Characterization of low molecular weight Fe3+-reducing compounds produced by fungi and mediation of Fenton reaction to degrade polysaccharides and lignin

Os fungos de decomposição branca e parda produzem enzimas para degradar os componentes da madeira, os primeiros produzem enzimas hidrolíticas e oxidativas enquanto os outros produzem principalmente enzimas hidrolíticas. A degradação de polissacarídeos e lignina por fungos de decomposição parda e branca seletiva, respectivamente, não ocorre na região imediata à hifa, e, também, não pode ser explicada unicamente por ação enzimática devido a impermeabilidade das mesmas na parede celular. Neste trabalho estudou-se o sistema degradativo não enzimático envolvendo compostos de baixa massa molar (CBMM) redutores de ferro em fungos degradadores de madeira. O fungo de decomposição parda, Wolfiporia cocos e o de decomposição branca seletiva, Perenniporia medulla-panis foram cultivados em diferentes concentrações de ferro, e a atividade redutora de Fe3+ micelial e a extracelular, assim como a produção de quelantes específicos de ferro, como derivados de ácido hidroxâmico e de catecol, foram induzidas em condição de deficiência de ferro. Os géis de SDS-PAGE dos extratos fúngicos mostraram várias proteínas negativamente reguladas por ferro em P. medulla-panis e W. cocos, principalmente para proteínas entre 10 - 30 kDa. Quando os fungos foram cultivados em diferentes fontes de carbono simples com e sem suplementação de celulose microcristalina e deficiência de ferro, produziram CBMM redutores de Fe3+, os quais tiveram a produção estimulada nos meios com celulose. Análises de eletroforese capilar dos compostos quelantes de metal extraídos dos meios que proporcionaram a maior atividade redutora (Lornitina/ celulose para P. medulla-panis e glicose/celulose para W. cocos) na presença e ausência de ferro, confirmaram que, principalmente P. medulla-panis produz compostos extracelulares que são regulados por ferro. Os CBMM purificados das frações < 5 kDa apresentaram atividade redutora de Fe3+ em pH 2,0 mesmo quando ácido oxálico foi adicionado na concentração 20 vezes maior que a concentração de Fe3+. Em pH 4,5, a atividade redutora foi detectada até uma concentração de ácido oxálico 10 vezes superior a de Fe3+. Em ambos os casos a atividade redutora foi observada quando Fe3+ estava presente na forma livre ou complexada como Fe(oxalato)+. Dentre os vários CBMM produzidos por P. medulla-panis e W. cocos aqueles com atividade redutora foram o ácido 4-hidroxi-fenilacético, 1,2-dihidroxi-3-metil-benzeno, 1,2,3-trihidroxi-benzeno e o ácido 4- hidroxi-cinâmico para W. cocos, e para P. medulla-panis os principais foram 1,2-dihidroxibenzeno e 1,2,3-trihidroxi-benzeno. Além desses compostos, ambos os fungos produziram peptídeos de baixa massa molar com atividade redutora. Os CBMM redutores de Fe3+ de P. medulla-panis (Pmp) e de W. cocos (Wc) foram utilizados na ausência e presença de Fe3+ e H2O2 (reação de Fenton mediada) para oxidar polissacarídeos e lignina in vitro. Verificou-se que os maiores níveis de oxidação foram obtidos nas reações de Fenton mediadas (Wc-Fe3+/H2O2 e Pmp-Fe3+/H2O2). A degradação da celulose por estes sistemas foi caracterizada por uma rápida e extensiva despolimerização, seguida de significativa oxidação. Análises dos monômeros de lignina liberados de conífera tratada e não tratada após 13C-TMAH termoquimólise indicaram oxidação da lignina pelos sistemas Wc-Fe3+/H2O2 e Pmp- Fe3+/H2O2, principalmente por desmetoxilação e/ou desmetilação. A ação sinérgica entre os CBMM redutores de Fe3+ e as enzimas ligninolíticas ficou evidenciada para os fungos de decomposição branca Lentinula edodes, P. medulla-panis e Trametes versicolor através da oxidação do corante Azure B.Brown and white rot fungi produce enzymes to degrade wood. The former produce hydrolytic and oxidative enzymes while the latter produce mainly hydrolytic enzymes. The degradation of polysaccharides and lignin by brown and white-rot fungi, respectively, do not occur next to the fungal hyphae and cannot be explained only by the enzymatic action due to the small pore size of sound wood. In this work, it was studied a non-enzymatic degradative system involving low molecular weight compounds (LMWC) with Fe3+-reducing activity in wood decay fungi. The brown rot fungus Wolfiporia cocos and the selective white rot Perenniporia medulla-panis were grown under varying concentration of iron. The micelial and extracellular Fe3+-reducing activity as well as the production of specific iron chelators (catechol and hydroxamate derivatives) were induced under iron starvation. SDS-PAGE gels of cellular proteins showed several proteins negatively iron-regulated in P. medulla-panis and in W. cocos, especially for proteins of 10 - 30 kDa. When the fungi were grown with different simple carbon source with and without microcrystalline cellulose supplementation and under iron restriction, they produced LMWC with Fe3+-reducing activity, which production was stimulated in the presence of cellulose. Capillary electrophoresis analyses of metal chelating compounds extracted from the growth media that promoted the highest Fe3+-reducing activity (L-ornithine/cellulose for P. medulla-panis and glucose/cellulose for W. cocos) in the presence and absence of iron, confirmed that, especially P. medulla-panis produces extracellular compounds that are iron-regulated. LMWC purified from these media showed Fe3+-reducing activity at pH 2.0 even when oxalic acid was added up to 20 fold the iron concentration. At pH 4.5, the Fe3+-reducing activity was detected at an oxalic acid concentration up to 10 fold the iron concentration. In both cases the LMWC were capable of reducing Fe3+ only when it was in its free form or complexed with oxalate to form Fe3+-monooxalate complex (Fe(C2O4)+). Among the several LMWC produced by P. medulla-panis and W. cocos those with Fe3+-reducing capability were 4-hydroxy-phenylacetic acid, 1,2- dihydroxy-methyl-benzene, 1,2,3-trihydroxy-benzene and 4-hydroxy-cinnamic acid to W. cocos and 1,2-dihydroxy-benzene, and 1,2,3-tri-hydroxy-benzene to P. medulla-panis. Both fungi also produce low molecular weight peptides with Fe3+-reducing capability. The purified LMWC with Fe3+-reducing activity from P. medulla-panis (Pmp) and from W. cocos (Wc) were utilized in the presence and absence of Fe3+ and H2O2 (mediated Fenton reaction) to oxidize polysaccharides and lignin in vitro. The highest oxidation levels were obtained with mediated Fenton reactions (Wc-Fe3+/H2O2 e Pmp-Fe3+/H2O2). Cellulose degradation by these systems was characterized by a rapid and extensive depolymerization followed by significant oxidation. Analyses of the lignin monomers released from treated and untreated softwood after 13C-TMAH thermochemolysis indicated lignin oxidation by the Wc-Fe3+/H2O2 and Pmp-Fe3+/H2O2 systems, mainly by demethoxylation and/or demethylation. The synergistic action between LMWC with Fe3+-reducing activity and the ligninolytic enzymes was evidenced to the white rot fungi Lentinula edodes, P. medulla-panis and Trametes versicolor with Azure B oxidation assays

Characterization of low molecular weight Fe3+-reducing compounds produced by fungi and mediation of Fenton reaction to degrade polysaccharides and lignin

Os fungos de decomposição branca e parda produzem enzimas para degradar os componentes da madeira, os primeiros produzem enzimas hidrolíticas e oxidativas enquanto os outros produzem principalmente enzimas hidrolíticas. A degradação de polissacarídeos e lignina por fungos de decomposição parda e branca seletiva, respectivamente, não ocorre na região imediata à hifa, e, também, não pode ser explicada unicamente por ação enzimática devido a impermeabilidade das mesmas na parede celular. Neste trabalho estudou-se o sistema degradativo não enzimático envolvendo compostos de baixa massa molar (CBMM) redutores de ferro em fungos degradadores de madeira. O fungo de decomposição parda, Wolfiporia cocos e o de decomposição branca seletiva, Perenniporia medulla-panis foram cultivados em diferentes concentrações de ferro, e a atividade redutora de Fe3+ micelial e a extracelular, assim como a produção de quelantes específicos de ferro, como derivados de ácido hidroxâmico e de catecol, foram induzidas em condição de deficiência de ferro. Os géis de SDS-PAGE dos extratos fúngicos mostraram várias proteínas negativamente reguladas por ferro em P. medulla-panis e W. cocos, principalmente para proteínas entre 10 - 30 kDa. Quando os fungos foram cultivados em diferentes fontes de carbono simples com e sem suplementação de celulose microcristalina e deficiência de ferro, produziram CBMM redutores de Fe3+, os quais tiveram a produção estimulada nos meios com celulose. Análises de eletroforese capilar dos compostos quelantes de metal extraídos dos meios que proporcionaram a maior atividade redutora (Lornitina/ celulose para P. medulla-panis e glicose/celulose para W. cocos) na presença e ausência de ferro, confirmaram que, principalmente P. medulla-panis produz compostos extracelulares que são regulados por ferro. Os CBMM purificados das frações < 5 kDa apresentaram atividade redutora de Fe3+ em pH 2,0 mesmo quando ácido oxálico foi adicionado na concentração 20 vezes maior que a concentração de Fe3+. Em pH 4,5, a atividade redutora foi detectada até uma concentração de ácido oxálico 10 vezes superior a de Fe3+. Em ambos os casos a atividade redutora foi observada quando Fe3+ estava presente na forma livre ou complexada como Fe(oxalato)+. Dentre os vários CBMM produzidos por P. medulla-panis e W. cocos aqueles com atividade redutora foram o ácido 4-hidroxi-fenilacético, 1,2-dihidroxi-3-metil-benzeno, 1,2,3-trihidroxi-benzeno e o ácido 4- hidroxi-cinâmico para W. cocos, e para P. medulla-panis os principais foram 1,2-dihidroxibenzeno e 1,2,3-trihidroxi-benzeno. Além desses compostos, ambos os fungos produziram peptídeos de baixa massa molar com atividade redutora. Os CBMM redutores de Fe3+ de P. medulla-panis (Pmp) e de W. cocos (Wc) foram utilizados na ausência e presença de Fe3+ e H2O2 (reação de Fenton mediada) para oxidar polissacarídeos e lignina in vitro. Verificou-se que os maiores níveis de oxidação foram obtidos nas reações de Fenton mediadas (Wc-Fe3+/H2O2 e Pmp-Fe3+/H2O2). A degradação da celulose por estes sistemas foi caracterizada por uma rápida e extensiva despolimerização, seguida de significativa oxidação. Análises dos monômeros de lignina liberados de conífera tratada e não tratada após 13C-TMAH termoquimólise indicaram oxidação da lignina pelos sistemas Wc-Fe3+/H2O2 e Pmp- Fe3+/H2O2, principalmente por desmetoxilação e/ou desmetilação. A ação sinérgica entre os CBMM redutores de Fe3+ e as enzimas ligninolíticas ficou evidenciada para os fungos de decomposição branca Lentinula edodes, P. medulla-panis e Trametes versicolor através da oxidação do corante Azure B.Brown and white rot fungi produce enzymes to degrade wood. The former produce hydrolytic and oxidative enzymes while the latter produce mainly hydrolytic enzymes. The degradation of polysaccharides and lignin by brown and white-rot fungi, respectively, do not occur next to the fungal hyphae and cannot be explained only by the enzymatic action due to the small pore size of sound wood. In this work, it was studied a non-enzymatic degradative system involving low molecular weight compounds (LMWC) with Fe3+-reducing activity in wood decay fungi. The brown rot fungus Wolfiporia cocos and the selective white rot Perenniporia medulla-panis were grown under varying concentration of iron. The micelial and extracellular Fe3+-reducing activity as well as the production of specific iron chelators (catechol and hydroxamate derivatives) were induced under iron starvation. SDS-PAGE gels of cellular proteins showed several proteins negatively iron-regulated in P. medulla-panis and in W. cocos, especially for proteins of 10 - 30 kDa. When the fungi were grown with different simple carbon source with and without microcrystalline cellulose supplementation and under iron restriction, they produced LMWC with Fe3+-reducing activity, which production was stimulated in the presence of cellulose. Capillary electrophoresis analyses of metal chelating compounds extracted from the growth media that promoted the highest Fe3+-reducing activity (L-ornithine/cellulose for P. medulla-panis and glucose/cellulose for W. cocos) in the presence and absence of iron, confirmed that, especially P. medulla-panis produces extracellular compounds that are iron-regulated. LMWC purified from these media showed Fe3+-reducing activity at pH 2.0 even when oxalic acid was added up to 20 fold the iron concentration. At pH 4.5, the Fe3+-reducing activity was detected at an oxalic acid concentration up to 10 fold the iron concentration. In both cases the LMWC were capable of reducing Fe3+ only when it was in its free form or complexed with oxalate to form Fe3+-monooxalate complex (Fe(C2O4)+). Among the several LMWC produced by P. medulla-panis and W. cocos those with Fe3+-reducing capability were 4-hydroxy-phenylacetic acid, 1,2- dihydroxy-methyl-benzene, 1,2,3-trihydroxy-benzene and 4-hydroxy-cinnamic acid to W. cocos and 1,2-dihydroxy-benzene, and 1,2,3-tri-hydroxy-benzene to P. medulla-panis. Both fungi also produce low molecular weight peptides with Fe3+-reducing capability. The purified LMWC with Fe3+-reducing activity from P. medulla-panis (Pmp) and from W. cocos (Wc) were utilized in the presence and absence of Fe3+ and H2O2 (mediated Fenton reaction) to oxidize polysaccharides and lignin in vitro. The highest oxidation levels were obtained with mediated Fenton reactions (Wc-Fe3+/H2O2 e Pmp-Fe3+/H2O2). Cellulose degradation by these systems was characterized by a rapid and extensive depolymerization followed by significant oxidation. Analyses of the lignin monomers released from treated and untreated softwood after 13C-TMAH thermochemolysis indicated lignin oxidation by the Wc-Fe3+/H2O2 and Pmp-Fe3+/H2O2 systems, mainly by demethoxylation and/or demethylation. The synergistic action between LMWC with Fe3+-reducing activity and the ligninolytic enzymes was evidenced to the white rot fungi Lentinula edodes, P. medulla-panis and Trametes versicolor with Azure B oxidation assays

Cellulose accessibility limits the effectiveness of minimum cellulase loading on the efficient hydrolysis of pretreated lignocellulosic substrates

A range of lignocellulosic feedstocks (including agricultural, softwood and hardwood substrates) were pretreated with either sulfur dioxide-catalyzed steam or an ethanol organosolv procedure to try to establish a reliable assessment of the factors governing the minimum protein loading that could be used to achieve efficient hydrolysis. A statistical design approach was first used to define what might constitute the minimum protein loading (cellulases and β-glucosidase) that could be used to achieve efficient saccharification (defined as at least 70% glucan conversion) of the pretreated substrates after 72 hours of hydrolysis. The likely substrate factors that limit cellulose availability/accessibility were assessed, and then compared with the optimized minimum amounts of protein used to obtain effective hydrolysis. The optimized minimum protein loadings to achieve efficient hydrolysis of seven pretreated substrates ranged between 18 and 63 mg protein per gram of glucan. Within the similarly pretreated group of lignocellulosic feedstocks, the agricultural residues (corn stover and corn fiber) required significantly lower protein loadings to achieve efficient hydrolysis than did the pretreated woody biomass (poplar, douglas fir and lodgepole pine). Regardless of the substantial differences in the source, structure and chemical composition of the feedstocks, and the difference in the pretreatment technology used, the protein loading required to achieve efficient hydrolysis of lignocellulosic substrates was strongly dependent on the accessibility of the cellulosic component of each of the substrates. We found that cellulose-rich substrates with highly accessible cellulose, as assessed by the Simons' stain method, required a lower protein loading per gram of glucan to obtain efficient hydrolysis compared with substrates containing less accessible cellulose. These results suggest that the rate-limiting step during hydrolysis is not the catalytic cleavage of the cellulose chains per se, but rather the limited accessibility of the enzymes to the cellulose chains due to the physical structure of the cellulosic substrate.Forestry, Faculty ofReviewedFacult

Access to cellulose limits the efficiency of enzymatic hydrolysis: the role of amorphogenesis

The efficient enzymatic saccharification of cellulose at low cellulase (protein) loadings continues to be a challenge for commercialization of a process for bioconversion of lignocellulose to ethanol. Currently, effective pretreatment followed by high enzyme loading is needed to overcome several substrate and enzyme factors that limit rapid and complete hydrolysis of the cellulosic fraction of biomass substrates. One of the major barriers faced by cellulase enzymes is their limited access to much of the cellulose that is buried within the highly ordered and tightly packed fibrillar architecture of the cellulose microfibrils. Rather than a sequential 'shaving' or 'planing' of the cellulose fibrils from the outside, it has been suggested that these inaccessible regions are disrupted or loosened by non-hydrolytic proteins, thereby increasing the cellulose surface area and making it more accessible to the cellulase enzyme complex. This initial stage in enzymatic saccharification of cellulose has been termed amorphogenesis. In this review, we describe the various amorphogenesis-inducing agents that have been suggested, and their possible role in enhancing the enzymatic hydrolysis of cellulose.Forestry, Faculty ofWood Science, Department ofReviewedFacult

The enzymatic hydrolysis of pretreated pulp fibers predominantly involves “peeling/erosion” modes of action

Background: There is still considerable debate regarding the actual mechanism by which a “cellulase mixture” deconstructs cellulosic materials, with accessibility to the substrate at the microscopic level being one of the major restrictions that limits fast, complete cellulose hydrolysis. In the work reported here we tried to determine the predominant mode of action, at the fiber level, of how a cellulase mixture deconstructs pretreated softwood and hardwood pulp fibers. Quantitative changes in the pulp fibers derived from different pretreated biomass substrates were monitored throughout the course of enzymatic hydrolysis to see if the dominant mechanisms involved either the fragmentation/cutting of longer fibers to shorter fibers or their “peeling/delamination/erosion,” or if both cutting and peeling mechanisms occurred simultaneously. Results: Regardless of the source of biomass, the type of pretreatment and the chemical composition of the substrate, under typical hydrolysis conditions (50°C, pH 4.8, mixing) longer pulp fibers (fiber length >200 μm) were rapidly broken down until a relatively constant fiber length of 130 to 160 μm was reached. In contrast, shorter fibers with an initial average fiber length of 130 to 160 μm showed no significant change in length despite their substantial hydrolysis. The fragmentation/cutting mode of deconstruction was only observed on longer fibers at early stages of hydrolysis. Although the fiber fragmentation mode of deconstruction was not greatly influenced by enzyme loading, it was significantly inhibited by glucose and was mainly observed during initial mixing of the enzyme and substrate. In contrast, significant changes in the fiber width occurred throughout the course of hydrolysis for all of the substrates, suggesting that fiber width may limit the rate and extent of cellulose hydrolysis. Conclusion: It appears that, at the fiber level, pretreated pulp fibers are hydrolyzed through a two-step mode of action involving an initial rapid fragmentation followed by simultaneous swelling and peeling/erosion of the fragmented fibers. This latter mechanism is the predominant mode of action involved in effectively hydrolyzing the cellulose present in pretreated wood substrates.Other UBCReviewedFacult

Use of substructure-specific carbohydrate binding modules to track changes in cellulose accessibility and surface morphology during the amorphogenesis step of enzymatic hydrolysis

Background: Cellulose amorphogenesis, described as the non-hydrolytic “opening up” or disruption of a cellulosic substrate, is becoming increasingly recognized as one of the key steps in the enzymatic deconstruction of cellulosic biomass when used as a feedstock for fuels and chemicals production. Although this process is thought to play a major role in facilitating hydrolysis, the lack of quantitative techniques capable of accurately describing the molecular-level changes occurring in the substrate during amorphogenesis has hindered our understanding of this process. Results: In this work, techniques for measuring changes in cellulose accessibility are reviewed and a new quantitative assay method is described. Carbohydrate binding modules (CBMs) with specific affinities for crystalline (CBM2a) or amorphous (CBM44) cellulose were used to track specific changes in the surface morphology of cotton fibres during amorphogenesis. The extents of phosphoric acid-induced and Swollenin-induced changes to cellulose accessibility were successfully quantified using this technique. Conclusions: The adsorption of substructure-specific CBMs can be used to accurately quantify the extent of changes to cellulose accessibility induced by non-hydrolytic disruptive proteins. The technique provided a quick, accurate and quantitative measure of the accessibility of cellulosic substrates. Expanding the range of CBMs used for adsorption studies to include those specific for such compounds as xylan or mannan should also allow for the accurate quantitative tracking of the accessibility of these and other polymers within the lignocellulosic biomass matrix.Forestry, Faculty ofWood Science, Department ofOther UBCReviewedFacult