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1.10.3.2: laccase

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

Word Map on EC 1.10.3.2

Reaction

4 benzenediol +

O2
= 4 benzosemiquinone + 2 H2O

Synonyms

ATM, benzenediol oxygen oxidoreductase, benzenediol-oxygen oxidoreductase, benzenediol: oxygen oxidoreductase, benzenediol:O2 oxidoreductase, Benzenediol:oxygen oxidoreductase, blue laccase, blue multicopper oxidase, CcLCC6, CotA, CotA laccase, CotA-laccase, CotA-type laccase, CueO, DA2_0547, DcLac1, DcLac2, Diphenol oxidase, EpoA, Ery4, Ery4 laccase, FpLcc1, FpLcc2, GMET_RS10855, Hvo_B0205, LAC, Lac I, Lac II, Lac-3.5, Lac-4.8, LAC1, Lac2, Lac2a, LAC3, Lac4, LacA, Lacc, laccase, laccase 1, laccase 3, laccase A, Laccase allele OR, Laccase allele TS, laccase CueO, laccase POXA3b, laccase-2, laccase2, LacCh, lacTT, LacTv, LacZ1, Lcc, lcc1, Lcc2, Lcc3, Lcc4, Lcc9, LccA, lccdelta, lccgamma, Ligninolytic phenoloxidase, MAL, McoP, MmPPO laccase, MmPPOA, More, MSK laccase, multicopper oxidase, p-benzenediol:dioxygen oxidoreductase, p-diphenol dioxygen oxidoreductase, p-diphenol oxidase, p-diphenol: dioxygen oxidoreductase, p-diphenol:dioxygenoxidoreductase, p-diphenol:O2 oxidoreductase, p-diphenol:oxygen oxidoreductase, p-diphenol:oxygen-oxidoreductase, PCL, phenol oxidase, PM1 laccase, polyphenol oxidase A, POXA1b, POXA1w, POXA2, POXA3 laccase, POXA3a, POXA3b, POXC, PpoA, PsLac1, PsLac2, rlac1338, SLAC, SN4LAC, spore coat A protein, spore coat protein A, SRL1, SvLAC13, SvLAC15, SvLAC50, SvLAC52, SvLAC9, TaLac1, ThL, TTC1370, TthLAC, two-domain laccase, urishiol oxidase, urushiol oxidase, Wlac, YacK, yellow laccase

ECTree

     1 Oxidoreductases
         1.10 Acting on diphenols and related substances as donors
             1.10.3 With oxygen as acceptor
                1.10.3.2 laccase

Engineering

Engineering on EC 1.10.3.2 - laccase

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PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
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
M502F
M502L
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
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
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
E106F
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)
H165A/R240H
M199A
M199G
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/R240H
Y230A
M199A
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]
-
Y230A
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
P394H
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
-
P394H
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
additional information