Degradation Of Cellulosic Biomass Research Articles

Cloning, expression and purification of cellobiohydrolase gene from Caldicellulosiruptor bescii for efficient saccharification of plant biomass

Anthropogenic activities have led to a drastic shift from natural fuels to alternative renewable energy reserves that demand heat-stable cellulases. Cellobiohydrolase is an indispensable member of cellulases that play a critical role in the degradation of cellulosic biomass. This article details the process of cloning the cellobiohydrolase gene from the thermophilic bacterium Caldicellulosiruptor bescii and expressing it in Escherichia coli (BL21) CondonPlus DE3-(RIPL) using the pET-21a(+) expression vector. Multi-alignments and structural modeling studies reveal that recombinant CbCBH contained a conserved cellulose binding domain III. The enzyme's catalytic site included Asp-372 and Glu-620, which are either involved in substrate or metal binding. The purified CbCBH, with a molecular weight of 91.8 kDa, displayed peak activity against pNPC (167.93 U/mg) at 65°C and pH 6.0. Moreover, it demonstrated remarkable stability across a broad temperature range (60–80°C) for 8 h. Additionally, the Plackett-Burman experimental model was employed to assess the saccharification of pretreated sugarcane bagasse with CbCBH, aiming to evaluate the cultivation conditions. The optimized parameters, including a pH of 6.0, a temperature of 55°C, a 24-hour incubation period, a substrate concentration of 1.5% (w/v), and enzyme activity of 120 U, resulted in an observed saccharification efficiency of 28.45%. This discovery indicates that the recombinant CbCBH holds promising potential for biofuel sector.

Impact of Synergy Partner Cel7B on Cel7A Binding Rates: Insights from Single-Molecule Data.

Enzymatic degradation of cellulosic biomass is a well-established route for the sustainable production of biofuels, chemicals, and materials. A strategy employed by nature and industry to achieve an efficient degradation of cellulose is that cellobiohydrolases (or exocellulases), such as Cel7A, work synergistically with endoglucanases, such as Cel7B, to achieve the complete degradation of cellulose. However, a complete mechanistic understanding of this exo-endo synergy is still lacking. Here, we used single-molecule fluorescence microscopy to quantify the binding kinetics of Cel7A on cellulose when it is acting alone on the cellulose fibrils and in the presence of its synergy partner, the endoglucanase Cel7B. To this end, we used a fluorescently tagged Cel7A and studied its binding in the presence of the unlabeled Cel7B. This provided the single-molecule data necessary for the estimation of the rate constants of association kON and dissociation kOFF of Cel7A for the substrate. We show that the presence of Cel7B does not impact the dissociation rate constant, kOFF. But, the association rate of Cel7A decreases by a factor of 2 when Cel7B is present at a molar proportion of 10:1. This ratio has previously been shown to lead to synergy. This decrease in association rate is observed in a wide range of total enzyme concentrations, from sub nM to μM concentrations. This decrease in kON is consistent with the formation of cellulase clusters recently observed by others using atomic force microscopy.

Enzymatic degradation of cellulose in soil: A review

Cellulose degradation is a critical process in soil ecosystems, playing a vital role in nutrient cycling and organic matter decomposition. Enzymatic degradation of cellulosic biomass is the most sustainable and green method of producing liquid biofuel. It has gained intensive research interest with future perspective as the majority of terrestrial lignocellulose biomass has a great potential to be used as a source of bioenergy. However, the recalcitrant nature of lignocellulose limits its use as a source of energy. Noteworthy enough, enzymatic conversion of cellulose biomass could be a leading future technology. Fungal enzymes play a central role in cellulose degradation. Our understanding of fungal cellulases has substantially redirected in the past few years with the discovery of a new class of enzymes and Cellulosome. Efforts have been made from time to time to develop an economically viable method of cellulose degradation. This review provides insights into the current state of knowledge regarding cellulose degradation in soil and identifies areas where further research is needed.

Prospects of soil microbiome application for lignocellulosic biomass degradation: An overview.

Sustainable and practically viable biofuels production technology using lignocellulosic biomass is still seeking its way of implementation owing to some major issues involved therein. Unavailability of efficient microbial sources for the degradation of cellulosic biomass is one of the major roadblocks in biomass to biofuels production technology. In this context, utilization of microbiomes to degrade lignocellulaosic biomass is emerging as a rapid and effective approach that can fulfill the requirements of biomass based biofuels production technology. Therefore, the present review is targeted to explore soil metagenomic approach to improve the lignocellulosic biomass degradation processing for the cost-effective and eco-friendly application. Soil microbiomes consist of rich microbial community along with high probability of cellulolytic microbes, and can be identified by culture independent metagenomics method which can be structurally and functionally explored via genomic library. Therefore, in depth analysis and discussion have also been made via structural & functional metagenomics tools along with their contribution to genomic library. Additionally, the present review highlights currently existing bottlenecks along with their feasible solutions. This review will help to understand the basic research as well as industrial concept for the process improvement based on soil microbiome mediated lignocellulosic biomass degradation, and this may likely to implement for the low-cost commercial biofuels production technology.

The expression of alternative sigma-I7 factor induces the transcription of cellulosomal genes in the cellulolytic bacterium Clostridium thermocellum

The composition of cellulosomal carbohydrate-active enzymes (CAZymes) secreted from a cellulolytic bacterium Clostridium thermocellum varies depending on the cellulosic substrate used during cultivation. C. thermocellum detects the polysaccharides in cellulosic material via anti-sigma factors and expresses the appropriate CAZyme gene via alternative sigma factors, SigIs. Previous studies on the regulation of CAZyme gene expression via SigIs in C. thermocellum have been conducted in vitro or in a heterologous host, because of the limited genetic tools available for C. thermocellum. To characterize the in vivo function of SigIs, in the present study, we established a sigI7 gene expression strain of C. thermocellum. Transcriptome analysis of this strain revealed that SigI7 induced the expression of cellulosomal CAZyme genes and cellulosomal scaffold genes. However, there was a decrease in the degradation ability of the exoproteome from the sigI7 expression strain; the product of the downregulated gene, Clo1313_1002, rescued the activity of the C. thermocellum exoproteome from the sigI7 expression strain. In this study, we demonstrate the in vivo function of SigI7 and discuss the CAZymes that are important for cellulosic biomass degradation by C. thermocellum.

Mangrove algae as sustainable microbial cell factory for cellulosic biomass degradation and lipid production

Stress factors’ effects on the induction of lipid synthesis of microalgaeShakirov Zair Saatovich, Khalilov Ilkhom Mamatkulovich, Khujamshukurov Nortoji

Improvement of catalytic performance of endoglucanase CgEndo from Colletotrichum graminicola by site-directed mutagenesis

In order to improve the catalytic efficiency of cellulase for more effective utilization of lignocellulose, a novel endoglucanase (CgEndo) from Colletotrichum graminicola was expressed by Pichia pastoris X33 and modified by site-directed mutagenesis. Two mutants, Y63S and N20D/S113T, with 62.31% and 57.14% increased enzyme activities were obtained, respectively. On this basis, their biochemical properties, kinetic parameters, structural information as well as the application in biomass degradation were investigated and compared with the wild-type CgEngo. The results indicated that the mutation Y63S and N20D/S113T resulted in an improvement of proximity between enzyme and substrate through conformational changes of the catalytic region, which might contribute to the higher enzyme activities and catalysis efficiency (Kcat/Km) of Y63S and N20D/S113T. These findings laid important foundation for the further engineering of this endoglucanase and practical application in efficient degradation of cellulosic biomass in nature.

On-Line Raman Measurement of the Radiation-Enhanced Reaction of Cellobiose with Hydrogen Peroxide.

Production of a chemical feedstock as a secondary product from a commercial nuclear reactor can increase the economic viability of the reactor and enable the deployment of nuclear energy as part of the low-carbon energy grid. Currently, commercial nuclear reactors produce underutilized energy in the form of neutrons and gamma photons. This excess energy can be exploited to drive chemical reactions, increasing the fraction of utilized energy in reactors and providing a valuable secondary product from the reactor. Gamma degradation of cellulosic biomass has been studied previously. However, real-time, on-line monitoring of the breakdown of biomass materials under gamma radiation has not been demonstrated. Here, we demonstrate on-line monitoring of the reaction of cellobiose with hydrogen peroxide under gamma radiation using Raman spectroscopy, providing in situ quantification of organic and inorganic system components.

Synergistic Action of a Lytic Polysaccharide Monooxygenase and a Cellobiohydrolase from Penicillium funiculosum in Cellulose Saccharification under High-Level Substrate Loading.

Lytic polysaccharide monooxygenases (LPMOs) are crucial industrial enzymes required in the biorefinery industry as well as in the natural carbon cycle. These enzymes, known to catalyze the oxidative cleavage of glycosidic bonds, are produced by numerous bacterial and fungal species to assist in the degradation of cellulosic biomass. In this study, we annotated and performed structural analysis of an uncharacterized LPMO from Penicillium funiculosum (PfLPMO9) based on computational methods in an attempt to understand the behavior of this enzyme in biomass degradation. PfLPMO9 exhibited 75% and 36% sequence identities with LPMOs from Thermoascus aurantiacus (TaLPMO9A) and Lentinus similis (LsLPMO9A), respectively. Furthermore, multiple fungal genetic manipulation tools were employed to simultaneously overexpress LPMO and cellobiohydrolase I (CBH1) in a catabolite-derepressed strain of Penicillium funiculosum, PfMig188 (an engineered variant of P. funiculosum), to improve its saccharification performance toward acid-pretreated wheat straw (PWS) at 20% substrate loading. The resulting transformants showed improved LPMO and CBH1 expression at both the transcriptional and translational levels, with ∼200% and ∼66% increases in ascorbate-induced LPMO and Avicelase activities, respectively. While the secretome of PfMig88 overexpressing LPMO or CBH1 increased the saccharification of PWS by 6% or 13%, respectively, over the secretome of PfMig188 at the same protein concentration, the simultaneous overexpression of these two genes led to a 20% increase in saccharification efficiency over that observed with PfMig188, which accounted for 82% saccharification of PWS under 20% substrate loading.IMPORTANCE The enzymatic hydrolysis of cellulosic biomass by cellulases continues to be a significant bottleneck in the development of second-generation biobased industries. While increasing efforts are being made to obtain indigenous cellulases for biomass hydrolysis, the high production cost of this enzyme remains a crucial challenge affecting its wide availability for the efficient utilization of cellulosic materials. This is because it is challenging to obtain an enzymatic cocktail with balanced activity from a single host. This report describes the annotation and structural analysis of an uncharacterized lytic polysaccharide monooxygenase (LPMO) gene in Penicillium funiculosum and its impact on biomass deconstruction upon overexpression in a catabolite-derepressed strain of P. funiculosum Cellobiohydrolase I (CBH1), which is the most important enzyme produced by many cellulolytic fungi for the saccharification of crystalline cellulose, was further overexpressed simultaneously with LPMO. The resulting secretome was analyzed for enhanced LPMO and exocellulase activities and the corresponding improvement in saccharification performance (by ∼20%) under high-level substrate loading using a minimal amount of protein.

Heteropoly Acid-Based Catalysts for Hydrolytic Depolymerization of Cellulosic Biomass.

Cellulose is the most abundant source of biomass, which can be converted into monosaccharide or other chemical platform molecules for the sustainable production of chemicals and fuels. Acid catalysts can promote hydrolytic degradation of cellulose into valuable platform molecules, which is of great significance in the development of chemicals and biofuels. However, there are still some shortcomings and limitations of the catalysts for the hydrolytic degradation of cellulosic biomass. Heteropoly acid (HPA), as a green catalyst, seems to be more conducive to the degradation of cellulosic biomass due to its extreme acidity. HPAs can be designed in homogeneous and heterogeneous systems. Moreover, they can be easily separated from the products in both systems by a simple extraction process. According to the unique properties of HPAs (e.g., good solubility, high thermal stability, and strong acidity), using heteropoly acid-based catalysts to depolymerize and convert cellulose into value-added chemicals and biofuels has become one of the most remarkable processes in chemistry for sustainability. In this review, the characteristics, advantages, and applications of HPAs in different categories for cellulose degradation, especially hydrolysis hydrolytic degradation, are summarized. Moreover, the mechanisms of HPAs catalysts in the effective degradation of cellulosic biomass are discussed. This review provides more avenues for the development of renewed and robust HPAs for cellulose degradation in the future.

Thermostable Mutants of Glycoside Hydrolase Family 6 Cellobiohydrolase from the Basidiomycete Phanerochaete chrysosporium.

Thermal inactivation of saccharifying enzymes is a crucial issue for the efficient utilization of cellulosic biomass as a renewable resource. Cellobiohydrolases (CBHs) are a kind of cellulase. In general, CBHs belonging to glycoside hydrolase (GH) family 6 (Cel6) act synergistically with CBHs of GH family 7 (Cel7) and other carbohydrate-active enzymes during the degradation of cellulosic biomass. However, while the catalytic rate of enzymes generally becomes faster at higher temperatures, Cel6 CBHs are inactivated at lower temperatures than Cel7 CBHs, and this represents a limiting factor for industrial utilization. In this study, we produced a series of mutants of the glycoside hydrolase family 6 cellobiohydrolase Pc Cel6A from the fungus Phanerochaete chrysosporium , and compared their thermal stability. Eight mutants from a random mutagenesis library and one rationally designed mutant were selected as candidate thermostable mutants and produced by heterologous expression in the yeast Pichia pastoris . Comparison of the hydrolytic activities at 50 and 60 °C indicated that the thermal stability of Pc Cel6A is influenced by the number and position of cysteine residues that are not involved in disulfide bonds.

Draft genome sequence of Parvularcula flava strain NH6-79T, revealing its role as a cellulolytic enzymes producer.

To date, the genus Parvularcula consists of 6 species and no potential application of this genus was reported. Current study presents the genome sequence of Parvularcula flava strain NH6-79T and its cellulolytic enzyme analysis. The assembled draft genome of strain NH6-79T consists of 9 contigs and 7 scaffolds with 3.68 Mbp in size and GC content of 59.87%. From a total of 3,465 genes predicted, 96 of them are annotated as glycoside hydrolases (GHs). Within these GHs, 20 encoded genes are related to cellulosic biomass degradation, including 12 endoglucanases (5 GH10, 4 GH5, and 3 GH51), 2 exoglucanases (GH9) and 6 β-glucosidases (GH3). In addition, highest relative enzyme activities (endoglucanase, exoglucanase, and β-glucosidase) were observed at 27th hour when the strain was cultured in the carboxymethyl cellulose/Avicel®-containing medium for 45h. The combination of genome analysis with experimental studies indicated the ability of strain NH6-79T to produce extracellular endoglucanase, exoglucanase, and β-glucosidase. These findings suggest the potential of Parvularcula flava strain NH6-79T in cellulose-containing biomass degradation and that the strain could be used in cellulosic biorefining process.

Microbial cellulolytic enzymes: diversity and biotechnology with reference to lignocellulosic biomass degradation

Lignocellulosic biomass is the earth’s most abundant renewable feedstock alternative that comprises of cellulose, hemi-cellulose and lignin. The synergistic action of cellulolytic/xylanolytic enzymes produced by lignocellulolytic micro-organisms such as bacteria, algae and fungi are capable of robust cellulosic biomass deconstruction. Most of the microorganisms dwelling in extreme environmental habitats such as rumen environment, hot/cold springs, deep ocean trenches, acidic/alkaline pH environment have been considered as an attractive producers of hemi/cellulolytic lignocellulolytic and other biotechnological enzymes with enhanced bio-chemical properties essential for industrial bioconversion processes. However, the potential microbial sources of cellulolytic enzymes and the underlying mechanism to achieve this is not fully elucidated. In this review article, first we detail the composition of lignocellulosic biomass. Next, we describe the structure and functions of divergent hydrolytic enzymes (cellulolytic and xylanolytic enzymes) involved in cellulosic biomass degradation. Third, we analyze, outline and unveil the prospective source of microbes encoding exceptionally diverse set of biotechnologically relevant cellulolytic enzymes which are critical to answer the specific ecological question of by whom, where and how cellulosic biomass is degraded in the environment. Finally, this review article features the snapshot about the future developments and perspectives on microbial enzymes, high-throughput techniques and molecular tools that could be exploited to derive those enzymes from the potential sources.

Effect of C-6 Methylol Groups on Substrate Recognition of Glucose/Xylose Mixed Oligosaccharides by Cellobiose Dehydrogenase from the Basidiomycete Phanerochaete chrysosporium.

Cellobiose dehydrogenase (CDH) is a flavocytochrome catalyzing oxidation of the reducing end of cellobiose and cellooligosaccharides, and has a key role in the degradation of cellulosic biomass by filamentous fungi. Here, we use a lineup of glucose/xylose-mixed β-1,4-linked disaccharides and trisaccharides, enzymatically synthesized by means of the reverse reaction of cellobiose phosphorylase and cellodextrin phosphorylase, to investigate the substrate recognition of CDH. We found that CDH utilizes β-D-xylopyranosyl-(1→4)-D-glucopyranose (Xyl-Glc) as an electron donor with similar K m and k cat values to cellobiose. β-D-Glucopyranosyl-(1→4)-D-xylopyranose (Glc-Xyl) shows a higher K m value, while xylobiose does not serve as a substrate. Trisaccharides show similar behavior; i.e., trisaccharides with cellobiose and Xyl-Glc units at the reducing end show similar kinetics, while the enzyme was less active towards those with Glc-Xyl, and inactive towards those with xylobiose. We also use docking simulation to evaluate substrate recognition of the disaccharides, and we discuss possible molecular mechanisms of substrate recognition by CDH.

A Biological Nanomachine at Work: Watching the Cellulosome Degrade Crystalline Cellulose.

The cellulosome is a supramolecular multienzymatic protein complex that functions as a biological nanomachine of cellulosic biomass degradation. How the megadalton-size cellulosome adapts to a solid substrate is central to its mechanism of action and is also key for its efficient use in bioconversion applications. We report time-lapse visualization of crystalline cellulose degradation by individual cellulosomes from Clostridium thermocellum by atomic force microscopy. Upon binding to cellulose, the cellulosomes switch to elongated, even filamentous shapes and morph these dynamically at below 1 min time scale according to requirements of the substrate surface under attack. Compared with noncomplexed cellulases that peel off material while sliding along crystalline cellulose surfaces, the cellulosomes remain bound locally for minutes and remove the material lying underneath. The consequent roughening up of the surface leads to an efficient deconstruction of cellulose nanocrystals both from the ends and through fissions within. Distinct modes of cellulose nanocrystal deconstruction by nature’s major cellulase systems are thus revealed.

Discovery, activity and characterisation of an AA10 lytic polysaccharide oxygenase from the shipworm symbiont Teredinibacter turnerae

BackgroundThe quest for novel enzymes for cellulosic biomass-degradation has recently been focussed on lytic polysaccharide monooxygenases (LPMOs/PMOs), Cu-containing proteins that catalyse the oxidative degradation of otherwise recalcitrant polysaccharides using O2 or H2O2 as a co-substrate.ResultsAlthough classical saprotrophic fungi and bacteria have been a rich source of lytic polysaccharide monooxygenases (LPMOs), we were interested to see if LPMOs from less evident bio-environments could be discovered and assessed for their cellulolytic activity in a biofuel context. In this regard, the marine shipworm Lyrodus pedicellatus represents an interesting source of new enzymes, since it must digest wood particles ingested during its natural tunnel boring behaviour and plays host to a symbiotic bacterium, Teredinibacter turnerae, the genome of which has revealed a multitude of enzymes dedicated to biomass deconstruction. Here, we show that T. turnerae encodes a cellulose-active AA10 LPMO. The 3D structure, at 1.4 Å resolution, along with its EPR spectrum is distinct from other AA10 polysaccharide monooxygenases insofar as it displays a “histidine-brace” catalytic apparatus with changes to the surrounding coordination sphere of the copper. Furthermore, TtAA10A possesses a second, surface accessible, Cu site 14 Å from the classical catalytic centre. Activity measurements show that the LPMO oxidises cellulose and thereby significantly augments the rate of degradation of cellulosic biomass by classical glycoside hydrolases.ConclusionShipworms are wood-boring marine molluscs that can live on a diet of lignocellulose. Bacterial symbionts of shipworms provide many of the enzymes needed for wood digestion. The shipworm symbiont T. turnerae produces one of the few LPMOs yet described from the marine environment, notably adding to the capability of shipworms to digest recalcitrant polysaccharides.

Secretomic analyses of Ruminiclostridium papyrosolvens reveal its enzymatic basis for lignocellulose degradation

BackgroundEfficient biotechnological conversion of lignocellulosic biomass to valuable products, such as transportation biofuels, is ecologically attractive, yet requires substantially improved mechanistic understanding and optimization to become economically feasible. Cellulolytic clostridia, such as Ruminiclostridium papyrosolvens (previously Clostridium papyrosolvens), produce a wide variety of carbohydrate-active enzymes (CAZymes) including extracellular multienzyme complexes—cellulosomes with different specificities for enhanced cellulosic biomass degradation. Identification of the secretory components, especially CAZymes, during bacterial growth on lignocellulose and their influence on bacterial catalytic capabilities provide insight into construction of potent cellulase systems of cell factories tuned or optimized for the targeted substrate by matching the type and abundance of enzymes and corresponding transporters.ResultsIn this study, we firstly predicted a total of 174 putative CAZymes from the genome of R. papyrosolvens, including 74 cellulosomal components. To explore profile of secreted proteins involved in lignocellulose degradation, we compared the secretomes of R. papyrosolvens grown on different substrates using label-free quantitative proteomics. CAZymes, extracellular solute-binding proteins (SBPs) of transport systems and proteins involved in spore formation were enriched in the secretome of corn stover for lignocellulose degradation. Furthermore, compared with free CAZymes, complex CAZymes (cellulosomal components) had larger fluctuations in variety and abundance of enzymes among four carbon sources. In particular, cellulosomal proteins encoded by the cip-cel operon and the xyl-doc gene cluster had the highest abundance with corn stover as substrate. Analysis of differential expression of CAZymes revealed a substrate-dependent secretion pattern of CAZymes, which was consistent with their catalytic activity from each secretome determined on different cellulosic substrates. The results suggest that the expression of CAZymes is regulated by the type of substrate in the growth medium.ConclusionsIn the present study, our results demonstrated the complexity of the lignocellulose degradation systems of R. papyrosolvens and showed the potency of its biomass degradation activity. Differential proteomic analyses and activity assays of CAZymes secreted by R. papyrosolvens suggested a distinct environment-sensing strategy for cellulose utilization in which R. papyrosolvens modulated the composition of the CAZymes, especially cellulosome, according to the degradation state of its natural substrate.

Multifunctional elastin-like polypeptide renders \u03b2-glucosidase enzyme phase transition and high stability

BackgroundIn the enzymatic conversion of biomass, it becomes an important issue to efficiently and cost-effectively degrade cellulose into fermentable glucose. β-Glucosidase (Bgluc), an essential member of cellulases, plays a critical role in cellulosic biomass degradation. The difficulty in improving the stability of Bgluc has been a bottleneck in the enzyme-dependent cellulose degradation. The traditional method of protein purification, however, leads to higher production cost and a decrease in activity. To simplify and efficiently purify Bgluc with modified special properties, Bgluc-tagged ELP and His with defined phase transitions was designed to facilitate the process.ResultsHere, a novel binary ELP and His tag was fused with Bgluc from termite Coptotermes formosanus to construct a Bgluc–linker–ELP–His recombinant fusion protein (BglucLEH). The recombinant plasmid Bgluc expressing a His tag (BglucH) was also constructed. The BglucLEH and BglucH were expressed in E. coli BL21 and purified using inverse transition cycling (ITC) or Ni-NTA resin. The optimum salt concentration for the ITC purification of BglucLEH was 0.5 M (NH4)2SO4 and the specific activity of BglucLEH purified by ITC was 75.5 U/mg for substrate p-NPG, which was slightly higher than that of BglucLEH purified by Ni-NTA (68.2 U/mg). The recovery rate and purification fold of BglucLEH purified by ITC and Ni-NTA were 77.8%, 79.1% and 12.60, 11.60, respectively. The results indicated that purification with ITC was superior to the traditional Ni-NTA. The Km of BglucLEH and BglucH for p-NPG was 5.27 and 5.73 mM, respectively. The Kcat/Km (14.79 S−1 mM−1) of BglucLEH was higher than that of BglucH (12.10 S−1 mM−1). The effects of ELP tag on the enzyme activity, secondary structure and protein stability were also studied. The results showed that ELP tag did not affect the secondary structure or enzyme activity of Bgluc. More importantly, ELP improved the protein stability in harsh conditions such as heating and exposure to denaturant.ConclusionThe Bgluc–linker–ELP–His system shows wide application prospect in maintaining the activity, efficient purification and improving the stability of Bgluc. These properties of BglucLEH make it an interesting tool to reduce cost, to improve the efficiency of biocatalyst and potentially to enhance the degradation of lignocellulosic biomass.

Novel Ethanol- and 5-Hydroxymethyl Furfural-Stimulated β-Glucosidase Retrieved From a Brazilian Secondary Atlantic Forest Soil Metagenome.

Beta-glucosidases are key enzymes involved in lignocellulosic biomass degradation for bioethanol production, which complete the final step during cellulose hydrolysis by converting cellobiose into glucose. Currently, industry requires enzymes with improved catalytic performance or tolerance to process-specific parameters. In this sense, metagenomics has become a powerful tool for accessing and exploring the biochemical biodiversity present in different natural environments. Here, we report the identification of a novel β-glucosidase from metagenomic DNA isolated from soil samples enriched with decaying plant matter from a Secondary Atlantic Forest region. For this, we employed a functional screening approach using an optimized and synthetic broad host-range vector for library production. The novel β-glucosidase – named Lfa2 – displays three GH3-family conserved domains and conserved catalytic amino acids D283 and E487. The purified enzyme was most active in pH 5.5 and at 50°C, and showed hydrolytic activity toward several pNP synthetic substrates containing β-glucose, β-galactose, β-xylose, β-fucose, and α-arabinopyranose, as well as toward cellobiose. Lfa2 showed considerable glucose tolerance, exhibiting an IC50 of 300 mM glucose and 30% of remaining activity in 600 mM glucose. In addition, Lfa2 retained full or slightly enhanced activity in the presence of several metal ions. Further, β-glucosidase activity was increased by 1.7-fold in the presence of 10% (v/v) ethanol, a concentration that can be reached in conventional fermentation processes. Similarly, Lfa2 showed 1.7-fold enhanced activity at high concentrations of 5-hydroxymethyl furfural, one of the most important cellulase inhibitors in pretreated sugarcane bagasse hydrolysates. Moreover, the synergistic effect of Lfa2 on Bacillus subtilis GH5-CBM3 endoglucanase activity was demonstrated by the increased production of glucose (1.6-fold). Together, these results indicate that β-glucosidase Lfa2 is a promissory enzyme candidate for utilization in diverse industrial applications, such as cellulosic biomass degradation or flavor enhancement in winemaking and grape processing.

Review] Degradation of Cellulosic Biomass and Production of Chemicals by <i>Clostridium</i>

Review] Degradation of Cellulosic Biomass and Production of Chemicals by <i>Clostridium</i>