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Production of Trametes pubescens laccase under submerged and semi-solid culture conditions on agro-industrial wastes.

Gonzalez JC, Medina SC, Rodriguez A, Osma JF, Alméciga-Díaz CJ, Sánchez OF - PLoS ONE (2013)

Bottom Line: The crude extracts presented two laccase isoforms with molecular mass of 120 (Lac1) and 60 kDa (Lac2).Regardless of the substrate, enzymatic crude extract and purified fractions behaved similarly at different temperatures and pHs, most of them presented the maximum activity at 55 °C and a pH range between 2 and 3.In addition, they showed similar stability and electro-chemical properties.

View Article: PubMed Central - PubMed

Affiliation: Chemical Engineering Department, Universidad de los Andes, Bogotá, Colombia.

ABSTRACT
Laccases are copper-containing enzymes involved in the degradation of lignocellulosic materials and used in the treatment of phenol-containing wastewater. In this study we investigated the effect of culture conditions, i.e. submerged or semi-solid, and copper supplementation on laccase production by Trametespubescens grown on coffee husk, soybean pod husk, or cedar sawdust. The highest specific laccase activity was achieved when the culture was conducted under submerged conditions supplemented with copper (5 mM), and using coffee husk as substrate. The crude extracts presented two laccase isoforms with molecular mass of 120 (Lac1) and 60 kDa (Lac2). Regardless of the substrate, enzymatic crude extract and purified fractions behaved similarly at different temperatures and pHs, most of them presented the maximum activity at 55 °C and a pH range between 2 and 3. In addition, they showed similar stability and electro-chemical properties. At optimal culture conditions laccase activity was 7.69 ± 0.28 U mg(-1) of protein for the crude extract, and 0.08 ± 0.001 and 2.86 ± 0.05 U mg(-1) of protein for Lac1 and Lac2, respectively. In summary, these results show the potential of coffee husk as an important and economical growth medium to produce laccase, offering a new alternative use for this common agro-industrial byproduct.

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Purification of lacasses produced by submerged culture of T. pubescens using coffee husk as substrate.All samples were analyzed by SDS-PAGE under non-reducing conditions and stained with ABTS. No heat denaturation of the samples was conducted. (a) The crude extract (1) was centrifuged and filtered through a Whatman No. 1 filter (2), 0.45 µM membrane (3) 0.22 µm membrane (4), and ultrafiltrated through a 10 kDa cut-off membrane, permeate (5) and retentate (6). (b) UF retentate was purified by anionic exchange chromatography. Lac1 represent the unbound fraction, while Lac2 represent the eluted fraction. The arrow shows the point when protein elution was started. (c) Purified fractions (1) crude extract, (2) Lac1 and (3) Lac2. MW: Molecular Weight. Similar results were observed for crude extracts produced by using soybean pod husk and cedar sawdust.
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pone-0073721-g002: Purification of lacasses produced by submerged culture of T. pubescens using coffee husk as substrate.All samples were analyzed by SDS-PAGE under non-reducing conditions and stained with ABTS. No heat denaturation of the samples was conducted. (a) The crude extract (1) was centrifuged and filtered through a Whatman No. 1 filter (2), 0.45 µM membrane (3) 0.22 µm membrane (4), and ultrafiltrated through a 10 kDa cut-off membrane, permeate (5) and retentate (6). (b) UF retentate was purified by anionic exchange chromatography. Lac1 represent the unbound fraction, while Lac2 represent the eluted fraction. The arrow shows the point when protein elution was started. (c) Purified fractions (1) crude extract, (2) Lac1 and (3) Lac2. MW: Molecular Weight. Similar results were observed for crude extracts produced by using soybean pod husk and cedar sawdust.

Mentions: Laccase purification was conducted using the crude extracts that presented the highest activities. For the SmC, crude extracts obtained with soybean pod husk + Cu+2 0.5 mM, coffee husk + Cu+2 5 mM, and cedar sawdust + Cu+2 5 mM were selected; while for SSC crude extracts obtained with soybean pod husk + Cu+2 2 mM, coffee husk + Cu+2 0 mM, and cedar sawdust + Cu+2 5 mM were selected. The electrophoretic analysis of permeates from 0.45 and 0.22 µm filtrations and the UF retentate showed the presence of two proteins with laccase activity, while no activity was observed within the ultrafiltrated sample (Figure 2a). These proteins presented a molecular mass of about 120 kDa and 60 kDa (Figure 2a). The purification of the UF retentate by ionic exchange chromatography confirmed the presence of two different fractions with laccase activity (Figure 2b). The SDS-PAGE analysis of these fractions, under non-reducing conditions, showed that one protein with laccase activity was presented on each fraction (Figure 2b), and were named Lac1 (120 kDa) and Lac2 (60 kDa). Considering that the conditions used to measure the enzyme activity were those suggested for laccase activity, and that H2O2 or manganese were not added during the reaction for measuring the enzyme activity, these results suggest that both proteins are laccases but not peroxidases.


Production of Trametes pubescens laccase under submerged and semi-solid culture conditions on agro-industrial wastes.

Gonzalez JC, Medina SC, Rodriguez A, Osma JF, Alméciga-Díaz CJ, Sánchez OF - PLoS ONE (2013)

Purification of lacasses produced by submerged culture of T. pubescens using coffee husk as substrate.All samples were analyzed by SDS-PAGE under non-reducing conditions and stained with ABTS. No heat denaturation of the samples was conducted. (a) The crude extract (1) was centrifuged and filtered through a Whatman No. 1 filter (2), 0.45 µM membrane (3) 0.22 µm membrane (4), and ultrafiltrated through a 10 kDa cut-off membrane, permeate (5) and retentate (6). (b) UF retentate was purified by anionic exchange chromatography. Lac1 represent the unbound fraction, while Lac2 represent the eluted fraction. The arrow shows the point when protein elution was started. (c) Purified fractions (1) crude extract, (2) Lac1 and (3) Lac2. MW: Molecular Weight. Similar results were observed for crude extracts produced by using soybean pod husk and cedar sawdust.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3760920&req=5

pone-0073721-g002: Purification of lacasses produced by submerged culture of T. pubescens using coffee husk as substrate.All samples were analyzed by SDS-PAGE under non-reducing conditions and stained with ABTS. No heat denaturation of the samples was conducted. (a) The crude extract (1) was centrifuged and filtered through a Whatman No. 1 filter (2), 0.45 µM membrane (3) 0.22 µm membrane (4), and ultrafiltrated through a 10 kDa cut-off membrane, permeate (5) and retentate (6). (b) UF retentate was purified by anionic exchange chromatography. Lac1 represent the unbound fraction, while Lac2 represent the eluted fraction. The arrow shows the point when protein elution was started. (c) Purified fractions (1) crude extract, (2) Lac1 and (3) Lac2. MW: Molecular Weight. Similar results were observed for crude extracts produced by using soybean pod husk and cedar sawdust.
Mentions: Laccase purification was conducted using the crude extracts that presented the highest activities. For the SmC, crude extracts obtained with soybean pod husk + Cu+2 0.5 mM, coffee husk + Cu+2 5 mM, and cedar sawdust + Cu+2 5 mM were selected; while for SSC crude extracts obtained with soybean pod husk + Cu+2 2 mM, coffee husk + Cu+2 0 mM, and cedar sawdust + Cu+2 5 mM were selected. The electrophoretic analysis of permeates from 0.45 and 0.22 µm filtrations and the UF retentate showed the presence of two proteins with laccase activity, while no activity was observed within the ultrafiltrated sample (Figure 2a). These proteins presented a molecular mass of about 120 kDa and 60 kDa (Figure 2a). The purification of the UF retentate by ionic exchange chromatography confirmed the presence of two different fractions with laccase activity (Figure 2b). The SDS-PAGE analysis of these fractions, under non-reducing conditions, showed that one protein with laccase activity was presented on each fraction (Figure 2b), and were named Lac1 (120 kDa) and Lac2 (60 kDa). Considering that the conditions used to measure the enzyme activity were those suggested for laccase activity, and that H2O2 or manganese were not added during the reaction for measuring the enzyme activity, these results suggest that both proteins are laccases but not peroxidases.

Bottom Line: The crude extracts presented two laccase isoforms with molecular mass of 120 (Lac1) and 60 kDa (Lac2).Regardless of the substrate, enzymatic crude extract and purified fractions behaved similarly at different temperatures and pHs, most of them presented the maximum activity at 55 °C and a pH range between 2 and 3.In addition, they showed similar stability and electro-chemical properties.

View Article: PubMed Central - PubMed

Affiliation: Chemical Engineering Department, Universidad de los Andes, Bogotá, Colombia.

ABSTRACT
Laccases are copper-containing enzymes involved in the degradation of lignocellulosic materials and used in the treatment of phenol-containing wastewater. In this study we investigated the effect of culture conditions, i.e. submerged or semi-solid, and copper supplementation on laccase production by Trametespubescens grown on coffee husk, soybean pod husk, or cedar sawdust. The highest specific laccase activity was achieved when the culture was conducted under submerged conditions supplemented with copper (5 mM), and using coffee husk as substrate. The crude extracts presented two laccase isoforms with molecular mass of 120 (Lac1) and 60 kDa (Lac2). Regardless of the substrate, enzymatic crude extract and purified fractions behaved similarly at different temperatures and pHs, most of them presented the maximum activity at 55 °C and a pH range between 2 and 3. In addition, they showed similar stability and electro-chemical properties. At optimal culture conditions laccase activity was 7.69 ± 0.28 U mg(-1) of protein for the crude extract, and 0.08 ± 0.001 and 2.86 ± 0.05 U mg(-1) of protein for Lac1 and Lac2, respectively. In summary, these results show the potential of coffee husk as an important and economical growth medium to produce laccase, offering a new alternative use for this common agro-industrial byproduct.

Show MeSH
Related in: MedlinePlus