Abstract:Holocellulase production by Aspergillus niger using raw sugarcane bagasse (rSCB) as the enzyme-inducing substrate is hampered by the intrinsic recalcitrance of this material. Here we report that mild hydrothermal pretreatment of rSCB increases holocellulase secretion by A. niger. Quantitative proteomic analysis revealed that pretreated solids (PS) induced a pronounced up-regulation of endoglucanases and cellobiohydrolases compared to rSCB, which resulted in a 10.1-fold increase in glucose release during SCB sa… Show more
“…2 C). The proteins without signal peptides in the secretome may be extracellular proteins secreted by alternative secretion pathways or intracellular proteins leaked as earlier reported [ 9 , 18 ]. Fig.…”
Section: Resultsmentioning
confidence: 91%
“…The higher number of xylan-degrading enzymes detected across the different secretomes was similar to that reported for Aspergillus niger cultivated on pretreated SCB. This diversity in xylanases has been attributed to the complexity of the acetylated glucuronoarabinoxylan present in SCB [ 9 , 19 ]. Furthermore, amidst the CAZymes detected were pectin-degrading enzymes that included rhamnogalacturonase (GH28) and rhamnogalacturonan acetylesterase (CE12), both responsible for degradation of rhamnogalacturonan I, the main pectic component in sugarcane cell walls [ 20 ].…”
Background
Sugarcane bagasse (SCB) is an abundant feedstock for second-generation bioethanol production. This complex biomass requires an array of carbohydrate active enzymes (CAZymes), mostly from filamentous fungi, for its deconstruction to monomeric sugars for the production of value-added fuels and chemicals. In this study, we evaluated the repertoire of proteins in the secretome of a catabolite repressor-deficient strain of Penicillium funiculosum, PfMig188, in response to SCB induction and examined their role in the saccharification of SCB.
Results
A systematic approach was developed for the cultivation of the fungus with the aim of producing and understanding arrays of enzymes tailored for saccharification of SCB. To achieve this, the fungus was grown in media supplemented with different concentrations of pretreated SCB (0–45 g/L). The profile of secreted proteins was characterized by enzyme activity assays and liquid chromatography–tandem mass spectrometry (LC–MS/MS). A total of 280 proteins were identified in the secretome of PfMig188, 46% of them being clearly identified as CAZymes. Modulation of the cultivation media with SCB up to 15 g/L led to sequential enhancement in the secretion of hemicellulases and cell wall-modifying enzymes, including endo-β-1,3(4)-glucanase (GH16), endo-α-1,3-glucanase (GH71), xylanase (GH30), β-xylosidase (GH5), β-1,3-galactosidase (GH43) and cutinase (CE5). There was ~ 122% and 60% increases in β-xylosidase and cutinase activities, respectively. There was also a 36% increase in activities towards mixed-linked glucans. Induction of these enzymes in the secretome improved the saccharification performance to 98% (~ 20% increase over control), suggesting their synergy with core cellulases in accessing the recalcitrant region of SCB.
Conclusion
Our findings provide an insight into the enzyme system of PfMig188 for degradation of complex biomass such as SCB and highlight the importance of adding SCB to the culture medium to optimize the secretion of enzymes specific for the saccharification of sugarcane bagasse.
“…2 C). The proteins without signal peptides in the secretome may be extracellular proteins secreted by alternative secretion pathways or intracellular proteins leaked as earlier reported [ 9 , 18 ]. Fig.…”
Section: Resultsmentioning
confidence: 91%
“…The higher number of xylan-degrading enzymes detected across the different secretomes was similar to that reported for Aspergillus niger cultivated on pretreated SCB. This diversity in xylanases has been attributed to the complexity of the acetylated glucuronoarabinoxylan present in SCB [ 9 , 19 ]. Furthermore, amidst the CAZymes detected were pectin-degrading enzymes that included rhamnogalacturonase (GH28) and rhamnogalacturonan acetylesterase (CE12), both responsible for degradation of rhamnogalacturonan I, the main pectic component in sugarcane cell walls [ 20 ].…”
Background
Sugarcane bagasse (SCB) is an abundant feedstock for second-generation bioethanol production. This complex biomass requires an array of carbohydrate active enzymes (CAZymes), mostly from filamentous fungi, for its deconstruction to monomeric sugars for the production of value-added fuels and chemicals. In this study, we evaluated the repertoire of proteins in the secretome of a catabolite repressor-deficient strain of Penicillium funiculosum, PfMig188, in response to SCB induction and examined their role in the saccharification of SCB.
Results
A systematic approach was developed for the cultivation of the fungus with the aim of producing and understanding arrays of enzymes tailored for saccharification of SCB. To achieve this, the fungus was grown in media supplemented with different concentrations of pretreated SCB (0–45 g/L). The profile of secreted proteins was characterized by enzyme activity assays and liquid chromatography–tandem mass spectrometry (LC–MS/MS). A total of 280 proteins were identified in the secretome of PfMig188, 46% of them being clearly identified as CAZymes. Modulation of the cultivation media with SCB up to 15 g/L led to sequential enhancement in the secretion of hemicellulases and cell wall-modifying enzymes, including endo-β-1,3(4)-glucanase (GH16), endo-α-1,3-glucanase (GH71), xylanase (GH30), β-xylosidase (GH5), β-1,3-galactosidase (GH43) and cutinase (CE5). There was ~ 122% and 60% increases in β-xylosidase and cutinase activities, respectively. There was also a 36% increase in activities towards mixed-linked glucans. Induction of these enzymes in the secretome improved the saccharification performance to 98% (~ 20% increase over control), suggesting their synergy with core cellulases in accessing the recalcitrant region of SCB.
Conclusion
Our findings provide an insight into the enzyme system of PfMig188 for degradation of complex biomass such as SCB and highlight the importance of adding SCB to the culture medium to optimize the secretion of enzymes specific for the saccharification of sugarcane bagasse.
“…The bioconversion process of this biomass requires several arrays of lignocellulolytic enzymes which act synergistically to deconstruct the tightly packed polymeric carbohydrate components into monomeric sugars that can subsequently be converted to fuels and valuable chemicals. Previous studies have shown that several biomass conversion processes rely on biomass pretreatment before enzymatic hydrolysis, to remove lignin and reduce the recalcitrance nature of the biopolymer 10 – 12 . However, this process often leads to a reduction in the hemicellulose content 13 .…”
The production of second-generation fuels from lignocellulosic residues such as sugarcane bagasse (SCB) requires the synergistic interaction of key cellulose-degrading enzymes and accessory proteins for their complete deconstruction to useful monomeric sugars. Here, we recombinantly expressed and characterized unknown GH5 xylanase from P. funiculosum (PfXyn5) in Pichia pastoris, which was earlier found in our study to be highly implicated in SCB saccharification. The PfXyn5 has a molecular mass of ~ 55 kDa and showed broad activity against a range of substrates like xylan, xyloglucan, laminarin and p-nitrophenyl-β-d-xylopyranoside, with the highest specific activity of 0.7 U/mg against xylan at pH 4.5 and 50 °C. Analysis of the degradation products of xylan and SCB by PfXyn5 showed significant production of xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from two (DP2) to six (DP6), thus, suggesting that the PfXyn5 is an endo-acting enzyme. The enzyme synergistically improved the saccharification of SCB when combined with the crude cellulase cocktail of P. funiculosum with a degree of synergism up to 1.32. The PfXyn5 was further expressed individually and simultaneously with a notable GH16 endoglucanase (PfEgl16) in a catabolite-derepressed strain of P. funiculosum, PfMig188, and the saccharification efficiency of the secretomes from the resulting transformants were investigated on SCB. The secretome of PfMig188 overexpressing Xyn5 or Egl16 increased the saccharification of SCB by 9% or 7%, respectively, over the secretome of PfMig188, while the secretome of dual transformant increased SCB saccharification by ~ 15% at the same minimal protein concentration.
“…The bioconversion process of this biomass requires several arrays of lignocellulolytic enzymes which acts synergistically to deconstruct the tightly packed polymeric carbohydrate components into monomeric sugars that can subsequently be converted to fuels and valuable chemicals. Previous studies have shown that several biomass conversion processes rely on biomass pretreatment before enzymatic hydrolysis, to remove lignin and to reduce recalcitrance nature of the biopolymer [9][10][11] . However, this process often leads to reduction in the hemicellulose content 12 .…”
Section: Introductionmentioning
confidence: 99%
“…However, this process often leads to reduction in the hemicellulose content 12 . Owing to this challenge, several mild pretreatment techniques are getting developed so as to reduce the loss in hemicellulose content of lignocellulosic biomass thereby giving attention to more characterization of hemicellulases and notable accessory enzymes bene cial to biomass deconstruction 9 . Hemicellulases such as xylanases, lichenases and laminarases which are endoglucanases active on mixed-glucans have earlier been reported to improve the hydrolysis of xylan and cellulose thereby contributing to the reduction of enzyme dosage for complete biomass sacchari cation 12,13 .…”
The production of second-generation fuels from lignocellulosic residues such as sugarcane bagasse (SCB) requires the synergistic interaction of key cellulose degrading enzymes and accessory proteins for their complete deconstruction to useful monomeric sugars. Here, we recombinantly expressed and characterized an unknown GH5 xylanase from P. funiculosum (PfXyn5) in Pichia pastoris, which was earlier found in our study to be highly implicated in SCB saccharification. The PfXyn5 has a molecular mass of ~ 55 kDa and showed broad activity against a range of substrates like xylan, xyloglucan, laminarin and p-nitrophenyl-β-d-xylopyranoside, with highest specific activity of 0.7 U/mg against xylan at pH 4.5 and 50 oC. Analysis of the degradation products of xylan and SCB by PfXyn5 showed significant production of xylooligosaccharides (XOS) with a degree of polymerization (DP) ranging from two (DP2) to six (DP6), thus, suggesting that the PfXyn5 is an endo-acting enzyme. The enzyme synergistically improved the saccharification of SCB when combined with crude cellulase cocktail of P. funiculosum with degree of synergism up to 1.32. The PfXyn5 was further expressed individually and simultaneously with a notable GH16 endoglucanase (PfEgl16) in a catabolite-derepressed strain of P. funiculosum, PfMig188, and the saccharification efficiency of the secretomes from the resulting transformants were investigated on SCB. The secretome of PfMig188 overexpressing Xyn5 or Egl16 increased the saccharification of SCB by 9% and 7%, respectively, over the secretome of PfMig188 while the secretome of dual transformant increased SCB saccharification by ~ 15% at the same minimal protein concentration.
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