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Differential proteomic analysis of the secretome of Irpex lacteus and other white-rot fungi during wheat straw pretreatment.

Salvachúa D, Martínez AT, Tien M, López-Lucendo MF, García F, de Los Ríos V, Martínez MJ, Prieto A - Biotechnol Biofuels (2013)

Bottom Line: Identifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production.In contrast, a significant increase of β-glucosidase production was observed when I. lacteus grew in liquid cultures.P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain. mjmartinez@cib.csic.es.

ABSTRACT

Background: Identifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production. Irpex lacteus is an efficient microorganism for wheat straw pretreatment, yielding easily hydrolysable products with high sugar content. Thus, this fungus was selected to investigate the enzymatic system involved in lignocellulose decay, and its secretome was compared to those from Phanerochaete chrysosporium and Pleurotus ostreatus which produced different degradation patterns when growing on wheat straw. Extracellular enzymes were analyzed through 2D-PAGE, nanoLC/MS-MS, and homology searches against public databases.

Results: In wheat straw, I. lacteus secreted proteases, dye-decolorizing and manganese-oxidizing peroxidases, and H2O2 producing-enzymes but also a battery of cellulases and xylanases, excluding those implicated in cellulose and hemicellulose degradation to their monosaccharides, making these sugars poorly available for fungal consumption. In contrast, a significant increase of β-glucosidase production was observed when I. lacteus grew in liquid cultures. P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases.

Conclusion: The protein pattern secreted during I. lacteus growth in wheat straw plus the differences observed among the different secretomes, justify the fitness of I. lacteus for biopretreatment processes in 2G-ethanol production. Furthermore, all these data give insight into the biological degradation of lignocellulose and suggest new enzyme mixtures interesting for its efficient hydrolysis.

No MeSH data available.


Related in: MedlinePlus

2D-gels from I. lacteus secretomes released under SSF and SmF conditions. (a-c) 7, 14 and 21-d wheat straw SSF cultures, respectively; (d) 21-d wheat straw SSF cultures with Mn+2 supplementation; and (e) 21-d SmF cultures in CSS medium. The spots analyzed are numbered on the gels and the proteins identified detailed in Table 2.
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Figure 1: 2D-gels from I. lacteus secretomes released under SSF and SmF conditions. (a-c) 7, 14 and 21-d wheat straw SSF cultures, respectively; (d) 21-d wheat straw SSF cultures with Mn+2 supplementation; and (e) 21-d SmF cultures in CSS medium. The spots analyzed are numbered on the gels and the proteins identified detailed in Table 2.

Mentions: I. lacteus degrades simultaneously all components of wheat straw (Table 1). The biopretreated product keeps high sugar concentration with improved accessibility for further enzymatic hydrolysis aimed to second-generation ethanol production [3]. In order to study the major enzymes involved in the degradation of wheat straw and to investigate their variations over the time, the secretome of I. lacteus after 7, 14, and 21-d SSF was isolated and a comparative analysis, using 2D-PAGE, was performed. The spot pattern proved to be highly reproducible in replicate cultures. Gels from control cultures (without fungus) did not show any spot (data not shown). Most proteins focused in a pH range of 3–6 and had molecular masses from 37 to 100 kDa, a profile similar to those reported for other basidiomycetes [14]. The evolution of enzymes release, concerning both the number of different molecular species and the amount of the proteins detected at different growth stages, can be observed by simple visual inspection of the gels images (Figure 1a-c). The one from the 21-d secretome did not only display the maximum spots number, but also contained all spots detected in the gels of samples from 7-d and 14-d SSF. Then, the spots from the 21-d gel (Figure 1c), were chosen to be excised, in-gel digested, and subjected to MS/MS analysis for protein identification.


Differential proteomic analysis of the secretome of Irpex lacteus and other white-rot fungi during wheat straw pretreatment.

Salvachúa D, Martínez AT, Tien M, López-Lucendo MF, García F, de Los Ríos V, Martínez MJ, Prieto A - Biotechnol Biofuels (2013)

2D-gels from I. lacteus secretomes released under SSF and SmF conditions. (a-c) 7, 14 and 21-d wheat straw SSF cultures, respectively; (d) 21-d wheat straw SSF cultures with Mn+2 supplementation; and (e) 21-d SmF cultures in CSS medium. The spots analyzed are numbered on the gels and the proteins identified detailed in Table 2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3750859&req=5

Figure 1: 2D-gels from I. lacteus secretomes released under SSF and SmF conditions. (a-c) 7, 14 and 21-d wheat straw SSF cultures, respectively; (d) 21-d wheat straw SSF cultures with Mn+2 supplementation; and (e) 21-d SmF cultures in CSS medium. The spots analyzed are numbered on the gels and the proteins identified detailed in Table 2.
Mentions: I. lacteus degrades simultaneously all components of wheat straw (Table 1). The biopretreated product keeps high sugar concentration with improved accessibility for further enzymatic hydrolysis aimed to second-generation ethanol production [3]. In order to study the major enzymes involved in the degradation of wheat straw and to investigate their variations over the time, the secretome of I. lacteus after 7, 14, and 21-d SSF was isolated and a comparative analysis, using 2D-PAGE, was performed. The spot pattern proved to be highly reproducible in replicate cultures. Gels from control cultures (without fungus) did not show any spot (data not shown). Most proteins focused in a pH range of 3–6 and had molecular masses from 37 to 100 kDa, a profile similar to those reported for other basidiomycetes [14]. The evolution of enzymes release, concerning both the number of different molecular species and the amount of the proteins detected at different growth stages, can be observed by simple visual inspection of the gels images (Figure 1a-c). The one from the 21-d secretome did not only display the maximum spots number, but also contained all spots detected in the gels of samples from 7-d and 14-d SSF. Then, the spots from the 21-d gel (Figure 1c), were chosen to be excised, in-gel digested, and subjected to MS/MS analysis for protein identification.

Bottom Line: Identifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production.In contrast, a significant increase of β-glucosidase production was observed when I. lacteus grew in liquid cultures.P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases.

View Article: PubMed Central - HTML - PubMed

Affiliation: Centro de Investigaciones Biológicas, CSIC, Ramiro de Maeztu 9, E-28040 Madrid, Spain. mjmartinez@cib.csic.es.

ABSTRACT

Background: Identifying new high-performance enzymes or enzyme complexes to enhance biomass degradation is the key for the development of cost-effective processes for ethanol production. Irpex lacteus is an efficient microorganism for wheat straw pretreatment, yielding easily hydrolysable products with high sugar content. Thus, this fungus was selected to investigate the enzymatic system involved in lignocellulose decay, and its secretome was compared to those from Phanerochaete chrysosporium and Pleurotus ostreatus which produced different degradation patterns when growing on wheat straw. Extracellular enzymes were analyzed through 2D-PAGE, nanoLC/MS-MS, and homology searches against public databases.

Results: In wheat straw, I. lacteus secreted proteases, dye-decolorizing and manganese-oxidizing peroxidases, and H2O2 producing-enzymes but also a battery of cellulases and xylanases, excluding those implicated in cellulose and hemicellulose degradation to their monosaccharides, making these sugars poorly available for fungal consumption. In contrast, a significant increase of β-glucosidase production was observed when I. lacteus grew in liquid cultures. P. chrysosporium secreted more enzymes implicated in the total hydrolysis of the polysaccharides and P. ostreatus produced, in proportion, more oxidoreductases.

Conclusion: The protein pattern secreted during I. lacteus growth in wheat straw plus the differences observed among the different secretomes, justify the fitness of I. lacteus for biopretreatment processes in 2G-ethanol production. Furthermore, all these data give insight into the biological degradation of lignocellulose and suggest new enzyme mixtures interesting for its efficient hydrolysis.

No MeSH data available.


Related in: MedlinePlus