Construction of a Bacterial Cellulase Cocktail for Saccharification of Regenerated Cellulose and Pretreated Corn Stover

To apply bacterial cellulases for efficient saccharification of biomass, three Clostridium thermocellum cellulases and a Thermoanaerobacter brockii β-1,4-glucosidase were synthesized in Escherichia coli, and the proportions among them were optimized. When the activities of CelD, CBHA, CBH48Y, and CglT were set at 554, 0.91, 0.91, and 856 mU per assay, respectively, the percent conversion of regenerated cellulose (0.92 g/L) reached 80.9% within 24 h at 60 °C without shaking. Meanwhile, the percent conversion of pretreated corn stover (0.62 g/L) reached 70.1%. Gradually raising the loads of regenerated cellulose from 0.92 to 4.58 g/L resulted in a linear increase in glucose production from 870 to 3208 μg (R2=0.997), as well as a decrease in the percent conversion from 80.9% to 59.6%. These findings suggested that the cellulase cocktail is efficient in saccharification of regenerated cellulose, as well as pretreated corn stover, and has potential applications in the biofuels industry

Characterization of a Salt-Tolerant and Cold-Adapted Xylanase from Bacillus cellulosilyticus

A xylanase (Xyn10A) gene from the saline-alkali-tolerant microorganism Bacillus cellulosilyticus DSM 2522 was cloned and expressed in Escherichia coli BL21 (DE3). The open reading frame was composed of 1008 base pairs, and it encoded 335 amino acid residues belonging to glycosyl hydrolase family 10. The optimal temperature and pH of the purified Xyn10A were 40 °C and 8.0, respectively. The Xyn10A was sensitive to heat and showed obvious cold-adapted activity, retaining 38.3%, 55.7%, and 82.9% of the optimal activity at 4, 20, and 30 °C, respectively. Xyn10A also showed a high level of NaCl tolerance. The highest activity was observed with 1.5 M NaCl. The specific enzyme activity of Xyn10A was as much as 163.8 U/mg. Kinetic assays showed that Km, Vmax, and Kcat were 2.56 mg/mL, 202.5 μM/min/mg, and 132.6 /s, respectively. Additionally, the main hydrolysis products using birchwood xylan as substrate were xylobiose, xylotriose, and xylotetraose, as determined by thin layer chromatography analysis. As a cold-adapted and salt-tolerant enzyme, Xyn10A is an ideal candidate for further research and biotechnological applications

Isolation of <i>Lactococcus</i> sp. X1 from Termite Gut, and Its Application in Lactic Acid Production

The production of lactic acid (LA) from lignocellulosic biomass is an important route for the exploitation of renewable resources; nevertheless, effective LA production from this feedstock is challenged by several limitations, such as pentose and oligosaccharide utilization. In this study, a new strain, Lactococcus sp. X1, which is capable of fermenting glucose, xylose, and several disaccharides to produce L-lactic acid, was isolated from the gut of a wood-feeding termite, Coptotermes formosanus. Compared to conventional lactic acid bacteria, Lactococcus sp. X1 requires less complex nitrogen sources, which might in turn reduce the cost of LA production. In addition, Lactococcus sp. X1 was able to completely ferment 50 g/L of glucose within 3 days, giving a high LA yield of 99.9%, and its LA yield from 50 g/L of pretreated corncob reached up to 0.34 g/g substrates in the presence of a commercial cellulase. Strain X1 was also capable of excreting two kinds of nutritional factors, namely biotin and vitamin C, indicating its crucial role in the nourishment of the termite. In conclusion, Lactococcus sp. X1 is a new lactic acid bacterium, which may hold promise for application in cost-effective LA production as well as in the field of food additives

Isolation of Lactococcus sp. X1 from Termite Gut, and Its Application in Lactic Acid Production

The production of lactic acid (LA) from lignocellulosic biomass is an important route for the exploitation of renewable resources; nevertheless, effective LA production from this feedstock is challenged by several limitations, such as pentose and oligosaccharide utilization. In this study, a new strain, Lactococcus sp. X1, which is capable of fermenting glucose, xylose, and several disaccharides to produce L-lactic acid, was isolated from the gut of a wood-feeding termite, Coptotermes formosanus. Compared to conventional lactic acid bacteria, Lactococcus sp. X1 requires less complex nitrogen sources, which might in turn reduce the cost of LA production. In addition, Lactococcus sp. X1 was able to completely ferment 50 g/L of glucose within 3 days, giving a high LA yield of 99.9%, and its LA yield from 50 g/L of pretreated corncob reached up to 0.34 g/g substrates in the presence of a commercial cellulase. Strain X1 was also capable of excreting two kinds of nutritional factors, namely biotin and vitamin C, indicating its crucial role in the nourishment of the termite. In conclusion, Lactococcus sp. X1 is a new lactic acid bacterium, which may hold promise for application in cost-effective LA production as well as in the field of food additives

New Insights into the Co-Occurrences of Glycoside Hydrolase Genes among Prokaryotic Genomes through Network Analysis

Glycoside hydrolase (GH) represents a crucial category of enzymes for carbohydrate utilization in most organisms. A series of glycoside hydrolase families (GHFs) have been classified, with relevant information deposited in the CAZy database. Statistical analysis indicated that most GHFs (134 out of 154) were prone to exist in bacteria rather than archaea, in terms of both occurrence frequencies and average gene numbers. Co-occurrence analysis suggested the existence of strong or moderate-strong correlations among 63 GHFs. A combination of network analysis by Gephi and functional classification among these GHFs demonstrated the presence of 12 functional categories (from group A to L), with which the corresponding microbial collections were subsequently labeled, respectively. Interestingly, a progressive enrichment of particular GHFs was found among several types of microbes, and type-L as well as type-E microbes were deemed as functional intensified species which formed during the microbial evolution process toward efficient decomposition of lignocellulose as well as pectin, respectively. Overall, integrating network analysis and enzymatic functional classification, we were able to provide a new angle of view for GHs from known prokaryotic genomes, and thus this study is likely to guide the selection of GHs and microbes for efficient biomass utilization

Expression and characterization of a novel metagenome-derived cellulase Exo2b and its application to improve cellulase activity in Trichoderma reesei

A metagenomic fosmid library containing 1 x 10(5) clones was constructed from a biogas digester fed with pig ordure and rice straw. In total, 121 clones with activity of 4-methylumbelliferyl-cellobiosidase were screened from the metagenomic library. A novel GH5 cellulase gene exo2b was identified from a sequenced clone EXO02C10 and expressed in Escherichia coli BL21. The corresponding recombinant Exo2b protein showed high specific activity toward both carboxymethylcellulose (CMC; 260 U/mg protein) and beta-d-glucan from barley (849 U/mg), with an optimal pH and temperature of 7.5 and 58 A degrees C, respectively. Exo2b showed stable activity at a wide pH range from 5.5 to 9.0 and was highly thermostable at 60 A degrees C in the presence of 60 mM cysteine. Residual activity was maintained at nearly 100% when Exo2b was incubated at 60 A degrees C for 15 h. A thin-layer chromatography analysis of the hydrolysis products confirmed that Exo2b was an endo-beta-1,4-glucanase and it could also produce oligosaccharide smaller than cellotetraose. The fragment encoding the Exo2b catalytic domain was then fused with the cbh1 gene from Trichoderma reesei, and the fused gene was successfully expressed in T. reesei Rut-C30. Compared to that of the parent strain, the filter paper activity and CMCase activity of the secreted proteins of a selected transformant A1 increased by 24% and 18%, respectively. Besides, the glucose concentration from the hydrolysis of pretreated corn stover by the A1 secreted proteins increased by 19.8%. The present study demonstrated the potential application of metagenome originated cellulase genes to modify cellulase producing fungi

Genomics and biochemistry investigation on the metabolic pathway of milled wood and alkali lignin-derived aromatic metabolites of Comamonas serinivorans SP-35

Abstract Background The efficient depolymerization and utilization of lignin are one of the most important goals for the renewable use of lignocelluloses. The degradation and complete mineralization of lignin by bacteria represent a key step for carbon recycling in land ecosystems as well. However, many aspects of this process remain unclear, for example, the complex network of metabolic pathways involved in the degradation of lignin and the catabolic pathway of intermediate aromatic metabolites. To address these subjects, we characterized the deconstruction and mineralization of lignin with milled wood lignin (MWL, the most representative molecule of lignin in its native state) and alkali lignin (AL), and elucidated metabolic pathways of their intermediate metabolites by a bacterium named Comamonas serinivorans SP-35. Results The degradation rate of MWL reached 30.9%, and its particle size range was decreased from 6 to 30 µm to 2–4 µm—when cultured with C. serinivorans SP35 over 7 days. FTIR analysis showed that the C–C and C–O–C bonds between the phenyl propane structures of lignin were oxidized and cleaved and the side chain structure was modified. More than twenty intermediate aromatic metabolites were identified in the MWL and AL cultures based on GC–MS analysis. Through genome sequencing and annotation, and from GC–MS analysis, 93 genes encoding 33 enzymes and 5 regulatory factors that may be involved in lignin degradation were identified and more than nine metabolic pathways of lignin and its intermediates were predicted. Of particular note is that the metabolic pathway to form the powerful antioxidant 3,4-dihydroxyphenylglycol is described for the first time in bacteria. Conclusion Elucidation of the β-aryl ether cleavage pathway in the strain SP-35 indicates that the β-aryl ether catabolic system is not only present in the family of Sphingomonadaceae, but also other species of bacteria kingdom. These newly elucidated catabolic pathways of lignin in strain SP-35 and the enzymes responsible for them provide exciting biotechnological opportunities for lignin valorization in future

Barcın Höyük. A Neolithic Village in the Yenişehir Valley

Additional file 4: Figure S2. Percentages of unigenes that assigned as termite gene and protistan gene from the four termite digestive organs: salivary gland (SG), foregut (FG), midgut (MG) and hindgut (HG)

Insight into Dominant Cellulolytic Bacteria from Two Biogas Digesters and Their Glycoside Hydrolase Genes

<div><p>Diverse cellulolytic bacteria are essential for maintaining high lignocellulose degradation ability in biogas digesters. However, little was known about functional genes and gene clusters of dominant cellulolytic bacteria in biogas digesters. This is the foundation to understand lignocellulose degradation mechanisms of biogas digesters and apply these gene resource for optimizing biofuel production. A combination of metagenomic and 16S rRNA gene clone library methods was used to investigate the dominant cellulolytic bacteria and their glycoside hydrolase (GH) genes in two biogas digesters. The 16S rRNA gene analysis revealed that the dominant cellulolytic bacteria were strains closely related to <i>Clostridium straminisolvens</i> and an uncultured cellulolytic bacterium designated BG-1. To recover GH genes from cellulolytic bacteria in general, and BG-1 in particular, a refined assembly approach developed in this study was used to assemble GH genes from metagenomic reads; 163 GH-containing contigs ≥ 1 kb in length were obtained. Six recovered GH5 genes that were expressed in <i>E</i>. <i>coli</i> demonstrated multiple lignocellulase activities and one had high mannanase activity (1255 U/mg). Eleven fosmid clones harboring the recovered GH-containing contigs were sequenced and assembled into 10 fosmid contigs. The composition of GH genes in the 163 assembled metagenomic contigs and 10 fosmid contigs indicated that diverse GHs and lignocellulose degradation mechanisms were present in the biogas digesters. In particular, a small portion of BG-1 genome information was recovered by PhyloPythiaS analysis. The lignocellulase gene clusters in BG-1 suggested that it might use a possible novel lignocellulose degradation mechanism to efficiently degrade lignocellulose. Dominant cellulolytic bacteria of biogas digester possess diverse GH genes, not only in sequences but also in their functions, which may be applied for production of biofuel in the future.</p></div

The characterization of six GH5 genes expressed in <i>E</i>.<i>coli</i>.

<p>¹Nearest neighbor represented nearest protein sequence for each GH5 genes.</p><p>²All activity assays were performed at pH7.4 and 50°C. The substrates used to determine activities of endoglucanase, mannanase, xylanase and exocellulase were CMC, locust bean gum, xylan (beechwood) and pNPC. The unit of enzyme activity is U/mg protein.</p><p>³Activity was not detected.</p><p>⁴Activity was less than 1 U/mg protein.</p><p>⁵Cel5 and Cel6 were also mentioned as man1 and en2 in elsewhere (Yan <i>et al</i>., 2013), but were not expressed before.</p><p>The characterization of six GH5 genes expressed in <i>E</i>.<i>coli</i>.</p