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Engineering a fungal peroxidase that degrades lignin at very acidic pH.

Fernández-Fueyo E, Ruiz-Dueñas FJ, Martínez AT - Biotechnol Biofuels (2014)

Bottom Line: The variants obtained largely maintain the acidic stability and strong Mn(2+)-oxidizing activity of the parent enzyme, and the ability to oxidize veratryl alcohol and Reactive Black 5 (two simple VP substrates) was introduced.The engineered peroxidases present more acidic optimal pH than the best VP from P. ostreatus, enabling higher catalytic efficiency oxidizing lignins, by lowering the reaction pH, as shown using a nonphenolic model dimer.A peroxidase that degrades lignin at very acidic pH could be obtained by engineering an exposed catalytic site, able to oxidize the bulky and recalcitrant lignin polymers, in a different peroxidase type selected because of its high stability at acidic pH.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain ; Department of Biotechnology, TU Delft, Julianalaan 136, 2628 BL Delft, Netherlands.

ABSTRACT

Background: Ligninolytic peroxidases are divided into three families: manganese peroxidases (MnPs), lignin peroxidases (LiPs), and versatile peroxidases (VPs). The latter two are able to degrade intact lignins, as shown using nonphenolic lignin model compounds, with VP oxidizing the widest range of recalcitrant substrates. One of the main limiting issues for the use of these two enzymes in lignocellulose biorefineries (for delignification and production of cellulose-based products or modification of industrial lignins to added-value products) is their progressive inactivation under acidic pH conditions, where they exhibit the highest oxidative activities.

Results: In the screening of peroxidases from basidiomycete genomes, one MnP from Ceriporiopsis subvermispora was found to have a remarkable acidic stability. The crystal structure of this enzyme recently became available and, after comparison with Pleurotus ostreatus VP and Phanerochaete chrysosporium LiP structures, it was used as a robust scaffold to engineer a stable VP by introducing an exposed catalytic tryptophan, with different protein environments. The variants obtained largely maintain the acidic stability and strong Mn(2+)-oxidizing activity of the parent enzyme, and the ability to oxidize veratryl alcohol and Reactive Black 5 (two simple VP substrates) was introduced. The engineered peroxidases present more acidic optimal pH than the best VP from P. ostreatus, enabling higher catalytic efficiency oxidizing lignins, by lowering the reaction pH, as shown using a nonphenolic model dimer.

Conclusions: A peroxidase that degrades lignin at very acidic pH could be obtained by engineering an exposed catalytic site, able to oxidize the bulky and recalcitrant lignin polymers, in a different peroxidase type selected because of its high stability at acidic pH. The potential of this type of engineered peroxidases as industrial biocatalysts in lignocellulose biorefineries is strongly enhanced by the possibility to perform the delignification (or lignin modification) reactions under extremely acidic pH conditions (below pH 2), resulting in enhanced oxidative power of the enzymes.

No MeSH data available.


Related in: MedlinePlus

Optimal pH for VA (left) and RB5 (right) oxidation by S168W (A and B) and S168W-environment (C and D) variants ofC. subvermisporaMnP6, and nativeP. ostreatusVP1 (E and F). Activities for 6 mM VA (A,C,andE) and 15 μM RB5 (B,D, andF) oxidation by the S168W (A andB) and S168W-environment (C andD) variants, and native VP1 (E andF) were estimated in 100 mM BR buffer of pH 1.6 to 5.0. Means from triplicate experiments.
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Fig5: Optimal pH for VA (left) and RB5 (right) oxidation by S168W (A and B) and S168W-environment (C and D) variants ofC. subvermisporaMnP6, and nativeP. ostreatusVP1 (E and F). Activities for 6 mM VA (A,C,andE) and 15 μM RB5 (B,D, andF) oxidation by the S168W (A andB) and S168W-environment (C andD) variants, and native VP1 (E andF) were estimated in 100 mM BR buffer of pH 1.6 to 5.0. Means from triplicate experiments.

Mentions: Optimal VA oxidation by the S168W variant took place under the most acidic conditions assayed, that is, at pH 1.6 (Figure 5A) (where the variant retained 50% residual activity after 24-h incubation at 4°C). With respect to pH 3.0 used in the standard assay, we found that the activity increased over fourfold, sixfold, and eightfold when the reaction was measured at pH 2.5, 2.0, and 1.6, respectively. On the other hand, optimal RB5 oxidation was at pH 2.5 (Figure 5B), the activity being 1.7-fold higher than at pH 3.5 (used in the standard assay) and over 28-fold higher than at the optimal pH for VA oxidation (pH 1.6). When these optimal pH values were compared with those of P. ostreatus VP1 (Figure 5E and F), a clear displacement to more acidic pH optima was observed for both VA and RB5 oxidation by the MnP6 variant. However, the S168W-environment variant did not show such displacement in the VA (Figure 5C) and RB5 (Figure 5D) oxidation optima. Since both variants are relatively stable at pH 1.6 (around 50% residual activity after 24 h), the less acidic optimal pH of the S168W-environment variant oxidizing VA (and RB5) is explained by the presence of a VP-type surface environment around the introduced catalytic tryptophan.Figure 5


Engineering a fungal peroxidase that degrades lignin at very acidic pH.

Fernández-Fueyo E, Ruiz-Dueñas FJ, Martínez AT - Biotechnol Biofuels (2014)

Optimal pH for VA (left) and RB5 (right) oxidation by S168W (A and B) and S168W-environment (C and D) variants ofC. subvermisporaMnP6, and nativeP. ostreatusVP1 (E and F). Activities for 6 mM VA (A,C,andE) and 15 μM RB5 (B,D, andF) oxidation by the S168W (A andB) and S168W-environment (C andD) variants, and native VP1 (E andF) were estimated in 100 mM BR buffer of pH 1.6 to 5.0. Means from triplicate experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4364632&req=5

Fig5: Optimal pH for VA (left) and RB5 (right) oxidation by S168W (A and B) and S168W-environment (C and D) variants ofC. subvermisporaMnP6, and nativeP. ostreatusVP1 (E and F). Activities for 6 mM VA (A,C,andE) and 15 μM RB5 (B,D, andF) oxidation by the S168W (A andB) and S168W-environment (C andD) variants, and native VP1 (E andF) were estimated in 100 mM BR buffer of pH 1.6 to 5.0. Means from triplicate experiments.
Mentions: Optimal VA oxidation by the S168W variant took place under the most acidic conditions assayed, that is, at pH 1.6 (Figure 5A) (where the variant retained 50% residual activity after 24-h incubation at 4°C). With respect to pH 3.0 used in the standard assay, we found that the activity increased over fourfold, sixfold, and eightfold when the reaction was measured at pH 2.5, 2.0, and 1.6, respectively. On the other hand, optimal RB5 oxidation was at pH 2.5 (Figure 5B), the activity being 1.7-fold higher than at pH 3.5 (used in the standard assay) and over 28-fold higher than at the optimal pH for VA oxidation (pH 1.6). When these optimal pH values were compared with those of P. ostreatus VP1 (Figure 5E and F), a clear displacement to more acidic pH optima was observed for both VA and RB5 oxidation by the MnP6 variant. However, the S168W-environment variant did not show such displacement in the VA (Figure 5C) and RB5 (Figure 5D) oxidation optima. Since both variants are relatively stable at pH 1.6 (around 50% residual activity after 24 h), the less acidic optimal pH of the S168W-environment variant oxidizing VA (and RB5) is explained by the presence of a VP-type surface environment around the introduced catalytic tryptophan.Figure 5

Bottom Line: The variants obtained largely maintain the acidic stability and strong Mn(2+)-oxidizing activity of the parent enzyme, and the ability to oxidize veratryl alcohol and Reactive Black 5 (two simple VP substrates) was introduced.The engineered peroxidases present more acidic optimal pH than the best VP from P. ostreatus, enabling higher catalytic efficiency oxidizing lignins, by lowering the reaction pH, as shown using a nonphenolic model dimer.A peroxidase that degrades lignin at very acidic pH could be obtained by engineering an exposed catalytic site, able to oxidize the bulky and recalcitrant lignin polymers, in a different peroxidase type selected because of its high stability at acidic pH.

View Article: PubMed Central - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain ; Department of Biotechnology, TU Delft, Julianalaan 136, 2628 BL Delft, Netherlands.

ABSTRACT

Background: Ligninolytic peroxidases are divided into three families: manganese peroxidases (MnPs), lignin peroxidases (LiPs), and versatile peroxidases (VPs). The latter two are able to degrade intact lignins, as shown using nonphenolic lignin model compounds, with VP oxidizing the widest range of recalcitrant substrates. One of the main limiting issues for the use of these two enzymes in lignocellulose biorefineries (for delignification and production of cellulose-based products or modification of industrial lignins to added-value products) is their progressive inactivation under acidic pH conditions, where they exhibit the highest oxidative activities.

Results: In the screening of peroxidases from basidiomycete genomes, one MnP from Ceriporiopsis subvermispora was found to have a remarkable acidic stability. The crystal structure of this enzyme recently became available and, after comparison with Pleurotus ostreatus VP and Phanerochaete chrysosporium LiP structures, it was used as a robust scaffold to engineer a stable VP by introducing an exposed catalytic tryptophan, with different protein environments. The variants obtained largely maintain the acidic stability and strong Mn(2+)-oxidizing activity of the parent enzyme, and the ability to oxidize veratryl alcohol and Reactive Black 5 (two simple VP substrates) was introduced. The engineered peroxidases present more acidic optimal pH than the best VP from P. ostreatus, enabling higher catalytic efficiency oxidizing lignins, by lowering the reaction pH, as shown using a nonphenolic model dimer.

Conclusions: A peroxidase that degrades lignin at very acidic pH could be obtained by engineering an exposed catalytic site, able to oxidize the bulky and recalcitrant lignin polymers, in a different peroxidase type selected because of its high stability at acidic pH. The potential of this type of engineered peroxidases as industrial biocatalysts in lignocellulose biorefineries is strongly enhanced by the possibility to perform the delignification (or lignin modification) reactions under extremely acidic pH conditions (below pH 2), resulting in enhanced oxidative power of the enzymes.

No MeSH data available.


Related in: MedlinePlus