RT-CaCCO process: an improved CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature.
Shiroma, R., Park, J. Y., Al-Haq, M. I., Arakane, M., Ike, M. & Tokuyasu, K. (2011). Bioresource Technology, 102(3), 2943-2949.
We improved the CaCCO process for rice straw by its incorporation with a step of lime pretreatment at room temperature (RT). We firstly optimized the RT-lime pretreatment for the lignocellulosic part. When the ratio of lime/dry-biomass was 0.2 (w/w), the RT lime-pretreatment for 7-d resulted in an effect on the enzymatic saccharification of cellulose and xylan equivalent to that of the pretreatment at 120°C for 1 h. Sucrose, starch and β-1,3-1,4-glucan, which could be often detected in rice straw, were mostly stable under the RT-lime pretreatment condition. Then, the pretreatment condition in the conventional CaCCO process was modified by the adaptation of the optimized RT lime-pretreatment, resulting in significantly better carbohydrate recoveries via enzymatic saccharification than those of the CaCCO process (120°C for 1 h). Thus, the improved CaCCO process (the RT-CaCCO process) could preserve/pretreat the feedstock at RT in a wet form with minimum loss of carbohydrates.
Real-time imaging of cellulose reorientation during cell wall expansion in Arabidopsis roots.
Anderson, C. T., Carroll, A., Akhmetova, L. & Somerville, C. (2010). Plant Physiology, 152(2), 787-796.
Cellulose forms the major load-bearing network of the plant cell wall, which simultaneously protects the cell and directs its growth. Although the process of cellulose synthesis has been observed, little is known about the behavior of cellulose in the wall after synthesis. Using Pontamine Fast Scarlet 4B, a dye that fluoresces preferentially in the presence of cellulose and has excitation and emission wavelengths suitable for confocal microscopy, we imaged the architecture and dynamics of cellulose in the cell walls of expanding root cells. We found that cellulose exists in Arabidopsis (Arabidopsis thaliana) cell walls in large fibrillar bundles that vary in orientation. During anisotropic wall expansion in wild-type plants, we observed that these cellulose bundles rotate in a transverse to longitudinal direction. We also found that cellulose organization is significantly altered in mutants lacking either a cellulose synthase subunit or two xyloglucan xylosyltransferase isoforms. Our results support a model in which cellulose is deposited transversely to accommodate longitudinal cell expansion and reoriented during expansion to generate a cell wall that is fortified against strain from any direction.
Characterization and three-dimensional structures of two distinct bacterial xyloglucanases from families GH5 and GH12.
Gloster, T. M., Ibatullin, F. M., Macauley, K., Eklöf, J. M., Roberts, S., Turkenburg, J. P., Bjørnvad, M. E., Jørgensen, P. L., Danielsen, S., Johansen, K. S., Borchert, T. V., Wilson, K. S., Brumer, H. & Davies, G. J. (2007). Journal of Biological Chemistry, 282(26), 19177-19189.
The plant cell wall is a complex material in which the cellulose microfibrils are embedded within a mesh of other polysaccharides, some of which are loosely termed “hemicellulose.” One such hemicellulose is xyloglucan, which displays a β-1,4-linked D-glucose backbone substituted with xylose, galactose, and occasionally fucose moieties. Both xyloglucan and the enzymes responsible for its modification and degradation are finding increasing prominence, reflecting both the drive for enzymatic biomass conversion, their role in detergent applications, and the utility of modified xyloglucans for cellulose fiber modification. Here we present the enzymatic characterization and three-dimensional structures in ligand-free and xyloglucan-oligosaccharide complexed forms of two distinct xyloglucanases from glycoside hydrolase families GH5 and GH12. The enzymes, Paenibacillus pabuli XG5 and Bacillus licheniformis XG12, both display open active center grooves grafted upon their respective (β/α)8 and β-jelly roll folds, in which the side chain decorations of xyloglucan may be accommodated. For the β-jelly roll enzyme topology of GH12, binding of xylosyl and pendant galactosyl moieties is tolerated, but the enzyme is similarly competent in the degradation of unbranched glucans. In the case of the (β/ α)8 GH5 enzyme, kinetically productive interactions are made with both xylose and galactose substituents, as reflected in both a high specific activity on xyloglucan and the kinetics of a series of aryl glycosides. The differential strategies for the accommodation of the side chains of xyloglucan presumably facilitate the action of these microbial hydrolases in milieus where diverse and differently substituted substrates may be encountered.
Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module.
Gunnarsson, L. C., Montanier, C., Tunnicliffe, R. B., Williamson, M. P., Gilbert, H. J., Nordberg, K. E. & Ohlin, M. (2007). Biochem. J, 406(2), 209-214.
Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.
Recombinant expression and enzymatic characterization of PttCel9A, a KOR homologue from Populus tremula x tremuloides.
Master, E. R., Rudsander, U. J., Zhou, W., Henriksson, H., Divne, C., Denman, S., Wilson D. B. & Teeri, T. T. (2004). Biochemistry, 43(31), 10080-10089.
PttCel9A is a membrane-bound, family 9 glycosyl hydrolase from Populus tremula x tremuloides that is upregulated during secondary cell wall synthesis. The catalytic domain of PttCel9A, Δ1-105PttCel9A, was purified, and its activity was compared to TfCel9A and TfCel9B from Thermobifida fusca. Since aromatic amino acids involved in substrate binding at subsites −4, −3, and −2 are missing in PttCel9A, the activity of TfCel9A mutant enzymes W256S, W209A, and W313G was also investigated. Δ1-105PttCel9A hydrolyzed a comparatively narrow range of polymeric substrates, and the preferred substrate was (carboxymethyl)cellulose 4M. Moreover, Δ1-105PttCel9A did not hydrolyze oligosaccharides shorter than cellopentaose, whereas TfCel9A and TfCel9B hydrolyzed cellotetraose and cellotriose, respectively. These data suggest that the preferred substrates of PttCel9A are long, low-substituted, soluble cellulosic polymers. At 30°C and pH 6.0, the kcat for cellohexaose of Δ1-105PttCel9A, TfCel9A, and TfCel9B were 0.023 ± 0.001, 16.9 ± 2.0, and 1.3 ± 0.2, respectively. The catalytic efficiency (kcat/km) of TfCel9B was 39% of that of TfCel9A, whereas the catalytic efficiency of Δ1-105PttCel9A was 0.04% of that of TfCel9A. Removing tryptophan residues at subsites −4, −3, and −2 decreased the efficiency of cellohexaose hydrolysis by TfCel9A. Mutation of W313 to G had the most drastic effect, producing a mutant enzyme with 1% of the catalytic efficiency of TfCel9A. The apparent narrow substrate range and catalytic efficiency of PttCel9A are correlated with a lack of aromatic amino acids in the substrate binding cleft and may be necessary to prevent excessive hydrolysis of cell wall polysaccharides during cell wall formation.
Neoglycolipid-Based “Designer” Oligosaccharide Microarrays to Define β-Glucan Ligands for Dectin-1.
Palma, A. S., Zhang, Y., Childs, R. A., Campanero-Rhodes, M. A., Liu, Y., Feizi, T. & Chai, W. (2012). Carbohydrate Microarrays, 808(2), 337-359.
In this chapter, we describe the key steps of the “designer” oligosaccharide microarray approach we followed to prove the carbohydrate binding activity and define the oligosaccharide ligands for Dectin-1, an atypical C-type lectin-like signaling receptor of the mammalian innate immune system with a key role in anti-fungal immunity. The term “designer” microarray, which we introduced in the course of the Dectin-1 study refers to a microarray of oligosaccharide probes generated from ligand-bearing glycoconjugates to reveal the oligosaccharide ligands they harbor, so that these can be isolated and characterized. Oligosaccharide probes were generated from two polysaccharides, one that was bound by Dectin-1 and known to be rich in β1,3-glucose sequence and another that was not bound and was rich in β1,6-glucose sequence and served as a negative control. The approach involved: classic ELISA-type binding assays to select the polysaccharides; partial depolymerization of the polysaccharides by chemical hydrolysis; fractionation by size of the glucan oligosaccharides obtained and determination of their chain lengths by mass spectrometry; detection of Dectin-1 ligand-positive and ligand-negative oligosaccharides using the neoglycolipid (NGL) technology; methylation analysis of oligosaccharides to derive glucose linkage information, and incorporation of the newly generated glucan oligosaccharide probes into microarrays encompassing diverse mammalian-type and exogenous sequences for microarray analysis of Dectin-1.
Cloning of an endoglycanase gene from Paenibacillus cookii and characterization of the recombinant enzyme.
Shinoda, S., Kanamasa, S. & Arai, M. (2012). Biotechnology Letters, 34(2), 281-286.
An endoglycanase gene of Paenibacillus cookii SS-24 was cloned and sequenced. This Pgl8A gene had an open reading frame of 1,230 bp that encoded a putative signal sequence (31 amino acids) and mature enzyme (378 amino acids: 41,835 Da). The enzyme was most homologous to a β-1,3-1,4-glucanase of Bacillus circulans WL-12 with 84% identity. The recombinant enzyme hydrolyzed carboxymethyl cellulose, swollen celluloses, chitosan and lichenan but not Avicel, chitin powder or xylan. With chitosan as the substrate, the optimum temperature and hydrolysis products of the recombinant enzyme varied at pH 4.0 and 8.0. This is the first report that characterizes chitosanase activity under different pH conditions.
Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: biological implications for cell wall metabolism.
Baumann, M. J., Eklöf, J. M., Michel, G., Kallas, Å. M., Teeri, T. T., Czjzek, M. & Brumer, H. (2007). The Plant Cell, 19(6), 1947-1963.
High-resolution, three-dimensional structures of the archetypal glycoside hydrolase family 16 (GH16) endo-xyloglucanases Tm-NXG1 and Tm-NXG2 from nasturtium (Tropaeolum majus) have been solved by x-ray crystallography. Key structural features that modulate the relative rates of substrate hydrolysis to transglycosylation in the GH16 xyloglucan-active enzymes were identified by structure–function studies of the recombinantly expressed enzymes in comparison with data for the strict xyloglucan endo-transglycosylase Ptt-XET16-34 from hybrid aspen (Populus tremula × Populus tremuloides). Production of the loop deletion variant Tm-NXG1-ΔYNIIG yielded an enzyme that was structurally similar to Ptt-XET16-34 and had a greatly increased transglycosylation:hydrolysis ratio. Comprehensive bioinformatic analyses of XTH gene products, together with detailed kinetic data, strongly suggest that xyloglucanase activity has evolved as a gain of function in an ancestral GH16 XET to meet specific biological requirements during seed germination, fruit ripening, and rapid wall expansion.
A comparative study on structure–function relations of mixed-linkage (1→ 3),(1→ 4) linear β-D-glucans.
Lazaridou, A., Biliaderis, C. G., Micha-Screttas, M. & Steele, B. R. (2004). Food Hydrocolloids, 18(5), 837-855.
The effects of fine structure and molecular size on the rheological properties of six mixed-linkage (1→3), (1→4)-β-D-glucans (β-glucans) in the solution and gel state were studied. Molecular size characterization was carried out with high-performance size exclusion chromatography combined with a refractive index detector. Samples were divided into two groups according to the values of apparent molecular weight (Mw) of the peak fraction of the main eluting peak calculated as 200×103 for an oat, a barley, and a wheat β-glucan and ∼100×103 for an oat and a barley β-glucan, and a lichenan sample. All polysaccharides analyzed by 2D NMR spectroscopy and high-performance anion-exchange chromatography of the cellulosic oligomers released by the action of lichenase showed the typical fine structure of mixed-linkage linear (1→3), (1→4)-β-D-glucan. Following lichenase digestion of β-glucans, the molar ratios of tri- to tetrasaccharides (DP3/DP4) were found to follow the order of lichenan (24.5)>wheat (3.7)>barley (2.8–3.0)>oat (2.1). Differences in critical concentration (c* *), viscosity, viscoelastic and shear thinning properties among samples were dependent mainly on differences in molecular size of the polymeric chains as well as on the β-glucan fine structure. All β-glucan isolates were able to form gels, as probed by dynamic rheometry; with decreasing molecular size and increasing DP3/DP4 ratio, the gelation time decreased and the gelation rate (IE=[d log G'/dt ]max) increased. Differential scanning calorimetry (DSC) showed that cereal β-glucan gels exhibit rather broad endothermic gel→sol transitions at 55–80°C, while lichenan gels give a sharper transition, implying a more cooperative process. The DSC kinetic data showed similar responses to that from dynamic rheometry; the rate of development of the endotherm increased with increasing DP3/DP4 ratio of the polysaccharide. Furthermore, the storage modulus (G′) and the apparent melting enthalpy values (plateau ΔH) increased with decreasing molecular size and with increasing DP3/DP4 ratio. The melting temperature of the gel network, as determined by DSC and dynamic rheometry, was found to increase with the molecular size and the DP3/DP4 ratio of β-glucans; the Tm for lichenan was ∼89°C and for cereal β-glucans varied in the narrow range of ∼65–72°C. Large deformation mechanical tests (compression mode) up to failure revealed an increase in strength and a decrease in brittleness of mixed-linkage β-glucan gels with increasing DP3/DP4 ratio and molecular size of the polysaccharide.
Evaluation of structure in the formation of gels by structurally diverse (1→3)(1→4)-β-D-glucans from four cereal and one lichen species.
Tosh, S. M., Brummer, Y., Wood, P. J., Wang, Q. & Weisz, J. (2004). Carbohydrate Polymers, 57(3), 249-259.
The (1→3)(1→4)-β-D-glucans from four cereal sources (oats, wheat, barley and rye) and one lichen source (Icelandic moss) were used to test two proposed structurally based hypotheses about the gelling mechanism of these polymers. Structures were evaluated using high performance anion exchange chromatography of the oligosaccharide fragments released by a (1→3)(1→4)-β-D-glucan-4-glucanohydrolase. This determined the relative amounts of cellodextrin units, of different degrees of polymerisation, which are joined by β-(1→3) linkages in the intact polysaccharide chain. Oat β-glucan had the lowest β-(1→3)-linked cellotriosyl unit content and lichenan had the highest. Strong correlations were found between the fraction of β-(1→3)-linked cellotriosyl units in the β-glucans and the elasticity of 6% gels in water, as measured by dynamic rheometry. Differential scanning calorimetry showed that the β-(1→3)-linked cellotriosyl unit content was also correlated with the onset and peak temperatures when 6% β-glucan gels were melted. No correlation was found between the longer (DP 6–9) β-(1→3)-linked cellodextrin oligosaccharide content and either the gel elasticity or melting characteristics. These findings are consistent with a model in which runs of consecutive β-(1→3)-linked cellotriosyl units form the junction zones in the gel network, but not with a model in which longer β-(1→3)-linked cellodextrins associate, as in cellulose fibres, to produce the gel network. Microscopic images of the β-glucan gels from the five species revealed that the microstructure was not homogeneous in any of the samples, which may be related to the variability in the enthalpy of melting of gels. There was a coarsening of gel structure as the β-(1→3)-linked cellotriosyl unit content increased.
A fibrolytic potential in the human ileum mucosal microbiota revealed by functional metagenomics.
Patrascu, O., Béguet-Crespel, F., Marinelli, L., Le Chatelier, E., Abraham, A., Leclerc, M., Klopp, C., Terrapon, N., Henrissat, B., Blottière, H. M., Doré, J. & Christel Béra-Maillet. (2017). Scientific Reports, 7, 40248.
The digestion of dietary fibers is a major function of the human intestinal microbiota. So far this function has been attributed to the microorganisms inhabiting the colon, and many studies have focused on this distal part of the gastrointestinal tract using easily accessible fecal material. However, microbial fermentations, supported by the presence of short-chain fatty acids, are suspected to occur in the upper small intestine, particularly in the ileum. Using a fosmid library from the human ileal mucosa, we screened 20,000 clones for their activities against carboxymethylcellulose and xylans chosen as models of the major plant cell wall (PCW) polysaccharides from dietary fibres. Eleven positive clones revealed a broad range of CAZyme encoding genes from Bacteroides and Clostridiales species, as well as Polysaccharide Utilization Loci (PULs). The functional glycoside hydrolase genes were identified, and oligosaccharide break-down products examined from different polysaccharides including mixed-linkage β-glucans. CAZymes and PULs were also examined for their prevalence in human gut microbiome. Several clusters of genes of low prevalence in fecal microbiome suggested they belong to unidentified strains rather specifically established upstream the colon, in the ileum. Thus, the ileal mucosa-associated microbiota encompasses the enzymatic potential for PCW polysaccharide degradation in the small intestine.
Enhanced saccharification of reed and rice straws by the addition of β-1,3-1,4-glucanase with broad substrate specificity and calcium ion.
Kim, D. U., Kim, H. J., Jeong, Y. S., Na, H. B., Cha, Y. L., Koo, B. C., Kim, J., Yun, H. D., Lee, J. K. & Kim, H. (2015). Journal of the Korean Society for Applied Biological Chemistry, 58(1), 29-33.
The possibility of using additive enzymes to improve the saccharification of lignocellulosic substrates with commercial cellulolytic enzymes was studied. Reed (Phragmites communis) and rice (Oryza sativa) straw powders were pretreated with NaOH/steam via a high-temperature explosion system. The saccharification of untreated reed and rice straw powders by commercial enzymes (Celluclast 1.5 L + Novozym 188) was not significantly increased by the addition of xylanases (Xyn10J, XynX), a cellulase (Cel6H), and a β-1,3-1,4-glucanase (BGlc8H) with broad substrate specificity. The saccharification of the pretreated reed and rice straw powders by the commercial enzymes was increased by 10.4 and 4.8 %, respectively, by the addition of BGlc8H. In the presence of Ca2+ and BGlc8H, the saccharification of the pretreated reed and rice straw powders by the commercial enzymes was increased by 18.5 and 11.7 %, respectively. No such effect of Ca2+ was observed with Xyn10J, XynX, or Cel6H. The results suggest that the enzymatic conversion of lignocellulosic biomass to reducing sugars could be enhanced by certain additive enzymes such as β-1,3-1,4-glucanase, and that the enhancement could further be increased by Ca2+.
Suberin regulates the production of cellulolytic enzymes in Streptomyces scabiei, the causal agent of potato common scab.
Padilla-Reynaud, R., Simao-Beaunoir, A. M., Lerat, S., Bernards, M. A. & Beaulieu, C. (2015). Microbes and environments, 30(3), 245-253.
Suberin, a major constituent of the potato periderm, is known to promote the production of thaxtomins, the key virulence factors of the common scab-causing agent Streptomyces scabiei. In the present study, we speculated that suberin affected the production of glycosyl hydrolases, such as cellulases, by S. scabiei, and demonstrated that suberin promoted glycosyl hydrolase activity when added to cellulose-, xylan-, or lichenin-containing media. Furthermore, secretome analyses revealed that the addition of suberin to a cellulose-containing medium increased the production of glycosyl hydrolases. For example, the production of 13 out of the 14 cellulases produced by S. scabiei in cellulose-containing medium was stimulated by the presence of suberin. In most cases, the transcription of the corresponding cellulase-encoding genes was also markedly increased when the bacterium was grown in the presence of suberin and cellulose. The level of a subtilase-like protease inhibitor was markedly decreased by the presence of suberin. We proposed a model for the onset of S. scabiei virulence mechanisms by both cellulose and suberin, the main degradation product of cellulose that acts as an inducer of thaxtomin biosynthetic genes, and suberin promoting the biosynthesis of secondary metabolites including thaxtomins.
Bonds broken and formed during the mixed-linkage glucan: xyloglucan endotransglucosylase reaction catalysed by Equisetum hetero-trans-β-glucanase.
Simmons, T. J. & Fry, S. C. (2017). Biochemical Journal, 474(7), 1055-1070.
Mixed-linkage glucan : xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-β-glucanase (HTG), which among land-plants is known only from Equisetum species. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's role in vivo and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1→3),(1→4)-β-D-glucan (MLG), with radioactive oligosaccharides of xyloglucan as acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography, HPLC and gel-permeation chromatography. Equisetum HTG consistently cleaved the MLG at the third consecutive β-( 1→4)-bond following (towards the reducing terminus) a β-( 1→3)-bond. It then formed a β-( 1→4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its XTH subfamily membership. Using size-homogeneous barley MLG as donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.
Characterization of Nicotiana tabacum plants expressing hybrid genes of cyanobacterial δ9 or δ12 acyl-lipid desaturases and thermostable lichenase.
Gerasymenko, I. M., Sakhno, L. A., Kyrpa, T. N., Ostapchuk, A. M., Hadjiev, T. A., Goldenkova-Pavlova, I. V. & Sheludko, Y. V. (2015). Russian Journal of Plant Physiology, 62(3), 283-291.
We established transgenic lines of Nicotiana tabacum expressing hybrid genes of Synechocystis sp. PCC 6803 Δ12 (desA) acyllipid desaturase and Synechococcus vulcanus δ9 (desC) acyllipid desaturase with or without sequence coding for transit peptide of Rubisco small subunit of Arabidopsis thaliana under control of a constitutive promoter. Reliable increase of linoleic acid portion (C18:2; δ9,12) accompanied with decrease of α-linolenic acid (C18:3; δ9,12,15) relative amount was detected for plants expressing hybrid desA::licBM3 gene. No reliable changes were detected in fatty acid profiles and unsaturation index of plants transformed with δ9 desaturase gene desC::licBM3 lacking signals of intracellular targeting while expression of this gene with Arabidopsis thaliana Rubisco small subunit transit peptide sequence caused growth of C18:3 α-linolenic acid part simultaneously with reduction of C18:2 linoleic acid part, as well as increase of unsat-uration index. No changes in relative amount of δ9-monounsaturated fatty acids were observed in any of studied lines. All plants expressing desaturase genes exhibited enhanced levels of superoxide dismutase (SOD) activity after cold treatment in contrast to control lines with suppressed SOD activity after cold treatment.
Improvement of enzyme activity of β-1,3-1,4-glucanase from Paenibacillus sp. X4 by error-prone PCR and structural insights of mutated residues.
Baek, S. C., Ho, T. H., Lee, H. W., Jung, W. K., Gang, H. S., Kang, L. W. & Kim, H. (2017). Applied Microbiology and Biotechnology, 101(10), 4073-4083.
β-1,3-1,4-Glucanase (BGlc8H) from Paenibacillus sp. X4 was mutated by error-prone PCR or truncated using termination primers to improve its enzyme properties. The crystal structure of BGlc8H was determined at a resolution of 1.8 Å to study the possible roles of mutated residues and truncated regions of the enzyme. In mutation experiments, three clones of EP 2-6, 2-10, and 5-28 were finally selected that exhibited higher specific activities than the wild type when measured using their crude extracts. Enzyme variants of BG2-6, BG2-10, and BG5-28 were mutated at two, two, and six amino acid residues, respectively. These enzymes were purified homogeneously by Hi-Trap Q and CHT-II chromatography. Specific activity of BG5-28 was 2.11-fold higher than that of wild-type BGwt, whereas those of BG2-6 and BG2-10 were 0.93- and 1.19-fold that of the wild type, respectively. The optimum pH values and temperatures of the variants were nearly the same as those of BGwt (pH 5.0 and 40°C, respectively). However, the half-life of the enzyme activity and catalytic efficiency (kcat/Km) of BG5-28 were 1.92- and 2.12-fold greater than those of BGwt at 40°C, respectively. The catalytic efficiency of BG5-28 increased to 3.09-fold that of BGwt at 60°C. These increases in the thermostability and catalytic efficiency of BG5-28 might be useful for the hydrolysis of β-glucans to produce fermentable sugars. Of the six mutated residues of BG5-28, five residues were present in mature BGlc8H protein, and two of them were located in the core scaffold of BGlc8H and the remaining three residues were in the substrate-binding pocket forming loop regions. In truncation experiments, three forms of C-terminal truncated BGlc8H were made, which comprised 360, 286, and 215 amino acid residues instead of the 409 residues of the wild type. No enzyme activity was observed for these truncated enzymes, suggesting the complete scaffold of the α6/α6-double-barrel structure is essential for enzyme activity.
Oligomerization triggered by foldon: a simple method to enhance the catalytic efficiency of lichenase and xylanase.
Wang, X., Ge, H., Zhang, D., Wu, S. & Zhang, G. (2017). BMC Biotechnology, 17(1), 57.
Background: Effective and simple methods that lead to higher enzymatic efficiencies are highly sough. Here we proposed a foldon-triggered trimerization of the target enzymes with significantly improved catalytic performances by fusing a foldon domain at the C-terminus of the enzymes via elastin-like polypeptides (ELPs). The foldon domain comprises 27 residues and can forms trimers with high stability. Results: Lichenase and xylanase can hydrolyze lichenan and xylan to produce value added products and biofuels, and they have great potentials as biotechnological tools in various industrial applications. We took them as the examples and compared the kinetic parameters of the engineered trimeric enzymes to those of the monomeric and wild type ones. When compared with the monomeric ones, the catalytic efficiency (kcat/Km) of the trimeric lichenase and xylanase increased 4.2- and 3.9- fold. The catalytic constant (kcat) of the trimeric lichenase and xylanase increased 1.8- fold and 5.0- fold than their corresponding wild-type counterparts. Also, the specific activities of trimeric lichenase and xylanase increased by 149% and 94% than those of the monomeric ones. Besides, the recovery of the lichenase and xylanase activities increased by 12.4% and 6.1% during the purification process using ELPs as the non-chromatographic tag. The possible reason is the foldon domain can reduce the transition temperature of the ELPs. Conclusion: The trimeric lichenase and xylanase induced by foldon have advantages in the catalytic performances. Besides, they were easier to purify with increased purification fold and decreased the loss of activities compared to their corresponding monomeric ones. Trimerizing of the target enzymes triggered by the foldon domain could improve their activities and facilitate the purification, which represents a simple and effective enzyme-engineering tool. It should have exciting potentials both in industrial and laboratory scales.
Characterization of two extracellular β-glucosidases produced from the cellulolytic fungus Aspergillus sp. YDJ216 and their potential applications for the hydrolysis of flavone glycosides.
Oh, J. M., Lee, J. P., Baek, S. C., Kim, S. G., Do Jo, Y., Kim, J. & Kim, H. (2018). International Journal of Biological Macromolecules, In Press.
A cellulolytic fungus YDJ216 was isolated from a compost and identified as an Aspergillus sp. strain. Two extracellular β-glucosidases, BGL1 and BGL2, were purified using ultrafiltration, ammonium sulfate fractionation, and High-Q chromatography. Molecular masses of BGL1 and BGL2 were estimated to be 97 and 45 kDa, respectively, by SDS-PAGE. The two enzymes eluted as one peak at 87 kDa by Sephacryl S-200 chromatography, and located at similar positions in a zymogram after intact gel electrophoresis, suggesting BGL1 and BGL2 might be monomeric and dimeric, respectively. The two enzymes showed similar enzymatic properties; they were optimally active at pH 4.0-4.5 and 60°C, and had similar half-lives at 70°C. Two enzymes also preferred p-nitrophenyl glucose (pNPG) with the same Km and hardly hydrolyzed cellobiose, suggesting BGL1 and BGL2 are aryl β-glucosidases. However, Vmax for pNPG of BGL1 (953.2 U/mg) was 14.3 times higher than that of BGL2 (66.5 U/mg). When tilianin (a flavone glycoside of acacetin) was reacted with both enzymes, inhibitory activity for monoamine oxidase, relating to oxidation of neurotransmitter amines, was increased closely to the degree obtained by acacetin. These results suggest that BGL1 and BGL2 could be used to hydrolyze flavone glycosides to improve their inhibitory activities.
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.
Performance, egg quality, nutrient digestibility, and excreta microbiota shedding in laying hens fed corn-soybean-meal-wheat-based diets supplemented with xylanase.
Lei, X. J., Lee, K. Y., & Kim, I. H. (2018). Poultry science, 97(6), 2071-2077.
The aim of this study was to evaluate the effects of dietary levels of xylanase on production performance, egg quality, nutrient digestibility, and excreta microbiota shedding of laying hens in a 12-week trial. Two-hundred-forty Hy-Line brown laying hens (44 wk old) were distributed according to a randomized block experimental design into one of 4 dietary treatments with 10 replicates of 6 birds each. The 4 dietary treatments were corn-soybean-meal-wheat-based diets supplemented with 0, 225, 450, or 900 U/kg xylanase. Daily feed intake, egg production, egg weight, egg mass, feed conversion ratio, and damaged egg rate showed no significant response to increasing xylanase supplementation during any phase (P > 0.05). No significant responses were observed for apparent total tract digestibility of dry matter, nitrogen, or gross energy (P > 0.05). A significant linear increase to increasing xylanase supplementation was seen for lactic acid bacteria numbers, although coliforms and Salmonella counts were not affected. Increasing the dietary xylanase resulted in a significant linear increase in eggshell thickness in wk 3, 6, 9, and 12 (P < 0.05). In addition, a significant linear increase occurred for Haugh unit and albumen height in wk 12 (P < 0.05). In summary, the inclusion of xylanase in corn-soybean-meal-wheat-based diets increased eggshell thickness, Haugh unit, albumen height, and excreta lactic acid bacteria count but had no effect on production performance or nutrient digestibility.
Double blind microarray-based polysaccharide profiling enables parallel identification of uncharacterized polysaccharides and carbohydrate-binding proteins with unknown specificities.
Salmeán, A. A., Guillouzo, A., Duffieux, D., Jam, M., Matard-Mann, M., Larocque, R., Pedersen, H. L., Michel, G., Czjzek, M., Willats, W. G. T. & Hervé, C. (2018). Scientific Reports, 8(1), 2500.
Marine algae are one of the largest sources of carbon on the planet. The microbial degradation of algal polysaccharides to their constitutive sugars is a cornerstone in the global carbon cycle in oceans. Marine polysaccharides are highly complex and heterogeneous, and poorly understood. This is also true for marine microbial proteins that specifically degrade these substrates and when characterized, they are frequently ascribed to new protein families. Marine (meta)genomic datasets contain large numbers of genes with functions putatively assigned to carbohydrate processing, but for which empirical biochemical activity is lacking. There is a paucity of knowledge on both sides of this protein/carbohydrate relationship. Addressing this ‘double blind’ problem requires high throughput strategies that allow large scale screening of protein activities, and polysaccharide occurrence. Glycan microarrays, in particular the Comprehensive Microarray Polymer Profiling (CoMPP) method, are powerful in screening large collections of glycans and we described the integration of this technology to a medium throughput protein expression system focused on marine genes. This methodology (Double Blind CoMPP or DB-CoMPP) enables us to characterize novel polysaccharide-binding proteins and to relate their ligands to algal clades. This data further indicate the potential of the DB-CoMPP technique to accommodate samples of all biological sources.
A novel method for simultaneous purification and immobilization of a xylanase-lichenase chimera via SpyTag/SpyCatcher spontaneous reaction.
Lin, Y., Jin, W., Wang, J., Cai, Z., Wua, S. & Zhang, G. (2018). Enzyme and Microbial Technology, 155, 29-36.
We generated a bifunctional enzyme chimera containing the xylanase and lichenase coupled with SpyTag between them. Meanwhile, we generated another chimera containing SpyCatcher and elastin-like polypeptides (ELPs). As ELPs could bond to the xylanase-lichenase chimera through SpyTag/SpyCatcher spontaneous reaction in mild condition, which would lead to the formation of a 3-arm star multifunctional chimera. We purified the xylanase-lichenase by the non-chromatographic purification tag of ELPs. Interestingly, 57.5% of the xylanase and 47.2% of the lichenase in chimera self-assembled into insoluble active particles during the process of purification, which could serve as immobilized bifunctional enzymes. Notably, the immobilized chimera xylanase-lichenase showed a remarkable stability even after 10 reaction cycles, which retained around 56% (lichenase) and 44% (xylanase) of their initial activities, respectively. Moreover, the enhanced thermostability of the immobilized enzymes was also achieved. After incubating at 60°C for 60 min, the residual activity of the immobilized lichenase was 35%, while the free one was only 24%. Unexpectedly, the free xylanase almost lost its activity when incubated at 55°C for 60 min, whereas the immobilized xylanase retained 10% of its activity. However, the catalytic efficiency (kcat/Km) of the free xylanase was 1.7-fold higher than the immobilized one, while the free lichenase was 1.1-fold higher than the immobilized one. This is among the first known reports that two enzymes are purified and immobilized in one-step. This novel strategy is easy to scale up and may meet the demands of biofuel industry. It would have great potentials in other biotechnological fields, such as the multifunctional biomaterials systems.
Characterization of truncated endo-β-1, 4-glucanases from a compost metagenomic library and their saccharification potentials.
Lee, J. P., Lee, H. W., Na, H. B., Lee, J. H., Hong, Y. J., Jeon, J. M., Kwon, F., Kim, S. K. & Kim, H. (2018). International Journal of Biological Macromolecules, 115, 554-562.
A gene encoding an endo-β-1,4-glucanase (Cel6H-f481) was cloned from a compost metagenomic library. The gene, cel6H-f481, was composed of 1446 bp to encode a fused protein of 481 amino acid residues (50,429 Da), i.e., 445 residues (Cel6H-445) from the metagenome, and 36 residues from the pUC19 vector at N-terminus. Cel6H-445 belonged to glycosyl hydrolase (GH) family 6 and showed 71% identity with Actinotalea fermentans endoglucanase with low coverage. Several active bands of truncated forms were observed by activity staining of the crude extract. Major truncated enzymes of 35 (Cel6H-p35) and 23 kDa (Cel6H-p23) were separated by HiTrap Q chromatography. The two enzymes had the same optimum temperature (50°C) and pH (5.5), but Cel6H-p35 was more thermostable than Cel6H-p23 and other GH6 endoglucanases reported. Both enzymes efficiently hydrolyzed carboxymethyl-cellulose (CMC) and barley β-glucan, but hardly hydrolyzed other substrates tested. The Vmax of Cel6H-p35 for CMC was 1.4 times greater than that of Cel6H-p23. The addition of the crude enzymes to a commercial enzyme set increased the saccharification of pretreated rice straw powder by up to 30.9%. These results suggest the N-terminal region of Cel6H-p35 contributes to thermostability and specific activity, and that the enzymes might be a useful additive for saccharification.