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N398D
-
twice better activity at pH 6 and 1.2fold improvement at pH 3
V159E
-
two-fold improvement in the laccase activity detected at pH 3 and 6. Lower thermotolerance than native enzyme
V159E/N398D
-
1.4fold higher activity than mutant enzyme V159E and 2.6fold higher activity than mutant enzyme N398D with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as substrate. Lower thermotolerance than native enzyme
V159E/N398D/I453F/M454L
-
parental-like activity at both pH values. Lower thermotolerance than native enzyme
V159E/N398D/I453L/M454L
-
1.3fold TAI at pH 3 and lower activity (0.8fold) than mutant enzyme V159E/N398D. Lower thermotolerance than native enzyme
N398D
-
twice better activity at pH 6 and 1.2fold improvement at pH 3
-
V159E
-
two-fold improvement in the laccase activity detected at pH 3 and 6. Lower thermotolerance than native enzyme
-
V159E/N398D
-
1.4fold higher activity than mutant enzyme V159E and 2.6fold higher activity than mutant enzyme N398D with 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) as substrate. Lower thermotolerance than native enzyme
-
V159E/N398D/I453F/M454L
-
parental-like activity at both pH values. Lower thermotolerance than native enzyme
-
V159E/N398D/I453L/M454L
-
1.3fold TAI at pH 3 and lower activity (0.8fold) than mutant enzyme V159E/N398D. Lower thermotolerance than native enzyme
-
D501G
-
better stability and catalytic efficiency than wild-type enzyme
D500G
in Pichia pastoris 9.3folds higher expression than wild-type enzyme
K316N
11.4-fold higher expression level. High dimerization of phenolic and decolorization of industrial dyes
L386Q/G417I
variant L2 harbours the insertion of two amino acids (Ser and Pro) after the N-terminal methionine, in addition to the L386Q/G417I substitutions. The mutant enzyme exhibits 9-fold higher guaiacol oxidation rates in the lysate compared to the strain expressing the wild-type enzyme
L386Q/G417I/V482G
variant L9 harbours the insertion of two amino acids (Ser and Pro) after the N-terminal methionine, in addition to the L386Q/G417I/V482G substitutions. The mutant enzyme exhibits 14-fold higher guaiacol oxidation rates in the lysate compared to the strain expressing the wild-type enzyme. The decline in relative activity from the maximum at pH 4 towards more alkaline pH values is sharper for the mutant enzyme compared to the wild-type with 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid). The mutant enzyme has an increased activity towards the lignin-like model compound guaiacol while retaining the very good thermostability and activity at neutral-to-basic pH of the wild-type enzyme
E188A
in comparison with the wild type, the mutant enzyme shows an increase in Km value and a decrease in kcat. In comparison with the wild type, the mutant enzyme shows increased stability to organis solvents (ethanol, methanol, 1-propanol)
E188I
in comparison with the wild type, the mutant enzyme shows an increase in kcat and catalytic efficiency values and a decrease in Km. In comparison with the wild type, the mutant enzyme shows increased stability to organis solvents (ethanol, methanol, 1-propanol)
E188K
in comparison with the wild type, the mutant enzyme shows an increase in Km value and a decrease in kcat. In comparison with the wild type, the mutant enzyme shows increased stability to organis solvents (ethanol, methanol, 1-propanol)
E188L
in comparison with the wild type, the mutant enzyme shows an increase in kcat and catalytic efficiency values and a decrease in Km. In comparison with the wild type, the mutant enzyme shows increased stability to organis solvents (ethanol, methanol, 1-propanol)
E188R
in comparison with the wild type, the mutant enzyme shows an increase in kcat and catalytic efficiency values and a decrease in Km, In comparison with the wild type, the mutant enzyme shows increased stability to organis solvents (ethanol, methanol, 1-propanol)
E188V
in comparison with the wild type, the mutant enzyme shows an increase in kcat and catalytic efficiency values and a decrease in Km. In comparison with the wild type, the mutant enzyme shows increased stability to organis solvents (ethanol, methanol, 1-propanol)
E188A
-
in comparison with the wild type, the mutant enzyme shows an increase in Km value and a decrease in kcat. In comparison with the wild type, the mutant enzyme shows increased stability to organis solvents (ethanol, methanol, 1-propanol)
-
E188I
-
in comparison with the wild type, the mutant enzyme shows an increase in kcat and catalytic efficiency values and a decrease in Km. In comparison with the wild type, the mutant enzyme shows increased stability to organis solvents (ethanol, methanol, 1-propanol)
-
E188L
-
in comparison with the wild type, the mutant enzyme shows an increase in kcat and catalytic efficiency values and a decrease in Km. In comparison with the wild type, the mutant enzyme shows increased stability to organis solvents (ethanol, methanol, 1-propanol)
-
E188R
-
in comparison with the wild type, the mutant enzyme shows an increase in kcat and catalytic efficiency values and a decrease in Km, In comparison with the wild type, the mutant enzyme shows increased stability to organis solvents (ethanol, methanol, 1-propanol)
-
E188V
-
in comparison with the wild type, the mutant enzyme shows an increase in kcat and catalytic efficiency values and a decrease in Km. In comparison with the wild type, the mutant enzyme shows increased stability to organis solvents (ethanol, methanol, 1-propanol)
-
H497A
-
copper center, no significant changes
I494A
-
site-directed mutagenesis at a hydrophobic residue in the vicinity of the type 1 copper site, the replacement of Ile494 by an alanine residue leads to significant changes in the enzyme, the mutant shows differences in the type 1 as well as in the type 2 copper centre compared to the wild-type enzyme
L386A
-
the site-directed mutation of Leu386, a hydrophobic residue in the vicinity of the type 1 copper site, to an alanine residue appears to cause only very subtle alterations in the properties of the enzyme indicating minimal changes in the structure of the copper centres
degradation
-
the highest biodegradation of the toxic organochlorine pesticide pentachlorophenol (PCP) is of 23% at pollutant concentration of 100 mg/l, which evidences that the thermostable enzyme acts directly in degradation of pentachlorophenol and may be a useful asset to remediate this pollutant
environmental protection
-
the highest biodegradation of the toxic organochlorine pesticide pentachlorophenol (PCP) is of 23% at pollutant concentration of 100 mg/l, which evidences that the thermostable enzyme acts directly in degradation of pentachlorophenol and may be a useful asset to remediate this pollutant
degradation
-
the highest biodegradation of the toxic organochlorine pesticide pentachlorophenol (PCP) is of 23% at pollutant concentration of 100 mg/l, which evidences that the thermostable enzyme acts directly in degradation of pentachlorophenol and may be a useful asset to remediate this pollutant
-
environmental protection
-
the highest biodegradation of the toxic organochlorine pesticide pentachlorophenol (PCP) is of 23% at pollutant concentration of 100 mg/l, which evidences that the thermostable enzyme acts directly in degradation of pentachlorophenol and may be a useful asset to remediate this pollutant
-
D360M
mutation at sites Cu5
D439A/M510L
140fold and 44fold increases in the kcat values for ABTS and 2,5-diaminotoluene, redox potential is 0.39 V
D439A/M510Q
redox potential of the mutant s 0.21 V
D439A/P444A
40fold increase in the ABTS-oxidizing activity, redox potential is 0.46 V
D439A/P444A/M510L
T1 copper in the triple mutant cannot be fully oxidized resulting in loss of enzymatic activities
D439A/P444A/M510Q
redox potential of the mutant was 0.26 V and high enzymatic activities are not attained
D507A
about 10% increase in specific activity activity
D507N
about 80% increase in specific activity activity
E506A
mutation results in the formation of a compensatory hydrogen bond network with one or two extra water molecules
E506D
about 20% decrease in specific activity activity
E506I
mutation results in the complete shutdown of the hydrogen bond network leading to loss of enzymatic activities
E506Q
mutation results in the hydrogen bond network without the proton transport function
G304K
mutant shows about 2.7fold increased the laccase activity. Movements of the regulatory loop combined with the changes of the methionine-rich region may uncover the T1 Cu site allowing greater access of the substrate
M355L/D360N
mutation at sites Cu5
M358S/M362S
mutation at sites Cu6
M358S/M362S/M364S/M368S
mutations at sites Cu6,7
M364S/M368S
mutation at sites Cu7
M510L
3.8-4.2 copper atoms per protein molecule, similar to wild-type, redox potential is 0.40 V
M510Q
3.8-4.2 copper atoms per protein molecule, similar to wild-type, redox potential is 0.23 V
P444A/D439A
mutation leads to a synergetic effect of the positive shift in the redox potential of the type I copper center and the increase in enzyme activity
P444A/M510L
enzymatic activities similar to wild-type
P444A/M510Q
3.4 copper atoms per protein molecule, similar to wild-type, redox potential is 0.21 V, no oxidizing activity of ABTS is observed
P444G
mutation results in positive shifts in the redox potential of this copper center and enhanced oxidase activity in CueO and in the region Pro357-His406 deletion mutant lacking a methionine-rich helical segment that covers the substrate-binding site
P444I
positive shift in the redox potential of this copper center and enhanced oxidase activity
P444L
positive shift in the redox potential of this copper center and enhanced oxidase activity
Q106F
-
mutation enhanced CueO oxidation activity
L559A
the C-terminal mutation affects the trinuclear site geometry of the mutant enzyme, which also shows 3-4fold reduced activity compared to the wild-type enzyme
K532A
site-directed mutagenesis, the mutant shows increased catalytic efficiency and altered substrate specificity compared to the wild-type enzyme
K532E
site-directed mutagenesis, the mutant shows increased catalytic efficiency and altered substrate specificity compared to the wild-type enzyme
K532R
site-directed mutagenesis, inactive mutant
P530A
site-directed mutagenesis, the mutant shows increased catalytic efficiency and altered substrate specificity compared to the wild-type enzyme
D205R
site-directed mutagenesis, the mutation in a highly conserved region perturbs the structural local environment in POXA1b, leading to a large rearrangement of the enzyme structure. The mutant shows highly reduced activity compared to the wild-type enzyme and is inactive with substrate syringaldazine
L466V/E467S/A468G
-
triple mutation: changes in pH optimum, redox potential, Km, kcat and fluoride inhibition
L470F
-
no significant changes
Y108A
Tyr108 does form an integral part of the active site and affects enzyme kinetics
Y108F
Tyr108 does form an integral part of the active site and affects enzyme kinetics
Y229A
over 10fold increase in activity
Y108A
-
Tyr108 does form an integral part of the active site and affects enzyme kinetics
-
Y108F
-
Tyr108 does form an integral part of the active site and affects enzyme kinetics
-
Y229A
-
over 10fold increase in activity
-
H165A/M199G
2.3fold increase in kcat/Km towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
M199G/H165A
2.3fold increase in kcat/Km for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). kcat/Km for 2,6-dimethoxyphenol is identical to kcat/Km of wilde-type enzyme. 2.7fold decrease in kcat/Km for K4[Fe(CN)6]. Drastic shift in the optimal pH of 2,6-dimethoxyphenol oxidation
M199G
-
5.4fold increase in kcat/Km for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). 4.7fold increase in kcat/Km for 2,6-dimethoxyphenol. 1.5fold decrease in kcat/Km for K4[Fe(CN)6]
-
L513F
-
no significant changes
V509L/S510E/G511A
-
triple mutation: changes in pH optimum, redox potential, Km, kcat and fluoride inhibition
D394E
mutant with the lower laccase activity displays a decreased decolorization efficiency as compared to the wild-type enzyme. Expressed in a lower level, about 50%, of the wild type enzyme. Optimum pH shifts towards the acidic value (0.5-1 units) relative to the wild type enzyme which has an optimal pH 6.0
D394M
mutant with the lower laccase activity displays a decreased decolorization efficiency as compared to the wild-type enzyme. Expressed in a lower level, about 50%, of the wild type enzyme. Optimum pH shifts towards the acidic value (0.5-1 units) relative to the wild type enzyme which has an optimal pH 6.0
D394R
mutant with the lower laccase activity displays a decreased decolorization efficiency as compared to the wild-type enzyme. Expressed in a lower level, about 16%, of the wild type enzyme. Optimum pH shifts towards the acidic value (0.5-1 units) relative to the wild type enzyme which has an optimal pH 6.0
D396A
mutant enzyme with higher catalytic efficiency decolorizes the synthetic dye more efficiently than the wild-type enzyme
D396E
mutant enzyme with higher catalytic efficiency decolorizes the synthetic dye more efficiently than the wild-type enzyme
D396M
mutant enzyme with higher catalytic efficiency decolorizes the synthetic dye more efficiently than the wild-type enzyme
K428E
-
1.3fold decrease in kcat/Km for the substrate guaiacol
K428L
-
1.6fold decrease in kcat/Km for the substrate guaiacol. 70% decrease in activity of mutant enzyme after 4 h at 80°C. 30% decrease in activity of wild-type enzyme after 4 h at 80°C
K428M
-
1.4fold increase in kcat/Km for the substrate guaiacol
K428R
-
1.3fold decrease in kcat/Km for the substrate guaiacol
M455L
mutation in T1 Cu site, incorporation of 3-4 copper atoms, similar to wild-type. Mutation results in an increase of 100 mV in the O2 reduction potential, while the enzymatic activity for ABTS oxidation is decreased
M456A
mutation in T1 Cu site, incorporation of 3-4 copper atoms, similar to wild-type
D394E
-
mutant with the lower laccase activity displays a decreased decolorization efficiency as compared to the wild-type enzyme. Expressed in a lower level, about 50%, of the wild type enzyme. Optimum pH shifts towards the acidic value (0.5-1 units) relative to the wild type enzyme which has an optimal pH 6.0
-
D394M
-
mutant with the lower laccase activity displays a decreased decolorization efficiency as compared to the wild-type enzyme. Expressed in a lower level, about 50%, of the wild type enzyme. Optimum pH shifts towards the acidic value (0.5-1 units) relative to the wild type enzyme which has an optimal pH 6.0
-
D394R
-
mutant with the lower laccase activity displays a decreased decolorization efficiency as compared to the wild-type enzyme. Expressed in a lower level, about 16%, of the wild type enzyme. Optimum pH shifts towards the acidic value (0.5-1 units) relative to the wild type enzyme which has an optimal pH 6.0
-
D396A
-
mutant enzyme with higher catalytic efficiency decolorizes the synthetic dye more efficiently than the wild-type enzyme
-
D396M
-
mutant enzyme with higher catalytic efficiency decolorizes the synthetic dye more efficiently than the wild-type enzyme
-
K428E
-
1.3fold decrease in kcat/Km for the substrate guaiacol
-
K428L
-
1.6fold decrease in kcat/Km for the substrate guaiacol. 70% decrease in activity of mutant enzyme after 4 h at 80°C. 30% decrease in activity of wild-type enzyme after 4 h at 80°C
-
K428M
-
1.4fold increase in kcat/Km for the substrate guaiacol
-
K428R
-
1.3fold decrease in kcat/Km for the substrate guaiacol
-
A240P
site-directed mutagenesis
D341N
site-directed mutagenesis the D3 domain coil, surface, the mutant shows H bonding with surrounding residue N340 in contrast to the wild-type enzyme
N208S |
site-directed mutagenesis in the D2 domain beta sheet, near D206 (responsible for binding phenolic substrates at the T1 site), the mutant shows increased H bonding with surrounding residues
N331D
site-directed mutagenesis the D3 domain beta sheet substrate binding loop, contiguous to F332 key residue of the binding pocket, the mutant shows increased H bonding with surrounding residues
R280H |
site-directed mutagenesis in the D2 domain end of distal beta sheet, surface, the mutant shows reduced H bonding with surrounding residues
A240P
-
site-directed mutagenesis
-
D341N
-
site-directed mutagenesis the D3 domain coil, surface, the mutant shows H bonding with surrounding residue N340 in contrast to the wild-type enzyme
-
D206A
site-directed mutagenesis, the Asn mutation leads to a significant shift of pH optimum for activity with 2,6-dimethoxyphenol, the mutant shows several fold increased activity compared to the wild-type enzyme
D206E
site-directed mutagenesis, the Asn mutation leads to a significant shift of pH optimum for activity with 2,6-dimethoxyphenol, the mutant shows several fold increased activity compared to the wild-type enzyme
D206N
site-directed mutagenesis, the Asn mutation leads to a significant shift of pH optimum for activity with 2,6-dimethoxyphenol, the mutant shows several fold increased activity compared to the wild-type enzyme
V281A/P309L/S318G/D232V
the expression of the optimized mutant enzyme increases by 22% compared to the unoptimized enzyme and the optimal reaction temperature of the mutant enzyme is 5°C higher than that of the recombinant wild-type enzyme rlac1338, and the optimal pH increases by 0.5 units. The thermal stability and pH stability of the mutant enzyme lac2-9 are improved. 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) is the most suitable substrate for the recombinant enzyme and mutant enzyme. In addition, the Km of the mutant strain lac2-9 (76 mM) is significantly lower, but the kcat/Km (0.618 /s*M) is significantly higher, and the specific enzyme activity (79.8 U/mg) increases by 3.5 times compared with the recombinant laccase (22.8 U/mg). Compared to the rlac1338, the degradation rates with the simultaneous addition of Ca2+ and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) of mutant enzyme lac2-9 for acid violet 7, bromophenol blue and coomassie brilliant blue significantly improved by 8.3, 3.4 and 3.4 times
M502F
-
site-directed mutagenesis, the mutation of the weak so-called axial ligand of the T1 copper site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme, the mutant shows 10% and 0.15-0.05% activity for the non-phenolic substrates and for the phenolic substrates, respectively, compared with the wild-type enzyme
M502F
-
mutations leads to an increase in the redox potential by approx. 100 mV, decrease in the catalytic efficiency, decrease in thermodynamic stability
M502F
-
replacement of Met502, which is weakly co-ordinating to the T1 copper, in CotA laccase by the non-co-ordinating residues leucine and phenylalanine allows the maintenance of the T1 copper geometry while causing an increase in the redox potential
M502L
-
copper center, no significant changes
M502L
-
site-directed mutagenesis, the mutation of the weak so-called axial ligand of the T1 copper site leads to an increase in the redox potential by approximately 100 mV relative to that of the wild-type enzyme, the mutant exhibits a twofold to fourfold decrease in the kcat
M502L
-
mutations leads to an increase in the redox potential by approx. 100 mV, decrease in the catalytic efficiency, decrease in thermodynamic stability
M502L
-
replacement of Met502, which is weakly co-ordinating to the T1 copper, in CotA laccase by the non-co-ordinating residues leucine and phenylalanine allows the maintenance of the T1 copper geometry while causing an increase in the redox potential
D439A
3.8-4.2 copper atoms per protein molecule, similar to wild-type, redox potential is 0.43 V compared to 0.36 for wild-type
D439A
positive shift in the redox potential of this copper center and enhanced oxidase activity
P444A
3.8-4.2 copper atoms per protein molecule, similar to wild-type, redox potential is 0.43 V
P444A
positive shift in the redox potential of this copper center and enhanced oxidase activity
E106F
-
site-directed mutagenesis with additional deletion of residues L351-G378, the mutant WlacD shows 3.5fold increased activity and enhanced thermal stability compared to the wild-type enzyme
E106F
-
site-directed mutagenesis, the mutant WlacS shows 2.2fold increased activity compared to the wild-type enzyme
E106F
-
mutation promotes both enzymatic activity and thermostability
H165A/R240H
1.8 fold increase in kcat/Km towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
H165A/R240H
4.8fold increase in kcat/Km for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). 15.1fold increase in kcat/Km for 2,6-dimethoxyphenol. 1.4fold decrease in kcat/Km for K4[Fe(CN)6]
M199A
2.4fold increase in kcat/Km for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). 1.4fold increase in kcat/Km for 2,6-dimethoxyphenol. 2.5fold decrease in kcat/Km for K4[Fe(CN)6]
M199A
2.4fold increase in kcat/Km towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
M199G
5.4fold increase in kcat/Km for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). 4.7fold increase in kcat/Km for 2,6-dimethoxyphenol. 1.5fold decrease in kcat/Km for K4[Fe(CN)6]
M199G
5.4fold increase in kcat/Km towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
M199G/R240H
1.8fold increase in kcat/Km for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). 1.5fold decrease in kcat/Km for 2,6-dimethoxyphenol. 2.4fold decrease in kcat/Km for K4[Fe(CN)6]. Combination of substitutions in the substrate-binding pocket and in the tunnel leading to Cu2+ ion increases the catalytic activity towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) by 5fold, and towards 2,6-dimethoxyphenol by 16fold
M199G/R240H
mutation increases catalytic activity of the enzyme towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) by 5fold, and towards 2,6-dimethoxyphenol by 16fold. Prospective 2D laccase variant with the highest activity under alkaline conditions
Y230A
4.02fold increase in kcat/Km for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). 2fold increase in kcat/Km for 2,6-dimethoxyphenol. 1.4fold decrease in kcat/Km for K4[Fe(CN)6]
Y230A
4.0fold increase in kcat/Km towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
M199A
-
2.4fold increase in kcat/Km for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). 1.4fold increase in kcat/Km for 2,6-dimethoxyphenol. 2.5fold decrease in kcat/Km for K4[Fe(CN)6]
-
M199A
-
2.4fold increase in kcat/Km towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
-
Y230A
-
4.02fold increase in kcat/Km for 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid). 2fold increase in kcat/Km for 2,6-dimethoxyphenol. 1.4fold decrease in kcat/Km for K4[Fe(CN)6]
-
Y230A
-
4.0fold increase in kcat/Km towards 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
-
P394H
site-directed mutagenesis
P394H
site-directed mutagenesis the D3 domain coil substrate binding loop, contiguous to H395, T1 Cu ligand, the mutant shows H bonding with surrounding residue S427 in contrast to the wild-type enzyme
P394H
-
site-directed mutagenesis
-
P394H
-
site-directed mutagenesis the D3 domain coil substrate binding loop, contiguous to H395, T1 Cu ligand, the mutant shows H bonding with surrounding residue S427 in contrast to the wild-type enzyme
-
additional information
deletion of the region Pro357-His406 comprising a methionine-rich helical segment that covers the substrate-binding site and replacement with a Gly-Gly linker. The scaffold of the CueO molecule and metal-binding sites are reserved in the mutant. The high thermostability of the protein molecule and its spectroscopic and magnetic properties are also conserved after truncation. The cuprous oxidase activity of the mutant is reduced to about 10% that of recombinant CueO due to the decrease in the affinity of the labile Cu site for Cu(I) ions
additional information
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deletion of the region Pro357-His406 comprising a methionine-rich helical segment that covers the substrate-binding site and replacement with a Gly-Gly linker. The scaffold of the CueO molecule and metal-binding sites are reserved in the mutant. The high thermostability of the protein molecule and its spectroscopic and magnetic properties are also conserved after truncation. The cuprous oxidase activity of the mutant is reduced to about 10% that of recombinant CueO due to the decrease in the affinity of the labile Cu site for Cu(I) ions
additional information
mutations at Pro444 to construct a second NH-S hydrogen bond between the backbone amide and coordinating Cys500 thiolate of the type I copper result in positive shifts in the redox potential of this copper center and enhanced oxidase activity in CueO. Pro444 mutations limit the incorporation of copper ions into the trinuclear copper center. The activities of both CueO and the region Pro357-His406 deletion mutant are also enhanced by mutations to break down the hydrogen bond between the imidazole group of His443 that is coordinated to the type I copper and the beta-carboxy group of Asp439 that is located in the outer sphere of the type I copper center. The characteristics of the Cu(II)-S(Cys) bond are only minimally perturbed by mutations involving formation or disruption of a hydrogen bond from the coordinating groups to the type I copper
additional information
-
mutations at Pro444 to construct a second NH-S hydrogen bond between the backbone amide and coordinating Cys500 thiolate of the type I copper result in positive shifts in the redox potential of this copper center and enhanced oxidase activity in CueO. Pro444 mutations limit the incorporation of copper ions into the trinuclear copper center. The activities of both CueO and the region Pro357-His406 deletion mutant are also enhanced by mutations to break down the hydrogen bond between the imidazole group of His443 that is coordinated to the type I copper and the beta-carboxy group of Asp439 that is located in the outer sphere of the type I copper center. The characteristics of the Cu(II)-S(Cys) bond are only minimally perturbed by mutations involving formation or disruption of a hydrogen bond from the coordinating groups to the type I copper
additional information
construction of a chimeric form of lcc1, CaMV35Sp:clcc1
additional information
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construction of a chimeric form of lcc1, CaMV35Sp:clcc1
additional information
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laccase Lcc4/1, composed of the N-terminus of the Lcc4 and the C-terminus of the Lcc1 laccases
additional information
laccase Lcc4/1, composed of the N-terminus of the Lcc4 and the C-terminus of the Lcc1 laccases
additional information
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laccase Lcc4/1, composed of the N-terminus of the Lcc4 and the C-terminus of the Lcc1 laccases. The fusion enzyme is more efficient, compared to Lcc1, in decolorizing RBBR and poly-R478, even if the latter is only in the presence of a redox mediator
additional information
laccase Lcc4/1, composed of the N-terminus of the Lcc4 and the C-terminus of the Lcc1 laccases. The fusion enzyme is more efficient, compared to Lcc1, in decolorizing RBBR and poly-R478, even if the latter is only in the presence of a redox mediator
additional information
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construction of a chimeric form of lcc1, CaMV35Sp:clcc1
-
additional information
elimination of the N-terminal sequence in mutant rMmPPOA-637 decreases the specific activity 15fold, which is partially restored in the presence of 1 M NaCl, and alters the secondary and tertiary structures and the pH dependence of optimal stability
additional information
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elimination of the N-terminal sequence in mutant rMmPPOA-637 decreases the specific activity 15fold, which is partially restored in the presence of 1 M NaCl, and alters the secondary and tertiary structures and the pH dependence of optimal stability
additional information
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elimination of the N-terminal sequence in mutant rMmPPOA-637 decreases the specific activity 15fold, which is partially restored in the presence of 1 M NaCl, and alters the secondary and tertiary structures and the pH dependence of optimal stability
-
additional information
deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL559 is practically inactive
additional information
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deletion of the last four amino acids dramatically affected the activity of the enzyme, as the deletion mutant delDSGL559 is practically inactive
additional information
construction and generation of C-terminal truncation mutants lacking 1, 2, 5, 8, 11, 14 or 18 amino acid residues
additional information
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construction and generation of C-terminal truncation mutants lacking 1, 2, 5, 8, 11, 14 or 18 amino acid residues
additional information
deletion of the C-terminal extension of 4 or 16 amino acids leads to truncated mutants which lose the high stability at pH 10, while they show an increased stability at pH 5.0, the thermostability is unaltered compared to the wild-type enzyme
additional information
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generation of a truncation mutant lacking the first 31 amino acid residues, the mutant protein displays a better thermostability, with a half life of over 400 min at 37°C, is less sensitive to chloride and more stable at pH 7.0 compared to the wild--type enzyme
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an alkaline laccase mutant form is chosen and further evolved for the synthesis of the C-N polydye at basic pHs. Over 11500 clones derived form 3 rounds of directed and focused evolution are screened through a high-throughput colorimetric assay, and a variant with 3.5-fold improved activity relative to that of the wild type is selected
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construction of a two-type domain laccase from a three-type domain laccase by eliminating the signal sequence domain
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replacement of the native PcL signal peptide by the alpha-factor preproleader, and subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The sequence encoding the evolved alpha-factor preproleader of 7A9 for secretion in Sacchaaromyces cerevisiae is replaced by the 24-amino-acid glucoamylase preprosequence from Aspergillus niger, under the Emericella nidulans gpd promoter and trpC terminator. Total laccase activity is enhanced 8000fold, the evolved alpha-factor preproleader improves secretion levels 40fold, and several mutations in mature laccase provide a 13.7fold increase in kcat. While the pH activity profile is shifted to more neutral values, the thermostability and the broad substrate specificity of PcL are retained. Mutational effect on laccase secretion, overview
additional information
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replacement of the native PcL signal peptide by the alpha-factor preproleader, and subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The sequence encoding the evolved alpha-factor preproleader of 7A9 for secretion in Sacchaaromyces cerevisiae is replaced by the 24-amino-acid glucoamylase preprosequence from Aspergillus niger, under the Emericella nidulans gpd promoter and trpC terminator. Total laccase activity is enhanced 8000fold, the evolved alpha-factor preproleader improves secretion levels 40fold, and several mutations in mature laccase provide a 13.7fold increase in kcat. While the pH activity profile is shifted to more neutral values, the thermostability and the broad substrate specificity of PcL are retained. Mutational effect on laccase secretion, overview
additional information
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replacement of the native PcL signal peptide by the alpha-factor preproleader, and subjected to six rounds of evolution coupled to a multiscreening assay based on the oxidation of natural and synthetic redox mediators at more neutral pHs. The sequence encoding the evolved alpha-factor preproleader of 7A9 for secretion in Sacchaaromyces cerevisiae is replaced by the 24-amino-acid glucoamylase preprosequence from Aspergillus niger, under the Emericella nidulans gpd promoter and trpC terminator. Total laccase activity is enhanced 8000fold, the evolved alpha-factor preproleader improves secretion levels 40fold, and several mutations in mature laccase provide a 13.7fold increase in kcat. While the pH activity profile is shifted to more neutral values, the thermostability and the broad substrate specificity of PcL are retained. Mutational effect on laccase secretion, overview
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enzyme adsorption to cellulose fibers from cotton and denim, attachment of PEG of 1.1-5.0 kDa to the purified enzyme, overview
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a variant deleted of the last 13 residues (C(DELTA)) and its 6His tagged counterpart (C(DELTA)6H) are active enzymes. The production of C(DELTA)6H results in the synthesis of a unusually high proportion of highly glycosylated forms of the enzyme therefore allowing the additional purification of a hyper-glycosylated form of C(DELTA)6H noted C(DELTA)6Hh. Properties of (C(DELTA)), C(DELTA)6H and C(DELTA)6Hh are compared. Catalytic efficiency of C(DELTA) (kcat/KM) decreases 4fold (with syringaldazine as substrate) and 10fold (with 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) as substrate) respectively. The catalytic parameters kcat and KM of C(DELTA)6H and C(DELTA)6Hh are strictly comparable revealing that over-glycosylation does not affect the enzyme catalytic efficiency. To the contrary, in vitro deglycosylation of laccase drastically reduces its activity
additional information
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a variant deleted of the last 13 residues (C(DELTA)) and its 6His tagged counterpart (C(DELTA)6H) are active enzymes. The production of C(DELTA)6H results in the synthesis of a unusually high proportion of highly glycosylated forms of the enzyme therefore allowing the additional purification of a hyper-glycosylated form of C(DELTA)6H noted C(DELTA)6Hh. Properties of (C(DELTA)), C(DELTA)6H and C(DELTA)6Hh are compared. Catalytic efficiency of C(DELTA) (kcat/KM) decreases 4fold (with syringaldazine as substrate) and 10fold (with 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) as substrate) respectively. The catalytic parameters kcat and KM of C(DELTA)6H and C(DELTA)6Hh are strictly comparable revealing that over-glycosylation does not affect the enzyme catalytic efficiency. To the contrary, in vitro deglycosylation of laccase drastically reduces its activity
additional information
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a variant deleted of the last 13 residues (C(DELTA)) and its 6His tagged counterpart (C(DELTA)6H) are active enzymes. The production of C(DELTA)6H results in the synthesis of a unusually high proportion of highly glycosylated forms of the enzyme therefore allowing the additional purification of a hyper-glycosylated form of C(DELTA)6H noted C(DELTA)6Hh. Properties of (C(DELTA)), C(DELTA)6H and C(DELTA)6Hh are compared. Catalytic efficiency of C(DELTA) (kcat/KM) decreases 4fold (with syringaldazine as substrate) and 10fold (with 2,2-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) as substrate) respectively. The catalytic parameters kcat and KM of C(DELTA)6H and C(DELTA)6Hh are strictly comparable revealing that over-glycosylation does not affect the enzyme catalytic efficiency. To the contrary, in vitro deglycosylation of laccase drastically reduces its activity
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additional information
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evaluation of the oxidation and decolorization of azo dyes by the enzyme through co-immobilization of enzyme and dye on an electrode, mechanism of electron transfer, overview