RESUMO
Treatment of whey protein isolate (WPI; 1 to 25% w/w) in heated κ-carrageenan (KC; 2% w/w) slurries with protease and/or transglutaminase modulated the properties of the hydrogels formed after cooling. Observation of peak compression stress and strain at gel rupture showed WPI incorporation at 1, 5 and 10% (w/w) significantly reduced the strength and deformability of 2% (w/w) KC gels. Treatment of WPI solutions in KC slurries with Alcalase 2.4L was shown by both SDS-Page and size exclusion HPLC to reduce protein/peptide molecular weight distributions below 10 kDa, with large portions below 1 kDa. This peptide size reduction within the KC matrix produced more translucent gels with a more organized wall and cell structure as observed by SEM, which resulted in gels with observed rupture stress/strain levels similar to 2% KC alone. Transglutaminase treatment of WPI-KC slurries showed the reverse behavior, reducing gel translucency, strength and deformability. At these loadings, WPI-KC gel strength/deformability appears to relate decreasing peptide size to gel behavior trending towards KC-only gels; suggesting peptide size modulation in protein-carbohydrate complexes will allow significant tailoring of texture for the delivery of protein/peptide rich gelled products.
Assuntos
Carragenina/química , Peptídeo Hidrolases/química , Transglutaminases/química , Proteínas do Soro do Leite/química , Biocatálise , Hidrogéis/química , Concentração de Íons de Hidrogênio , ReologiaRESUMO
This study compliments previous work where peroxidase was successfully used to crosslink corn fiber gum (CFG) with bovine serum albumin and improve CFG's emulsifying properties. Herein, an alternative type of enzyme, transglutaminase, was used to prepare conjugates of CFG and sodium caseinate. Additionally, the CFG was partially hydrolyzed by sulfuric acid and its crosslinking pattern with caseinate was evaluated. The interfacial crosslinking degree between caseinate and CFG increased after hydrolysis according to high performance size exclusion chromatography. The equilibrium interfacial tension of CFG hydrolysate-caseinate conjugate was lower than that of CFG-caseinate conjugate as the rearrangement rate of the CFG hydrolysate-caseinate conjugate was higher. The dilatational modulus of CFG hydrolysate decreased from that of CFG.
RESUMO
High internal phase emulsions (HIPE) prepared using whey protein microgels (WPMs) as a surfactant were demonstrated to have substantially higher stability than HIPEs prepared using similar loadings of non-gelled whey protein isolate (WPI) or Tween 20. Microgel colloids were prepared from WPI solutions by heat treatment at 85 °C in a narrow pH range (5.8-6.0) to particle sizes of approximately 90, 160 and 350 nm in diameter. ζ-potentials of the WPM increased in negativity with decreasing particle size from -7.4 ± 2.5 down to -21.1 ± 0.9 at 90 nm. All WPMs conferred high stability to corn oil based HIPE when used as an emulsifier. Light microscopy and cryo-scanning electron microscopy showed that both increasing WPM concentration and decreasing WPM particle size produced increasingly smaller and more hexagonally shaped corn oil emulsion droplets; WPI and Tween 20 based HIPE droplets were generally smaller and spherical in shape. The HIPE (75% w/w corn oil) produced with 1% (w/w) WPM as an emulsifier showed stability through 6 months storage at 4 °C at all WPM sizes tested, while the HIPE prepared with 1% (w/w) WPI or Tween 20 exhibited significant creaming. WPM and WPI based HIPE both showed thermal stability at 70 °C and 95 °C while the heating of Tween 20 based HIPE resulted in droplet coalescence and oil-phase separation. HIPE production with WPMs significantly improved the viscoelastic properties of the HIPE, imparting drastic increases in yield stress, critical stress, complex modulus and elastic modulus over HIPE prepared with WPI or Tween 20. The more rigid rheology of the WPM HIPE indicated by these data is likely the primary mechanism driving the improved stability of these emulsions.
Assuntos
Emulsificantes/química , Proteínas do Soro do Leite/química , Óleo de Milho/química , Emulsões/química , Géis/química , Concentração de Íons de Hidrogênio , Tamanho da Partícula , Polissorbatos/química , ReologiaRESUMO
The presence of xylan is a detriment to the enzymatic saccharification of cellulose in lignocelluloses. The inhibition of the processive cellobiohydrolase Cel7A by soluble wheat arabinoxylan is shown here to increase by 50% following enzymatic treatment with a commercially-purified α-L-arabinofuranosidase. The enhanced inhibitory effect was shown by T2 relaxation time measurements via low field NMR to coincide with an increasing degree of constraint put on the water in xylan solutions. Furthermore, quartz crystal micro-balance with dissipation experiments showed that α-L-arabinofuranosidase treatment considerably increased the rate and rigidity of arabinoxylan mass association with cellulose. These data also suggest significant xylan-xylan adlayer formation occurs following initial binding of debranched arabinoxylan. From this, we speculate the inhibitory effects of xylan to cellulases may result from reduced enzymatic access via the dense association of xylan with cellulose.
Assuntos
Celulose 1,4-beta-Celobiosidase/antagonistas & inibidores , Celulose 1,4-beta-Celobiosidase/metabolismo , Celulose/metabolismo , Glicosídeo Hidrolases/metabolismo , Triticum/química , Xilanos/metabolismo , Inibidores Enzimáticos/metabolismo , Espectroscopia de Ressonância Magnética , Ligação ProteicaRESUMO
BACKGROUND: Studies in bioconversions have continuously sought the development of processing strategies to overcome the "close physical association" between plant cell wall polymers thought to significantly contribute to biomass recalcitrance [Adv Space Res 18:251-265, 1996],[ Science 315:804-807, 2007]. To a lesser extent, studies have sought to understand biophysical factors responsible for the resistance of lignocelluloses to enzymatic degradation. Provided here are data supporting our hypothesis that the inhibitory potential of different cell wall polymers towards enzymatic cellulose hydrolysis is related to how much these polymers constrain the water surrounding them. We believe the entropy-reducing constraint imparted to polymer associated water plays a negative role by increasing the probability of detrimental interactions such as junction zone formation and the non-productive binding of enzymes. RESULTS: Selected commercial lignocellulose-derived polymers, including hemicelluloses, pectins, and lignin, showed varied potential to inhibit 24-h cellulose conversion by a mix of purified cellobiohydrolase I and ß-glucosidase. At low dry matter loadings (0.5% w/w), insoluble hemicelluloses were most inhibitory (reducing conversion relative to cellulose-only controls by about 80%) followed by soluble xyloglucan and wheat arabinoxylan (reductions of about 70% and 55%, respectively), while the lignin and pectins tested were the least inhibitory (approximately 20% reduction). Low field nuclear magnetic resonance (LF-NMR) relaxometry used to observe water-related proton relaxation in saturated polymer suspensions (10% dry solids, w/w) showed spin-spin, T2, relaxation time curves generally approached zero faster for the most inhibitory polymer preparations. The manner of this decline varied between polymers, indicating different biophysical aspects may differentially contribute to overall water constraint in each case. To better compare the LF-NMR data to inhibitory potential, T2 values from monocomponent exponential fits of relaxation curves were used as a measure of overall water constraint. These values generally correlated faster relaxation times (greater water constraint) with greater inhibition of the model cellulase system by the polymers. CONCLUSIONS: The presented correlation of cellulase inhibition and proton relaxation data provides support for our water constraint-biomass recalcitrance hypothesis. Deeper investigation into polymer-cellulose-cellulase interactions should help elucidate the types of interactions that may be propagating this correlation. If these observations can be verified to be more than correlative, the hypothesis and data presented suggest that a focus on water-polymer interactions and ways to alter them may help resolve key biological lignocellulose processing bottlenecks.
RESUMO
Microbial utilization of lignocellulose from plant cell walls is integral to carbon cycling on Earth. Correspondingly, secreted enzymes that initiate lignocellulose depolymerization serve a crucial step in the bioconversion of lignocellulosic biomass to fuels and chemicals. Genome and metagenome sequencing efforts that span the past decade reveal the diversity of enzymes that have evolved to transform lignocellulose from wood, herbaceous plants and grasses. Nevertheless, there are relatively few examples where 'omic' technologies have identified novel enzyme activities or combinations thereof that dramatically improve the economics of lignocellulose bioprocessing and utilization. A likely factor contributing to the discrepancy between sequence-based enzyme discovery and enzyme application is the common practice to screen enzyme candidates based on activity measurements using soluble model compounds. In this context, the development and application of imaging, physicochemical, and spectromicroscopic techniques that allow direct assessment of enzyme action on relevant lignocellulosic substrates is reviewed.
Assuntos
Bactérias/enzimologia , Lignina/análise , Lignina/metabolismo , Biomassa , Ensaios Enzimáticos , Lignina/química , Metagenoma , Plantas/química , Plantas/metabolismo , Solubilidade , Especificidade por SubstratoRESUMO
Crystalline cellulose Iß (Avicel) was chemically transformed into cellulose II and III(I) producing allomorphs with similar crystallinity indices (ATR-IR and XRD derived). Saccharifications by commercial cellulases at arrayed solids loadings showed cellulose III(I) was more readily hydrolysable and less susceptible to increased dry solids levels than cellulose Iß and II. Analysis by dynamic vapor sorption revealed cellulose II has a distinctively higher absorptive capacity than cellulose I and III(I). When equally hydrated (g water/g cellulose), low-field nuclear magnetic resonance (LF-NMR) relaxometry showed that cellulose II, on average, most constrained water while cellulase III(I) left the most free water. LF-NMR spin-spin relaxation time distribution profiles representing distinct water pools suggest cellulose III(I) had the most restricted pool and changes in water distribution during enzymatic saccharification were most dramatic with respect to cellulose III(I) compared to celluloses Iß and II.
Assuntos
Celulases/metabolismo , Celulose/química , Celulose/metabolismo , Biotransformação , Espectroscopia de Ressonância Magnética , Água/análiseRESUMO
Milliliter scale (ligno)cellulose saccharifications suggest general solute concentration and its impact on water availability plays a significant role in detrimental effects associated with high solids lignocellulose conversions. A microtumbler developed to enable free-fall mixing at dry solids loadings up to 35% (w/w) repeatedly produced known detrimental conversion trends on cellulose, xylan and pretreated lignocellulose with commercial enzymes. Despite this, high concentrations of insoluble nonhydrolysable dextrans did not depress saccharification extents in 5% (w/w) cellulose slurries suggesting mass transfer limitations may not significantly limit hydrolysis extents at high solids loadings. Interestingly, cellulose saccharification by purified cellulases showed increased conversions with increasing dry solids loadings. This prompted investigations into impacts the concentration of soluble species, such as sugar alcohols, low molecular weight enzyme preparation components, and monomer hydrolysis products, have on the hydrolysis environment. Such substances significantly depress conversion rates and were shown to correlatively lower water activity (A(w) ) in the hydrolysis environment while high insoluble solids concentrations did not. Furthermore, low-field NMR on concentrated slurries of insoluble complex carbohydrates, including the nonhydrolysable dextrans, showed all solids constrained water significantly more than high concentrations of soluble species (inhibitory) suggesting water constraint may not be as problematic an issue at high solids loadings compared to the availability of water in the system. Additionally, the introduction of soluble species lessened overall water constraint in high solids systems and appears to shift the distribution of water away from insoluble surfaces. This is potentially a critical issue for industrial processes operating at high dry solids levels.
Assuntos
Reatores Biológicos , Biotecnologia/métodos , Celulases/metabolismo , Glucose/metabolismo , Lignina/química , Água/química , Biomassa , Proteínas Fúngicas/metabolismo , Glucose/química , Hidrólise , Lignina/metabolismo , Espectroscopia de Ressonância Magnética , Álcoois Açúcares/química , Álcoois Açúcares/metabolismo , Xilose/química , Xilose/metabolismoRESUMO
Enzymatic conversion of oligomeric xylose and insoluble xylan remaining after effective pretreatment offers significant potential to improve xylan-to-xylose yields while minimizing yields of degredation products and fermentation inhibitors. In this work, a commercial enzyme cocktail is demonstrated to convert up to 70 % of xylo-oligomers found in dilute acid-pretreated hydrolyzate liquor at varying levels of dilution when supplemented with accessory enzymes targeting common side chains. Commercial enzyme cocktails are also shown to convert roughly 80 % of insoluble xylan remaining after effective high-solids, dilute acid pretreatment.
Assuntos
Ácidos/química , Xilanos/química , Zea mays/química , Aspergillus niger/enzimologia , Endo-1,4-beta-Xilanases/metabolismo , Hidrólise , Folhas de Planta/química , Caules de Planta/química , Solubilidade , Xilose/químicaRESUMO
Cell wall recalcitrance is the largest contributor to the high expense of lignocellulose conversion to biofuels (Himmel ME et al., Science 315:804-807, 2007). In response to this problem, researchers at the BioEnergy Science Center (BESC) are working to determine the contributing factors of biomass recalcitrance. The primary approach to this is screening large sample sets of genetic and environmental variants of model and feedstock plant species for differences in recalcitrance to combined hydrothermal pretreatment and enzymatic hydrolysis (Decker S et al., BioEnergy Res 2:179-192, 2009). To handle these large sample sets (up to several thousand samples per set), the BESC has developed high throughput screening systems to evaluate both cell wall composition and recalcitrance (Selig MJ et al., Biotechnol Lett 33:961-967, 2011; Selig MJ et al., Ind Biotechnol 6, 104-111, 2010). Molecular beam mass spectroscopy and high throughput, 2-stage acid hydrolysis are used to determine amounts and ratios of cell wall components such as lignin, cellulose, and xylan. Recalcitrance is measured by glucose and xylose release after high throughput hydrothermal pretreatment and enzymatic saccharification, screening large numbers (up to 1,000 s per week) of biomass samples (Selig MJ et al., Ind Biotechnol 6, 104-111, 2010; Sykes R et al., Methods Mol Biol 581, 169-183, 2009). Implementation of these high throughput techniques revealed additional concerns when screening biomass samples for recalcitrance, principal among these was the contribution of starch to glucose release quantitation in both compositional analysis and recalcitrance screening.
Assuntos
Biomassa , Biotecnologia/métodos , Parede Celular/química , Ensaios de Triagem em Larga Escala/métodos , Panicum/química , Amido/análise , Amilases/metabolismo , Biocombustíveis , Técnicas de Química Analítica/métodosRESUMO
Comparative studies between commercial Trichoderma reesei cellulase preparations show that, depending on the preparation and loading, total protein precipitation can be as high as 30 % under standard hydrolysis conditions used for lignocellulosic materials. ATR-IR and SDS-PAGE data verify precipitates are protein-based and contain key cell wall hydrolyzing enzymes. Precipitation increased considerably with incubation temperature; roughly 50-150 % increase from 40 to 50 °C and 800 % greater at 60 °C. All of the reported protein losses translated into significant, and often drastic, losses in activity on related 4-nitrophenyl substrates. In addition, supplementation with the non-ionic surfactant PEG 6,000 decreased precipitation up to 80 % in 24 h precipitation levels. Protein precipitation is potentially substantial during enzymatic hydrolysis of lignocelluloses and should be accounted for during lignocellulose conversion process design, particularly when enzyme recycling is considered.
Assuntos
Celulase/isolamento & purificação , Proteínas Fúngicas/isolamento & purificação , Lignina/química , Trichoderma/enzimologia , Biocombustíveis , Celulase/química , Precipitação Química , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Temperatura Alta , Hidrólise , Espectroscopia de Infravermelho com Transformada de Fourier , Trichoderma/metabolismoRESUMO
The analysis of structural glucan and xylan in lignocellulose was scaled down from original two-stage sulfuric acid hydrolysis methods (Moore WE and Johnson DB 1967 Procedures for the chemical analysis of wood and wood products. U.S. Forest Products Laboratory, U.S. Department of Agriculture., Madison, WI) and integrated into a recently-developed, high throughput pretreatment and enzymatic saccharification system. Novel 96×1.8 ml-well Hastelloy reactor plates (128×86×51 mm) based on previously described 96-well pretreatment reactor plates were paired with custom aluminum filler plates (128×86×18 mm) for use in Symyx Powdernium solids dispensing systems. The incorporation of glucose oxidase and xylose dehydrogenase linked assays to speed post-hydrolysis sugar analysis dramatically reduced the time for analysis of large lignocellulosic sample sets. The current system permits the determination of the glucan and xylan content of 96 replicates (per reactor plate) in under 6 h and parallel plate processing increases the analysis throughput substantially.
Assuntos
Glucanos/análise , Ensaios de Triagem em Larga Escala/métodos , Lignina/química , Xilanos/análise , Desidrogenases de Carboidrato/metabolismo , Glucose Oxidase/metabolismoRESUMO
The glycoside hydrolase family 5 endocellulase, E1 (Cel5A), from Acidothermus cellulolyticus was transformed into both Nicotiana tabacum and Zea mays with expression targeted to the cell wall under a constitutive promoter. Here we explore the possibility that in planta expression of endocellulases will allow these enzymes to access their substrates during cell wall construction, rendering cellulose more amenable to pretreatment and enzyme digestion. Tobacco and maize plants were healthy and developed normally compared with the wild type (WT). After thermochemical pretreatment and enzyme digestion, transformed plants were clearly more digestible than WT, requiring lower pretreatment severity to achieve comparable conversion levels. Furthermore, the decreased recalcitrance was not due to post-pretreatment residual E1 activity and could not be reproduced by the addition of exogenous E1 to the biomass prior to pretreatment, indicating that the expression of E1 during cell wall construction altered the inherent recalcitrance of the cell wall.
RESUMO
Transgenic down-regulation of the Pt4CL1 gene family encoding 4-coumarate:coenzyme A ligase (4CL) has been reported as a means for reducing lignin content in cell walls and increasing overall growth rates, thereby improving feedstock quality for paper and bioethanol production. Using hybrid poplar (Populus tremula × Populus alba), we applied this strategy and examined field-grown transformants for both effects on wood biochemistry and tree productivity. The reductions in lignin contents obtained correlated well with 4CL RNA expression, with a sharp decrease in lignin amount being observed for RNA expression below approximately 50% of the nontransgenic control. Relatively small lignin reductions of approximately 10% were associated with reduced productivity, decreased wood syringyl/guaiacyl lignin monomer ratios, and a small increase in the level of incorporation of H-monomers (p-hydroxyphenyl) into cell walls. Transgenic events with less than approximately 50% 4CL RNA expression were characterized by patches of reddish-brown discolored wood that had approximately twice the extractive content of controls (largely complex polyphenolics). There was no evidence that substantially reduced lignin contents increased growth rates or saccharification potential. Our results suggest that the capacity for lignin reduction is limited; below a threshold, large changes in wood chemistry and plant metabolism were observed that adversely affected productivity and potential ethanol yield. They also underline the importance of field studies to obtain physiologically meaningful results and to support technology development with transgenic trees.
Assuntos
Coenzima A Ligases/metabolismo , Lignina/química , Populus/enzimologia , RNA Antissenso/genética , Árvores/crescimento & desenvolvimento , Biomassa , Coenzima A Ligases/genética , Regulação da Expressão Gênica de Plantas , Dados de Sequência Molecular , Fenóis/análise , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/crescimento & desenvolvimento , Populus/genética , Populus/crescimento & desenvolvimento , Madeira/químicaRESUMO
In general, pretreatments are designed to enhance the accessibility of cellulose to enzymes, allowing for more efficient conversion. In this study, we have detected the penetration of major cellulases present in a commercial enzyme preparation (Spezyme CP) into corn stem cell walls following mild-, moderate- and high-severity dilute sulfuric acid pretreatments. The Trichoderma reesei enzymes, Cel7A (CBH I) and Cel7B (EG I), as well as the cell wall matrix components xylan and lignin were visualized within digested corn stover cell walls by immuno transmission electron microscopy (TEM) using enzyme- and polymer-specific antibodies. Low severity dilute-acid pretreatment (20 min at 100 degrees C) enabled <1% of the thickness of secondary cell walls to be penetrated by enzyme, moderate severity pretreatment at (20 min at 120 degrees C) allowed the enzymes to penetrate approximately 20% of the cell wall, and the high severity (20 min pretreatment at 150 degrees C) allowed 100% penetration of even the thickest cell walls. These data allow direct visualization of the dramatic effect dilute-acid pretreatment has on altering the condensed ultrastructure of biomass cell walls. Loosening of plant cell wall structure due to pretreatment and the subsequently improved access by cellulases has been hypothesized by the biomass conversion community for over two decades, and for the first time, this study provides direct visual evidence to verify this hypothesis. Further, the high-resolution enzyme penetration studies presented here provide insight into the mechanisms of cell wall deconstruction by cellulolytic enzymes.
Assuntos
Parede Celular/química , Celulase/análise , Zea mays/química , Cáusticos/farmacologia , Parede Celular/efeitos dos fármacos , Lignina/análise , Microscopia Imunoeletrônica/métodos , Ácidos Sulfúricos/farmacologia , Xilanos/análise , Zea mays/efeitos dos fármacosRESUMO
Pretreatment of corn stover with alkaline peroxide (AP) at pH 11.5 resulted in reduction of lignin content in the residual solids as a function of increasing batch temperature. Scanning electron microscopy of these materials revealed notably more textured surfaces on the plant cell walls as a result of the delignifying pretreatment. As expected, digestion of the delignified samples with commercial cellulase preparations showed an inverse relationship between the content of lignin present in the residual solids after pretreatment and the extent of both glucan and xylan conversion achievable. Digestions with purified enzymes revealed that decreased lignin content in the pretreated solids did not significantly impact the extent of glucan conversion achievable by cellulases alone. Not until purified xylanolytic activities were included with the cellulases were significant improvements in glucan conversion realized. In addition, an inverse relationship was observed between lignin content after pretreatment and the extent of xylan conversion achievable in a 24-h period with the xylanolytic enzymes in the absence of the cellulases. This observation, coupled with the direct relationship between enzymatic xylan and glucan conversion observed in a number of cases, suggests that the presence of lignins may not directly occlude cellulose present in lignocelluloses but rather impact cellulase action indirectly by its association with xylan.
Assuntos
Celulase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Lignina/química , Lignina/isolamento & purificação , Peróxidos/química , Zea mays/química , Zea mays/metabolismo , Cromatografia Líquida de Alta Pressão , Concentração de Íons de HidrogênioRESUMO
The gene encoding a glycosyl hydrolase family 3 xylan 1,4-beta-xylosidase, xlnD, was successfully cloned from Aspergillus niger strain ATCC 10864. The recombinant product was expressed in Aspergillus awamori, purified by column chromatography, and verified by matrix-assisted laser desorption ionization, tandem time of flight (MALDI-TOF/TOF) mass spectroscopy of tryptic digests. The T (max) was determined using differential scanning microcalorimetry (DSC) to be 78.2 degrees C; the K (m) and k (cat) were found to be 255 microM and 13.7 s(-1), respectively, using pNP-beta-D-xylopyranoside as substrate. End-product inhibition by D-xylose was also verified and shown to be competitive; the K (i) for this inhibition was estimated to be 3.3 mM. XlnD was shown to efficiently hydrolyze small xylo-oligomers to monomeric xylose, making it a critical hydrolytic activity in cases where xylose is to be recovered from biomass conversion processes. In addition, the presence of the XlnD was shown to synergistically enhance the ability of an endoxylanase, XynA from Thermomyces lanuginosus, to convert xylan present in selected pretreated lignocellulosic substrates. Furthermore, the addition of the XynA/XlnD complex was effective in enhancing the ability of a simplified cellulase complex to convert glucan present in the substrates.
Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/genética , Lignina/química , Engenharia de Proteínas/métodos , Xilosidases/química , Xilosidases/metabolismo , Adsorção , Aspergillus niger/classificação , Sítios de Ligação , Ativação Enzimática , Estabilidade Enzimática , Enzimas Imobilizadas/química , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Especificidade por Substrato , Xilosidases/isolamento & purificaçãoRESUMO
Two recombinant ferulic acid esterases from Penicillium funiculosum produced in Aspergillus awamori were evaluated for their ability to improve the digestibility of pretreated corn stover. The genes, faeA and faeB, were cloned from P. funiculosum and expressed in A. awamori using their native signal sequences. Both enzymes contain a catalytic domain connected to a family 1 carbohydrate-binding module by a threonine-rich linker peptide. Interestingly, the carbohydrate binding-module is N-terminal in FaeA and C-terminal in FaeB. The enzymes were purified to homogeneity using column chromatography, and their thermal stability was characterized by differential scanning microcalorimetry. We evaluated both enzymes for their potential to enhance the cellulolytic activity of purified Trichoderma reesei Cel7A on pretreated corn stover.
Assuntos
Aspergillus/genética , Aspergillus/metabolismo , Hidrolases de Éster Carboxílico/química , Hidrolases de Éster Carboxílico/metabolismo , Celulose/química , Penicillium/genética , Penicillium/metabolismo , Adsorção , Sítios de Ligação , Hidrolases de Éster Carboxílico/genética , Ativação Enzimática , Estabilidade Enzimática , Enzimas Imobilizadas/química , Penicillium/classificação , Ligação Proteica , Engenharia de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Especificidade da Espécie , Especificidade por SubstratoRESUMO
Significant increases in the depolymerization of corn stover cellulose by cellobiohydrolase I (Cel7A) from Trichoderma reesei were observed using small quantities of non-cellulolytic cell wall-degrading enzymes. Purified endoxylanase (XynA), ferulic acid esterase (FaeA), and acetyl xylan esterase (Axe1) all enhanced Cel7A performance on corn stover subjected to hot water pretreatment. In all cases, the addition of these activities improved the effectiveness of the enzymatic hydrolysis in terms of the quantity of cellulose converted per milligram of total protein. Improvement in cellobiose release by the addition of the non-cellulolytic enzymes ranged from a 13-84% increase over Cel7A alone. The most effective combinations included the addition of both XynA and Axe1, which synergistically enhance xylan conversions resulting in additional synergistic improvements in glucan conversion. Additionally, we note a direct relationship between enzymatic xylan removal in the presence of XynA and the enhancement of cellulose hydrolysis by Cel7A.
Assuntos
Aspergillus/enzimologia , Celulose 1,4-beta-Celobiosidase/metabolismo , Endo-1,4-beta-Xilanases/metabolismo , Esterases/metabolismo , Trichoderma/enzimologia , Zea mays/metabolismo , Celobiose/metabolismo , Endo-1,4-beta-Xilanases/isolamento & purificação , Esterases/isolamento & purificação , Hidrólise , Temperatura , Água , Xilanos/metabolismoRESUMO
Electron microscopy of lignocellulosic biomass following high-temperature pretreatment revealed the presence of spherical formations on the surface of the residual biomass. The hypothesis that these droplet formations are composed of lignins and possible lignin carbohydrate complexes is being explored. Experiments were conducted to better understand the formation of these "lignin" droplets and the possible implications they might have on the enzymatic saccharification of pretreated biomass. It was demonstrated that these droplets are produced from corn stover during pretreatment under neutral and acidic pH at and above 130 degrees C, and that they can deposit back onto the surface of residual biomass. The deposition of droplets produced under certain pretreatment conditions (acidic pH; T > 150 degrees C) and captured onto pure cellulose was shown to have a negative effect (5-20%) on the enzymatic saccharification of this substrate. It was noted that droplet density (per unit area) was greater and droplet size more variable under conditions where the greatest impact on enzymatic cellulose conversion was observed. These results indicate that this phenomenon has the potential to adversely affect the efficiency of enzymatic conversion in a lignocellulosic biorefinery.