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High purity Cellopentaose for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
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Versatile high resolution oligosaccharide microarrays for plant glycobiology and cell wall research.
Pedersen, H. L., Fangel, J. U., McCleary, B., Ruzanski, C., Rydahl, M. G., Ralet, M. C., Farkas, V., Von Schantz, L., Marcus, S. E., Andersen, M.C. F., Field, R., Ohlin, M., Knox, J. P., Clausen, M. H. & Willats, W. G. T. (2012). Journal of Biological Chemistry, 287(47), 39429-39438.
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.Hide Abstract
Biological cellulose saccharification using a coculture of Clostridium thermocellum and Thermobrachium celere strain A9.
Nhim, S., Waeonukul, R., Uke, A., Baramee, S., Ratanakhanokchai, K., Tachaapaikoon, C., Pason, P., Liu, Ya-Jun, & Kosugi, A. (2022). Applied microbiology and Biotechnology, 106(5), 2133-2145.
An anaerobic thermophilic bacterial strain, A9 (NITE P-03545), that secretes β-glucosidase was newly isolated from wastewater sediments by screening using esculin. The 16S rRNA gene sequence of strain A9 had 100% identity with that of Thermobrachium celere type strain JW/YL-NZ35. The complete genome sequence of strain A9 showed 98.4% average nucleotide identity with strain JW/YL-NZ35. However, strain A9 had different physiological properties from strain JW/YL-NZ35, which cannot secrete β-glucosidases or grow on cellobiose as the sole carbon source. The key β-glucosidase gene (TcBG1) of strain A9, which belongs to glycoside hydrolase family 1, was characterized. Recombinant β-glucosidase (rTcBG1) hydrolyzed cellooligosaccharides to glucose effectively. Furthermore, rTcBG1 showed high thermostability (at 60°C for 2 days) and high glucose tolerance (IC50 = 0.75 M glucose), suggesting that rTcBG1 could be used for biological cellulose saccharification in cocultures with Clostridium thermocellum. High cellulose degradation was observed when strain A9 was cocultured with C. thermocellum in a medium containing 50 g/l crystalline cellulose, and glucose accumulation in the culture supernatant reached 35.2 g/l. In contrast, neither a monoculture of C. thermocellum nor coculture of C. thermocellum with strain JW/YL-NZ35 realized efficient cellulose degradation or high glucose accumulation. These results show that the β-glucosidase secreted by strain A9 degrades cellulose effectively in combination with C. thermocellum cellulosomes and has the potential to be used in a new biological cellulose saccharification process that does not require supplementation with β-glucosidases.Hide Abstract
Inhibition of LPMOs by Fermented Persimmon Juice.
Tokin, R., Ipsen, J. Ø., Poojary, M. M., Jensen, P. E., Olsson, L. & Johansen, K. S. (2021). Biomolecules, 11(12), 1890.
Fermented persimmon juice, Kakishibu, has traditionally been used for wood and paper protection. This protective effect stems at least partially from inhibition of microbial cellulose degrading enzymes. The inhibitory effect of Kakishibu on lytic polysaccharide monooxygenases (LPMOs) and on a cocktail of cellulose hydrolases was studied, using three different cellulosic substrates. Dose dependent inhibition of LPMO activity by a commercial Kakishibu product was assessed for the well-characterized LPMO from Thermoascus aurantiacus TaAA9A, and the inhibitory effect was confirmed on five additional microbial LPMOs. The model tannin compound, tannic acid exhibited a similar inhibitory effect on TaAA9A as Kakishibu. It was further shown that both polyethylene glycol and tannase can alleviate the inhibitory effect of Kakishibu and tannic acid, indicating a likely mechanism of inhibition caused by unspecific tannin-protein interactions.Hide Abstract
Chromatographic analysis of oxidized cello-oligomers generated by lytic polysaccharide monooxygenases using dual electrolytic eluent generation.
Østby, H., Jameson, J. K., Costa, T., Eijsink, V. G. & Arntzen, M. Ø. (2021). Journal of Chromatography A, 462691.
Research on oligosaccharides, including the complicated product mixtures generated by lytic polysaccharide monooxygenases (LPMOs), is growing at a rapid pace. LPMOs are gaining major interest, and the ability to efficiently and accurately separate and quantify their native and oxidized products chromatographically is essential in furthering our understanding of these oxidative enzymes. Here we present a novel set of methods based on dual electrolytic eluent generation, where the conventional sodium acetate/sodium hydroxide (NaOAc/NaOH) eluents in high-performance anion-exchange chromatography (HPAEC) are replaced by electrolytically-generated potassium methane sulfonate/potassium hydroxide (KMSA/KOH). The new methods separate all compounds of interest within 24-45 minutes and with high sensitivity; limits of detection and quantification were in the range of 0.0001-0.0032 mM and 0.0002-0.0096 mM, respectively. In addition, an average of 3.5 times improvement in analytical CV was obtained. This chromatographic platform overcomes drawbacks associated with manual preparation of eluents and offers simplified operation and rapid method optimization, with increased precision for less abundant LPMO-derived products.Hide Abstract
Fast and specific peroxygenase reactions catalyzed by fungal mono-copper enzymes.
Rieder, L., Stepnov, A. A., Sørlie, M. & Eijsink, V. G. (2021). Biochemistry, In Press.
The copper-dependent lytic polysaccharide monooxygenases (LPMOs) are receiving attention because of their role in the degradation of recalcitrant biomass and their intriguing catalytic properties. The fundamentals of LPMO catalysis remain somewhat enigmatic as the LPMO reaction is affected by a multitude of LPMO- and co-substrate-mediated (side) reactions that result in a complex reaction network. We have performed kinetic studies with two LPMOs that are active on soluble substrates, NcAA9C and LsAA9A, using various reductants typically employed for LPMO activation. Studies with NcAA9C under “monooxygenase” conditions showed that the impact of the reductant on catalytic activity is correlated with the hydrogen peroxide-generating ability of the LPMO-reductant combination, supporting the idea that a peroxygenase reaction is taking place. Indeed, the apparent monooxygenase reaction could be inhibited by a competing H2O2-consuming enzyme. Interestingly, these fungal AA9-type LPMOs were found to have higher oxidase activity than bacterial AA10-type LPMOs. Kinetic analysis of the peroxygenase activity of NcAA9C on cellopentaose revealed a fast stoichiometric conversion of high amounts of H2O2 to oxidized carbohydrate products. A kcat value of 124 ± 27 s–1 at 4 °C is 20 times higher than a previously described kcat for peroxygenase activity on an insoluble substrate (at 25 °C) and some 4 orders of magnitude higher than typical “monooxygenase” rates. Similar studies with LsAA9A revealed differences between the two enzymes but confirmed fast and specific peroxygenase activity. These results show that the catalytic site arrangement of LPMOs provides a unique scaffold for highly efficient copper redox catalysis.Hide Abstract
Oligosaccharides production by enzymatic hydrolysis of banana pseudostem pulp.
Díaz, S., Ortega, Z., Benítez, A. N., Marrero, M. D., Carvalheiro, F., Duarte, L. C., Leonidas Matsakas, L., Eleni Krikigianni, E., Ulrika Rova, U., Christakopoulos, P. & Fernandes, M. C. (2021). Biomass Conversion and Biorefinery, 136, 1-12.
Banana production generates significant amounts of agricultural wastes, being fiber extraction one of the most relevant alternatives for their valorization. This process produces banana’s pseudostem pulp (BPP) as a byproduct, which shows an interesting composition for the biorefinery’s biochemical platform, with high polysaccharides (68%) and low lignin contents. This work deals with the enzymatic hydrolysis (EH) of raw and hydrothermally pre-treated BPP, focusing on the production of oligosaccharides (OS). Raw BPP hydrolysis with cellulase at different dosages rendered only 3.2% OS yields (OSY). Pectinase addition has not affected EH performance. On the other hand, EH of hydrothermally pre-treated BPP at 150°C and 170°C (P150 and P170) allowed to increase OSY up to 28% (P150, 1 FPU of cellulase/g dry biomass, 12 h), being 72% of the solubilized sugars in the form of cello-oligosaccharides. This last condition was subjected to a multi-stage EH strategy without improvements in OSY. An endo-glucanase was also tested, but obtained OSY were lower than cellulase results. Finally, obtained OS demonstrated to stimulate the growth of two Lactobacilli strains. The results show that BPP pre-treated under mild operational conditions is a good candidate for cello-oligosaccharides production by EH using 1 FPU/g DB of cellulase with a simple strategy.Hide Abstract
Four cellulose-active lytic polysaccharide monooxygenases from Cellulomonas species.
Li, J., Solhi, L., Goddard-Borger, E. D., Mathieu, Y., Wakarchuk, W. W., Withers, S. G. & Brumer, H. (2021). Biotechnology for Biofuels, 14(1), 1-19.
Background: The discovery of lytic polysaccharide monooxygenases (LPMOs) has fundamentally changed our understanding of microbial lignocellulose degradation. Cellulomonas bacteria have a rich history of study due to their ability to degrade recalcitrant cellulose, yet little is known about the predicted LPMOs that they encode from Auxiliary Activity Family 10 (AA10). Results: Here, we present the comprehensive biochemical characterization of three AA10 LPMOs from Cellulomonas flavigena (CflaLPMO10A, CflaLPMO10B, and CflaLPMO10C) and one LPMO from Cellulomonas fimi (CfiLPMO10). We demonstrate that these four enzymes oxidize insoluble cellulose with C1 regioselectivity and show a preference for substrates with high surface area. In addition, CflaLPMO10B, CflaLPMO10C, and CfiLPMO10 exhibit limited capacity to perform mixed C1/C4 regioselective oxidative cleavage. Thermostability analysis indicates that these LPMOs can refold spontaneously following denaturation dependent on the presence of copper coordination. Scanning and transmission electron microscopy revealed substrate-specific surface and structural morphological changes following LPMO action on Avicel and phosphoric acid-swollen cellulose (PASC). Further, we demonstrate that the LPMOs encoded by Cellulomonas flavigena exhibit synergy in cellulose degradation, which is due in part to decreased autoinactivation. Conclusions: Together, these results advance understanding of the cellulose utilization machinery of historically important Cellulomonas species beyond hydrolytic enzymes to include lytic cleavage. This work also contributes to the broader mapping of enzyme activity in Auxiliary Activity Family 10 and provides new biocatalysts for potential applications in biomass modification.Hide Abstract
Characterization and engineering of two new GH9 and GH48 cellulases from a Bacillus pumilus isolated from Lake Bogoria.
Ogonda, L. A., Saumonneau, A., Dion, M., Muge, E. K., Wamalwa, B. M., Mulaa, F. J. & Tellier, C. (2021). Biotechnology Letters, 1-10.
Objectives: To search for new alkaliphilic cellulases and to improve their efficiency on crystalline cellulose through molecular engineering. Results: Two novel cellulases, BpGH9 and BpGH48, from a Bacillus pumilus strain were identified, cloned and biochemically characterized. BpGH9 is a modular endocellulase belonging to the glycoside hydrolase 9 family (GH9), which contains a catalytic module (GH) and a carbohydrate-binding module belonging to class 3 and subclass c (CBM3c). This enzyme is extremely tolerant to high alkali pH and remains significantly active at pH 10. BpGH48 is an exocellulase, belonging to the glycoside hydrolase 48 family (GH48) and acts on the reducing end of oligo-β1,4 glucanes. A truncated form of BpGH9 and a chimeric fusion with an additional CBM3a module was constructed. The deletion of the CBM3c module results in a significant decline in the catalytic activity. However, fusion of CBM3a, although in a non native position, enhanced the activity of BpGH9 on crystalline cellulose. Conclusions: A new alkaliphilic endocellulase BpGH9, was cloned and engineered as a fusion protein (CBM3a-BpGH9), which led to an improved activity on crystalline cellulose.Hide Abstract
Engineering of cellobiose phosphorylase for the defined synthesis of cellotriose.
Ubiparip, Z., Moreno, D. S., Beerens, K. & Desmet, T. (2020). Applied Microbiology and Biotechnology, 104(19), 8327-8337.
Cellodextrins are non-digestible oligosaccharides that have attracted interest from the food industry as potential prebiotics. They are typically produced through the partial hydrolysis of cellulose, resulting in a complex mixture of oligosaccharides with a varying degree of polymerisation (DP). Here, we explore the defined synthesis of cellotriose as product since this oligosaccharide is believed to be the most potent prebiotic in the mixture. To that end, the cellobiose phosphorylase (CBP) from Cellulomonas uda and the cellodextrin phosphorylase (CDP) from Clostridium cellulosi were evaluated as biocatalysts, starting from cellobiose and α-D-glucose 1-phosphate as acceptor and donor substrate, respectively. The CDP enzyme was shown to rapidly elongate the chains towards higher DPs, even after extensive mutagenesis. In contrast, an optimised variant of CBP was found to convert cellobiose to cellotriose with a molar yield of 73%. The share of cellotriose within the final soluble cellodextrin mixture (DP2-5) was 82%, resulting in a cellotriose product with the highest purity reported to date. Interestingly, the reaction could even be initiated from glucose as acceptor substrate, which should further decrease the production costs.Hide Abstract
Characterization of two GH5 endoglucanases from termite microbiome using synthetic metagenomics.
Guerrero, E. B., de Villegas, R. M. D., Soria, M. A., Santangelo, M. P., Campos, E. & Talia, P. M. (2020). Applied Microbiology and Biotechnology, 104(19), 8351-8366.
Here, we characterize two novel GH5 endoglucanases (GH5CelA and GH5CelB) from an uncultured bacterium identified in termite gut microbiomes. Both genes were codon-optimized, synthetized, cloned, and expressed as recombinant proteins in Escherichia coli for subsequent purification. Both enzymes showed activity on the pNPC and barley β-glucan substrates, whereas GH5CelB also showed low activity on carboxymethyl cellulose. The optimum conditions for both enzymes were an acid pH (5) and moderate temperature (35 to 50°C). The enzymes differed in the kinetic profiles and patterns of the generated hydrolysis products. A structural-based modeling analysis indicated that both enzymes possess a typical (β/α)8-barrel fold characteristic of GH5 family, with some differential features in the active site cleft. Also, GH5CelB presents a putative secondary binding site. Furthermore, adjacent to the active site of GH5CelA and GH5CelB, a whole subdomain rarely found in GH5 family may participate in substrate binding and thermal stability. Therefore, GH5CelA may be a good candidate for the production of cello-oligosaccharides of different degrees of polymerization applicable for feed and food industries, including prebiotics. On the other hand, GH5CelB could be useful in an enzymatic cocktail for the production of lignocellulosic bioethanol, because of the production of glucose as a hydrolysis product.Hide Abstract
Promoting and Impeding Effects of Lytic Polysaccharide Monooxygenases on Glycoside Hydrolase Activity.
Keller, M. B., Badino, S. F., Blossom, B. M., McBrayer, B., Borch, K. & Westh, P. (2020). ACS Sustainable Chemistry & Engineering, 8(37), 14117-14126.
Lytic polysaccharide monooxygenases (LPMOs) have attracted attention due to their ability to boost cellulolytic enzyme cocktails for application in biorefineries. However, the interplay between LPMOs and individual glycoside hydrolases remains poorly understood. We investigated how the activity of two cellobiohydrolases (Cel7A and Cel6A) and an endoglucanase (Cel7B) from Trichoderma reesei were affected by a C1-oxidizing LPMO from Thielavia terrestrit (TtAA9). We quantified products from a mixture of LPMO and glycoside hydrolase and estimated separate contributions of products by each of the enzymes. Hereby, we assessed if an observed synergy reflected a promotion of the activity of hydrolase, LPMO, or both. We consistently found that TtAA9 affected the investigated hydrolases differently. It strongly impeded the turnover of the reducing end cellobiohydrolase, TrCel7A, moderately promoted the turnover of the nonreducing end cellobiohydrolase TrCel6A, and promoted the turnover of the endoglucanase, TrCel7B up to 5-fold. The promoting effect on the endoglucanase increased with hydrolysis extent, indicating that the promoting effect became more important as the recalcitrance of the substrate increased. Experiments with mixtures containing multiple glycoside hydrolases suggested that the LPMO primarily promoted the activity of the endoglucanase, whereas promotion of TrCel6A was secondary.Hide Abstract
Degradative GH5 β-1, 3-1, 4-glucanase PpBglu5A for glucan in Paenibacillus polymyxa KF-1.
Yuan, Y., Zhang, X., Zhang, H., Wang, W., Zhao, X., Gao, J. & Zhou, Y. (2020). Process Biochemistry, 98, 183-192.
A novel β-1,3-1,4-glucanase in the glycoside hydrolase family 5 (GH5) has been identified in the secretome of Paenibacillus polymyxa KF-1. The recombinant GH5 enzyme PpBglu5A shows broad substrate specificity, with strong lichenase activity, medium β-1,3-glucanase activity, and minimal cellulase activity. Barley β-glucan, lichenan, curdlan, and carboxymethyl cellulose are hydrolyzed to varying degrees by PpBglu5A, with the highest catalytic activity being observed with barley β-glucan. Hydrolysates from barley β-glucan or lichenan are primarily glucan oligosaccharides with degrees of polymerization from 2 to 4. PpBglu5A also hydrolyzes oat bran into oligosaccharides mainly consisted of di-, tri-, and tetra- oligosaccharides that are useful in the preparation of gluco-oligosaccharides. In addition to hydrolytic activity, transglycosylation was also observed with PpBglu5A and cellotriose as substrate. An in vitro assay indicated that the recombinant PpBglu5A has antifungal activity and can inhibit the growth of Canidia albicans. These results suggest that PpBglu5A exhibits unique properties and may be useful as an antifungal agent.Hide Abstract
Identification of a unique 1, 4-β-d-glucan glucohydrolase of glycoside hydrolase family 9 from Cytophaga hutchinsonii.
Jiang, N., Ma, X. D., Fu, L. H., Li, C. X., Feng, J. X. & Duan, C. J. (2020). Applied Microbiology and Biotechnology, 1-16.
Cytophaga hutchinsonii is an aerobic cellulolytic soil bacterium that rapidly digests crystalline cellulose. The predicted mechanism by which C. hutchinsonii digests cellulose differs from that of other known cellulolytic bacteria and fungi. The genome of C. hutchinsonii contains 22 glycoside hydrolase (GH) genes, which may be involved in cellulose degradation. One predicted GH with uncertain specificity, CHU_0961, is a modular enzyme with several modules. In this study, phylogenetic tree of the catalytic modules of the GH9 enzymes showed that CHU_0961 and its homologues formed a new group (group C) of GH9 enzymes. The catalytic module of CHU_0961 (CHU_0961B) was identified as a 1,4-β-D-glucan glucohydrolase (EC 184.108.40.206) that has unique properties compared with known GH9 cellulases. CHU_0961B showed highest activity against barley glucan, but low activity against other polysaccharides. Interestingly, CHU_0961B showed similar activity against ρ-nitrophenyl β-D-cellobioside (ρ-NPC) and ρ-nitrophenyl β-D-glucopyranoside. CHU_0961B released glucose from the nonreducing end of cello-oligosaccharides, ρ-NPC, and barley glucan in a nonprocessive exo-type mode. CHU_0961B also showed same hydrolysis mode against deacetyl-chitooligosaccharides as against cello-oligosaccharides. The kcat/Km values for CHU_0961B against cello-oligosaccharides increased as the degree of polymerization increased, and its kcat/Km for cellohexose was 750 times higher than that for cellobiose. Site-directed mutagenesis showed that threonine 321 in CHU_0961 played a role in hydrolyzing cellobiose to glucose. CHU_0961 may act synergistically with other cellulases to convert cellulose to glucose on the bacterial cell surface. The end product, glucose, may initiate cellulose degradation to provide nutrients for bacterial proliferation in the early stage of C. hutchinsonii growth.Hide Abstract
Improved cellulose X-ray diffraction analysis using Fourier series modeling.
Yao, W., Weng, Y. & Catchmark, J. M. (2020). Cellulose, 1-17.
This paper addresses two fundamental issues in the peak deconvolution method of cellulose XRD data analysis: there is no standard model for amorphous cellulose and common peak functions such as Gauss, Lorentz and Voigt functions do not fit the amorphous profile well. It first examines the effects of ball milling on three types of cellulose and results show that ball milling transforms all samples into a highly amorphous phase exhibiting nearly identical powder X-ray diffraction (XRD) profiles. It is hypothesized that short range order within a glucose unit and between adjacent units survives ball milling and generates the characteristic amorphous XRD profiles. This agrees well with cellulose I d-spacing measurements and oligosaccharide XRD analysis. The amorphous XRD profile is modeled using a Fourier series equation where the coefficients are determined using the nonlinear least squares method. A new peak deconvolution method then is proposed to analyze cellulose XRD data with the amorphous Fourier model function in conjunction with standard Voigt functions representing the crystalline peaks. The impact of background subtraction method has also been assessed. Analysis of several cellulose samples was then performed and compared to the conventional peak deconvolution methods with common peak fitting functions and background subtraction approach. Results suggest that prior peak deconvolution methods overestimate cellulose crystallinity.Hide Abstract
Lytic polysaccharide monooxygenases (LPMOs) facilitate cellulose nanofibrils production.
Moreau, C., Tapin-Lingua, S., Grisel, S., Gimbert, I., Le Gall, S., Meyer, V., Petit-Conil, M., Berrin, J, Cathala, B. & Villares, A. (2019). Biotechnology for Biofuels, 12(1), 1-13.
Background: Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that cleave polysaccharides through an oxidative mechanism. These enzymes are major contributors to the recycling of carbon in nature and are currently used in the biorefinery industry. LPMOs are commonly used in synergy with cellulases to enhance biomass deconstruction. However, there are few examples of the use of monocomponent LPMOs as a tool for cellulose fibrillation. In this work, we took advantage of the LPMO action to facilitate disruption of wood cellulose fibers as a strategy to produce nanofibrillated cellulose (NFC). Results: The fungal LPMO from AA9 family (PaLPMO9E) was used in this study as it displays high specificity toward cellulose and its recombinant production in bioreactor is easily upscalable. The treatment of birchwood fibers with PaLPMO9E resulted in the release of a mixture of C1-oxidized oligosaccharides without any apparent modification in fiber morphology and dimensions. The subsequent mechanical shearing disintegrated the LPMO-pretreated samples yielding nanoscale cellulose elements. Their gel-like aspect and nanometric dimensions demonstrated that LPMOs disrupt the cellulose structure and facilitate the production of NFC. Conclusions: This study demonstrates the potential use of LPMOs as a pretreatment in the NFC production process. LPMOs weaken fiber cohesion and facilitate fiber disruption while maintaining the crystallinity of cellulose.Hide Abstract
Identification and characterization of a hyperthermophilic GH9 cellulase from the Arctic Mid-Ocean Ridge vent field.
Stepnov, A. A., Fredriksen, L., Steen, I. H., Stokke, R. & Eijsink, V. G. (2019). PloS One, 14(9), e0222216.
A novel GH9 cellulase (AMOR_GH9A) was discovered by sequence-based mining of a unique metagenomic dataset collected at the Jan Mayen hydrothermal vent field. AMOR_GH9A comprises a signal peptide, a catalytic domain and a CBM3 cellulose-binding module. AMOR_GH9A is an exceptionally stable enzyme with a temperature optimum around 100°C and an apparent melting temperature of 105°C. The novel cellulase retains 64% of its activity after 4 hours of incubation at 95°C. The closest characterized homolog of AMOR_GH9A is TfCel9A, a processive endocellulase from the model thermophilic bacterium Thermobifida fusca (64.2% sequence identity). Direct comparison of AMOR_GH9A and TfCel9A revealed that AMOR_GH9A possesses higher activity on soluble and amorphous substrates (phosphoric acid swollen cellulose, konjac glucomannan) and has an ability to hydrolyse xylan that is lacking in TfCel9A.Hide Abstract
A lytic polysaccharide monooxygenase from Myceliophthora thermophila and its synergism with cellobiohydrolases in cellulose hydrolysis.
Zhou, H., Li, T., Yu, Z., Ju, J., Zhang, H., Tan, H., Li, K. & Yin, H. (2019). International Journal of Biological Macromolecules, 139, 570-576.
Lytic polysaccharide monooxygenases (LPMOs) have attracted vast attention because of their unique mechanism of oxidative degradation of carbohydrate polymers and the potential application in biorefineries. This study characterized a novel LPMO from Myceliophthora thermophila, denoted MtLPMO9L. The structure model of the enzyme indicated that it belongs to the C1-oxidizing LPMO, which has neither an extra helix in the L3 loop nor extra loop region in the L2 loop. This was confirmed subsequently by the enzymatic assays since MtLPMO9L only acts on cellulose and generates C1-oxidized cello-oligosaccharides. Moreover, synergetic experiments showed that MtLPMO9L significantly improves the efficiency of cellobiohydrolase (CBH) II. In contrast, the inhibitory rather than synergetic effect was observed when combining used MtLPMO9L and CBHI. Changing the incubation time and concentration ratio of MtLPMO9L and CBHI could attenuate the inhibitory effects. This discovery suggests a different synergy detail between MtLPMO9L and two CBHs, which implies that the composition of cellulase cocktails may need reconsideration.Hide Abstract
An actinobacteria lytic polysaccharide monooxygenase acts on both cellulose and xylan to boost biomass saccharification.
Corrêa, T. L. R., Júnior, A. T., Wolf, L. D., Buckeridge, M. S., dos Santos, L. V. & Murakami, M. T. (2019). Biotechnology for Biofuels, 12(1), 117.
Background: Lytic polysaccharide monooxygenases (LPMOs) opened a new horizon for biomass deconstruction. They use a redox mechanism not yet fully understood and the range of substrates initially envisaged to be the crystalline polysaccharides is steadily expanding to non-crystalline ones. Results: The enzyme KpLPMO10A from the actinomycete Kitasatospora papulosa was cloned and overexpressed in Escherichia coli cells in the functional form with native N-terminal. The enzyme can release oxidized species from chitin (C1-type oxidation) and cellulose (C1/C4-type oxidation) similarly to other AA10 members from clade II (subclade A). Interestingly, KpLPMO10A also cleaves isolated xylan (not complexed with cellulose, C4-type oxidation), a rare activity among LPMOs not described yet for the AA10 family. The synergistic effect of KpLPMO10A with Celluclast ® and an endo-β-1,4-xylanase also supports this finding. The crystallographic elucidation of KpLPMO10A at 1.6 Å resolution along with extensive structural analyses did not indicate any evident diference with other characterized AA10 LPMOs at the catalytic interface, tempting us to suggest that these enzymes might also be active on xylan or that the ability to attack both crystalline and non-crystalline substrates involves yet obscure mechanisms of substrate recognition and binding. Conclusions: This work expands the spectrum of substrates recognized by AA10 family, opening a new perspective for the understanding of the synergistic efect of these enzymes with canonical glycoside hydrolases to deconstruct ligno(hemi)cellulosic biomass.Hide Abstract
FGB1 and WSC3 are in planta‐induced β‐glucan‐binding fungal lectins with different functions.
Wawra, S., Fesel, P., Widmer, H., Neumann, U., Lahrmann, U., Becker, S., Hehemann, J. H., Langen, G. & & Zuccaro, A. (2019). New Phytologist, 222(3), 1493-1506.
In the root endophyte Serendipita indica, several lectin‐like members of the expanded multigene family of WSC proteins are transcriptionally induced in planta and are potentially involved in β-glucan remodeling at the fungal cell wall. Using biochemical and cytological approaches we show that one of these lectins, SiWSC3 with three WSC domains, is an integral fungal cell wall component that binds to long‐chain β1‐3‐glucan but has no affinity for shorter β1‐3‐ or β1‐6‐linked glucose oligomers. Comparative analysis with the previously identified β-glucan‐binding lectin SiFGB1 demonstrated that whereas SiWSC3 does not require β1‐6‐linked glucose for efficient binding to branched β1‐3‐glucan, SiFGB1 does. In contrast to SiFGB1, the multivalent SiWSC3 lectin can efficiently agglutinate fungal cells and is additionally induced during fungus-fungus confrontation, suggesting different functions for these two β-glucan‐binding lectins. Our results highlight the importance of the β-glucan cell wall component in plant–fungus interactions and the potential of β-glucan‐binding lectins as specific detection tools for fungi in vivo.Hide Abstract
Turning a potent family‐9 free cellulase into an operational cellulosomal component and vice versa.
Vita, N., Borne, R., Perret, S., de Philip, P. & Fierobe, H. P. (2019). The FEBS Journal, 286(17), 3359-3373.
Ruminiclostridium cellulolyticum and Lachnoclostridium phytofermentans are cellulolytic clostridia either producing extracellular multienzymatic complexes termed cellulosomes or secreting free cellulases respectively. In the free state, the cellulase Cel9A secreted by L. phytofermentans is much more active on crystalline cellulose than any cellulosomal family‐9 enzyme produced by R. cellulolyticum. Nevertheless, the incorporation of Cel9A in vitro in hybrid cellulosomes was formerly shown to generate artificial complexes with altered activity, whereas its incorporation in vivo in native R. cellulolyticum cellulosomes resulted in a strain displaying a weakened cellulolytic phenotype. In this study, we investigated why Cel9A is so potent in the free state but functions poorly as a cellulosomal component, in contrast to the most similar enzyme synthesized by R. cellulolyticum, Cel9G, weakly active in the free state but whose activity on crystalline cellulose is drastically increased in cellulosomes. We show that the removal of the C‐terminal moiety of Cel9A encompassing the two X2 modules and the family‐3b carbohydrate binding module (CBM3b), reduces its activity on crystalline cellulose. Grafting a dockerin module further diminishes the activity, but this truncated cellulosomal form of Cel9A displays important synergies in hybrid cellulosomes with the pivotal family‐48 cellulosomal enzyme of R. cellulolyticum. The exact inverse approach was applied to the cellulosomal Cel9G. Grafting the two X2 modules and the CBM3b of Cel9A to Cel9G strongly increases its activity on crystalline cellulose, to reach Cel9A activity levels. Altogether these data emphasize the specific features required to generate an efficient free or cellulosomal family‐9 cellulase.Hide Abstract