Hydrolysis Of Plant Biomass Research Articles

A novel glucose-tolerant β-glucosidase from the salivary gland of the termite Nasutitermes takasagoensis

Lignocellulose, a major constituent of plant biomass, is a potential feedstock for bioconversion. However, one of the main bottlenecks to produce bioethanol is the high cost of enzymes used for hydrolysis of insoluble substrates to the fermentable sugar glucose. Hence, research on cellulases that enable efficient hydrolysis of plant biomass is a key point. Among cellulases, β-glucosidase (EC 3.2.1.21) acts in the final step of cellulose saccharification, liberating glucose. Moreover, it reduces the inhibition of endoglucanase (EC 3.2.1.4) and cellobiohydrolase (EC 3.2.1.91) through hydrolysis of cellobiose and cello-oligosaccharides (Bhatia et al., 2002; Lynd et al., 2002). β-Glucosidase itself is also subjected to the product inhibition which makes the identification of β-glucosidases insensitive to or stimulated by glucose of special interest in bioconversion (Perezpons et al., 1995; Uchima et al., 2011, 2012). Interestingly, in nature, termites are insects with a great capacity/facility to degrade wood; 74‒99% of the cellulose ingested is hydrolyzed (Prins and Kreulen, 1991). The high efficiency of termites in the degradation of plant biomass motivated the development of the present work. The aim of this study is to analyze some features of an endogenous β-glucosidase from the salivary gland of the higher termite Nasutitermes takasagoensis, hereafter called G1sgNtBG1 (glycoside hydrolase family 1), that might be useful in bioethanol production. N. takasagoensis cDNA was used as a donor of the target gene encoding G1sgNtBG1 (GenBank accession no. AB508954). Escherichia coli DH5α was used for DNA manipulations, and Pichia pastoris strain KM71 (his4 aox1::ARG4, Invitrogen) was used as a host for heterologous expression of G1sgNtBG1. The episomal plasmid pBGP3 (Uchima and Arioka, 2012) was employed for the expression of mycand hexahistidine-tagged G1sgNtBG1 in P. pastoris; electroporation of the expression plasmid into P. pastoris was done according to the standard method (Cereghino and Cregg, 2000). Purification of G1sgNtBG1 was performed using a Ni2+-NTA Purification System following the manufacturer’s instructions (Qiagen). β-Glucosidase activity was routinely assayed using 10 μM of p-nitrophenyl-β-D-glucopyranoside (pNPG; Sigma, St. Louis, USA) as a substrate according to the method previously described (Uchima et al., 2011) and the release of p-nitrophenol (pNP) was measured at A410. G1sgNtBG1 activity was also determined by the release of glucose from saccharides and cello-oligosaccharides using glucose oxidase-mutarotase reagent (Glucose CII Test Wako; Wako Pure Chemical Co., Tokyo, Japan) according to the method previously deJ. Gen. Appl. Microbiol., 59, 141‒145 (2013)

Microbial d-xylonate production

d-Xylonic acid is a versatile platform chemical with reported applications as complexing agent or chelator, in dispersal of concrete, and as a precursor for compounds such as co-polyamides, polyesters, hydrogels and 1,2,4-butanetriol. With increasing glucose prices, d-xylonic acid may provide a cheap, non-food derived alternative for gluconic acid, which is widely used (about 80 kton/year) in pharmaceuticals, food products, solvents, adhesives, dyes, paints and polishes. Large-scale production has not been developed, reflecting the current limited market for d-xylonate. d-Xylonic acid occurs naturally, being formed in the first step of oxidative metabolism of d-xylose by some archaea and bacteria via the action of d-xylose or d-glucose dehydrogenases. High extracellular concentrations of d-xylonate have been reported for various bacteria, in particular Gluconobacter oxydans and Pseudomonas putida. High yields of d-xylonate from d-xylose make G. oxydans an attractive choice for biotechnical production. G. oxydans is able to produce d-xylonate directly from plant biomass hydrolysates, but rates and yields are reduced because of sensitivity to hydrolysate inhibitors. Recently, d-xylonate has been produced by the genetically modified bacterium Escherichia coli and yeast Saccharomyces cerevisiae and Kluyveromyces lactis. Expression of NAD+-dependent d-xylose dehydrogenase of Caulobacter crescentus in either E. coli or in a robust, hydrolysate-tolerant, industrial Saccharomyces cerevisiae strain has resulted in d-xylonate titres, which are comparable to those seen with G. oxydans, at a volumetric rate approximately 30 % of that observed with G. oxydans. With further development, genetically modified microbes may soon provide an alternative for production of d-xylonate at industrial scale.

Simulation Analysis of the Temperature Dependence of Lignin Structure and Dynamics

Lignins are hydrophobic, branched polymers that regulate water conduction and provide protection against chemical and biological degradation in plant cell walls. Lignins also form a residual barrier to effective hydrolysis of plant biomass pretreated at elevated temperatures in cellulosic ethanol production. Here, the temperature-dependent structure and dynamics of individual softwood lignin polymers in aqueous solution are examined using extensive (17 μs) molecular dynamics simulations. With decreasing temperature the lignins are found to transition from mobile, extended to glassy, compact states. The polymers are composed of blobs, inside which the radius of gyration of a polymer segment is a power-law function of the number of monomers comprising it. In the low temperature states the blobs are interpermeable, the polymer does not conform to Zimm/Stockmayer theory, and branching does not lead to reduction of the polymer size, the radius of gyration being instead determined by shape anisotropy. At high temperatures the blobs become spatially separated leading to a fractal crumpled globule form. The low-temperature collapse is thermodynamically driven by the increase of the translational entropy and density fluctuations of water molecules removed from the hydration shell, thus distinguishing lignin collapse from enthalpically driven coil-globule polymer transitions and providing a thermodynamic role of hydration water density fluctuations in driving hydrophobic polymer collapse. Although hydrophobic, lignin is wetted, leading to locally enhanced chain dynamics of solvent-exposed monomers. The detailed characterization obtained here provides insight at atomic detail into processes relevant to biomass pretreatment for cellulosic ethanol production and general polymer coil-globule transition phenomena.

Conformational Analyses of the Reaction Coordinate of Glycosidases

The enzymatic hydrolysis of the glycosidic bond is catalyzed by diverse enzymes generically termed glycoside hydrolases (hereafter GHs) or glycosidases. The many sequence-based families of glycosidases have served as a rich hunting ground for enzymologists for years. Not only are these enzymes of fundamental interest, providing paradigms for enzymatic catalysis that extend beyond the bounds of carbohydrate chemistry, but the enzymes themselves play myriad essential roles in diverse biological processes. The wide utility of glycosidases, from their industrial harnessing in the hydrolysis of plant biomass to their roles in human physiology and disease, has engendered a large scientific constituency with an interest in glycosidase chemistry. A fascinating thread of this research, and one with major impact on the design of enzyme inhibitors, is the conformational analysis of reaction pathways within the diverse families. These GH families provide a large pallet of enzymes with which chemists have attempted to depict the conformational landscape of glycosidase action. In this Account, we review three-dimensional insight into the conformational changes directed by glycosidases, primarily from structural observations of the stable enzyme-ligand species adjacent to the transition state (or states) and of enzyme-inhibitor complexes. We further show how recent computational advances dovetail with structural insight to provide a quantum mechanical basis for glycosidase action. The glycosidase-mediated hydrolysis of the acetal or ketal bond in a glycoside may occur with either inversion or retention of the configuration of the anomeric carbon. Inversion involves a single step and transition state, whereas retention, often referred to as the double displacement, is a two-step process with two transition states. The single transition state for the inverting enzymes and the two transition states (those flanking the covalent intermediate) in the double displacement have been shown to have substantial oxocarbenium ion character. The dissociative nature of these transition states results in significant relative positive charge accumulation on the pyranose ring. The delocalization of lone-pair electrons from the ring oxygen that stabilizes the cationic transition state implies that at, or close to, the transition states the pyranose will be distorted away from its lowest energy conformation to one that favors orbital overlap. Over the preceding decade, research has highlighted the harnessing of noncovalent interactions to aid this distortion of the sugar substrates from their lowest energy chair conformation to a variety of different boat, skew boat, and half-chair forms, each of which favors catalysis with a given enzyme and substrate. Crystallographic observation of stable species that flank the transition state (or states), of both retaining and inverting glycosidases, has allowed a description of their conformational itineraries, illustrating how enzymes facilitate the "electrophilic migration" of the anomeric center along the reaction coordinate. The blossoming of computational approaches, such as ab initio metadynamics, has underscored the quantum mechanical basis for glycoside hydrolysis. Conformational analyses highlight not only the itineraries used by enzymes, enabling their inhibition, but are also reflected in the nonenzymatic synthesis of glycosides, wherein chemists mimic strategies found in nature.

The α-glucuronidase Agu1 from Schizophyllum commune is a member of a novel glycoside hydrolase family (GH115)

Schizophyllum commune produces an α-glucuronidase that is active on polymeric xylan, while the ascomycete α-glucuronidases are only active on xylan oligomers. In this study, we have identified the gene (agu1) encoding this enzyme and confirmed the functionality by overexpression of the gene in S. commune and degradation of aldopentauronic acids, (MeGlcA)(3)-Xyl(4), in the cultivation medium of the transformants. Expression analysis demonstrated that agu1 is not co-regulated with the predominant xylanase-encoding gene (xynA) of S. commune. The detailed sequence analysis of Agu1 demonstrated that this gene belongs to a novel glycoside hydrolase family (GH115) that also contains candidate genes from ascomycete fungi and bacteria. Phylogenetic analysis showed that the fungal GH115 α-glucuronidases are distinctly separate from the prokaryotic clade and distributed over three branches. The identification of putative genes encoding this enzyme in industrial fungi, such as Aspergillus oryzae and Hypocrea jecorina, will provide a starting point for further analysis of the importance of this enzyme for the hydrolysis of plant biomass.

Batch and continuous culture-based selection strategies for acetic acid tolerance in xylose-fermenting Saccharomyces cerevisiae

Acetic acid tolerance of Saccharomyces cerevisiae is crucial for the production of bioethanol and other bulk chemicals from lignocellulosic plant-biomass hydrolysates, especially at a low pH. This study explores two evolutionary engineering strategies for the improvement of acetic acid tolerance of the xylose-fermenting S. cerevisiae RWB218, whose anaerobic growth on xylose at pH 4 is inhibited at acetic acid concentrations >1 g L(-1) : (1) sequential anaerobic, batch cultivation (pH 4) at increasing acetic acid concentrations and (2) prolonged anaerobic continuous cultivation without pH control, in which acidification by ammonium assimilation generates selective pressure for acetic acid tolerance. After c. 400 generations, the sequential-batch and continuous selection cultures grew on xylose at pH≤4 with 6 and 5 g L(-1) acetic acid, respectively. In the continuous cultures, the specific xylose-consumption rate had increased by 75% to 1.7 g xylose g(-1) biomass h(-1) . After storage of samples from both selection experiments at -80 °C and cultivation without acetic acid, they failed to grow on xylose at pH 4 in the presence of 5 g L(-1) acetic acid. Characterization in chemostat cultures with linear acetic acid gradients demonstrated an acetate-inducible acetic acid tolerance in samples from the continuous selection protocol.

Expression in Escherichia coli, refolding and crystallization of Aspergillus niger feruloyl esterase A using a serial factorial approach

Hydrolysis of plant biomass is achieved by the combined action of enzymes secreted by microorganisms and directed against the backbone and the side chains of plant cell wall polysaccharides. Among side chains degrading enzymes, the feruloyl esterase A (FAEA) specifically removes feruloyl residues. Thus, FAEA has potential applications in a wide range of industrial processes such as paper bleaching or bio-ethanol production. To gain insight into FAEA hydrolysis activity, we solved its crystal structure. In this paper, we report how the use of four consecutive factorial approaches (two incomplete factorials, one sparse matrix, and one full factorial) allowed expressing in Escherichia coli, refolding and then crystallizing Aspergillus niger FAEA in 6 weeks. Culture conditions providing the highest expression level were determined using an incomplete factorial approach made of 12 combinations of four E. coli strains, three culture media and three temperatures (full factorial: 36 combinations). Aspergillus niger FAEA was expressed in the form of inclusion bodies. These were dissolved using a chaotropic agent, and the protein was purified by affinity chromatography on Ni column under denaturing conditions. A suitable buffer for refolding the protein eluted from the Ni column was found using a second incomplete factorial approach made of 96 buffers (full factorial: 3840 combinations). After refolding, the enzyme was further purified by gel filtration, and then crystallized following a standard protocol: initial crystallization conditions were found using commercial crystallization screens based on a sparse matrix. Crystals were then optimized using a full factorial screen.

Alcoholic fermentation of carbon sources in biomass hydrolysates by Saccharomyces cerevisiae: current status

Fuel ethanol production from plant biomass hydrolysates by Saccharomyces cerevisiae is of great economic and environmental significance. This paper reviews the current status with respect to alcoholic fermentation of the main plant biomass-derived monosaccharides by this yeast. Wild-type S. cerevisiae strains readily ferment glucose, mannose and fructose via the Embden-Meyerhof pathway of glycolysis, while galactose is fermented via the Leloir pathway. Construction of yeast strains that efficiently convert other potentially fermentable substrates in plant biomass hydrolysates into ethanol is a major challenge in metabolic engineering. The most abundant of these compounds is xylose. Recent metabolic and evolutionary engineering studies on S. cerevisiae strains that express a fungal xylose isomerase have enabled the rapid and efficient anaerobic fermentation of this pentose. L: -Arabinose fermentation, based on the expression of a prokaryotic pathway in S. cerevisiae, has also been established, but needs further optimization before it can be considered for industrial implementation. In addition to these already investigated strategies, possible approaches for metabolic engineering of galacturonic acid and rhamnose fermentation by S. cerevisiae are discussed. An emerging and major challenge is to achieve the rapid transition from proof-of-principle experiments under 'academic' conditions (synthetic media, single substrates or simple substrate mixtures, absence of toxic inhibitors) towards efficient conversion of complex industrial substrate mixtures that contain synergistically acting inhibitors.

Purification of Carbohydrate-Containing Substrates from Plant Biomass Hydrolysates

Carbohydrate-containing substrates are produced in Russia by means of dilute acid hydrolysis of wood and vegetable residues. Hydrolysates contain 3 to 4% monosaccharides and nearly 1% impurities. The common schemes used to prepare the hydrolysate for fermentation include cooling by self-evaporation; inversion of oligosaccharides; neutralization of sulfuric (and organic) acids; addition of mineral food substances containing nutrient N, P, and K salts; aeration to coagulate colloidal substances; and sedimentation of residues. Our work led to the suggestion that a flocculation step using cationic polyelectrolytes be added to purify more completely the substrates from lignin-huminic substances (LHS). This method permits the transfer of a part of the dissolved and colloidal LHS into a suspended state and the removal of more than 90% of the suspended materials, thereby increasing the yield and quality of ethanol and fodder yeast. Three industrial plants have reported successful results using this method in the past few years. Flocculation has also proven itself capable of clearing waste culture liquids of their dispersed phases by extents of 30–60%, as measured by chemical oxygen demand, color depth, or lignin content parameters, prior to consideration of such wastewater for recycling.

Crystal structure of the catalytic domain of the beta-1,4-glycanase cex from Cellulomonas fimi.

beta-1,4-Glycanases, principally cellulases and xylanases, are responsible for the hydrolysis of plant biomass. The bifunctional beta-1,4-xylanase/glucanase Cex from the bacterium Cellulomonas fimi, one of a large family of cellulases/xylanases, depolymerizes oligosaccharides and releases a disaccharide unit from the substrate nonreducing end. Hydrolysis occurs with net retention of the anomeric configuration of the sugar through a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. The active site nucleophile, Glu233, has been unambiguously identified by trapping of such an intermediate [Tull et al. (1991) J. Biol. Chem. 266, 15621-15625] and the acid/base catalyst, Glu127, by detailed kinetic analysis of mutants [MacLeod et al. (1994) Biochemistry 33, 6371-6376]. However, little is known about the enzyme's overall folding and its active site architecture. We report here the high-resolution crystal structure of the catalytic domain of Cex. The atomic structure refinement results in a model that includes 2400 protein atoms and 45 water molecules, with an R-factor of 0.217 for data extending to 1.8-A resolution. The protein forms an eight-parallel-stranded alpha/beta-barrel, which is a novel folding pattern for a microbial beta-glycanase. The active site, inferred from the location of Glu233, Glu127, and other conserved residues, is an open cleft on the carboxy-terminal end of the alpha/beta-barrel. An extensive hydrogen-bonding network stabilizes the ionization states of the key residues; in particular, the Asp235-His205-Glu233 hydrogen-bonding network may play a role in modulating the ionization state of Glu233 and in controlling local charge balance during the reaction.

ChemInform Abstract: Ethanol Production from Sugars Derived from Plant Biomass by a Novel Fungus.

The concept of transforming renewable plant biomass, and in particular organic waste, to fuels or chemicals continues to attract attention as nonrenewable fossil fuel reserves decrease1,2. Cellulose and hemicellulose, which comprise more than 70% of plant biomass and are the two most abundant organic compounds in the biosphere, occur in the wastes from agriculture and forestry as well as from vegetable and fruit processing and municipal waste treatment. Recycling of these wastes to useful products helps to prevent pollution. After enzymatic or acid hydrolysis, plant biomass yields mostly cellobiose, xylose and glucose together with mannose, galactose and arabinose. Microorganisms which can ferment such a wide range of sugars, at high concentrations, into a high concentration of ethanol have not been reported previously. Here we report on a novel fungus, Paecilomyces sp. NF1, which is capable of fermenting all of the major sugars derived from hydrolysis of plant biomass to ethanol. In xylose fermentation, the ethanol yield of this fungus remained almost unchanged when the concentration of xylose was increased from 20 to 200 g l−1. The fungus also produced good yields of ethanol from glucose, fructose, cellobiose, arabinose, galactose, mannose and ribose, as well as from the simultaneous saccharification and fermentation (SSF) of cellulose with hemicellulose, and from acid hydrolysates of plant biomass.

Ethanol production from sugars derived from plant biomass by a novel fungus

The concept of transforming renewable plant biomass, and in particular organic waste, to fuels or chemicals continues to attract attention as nonrenewable fossil fuel reserves decrease1,2. Cellulose and hemicellulose, which comprise more than 70% of plant biomass and are the two most abundant organic compounds in the biosphere, occur in the wastes from agriculture and forestry as well as from vegetable and fruit processing and municipal waste treatment. Recycling of these wastes to useful products helps to prevent pollution. After enzymatic or acid hydrolysis, plant biomass yields mostly cellobiose, xylose and glucose together with mannose, galactose and arabinose. Microorganisms which can ferment such a wide range of sugars, at high concentrations, into a high concentration of ethanol have not been reported previously. Here we report on a novel fungus, Paecilomyces sp. NF1, which is capable of fermenting all of the major sugars derived from hydrolysis of plant biomass to ethanol. In xylose fermentation, the ethanol yield of this fungus remained almost unchanged when the concentration of xylose was increased from 20 to 200 g l−1. The fungus also produced good yields of ethanol from glucose, fructose, cellobiose, arabinose, galactose, mannose and ribose, as well as from the simultaneous saccharification and fermentation (SSF) of cellulose with hemicellulose, and from acid hydrolysates of plant biomass.

HPLC-analyses of plant biomass hydrolysis and fermentation solutions

In this work the analytical separation of biomass hydrolysis and fermentation products is described. Aminobonded phase and ion exchange materials (HPX 87H, HPX 42A, μ Spherogel Carbohydrate) were used as HPLC stationary phases for the determination of monomeric and oligomeric carbohydrates, carbohydrate degradation and fermentation products. The HPX 42A and the amino-bonded phase columns are especially suitable for the analysis of the gluco oligomers. The HPX 87H column separates the monomeric sugars and their degradation and fermentation products (e.g. furfurals and ethanol) very well. Using the μ Spherogel Carbohydrate column the lower gluco-oligomers as well as the monomeric sugars and their degradation and fermentation products, can be determined within 35 minutes.