to those of Thermobifida fusca cutinase. Addition of pectinase enhances the cotton fiber binding activities of cutinase-CBMCel6A and cutinase-CBMCenA by 40%, and 45%, respectively. A dramatic increase of up to 3fold is observed in the amount of fatty acids released from cotton fiber by the combination of cutinase-CBM fusion proteins with pectinase
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