Microbial β-glucosidases from cow rumen metagenome enhance the saccharification of lignocellulose in combination with commercial cellulase cocktail.
Del Pozo, M. V., Fernández-Arrojo, L., Gil-Martínez, J., Montesinos, A., Chernikova, T. N., Nechitaylo, T. Y., Waliszek, A., Tortajada, M., Rojas, A., Huws, S. A., Golyshina, O. V., Newbold, C. J., Polaina, J., Ferrer, M. & Golyshin, P. N. (2012). Biotechnology Biofuels, 5, 73.
Background: A complete saccharification of plant polymers is the critical step in the efficient production of bio-alcohols. Beta-glucosidases acting in the degradation of intermediate gluco-oligosaccharides produced by cellulases limit the yield of the final product. Results: In the present work, we have identified and then successfully cloned, expressed, purified and characterised 4 highly active beta-glucosidases from fibre-adherent microbial community from the cow rumen. The enzymes were most active at temperatures 45–55°C and pH 4.0-7.0 and exhibited high affinity and activity towards synthetic substrates such as p-nitrophenyl-beta-D-glucopyranoside (pNPbetaG) and pNP-beta-cellobiose, as well as to natural cello-oligosaccharides ranging from cellobiose to cellopentaose. The apparent capability of the most active beta-glucosidase, herein named LAB25g2, was tested for its ability to improve, at low dosage (31.25 units g-1 dry biomass, using pNPbetaG as substrate), the hydrolysis of pre-treated corn stover (dry matter content of 20%; 350 g glucan kg-1 dry biomass) in combination with a beta-glucosidase-deficient commercial Trichoderma reseei cellulase cocktail (5 units g-1 dry biomass in the basis of pNPbetaG). LAB25g2 increased the final hydrolysis yield by a factor of 20% (44.5 ± 1.7% vs. 34.5 ± 1.5% in control conditions) after 96–120 h as compared to control reactions in its absence or in the presence of other commercial beta-glucosidase preparations. The high stability (half-life higher than 5 days at 50°C and pH 5.2) and 2–38000 fold higher (as compared with reported beta-glucosidases) activity towards cello-oligosaccharides may account for its performance in supplementation assays. Conclusions: The results suggest that beta-glucosidases from yet uncultured bacteria from animal digestomes may be of a potential interest for biotechnological processes related to the effective bio-ethanol production in combination with low dosage of commercial cellulases.
Substrate specificities of glycosidases from Aspergillus species pectinase preparations on elderberry anthocyanins.
Pricelius, S., Murkovic, M., Souter, P. & Guebitz, G. M. (2009). Journal of Agricultural and Food Chemistry, 57(3), 1006-1012.
Attractive color is one of the most important sensory characteristics of fruit and berry products, and elderberry juice is widely used as natural colorant. When pectinase preparations were used in the production of elderberry juice for clarification, a concomitant decrease of anthocyanins and thus a color loss were observed. This paper demonstrates that this is due to side glycosidase activities contained in commercial pectinase preparations from Aspergillus sp. Using LC-MS, sequential deglycosylation of cyanidin-3-sambubioside, cy-3-glucoside, cy-3-sambubioside-5-glucoside, and cy-3,5-diglucoside was found to be catalyzed by specific glycosidases contained in the pectinase preparations. There was no big difference in the deglycosylation rate between monoglucosidic or diglucosidic anthocyanins. However, the degradation rate was decreased when rutinose was attached to cyanidin, whereas the structure of the aglycone itself had almost no influence. Pure β-glucosidases from Agrobacterium species and Aspergillus niger and the β-glucosidase N188 from A. niger did not show any conversion of anthocyanins, indicating the presence of specific glycosidases. Thus, an activity gel based assay was developed to detect anthocyanin-specific glycosidase activity in enzyme preparations, and according to LC-MS peptide mass mapping of digested bands, homologies to a β-glucosidase from Aspergillus kawachii were found.
Arabinose substitution degree in xylan positively affects lignocellulose enzymatic digestibility after various NaOH/ H2SO4 pretreatments in Miscanthus.
Li, F., Ren, S., Zhang, W., Xu, Z., Xie, G., Chen, Y., Tu, Y., Li, Q., Zhou, S., Li, Y., Tu, F., Liu, L., Wang, Y., Jiang, J., Qin, J., Li, S., Li, Q., Jing, H. C., Zhou, F., Gutterson, N. & Peng, L. (2013). Bioresource Technology, 130, 629-637.
Xylans are the major hemicelluloses in grasses, but their effects on biomass saccharification remain unclear. In this study, we examined the 79 representative Miscanthus accessions that displayed a diverse cell wall composition and varied biomass digestibility. Correlation analysis showed that hemicelluloses level has a strong positive effect on lignocellulose enzymatic digestion after NaOH or H2SO4 pretreatment. Characterization of the monosaccharide compositions in the KOH-extractable and non-KOH-extractable hemicelluloses indicated that arabinose substitution degree of xylan is the key factor that positively affects biomass saccharification. The xylose/arabinose ratio after individual enzyme digestion revealed that the arabinose in xylan is partially associated with cellulose in the amorphous regions, which negatively affects cellulose crystallinity for high biomass digestibility. The results provide insights into the mechanism of lignocellulose enzymatic digestion upon pretreatment, and also suggest a goal for the genetic modification of hemicelluloses towards the bioenergy crop breeding of Miscanthus and grasses.
A chromogenic assay for limit dextrinase and pullulanase activity.
Bøjstrup, M., Christensen, C. E., Windahl, M. S., Henriksen, A. & Hindsgaul, O. (2014). Analytical Biochemistry, 449, 45–51.
A new chromogenic substrate to assay the starch debranching enzymes limit dextrinase and pullulanase is described. The 2-chloro-4-nitrophenyl glycoside of a commercially available branched heptasaccharide (Glc-maltotriosyl-maltotriose) was found to be a suitable specific substrate for starch debranching enzymes and allows convenient assays of enzymatic activities in a format suited for high-throughput analysis. The kinetic parameters of these enzymes toward the synthesized substrate are determined, and the selectivity of the substrate in a complex cereal-based extract is established.
Conversion of Levoglucosan and Cellobiosan by Pseudomonas putida KT2440.
Linger, J. G., Hobdey, S. E., Franden, M. A., Fulk, E. M. & Beckham, G. T. (2016). Metabolic Engineering Communications, 3, 24-29.
Pyrolysis offers a straightforward approach for the deconstruction of plant cell wall polymers into bio-oil. Recently, there has been substantial interest in bio-oil fractionation and subsequent use of biological approaches to selectively upgrade some of the resulting fractions. A fraction of particular interest for biological upgrading consists of polysaccharide-derived substrates including sugars and sugar dehydration products such as levoglucosan and cellobiosan, which are two of the most abundant pyrolysis products of cellulose. Levoglucosan can be converted to glucose-6-phosphate through the use of a levoglucosan kinase (LGK), but to date, the mechanism for cellobiosan utilization has not been demonstrated. Here, we engineer the microbe Pseudomonas putida KT2440 to use levoglucosan as a sole carbon and energy source through LGK integration. Moreover, we demonstrate that cellobiosan can be enzymatically converted to levoglucosan and glucose with β-glucosidase enzymes from both Glycoside Hydrolase Family 1 and Family 3. β-glucosidases are commonly used in both natural and industrial cellulase cocktails to convert cellobiose to glucose to relieve cellulase product inhibition and to facilitate microbial uptake of glucose. Using an exogenous β-glucosidase, we demonstrate that the engineered strain of P. putida can grow on levoglucosan up to 60 g/L and can also utilize cellobiosan. Overall, this study elucidates the biological pathway to co-utilize levoglucosan and cellobiosan, which will be a key transformation for the biological upgrading of pyrolysis-derived substrates.
A comparative study on the activity of fungal lytic polysaccharide monooxygenases for the depolymerization of cellulose in soybean spent flakes.
Pierce, B. C., Agger, J. W., Zhang, Z., Wichmann, J. & Meyer, A. S. (2017). Carbohydrate Research, 449, 85-94.
Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes capable of the oxidative breakdown of polysaccharides. They are of industrial interest due to their ability to enhance the enzymatic depolymerization of recalcitrant substrates by glycoside hydrolases. In this paper, twenty-four lytic polysaccharide monooxygenases (LPMOs) expressed in Trichoderma reesei were evaluated for their ability to oxidize the complex polysaccharides in soybean spent flakes, an abundant and industrially relevant substrate. TrCel61A, a soy-polysaccharide-active AA9 LPMO from T. reesei, was used as a benchmark in this evaluation. In total, seven LPMOs demonstrated activity on pretreated soy spent flakes, with the products from enzymatic treatments evaluated using mass spectrometry and high performance anion exchange chromatography. The hydrolytic boosting effect of the top-performing enzymes was evaluated in combination with endoglucanase and beta-glucosidase. Two enzymes (TrCel61A and Aspte6) showed the ability to release more than 36% of the pretreated soy spent flake glucose – a greater than 75% increase over the same treatment without LPMO addition.
An ancient family of lytic polysaccharide monooxygenases with roles in arthropod development and biomass digestion.
Sabbadin , F., Hemsworth, G. R., Ciano, L., Henrissat, B., Dupree, P., Tryfona, T., Marques, R. D. S., Sweeney, S. T., Besser, K., Elias, L., Pesante, G., Li, Y., Dowle, A. A., Bates, R., Gomez, L. D., Simister, R., Davies, G. J., Walton, P. H., Bruce, N. C., McQueen-Mason, S. J. (2018). Nature Communications, In Press.
Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over twenty members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin and was recruited by early arthropods with possible roles in remodelling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia’s LPMOs, we propose that diversification of these enzymes towards cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.
Yeast lipids from cardoon stalks, stranded driftwood and olive tree pruning residues as possible extra sources of oils for producing biofuels and biochemicals.
Tasselli, G., Filippucci, S., Borsella, E., D’Antonio, S., Gelosia, M., Cavalaglio, G., Turchetti, B., Sannino, C., Onofri, A., Mastrolitti, S., De Bari, I., Cotana, F. & Bari, I. (2018). Biotechnology for Biofuels, 11(1), 147.
Background: Some lignocellulosic biomass feedstocks occur in Mediterranean Countries. They are still largely unexploited and cause considerable problems due to the lack of cost-effective harvesting, storage and disposal technologies. Recent studies found that some basidiomycetous yeasts are able to accumulate high amount of intracellular lipids for biorefinery processes (i.e., biofuels and biochemicals). Accordingly, the above biomass feedstocks could be used as carbon sources (after their pre-treatment and hydrolysis) for lipid accumulation by oleaginous yeasts. Results: Cardoon stalks, stranded driftwood and olive tree pruning residues were pre-treated with steam-explosion and enzymatic hydrolysis for releasing free mono- and oligosaccharides. Lipid accumulation tests were performed at two temperatures (20 and 25°C) using Leucosporidium creatinivorum DBVPG 4794, Naganishia adeliensis DBVPG 5195 and Solicoccozyma terricola DBVPG 5870. S. terricola grown on cardoon stalks at 20°C exhibited the highest lipid production (13.20 g/l), a lipid yield (28.95%) close to the maximum theoretical value and a lipid composition similar to that found in palm oil. On the contrary, N. adeliensis grown on stranded driftwood and olive tree pruning residues exhibited a lipid composition similar to those of olive and almonds oils. A predictive evaluation of the physical properties of the potential biodiesel obtainable by lipids produced by tested yeast strains has been reported and discussed. Conclusions: Lipids produced by some basidiomycetous yeasts grown on Mediterranean lignocellulosic biomass feedstocks could be used as supplementary sources of oils for producing biofuels and biochemicals.