Genomic dissection of Niallia sp. for potential application in lignocellulose hydrolysis and bioremediation.

Genus Niallia has recently been separated taxonomic group from the Bacillus based on conserved signature indels in the genome. Unlike bioremediation, its role in plant biomass hydrolysis has not garnered considerable attention. The present study investigates the genomic potential of a novel Niallia sp. CRN 25 for applications in lignocellulose hydrolysis, significant enzyme production, and bioremediation. The CRN 25 strain exhibits xylosidase, cellobiosidase, α-arabinosidase, and α-D-galactosidase activity as 0.03 U/ml whereas ß-D-glucosidase and glucuronidase as 0.06 U/ml and 0.01 U/ml, respectively. Further genome sequencing reveals nine copies of GH43 gene coding for hemicellulose-specific xylanase enzyme attached to the CBM 6 domain for increased processivity. The presence of ß-glucosidase and ß-galactosidase indicates the possible application of CRN 25 in facilitating the valorization of plant biomass into value-added products. Apart from this, genes of FMN-dependent NADH-azoreductase, cytochrome P450, and nitrate reductase, playing a crucial role in bioremediation processes, were annotated. Biosynthetic gene clusters (BGCs), responsible for synthesizing specialized metabolites of terpenes and lasso peptides, were also found in the genome. Conclusively genomic sketch of Niallia sp. CRN 25 reveals versatile metabolic potential for diverse environmental applications.

Biochemical characterization of an engineered bifunctional xylanase/feruloyl esterase and its synergistic effects with cellulase on lignocellulose hydrolysis.

Herein, the xylanase and feruloyl esterase domains of the xylanase/feruloyl esterase bifunctional enzyme (Xyn-Fae) from Prevotella ruminicola 23 were identified using N- and C-terminal truncation mutagenesis. In addition, a novel and more efficient xylanase/feruloyl esterase bifunctional enzyme XynII-Fae was constructed, and its synergistic action with a commercial cellulase for lignocellulose hydrolysis was studied. When 40% cellulase was replaced by XynII-Fae, the production of reducing sugars increased by 65% than that with the cellulase alone, and the conversions of xylan and glucan were increased by 125.1% and 54.3%, respectively. When 80% cellulase was substituted by XynII-Fae, up to 43.5 µg/mL ferulic acid and 418.7 µg/mL acetic acid were obtained. The XynII-Fae could also accelerate the hydrolysis of wheat straw and sugarcane bagasse with commercial cellulase. These results indicated that the synergistic action of XynII-Fae with cellulase could dramatically improve the hydrolysis efficiency of lignocellulose, showing the great potential for industrial applications.

Glucan Conversion and Membrane Recovery of Biomimetic Cellulosomes During Lignocellulosic Biomass Hydrolysis.

Enzyme immobilization has been identified as one way to recycle enzymes and reduce processing costs during enzymatic hydrolysis of lignocellulosic materials. However, most immobilization methods have not been attractive to lignocellulosic processing plants. In this study, cellulase enzymes were attached to a copolymer of glycidyl methacrylate (GMA) and poly(ethylene glycol) methyl ether methacrylate (PEGMA) to make polymer-enzyme conjugates (PECs) and facilitate recovery using a 50-kDa molecular weight cutoff membrane. Glucan conversion during biomass hydrolysis was investigated using new PECs and PECs recovered after an initial hydrolysis stage. Enzyme immobilization on PECs did not reduce effectiveness during the initial hydrolysis. Temperature and pH showed similar effects on free enzymes and PECs. PECs facilitated higher conversion rates than free enzymes at high biomass loadings. Recovered PECs were used to achieve approximately 100% glucan conversion in a subsequent hydrolysis step when supplemented with 40% of the free enzyme used in the first stage. The combination of PECs and membrane recovery has the potential to reduce hydrolysis cost during cellulosic bioprocessing.

Plant cell wall architecture guided design of CBM3-GH11 chimeras with enhanced xylanase activity using a tandem repeat left-handed ß-3-prism scaffold.

Effective use of plant biomass as an abundant and renewable feedstock for biofuel production and biorefinery requires efficient enzymatic mobilization of cell wall polymers. Knowledge of plant cell wall composition and architecture has been exploited to develop novel multifunctional enzymes with improved activity against lignocellulose, where a left-handed ß-3-prism synthetic scaffold (BeSS) was designed for insertion of multiple protein domains at the prism vertices. This allowed construction of a series of chimeras fusing variable numbers of a GH11 ß-endo-1,4-xylanase and the CipA-CBM3 with defined distances and constrained relative orientations between catalytic domains. The cellulose binding and endoxylanase activities of all chimeras were maintained. Activity against lignocellulose substrates revealed a rapid 1.6- to 3-fold increase in total reducing saccharide release and increased levels of all major oligosaccharides as measured by polysaccharide analysis using carbohydrate gel electrophoresis (PACE). A construct with CBM3 and GH11 domains inserted in the same prism vertex showed highest activity, demonstrating interdomain geometry rather than number of catalytic sites is important for optimized chimera design. These results confirm that the BeSS concept is robust and can be successfully applied to the construction of multifunctional chimeras, which expands the possibilities for knowledge-based protein design.

Thermostable endoglucanase gene derived by amplification from the genomic DNA of a cellulose-enriched mixed culture from mudspring water of Mt. Makiling, Laguna, Philippines.

Culture-independent molecular-based approaches can be used to identify genes of interest from environmental sources that have desirable properties such as thermo activity. For this study, a putative thermo stable endoglucanase gene was identified from a mixed culture resulting from the inoculation of Brock-CMcellulose (1%) broth with mudspring water from Mt. Makiling, Laguna, Philippines that had been incubated at 90 °C. Genomic DNA was extracted from the cellulose-enriched mixed culture and endo1949 forward and reverse primers were used to amplify the endoglucanase gene, which was cloned into pCR-script plasmid vector. Blastn alignment of the sequenced insert revealed 99.69% similarity to the glycosyl hydrolase, sso1354 (CelA1; Q97YG7) from Saccharolobus solfataricus. The endoglucanase gene (GenBank accession number MK984682) was determined to be 1,021 nucleotide bases in length, corresponding to 333 amino acids with a molecular mass of ~ 37 kDa. The endoglucanase gene was inserted into a pET21 vector and transformed in E. coli BL21 for expression. Partially purified recombinant Mt. Makiling endoglucanase (MM-Engl) showed a specific activity of 187.61 U/mg and demonstrated heat stability up to 80 °C. The thermo-acid stable endoglucanase can be used in a supplementary hydrolysis step to further hydrolyze the lignocellulosic materials that were previously treated under high temperature-dilute acid conditions, thereby enhancing the release of more glucose sugars for bioethanol production.

LPMO AfAA9_B and Cellobiohydrolase AfCel6A from A. fumigatus Boost Enzymatic Saccharification Activity of Cellulase Cocktail.

Cellulose is the most abundant polysaccharide in lignocellulosic biomass, where it is interlinked with lignin and hemicellulose. Bioethanol can be produced from biomass. Since breaking down biomass is difficult, cellulose-active enzymes secreted by filamentous fungi play an important role in degrading recalcitrant lignocellulosic biomass. We characterized a cellobiohydrolase (AfCel6A) and lytic polysaccharide monooxygenase LPMO (AfAA9_B) from Aspergillus fumigatus after they were expressed in Pichia pastoris and purified. The biochemical parameters suggested that the enzymes were stable; the optimal temperature was ~60 °C. Further characterization revealed high turnover numbers (kcat of 147.9 s-1 and 0.64 s-1, respectively). Surprisingly, when combined, AfCel6A and AfAA9_B did not act synergistically. AfCel6A and AfAA9_B association inhibited AfCel6A activity, an outcome that needs to be further investigated. However, AfCel6A or AfAA9_B addition boosted the enzymatic saccharification activity of a cellulase cocktail and the activity of cellulase Af-EGL7. Enzymatic cocktail supplementation with AfCel6A or AfAA9_B boosted the yield of fermentable sugars from complex substrates, especially sugarcane exploded bagasse, by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass by up to 95%. The synergism between the cellulase cocktail and AfAA9_B was enzyme- and substrate-specific, which suggests a specific enzymatic cocktail for each biomass.

Physico-chemical kinetic modelling of hydrolysis of a steam-explosion pre-treated corn stover: A two-step approach.

A physico-chemical kinetic model for the hydrolysis of pre-treated corn stover is proposed. This model takes into account two reactions in series, the hydrolysis of cellulose to cellobiose and the production of glucose from cellobiose. Experiments have been carried out with an industrial enzymatic cocktail from Trichoderma reesei containing endo and exoglucanases and a very low activity of ß-glucosidase. Kinetic parameters were calculated by fitting the proposed model to experimental data of cellulose and glucose concentrations with time. The kinetic parameters fulfilled all relevant statistical and physical criteria. The kinetic model has been validated with published saccharification data regarding differently pre-treated corn stover and enzymatic cocktail, in this case with a very high ß-glucosidase activity (as it is common in modern industrial cellulase cocktails). In both cases, the kinetic model proposed could be fitted very appropriately to cellulose hydrolysis data.

Distinct wall polymer deconstruction for high biomass digestibility under chemical pretreatment in Miscanthus and rice.

Miscanthus is a leading bioenergy crop and rice provides enormous biomass for biofuels. Using Calcofluor White staining, this work in situ observed an initial lignocellulose hydrolysis in two distinct Miscanthus accessions, rice cultivar (NPB), and Osfc16 mutant after mild chemical pretreatments. In comparison, the M. sin and Osfc16 respectively exhibited weak Calcofluor fluorescence compared to the M. sac and NPB during enzymatic hydrolysis, consistent with the high biomass saccharification detected in vitro. Using xyloglucan-directed monoclonal antibodies (mAbs), xyloglucan deconstruction was observed from initial cellulose hydrolysis, whereas the M. sin and Osfc16 exhibited relatively strong immunolabeling using xylan-directed mAb, confirming previous findings of xylan positive impacts on biomass saccharification. Furthermore, the M. sin showed quick disappearance of RG-I immunolabeling with varied HG labelings between acid and alkali pretreatments. Hence, this study demonstrated a quick approach to explore wall polymer distinct deconstruction for enhanced biomass saccharification under chemical pretreatment in bioenergy crops.

The effect of a lytic polysaccharide monooxygenase and a xylanase from Gloeophyllum trabeum on the enzymatic hydrolysis of lignocellulosic residues using a commercial cellulase.

Hydrolysis of lignocellulosic biomass depends on the concerted actions of cellulases and accessory proteins. In this work we examined the combined action of two auxiliary proteins from the brown rot fungus Gloeophyllum trabeum: a family AA9 lytic polysaccharide monooxygenase (GtLPMO) and a GH10 xylanase (GtXyn10A). The enzymes were produced in the heterologous host Pichia pastoris. In the presence of an electron source, GtLPMO increased the activity of a commercial cellulase on filter paper, and the xylanase activity of GtXyn10A on beechwood xylan. Mixtures of GtLPMO, GtXyn10A and Celluclast 1.5L were used for hydrolysis of pretreated wheat straw. Results showed that a mixture of 60% Celluclast 1.5L, 20% GtXyn10A and 20% GtLPMO increased total reducing sugar production by 54%, while the conversions of glucan to glucose and xylan to xylose were increased by 40 and 57%, respectively. This suggests that GtLPMO can contribute to lignocellulose hydrolysis, not only by oxidative activity on glycosidic bonds, but also to hemicellulose through the oxidation of xylosyl bonds in xylan. The concerted action of these auxiliary enzymes may significantly improve large-scale recovery of sugars from lignocellulose.

Strain Breeding Enhanced Heterologous Cellobiohydrolase Secretion by Saccharomyces cerevisiae in a Protein Specific Manner.

The yeast Saccharomyces cerevisiae has a long association with alcoholic fermentation industries and has received renewed interest as a biocatalyst for second-generation bioethanol production. Rational engineering strategies are used to create yeast strains for consolidated bioprocessing of lignocellulosic biomass. Although significant progress is made in this regard with the expression of different cellulolytic activities in yeast, cellobiohydrolase (CBH) titers remain well below ideal levels. Through classical breeding, S. cerevisiae strains with up to twofold increased CBH secretion titers is obtained in strains expressing a single gene copy. An increase of up to 3.5-fold in secreted cellobiohydrolase activity is subsequently shown for strains expressing the heterologous gene on a high copy episomal vector. To our knowledge, this is the first report of classical breeding being used to enhance heterologous protein secretion and also the most significant enhancement of CBH secretion in yeast yet reported. This enhanced secretion phenotype is specific for cellobiohydrolase I secretion, indicating that reporter protein properties might be a major determining factor for efficient protein secretion in yeast. By exploring the latent potential of different S. cerevisiae strains, the authors show that the allele pool of various strains is a valuable engineering resource to enhance secretion in yeast.

Insights into the functionality and stability of designer cellulosomes at elevated temperatures.

Enzymatic breakdown of lignocellulose is a major limiting step in second generation biorefineries. Assembly of the necessary activities into designer cellulosomes increases the productivity of this step by enhancing enzyme synergy through the proximity effect. However, most cellulosomal components are obtained from mesophilic microorganisms, limiting the applications to temperatures up to 50 °C. We hypothesized that a scaffoldin, comprising modular components of mainly mesophilic origin, can function at higher temperatures when combined with thermophilic enzymes, and the resulting designer cellulosomes could be employed in higher temperature reactions. For this purpose, we used a tetravalent scaffoldin constituted of three cohesins of mesophilic origin as well as a cohesin and cellulose-binding module derived from the thermophilic bacterium Clostridium thermocellum. The scaffoldin was combined with four thermophilic enzymes from Geobacillus and Caldicellulosiruptor species, each fused with a dockerin whose specificity matched one of the cohesins. We initially verified that the biochemical properties and thermal stability of the resulting chimeric enzymes were not affected by the presence of the mesophilic dockerins. Then we examined the stability of the individual single-enzyme-scaffoldin complexes and the full tetravalent cellulosome showing that all complexes are stable and functional for at least 6 h at 60 °C. Finally, within this time frame and conditions, the full complex appeared over 50 % more efficient in the hydrolysis of corn stover compared to the free enzymes. Overall, the results support the utilization of scaffoldin components of mesophilic origin at relatively high temperatures and provide a framework for the production of designer cellulosomes suitable for high temperature biorefinery applications.

Synthesis and characterization of new low-cost ILs based on butylammonium cation and application to lignocellulose hydrolysis.

Fourteen ionic liquids (ILs) were obtained and characterized by nuclear magnetic resonance and infra-red spectroscopy. One of these liquids, n-butylammonium acetate, was used in the treatment of coir fiber prior to acid hydrolysis. For this purpose, the fiber was pulped with 8% (w/w) sodium hydroxide for 6h under 2.5 atm pressure at 137°C and then treated with IL for 2h at 90°C. The samples were hydrolyzed in acetic acid at different concentrations and temperatures. The reducing sugar concentrations were determined in all samples, and the optimal hydrolysis conditions were established (32.2% acetic acid at 122.4°C). The reaction time was also studied, and the conversion was maximized at 3h. Under the best hydrolysis conditions, crude fiber, pulping fiber, and IL-treated fiber were hydrolyzed to yield 8.53%, 47.58%, and 89.75% of reducing sugars, respectively.

Rate-constraining changes in surface properties, porosity and hydrolysis kinetics of lignocellulose in the course of enzymatic saccharification.

BACKGROUND: Explaining the reduction of hydrolysis rate during lignocellulose hydrolysis is a challenge for the understanding and modelling of the process. This article reports the changes of cellulose and lignin surface areas, porosity and the residual cellulase activity during the hydrolysis of autohydrolysed wheat straw and delignified wheat straw. The potential rate-constraining mechanisms are assessed with a simplified kinetic model and compared to the observed effects, residual cellulase activity and product inhibition. RESULTS: The reaction rate depended exclusively on the degree of hydrolysis, while enzyme denaturation or time-dependent changes in substrate hydrolysability were absent. Cellulose surface area decreased linearly with hydrolysis, in correlation with total cellulose content. Lignin surface area was initially decreased by the dissolution of phenolics and then remained unchanged. The dissolved phenolics did not contribute to product inhibition. The porosity of delignified straw was decreased during hydrolysis, but no difference in porosity was detected during the hydrolysis of autohydrolysed straw. CONCLUSIONS: Although a hydrolysis-dependent increase of non-productive binding capacity of lignin was not apparent, the dependence of hydrolysis maxima on the enzyme dosage was best explained by partial irreversible product inhibition. Cellulose surface area correlated with the total cellulose content, which is thus an appropriate approximation of the substrate concentration for kinetic modelling. Kinetic models of cellulose hydrolysis should be simplified enough to include reversible and irreversible product inhibition and reduction of hydrolysability, as well as their possible non-linear relations to hydrolysis degree, without overparameterization of particular factors.

Subcritical hydrothermal pretreatment of olive mill solid waste for biofuel production.

The hydrothermal pretreatment of olive mill solid waste amended with 0.6M organic acids was studied at temperatures between 100 and 170°C. Acetic and formic acids which are endogenous intermediates of hemiacetyl splitting at subcritical conditions were tested. Formic acid, with smaller molecular size and lower pKa, was found to be more effective than acetic in the entire range of temperatures tested. Yield of enzymatic hydrolysis was significantly enhanced (>2 folds) at temperatures above 140°C. Concentration of aldehyde byproducts in the medium increased with temperature and pressure and addition of organic acids, however, the highest concentration detected (ca 1g/L) did not surpass values reported as inhibitory of sugars fermentation to ethanol by either yeast or bacteria. Aldehyde production was more affected by temperature than by acid addition. Concluding, addition of formic acid to hydrothermal pretreatment at relatively mild temperatures (140-170°C) and pressure (10-13 atm) improved saccharification yield while saving energy.