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.
Efficient chemoenzymatic oligosaccharide synthesis by reverse phosphorolysis using cellobiose phosphorylase and cellodextrin phosphorylase from Clostridium thermocellum.
Nakai, H., Hachem, M. A., Petersen, B. O., Westphal, Y., Mannerstedt, K., Baumann, M. J., Dilokpimol, A., Schols, H. A., Duus, J. Ø. & Svensson, B. (2010). Biochimie, 92(12), 1818-1826.
Inverting cellobiose phosphorylase (CtCBP) and cellodextrin phosphorylase (CtCDP) from Clostridium thermocellum ATCC27405 of glycoside hydrolase family 94 catalysed reverse phosphorolysis to produce cellobiose and cellodextrins in 57% and 48% yield from α-D-glucose 1-phosphate as donor with glucose and cellobiose as acceptor, respectively. Use of α-D-glucosyl 1-fluoride as donor increased product yields to 98% for CtCBP and 68% for CtCDP. CtCBP showed broad acceptor specificity forming β-glucosyl disaccharides with β-(1→4)- regioselectivity from five monosaccharides as well as branched β-glucosyl trisaccharides with β-(1→4)-regioselectivity from three (1→6)-linked disaccharides. CtCDP showed strict β-(1→4)-regioselectivity and catalysed linear chain extension of the three β-linked glucosyl disaccharides, cellobiose, sophorose, and laminaribiose, whereas 12 tested monosaccharides were not acceptors. Structure analysis by NMR and ESI-MS confirmed two β-glucosyl oligosaccharide product series to represent novel compounds, i.e. β-D-glucopyranosyl-[(1→4)-β-D-glucopyranosyl]n-(1→2)-D-glucopyranose, and β-D-glucopyranosyl-[(1→4)-β-D-glucopyranosyl]n-(1→3)-D-glucopyranose (n = 1–7). Multiple sequence alignment together with a modelled CtCBP structure, obtained using the crystal structure of Cellvibrio gilvus CBP in complex with glucose as a template, indicated differences in the subsite +1 region that elicit the distinct acceptor specificities of CtCBP and CtCDP. Thus Glu636 of CtCBP recognized the C1 hydroxyl of β-glucose at subsite +1, while in CtCDP the presence of Ala800 conferred more space, which allowed accommodation of C1 substituted disaccharide acceptors at the corresponding subsites +1 and +2. Furthermore, CtCBP has a short Glu496-Thr500 loop that permitted the C6 hydroxyl of glucose at subsite +1 to be exposed to solvent, whereas the corresponding longer loop Thr637–Lys648 in CtCDP blocks binding of C6-linked disaccharides as acceptors at subsite +1. High yields in chemoenzymatic synthesis, a novel regioselectivity, and novel oligosaccharides including products of CtCDP catalysed oligosaccharide oligomerisation using α-D-glucosyl 1-fluoride, all together contribute to the formation of an excellent basis for rational engineering of CBP and CDP to produce desired oligosaccharides.
Functional analyses of the digestive β-glucosidase of Formosan subterranean termites (Coptotermes formosanus).
Zhang, D., Allen, A. B. & Lax, A. R. (2012). Journal of Insect Physiology, 58(1), 205-210.
The research was to elucidate the function of the β-glucosidase of Formosan subterranean termites in vitro and in vivo. The gene transcript was detected predominantly in the salivary gland tissue, relative to the midgut and the hindgut of the foraging worker caste, indicating salivary glands were the major expression sites of the β-glucosidase. Using recombinant β-glucosidase produced in Escherichia coli, the enzyme showed higher affinity and activity toward cellobiose and cellotriose than other substrates tested. In assessing impacts of specific inhibitors, we found that the β-glucosidase could be irreversibly inactivated by conduritol B epoxide (CBE) but not gluconolactone. Termite feeding assays showed that the CBE treatment reduced the glucose supply in the midgut and resulted in the body weight loss while no effect was observed for the gluconolactone treatment. These findings highlighted that the β-glucosidase is one of the critical cellulases responsible for cellulose degradation and glucose production; inactivation of these digestive enzymes by specific inhibitors may starve the termite.
Isolation and properties of fungal β-glucosidases.
Korotkova, O. G., Semenova, M. V., Morozova, V. V., Zorov, I. N., Sokolova, L. M., Bubnova, T. M., Okunev, O. N. & Sinitsyn, A. P. (2009). Biochemistry (Moscow), 74(5), 569-577
Using chromatography on different matrixes, three β-glucosidases (120, 116, and 70 kDa) were isolated from enzymatic complexes of the mycelial fungi Aspergillus japonicus, Penicillium verruculosum, and Trichoderma reesei, respectively. The enzymes were identified by MALDI-TOF mass-spectrometry. Substrate specificity, kinetic parameters for hydrolysis of specific substrates, ability to catalyze the transglucosidation reaction, dependence of the enzymatic activity on pH and temperature, stability of the enzymes at different temperatures, adsorption ability on insoluble cellulose, and the influence of glucose on catalytic properties of the enzymes were investigated. According to the substrate specificity, the enzymes were shown to belong to two groups: i) β-glucosidase of A. japonicus exhibiting high specific activity to the low molecular weight substrates cellobiose and pNPG (the specific activity towards cellobiose was higher than towards pNPG) and low activity towards polysaccharide substrates (β-glucan from barley and laminarin); ii) β-glucosidases from P. verruculosum and T. reesei exhibiting relatively high activity to polysaccharide substrates and lower activity to low molecular weight substrates (activity to cellobiose was lower than to pNPG).
Fruit Fly Bioassay To Distinguish “Sweet” Sugar Structures.
Hodoniczky, J., Robinson, G. J., McGraw, E. A. & Rae, A. L. (2010). Journal of Agricultural and Food Chemistry, 58(24), 12885-12889.
Palatable response to dietary sugars plays a significant role in influencing metabolic health. New structures are being explored with beneficial health properties, although consumer acceptance relies heavily on desirable sensory properties. Despite the importance of behavioral responses, the ability to elucidate structure-preference relationships of sugars is lacking. A wild population of Drosophila melanogaster was used as a model to perform pairwise comparisons across structural groups to characterize a fruit fly bioassay for assessing sugar preference. Preference was successfully described in structurally relevant terms, particularly through the ability to directly test sugars of related structures in addition to standard sucrose comparisons. The fruit fly bioassay also provided the first report on the relative preference for the β-linked sugar alcohol, gentiobiitol. In making reference to well-known human preferences, the bioassay also raises opportunities for greater understanding of behavioral response to sugar structures in general.
Rational engineering of Lactobacillus acidophilus NCFM maltose phosphorylase into either trehalose or kojibiose dual specificity phosphorylase.
Nakai, H., Petersen, B. O., Westphal, Y., Dilokpimol, A., Hachem, M. A., Duus, J. Ø., Schols, H. A. & Svensson, B. (2010). Protein Engineering Design and Selection, 23(10), 781-787.
Lactobacillus acidophilus NCFM maltose phosphorylase (LaMP) of the (α/α)6-barrel glycoside hydrolase family 65 (GH65) catalyses both phosphorolysis of maltose and formation of maltose by reverse phosphorolysis with β-glucose 1-phosphate and glucose as donor and acceptor, respectively. LaMP has about 35 and 26% amino acid sequence identity with GH65 trehalose phosphorylase (TP) and kojibiose phosphorylase (KP) from Thermoanaerobacter brockii ATCC35047. The structure of L. brevis MP and multiple sequence alignment identified (α/α)6-barrel loop 3 that forms the rim of the active site pocket as a target for specificity engineering since it contains distinct sequences for different GH65 disaccharide phosphorylases. Substitution of LaMP His413–Glu421, His413–Ile418 and His413–Glu415 from loop 3, that include His413 and Glu415 presumably recognising the α-anomeric O-1 group of the glucose moiety at subsite +1, by corresponding segments from Ser426–Ala431 in TP and Thr419–Phe427 in KP, thus conferred LaMP with phosphorolytic activity towards trehalose and kojibiose, respectively. Two different loop 3 LaMP variants catalysed the formation of trehalose and kojibiose in yields superior of maltose by reverse phosphorolysis with (α1, α1)- and α-(1,2)-regioselectivity, respectively, as analysed by nuclear magnetic resonance. The loop 3 in GH65 disaccharide phosphorylase is thus a key determinant for specificity both in phosphorolysis and in regiospecific reverse phosphorolysis.
Identification of anomeric configuration of underivatized reducing glucopyranosyl-glucose disaccharides by tandem mass spectrometry and multivariate analysis.
Simões, J., Domingues, P., Reis, A., Nunes, F. M., Coimbra, M. A. & Domingues, M. R. M. (2007). Analytical Chemistry, 79(15), 5896-5905.
The possibility of discrimination of the anomeric configuration (α or β) of underivatized reducing glucopyranosyl-glucose disaccharides, using a hybrid mass spectrometer Q-TOF 2 (Micromass), a linear ion trap LXQ (Thermo), and a triple quadrupole Quattro (Micromass) with an electrospray source (ESI) was investigated. Differences observed in the relative abundances of specific product ions obtained from collisionally induced dissociation of the [M + Li]+ adducts were statistically analyzed, and discriminant analysis was performed. MANOVA has shown that anomeric configuration has influence on the combined dependent variables (relative abundances of m/z product ions) in all the three mass spectrometers used (Q-TOF 2, LIT, and QqQ). Discriminant analysis has shown that, in all instruments, it is possible to discriminate anomeric configurations and to build a diagnostic model. These diagnostic differences are even more relevant considering that no derivatization procedures are needed for obtaining this structural information. The Q-TOF 2 instrument has been shown to give data that allowed us to build a model with better discriminant power (Wilks' λ value of 0.014) followed by the QqQ instrument (Wilks' λ value of 0.029) and the LIT instrument (Wilks' λ value of 0.037).
Flavobacterium johnsoniae as a model organism for characterizing biopolymer utilization in oligotrophic freshwater environments.
Sack, E. L. W., van der Wielen, P. W. J. J. & van der Kooij, D. (2011). Applied and Environmental Microbiology, 77(19), 6931-6938.
Biopolymers are important substrates for heterotrophic bacteria in oligotrophic freshwater environments, but information on bacterial growth kinetics with biopolymers is scarce. The objective of this study was to characterize bacterial biopolymer utilization in these environments by assessing the growth kinetics of Flavobacterium johnsoniae strain A3, which is specialized in utilizing biopolymers at µg liter-1 levels. Growth of strain A3 with amylopectin, xyloglucan, gelatin, maltose, or fructose at 0 to 200 µg C liter-1 in tap water followed Monod or Teissier kinetics, whereas growth with laminarin followed Teissier kinetics. Classification of the specific affinity of strain A3 for the tested substrates resulted in the following affinity order: laminarin (7.9 × 10-2 liter·µg-1 of C·h-1) >> maltose > amylopectin ≈ gelatin ≈ xyloglucan > fructose (0.69 × 10-2 liter·µg-1 of C·h-1). No specific affinity could be determined for proline, but it appeared to be high. Extracellular degradation controlled growth with amylopectin, xyloglucan, or gelatin but not with laminarin, which could explain the higher affinity for laminarin. The main degradation products were oligosaccharides or oligopeptides, because only some individual monosaccharides and amino acids promoted growth. A higher yield and a lower ATP cell-1 level was achieved at ≤10 µg C liter-1 than at >10 µg C liter-1 with every substrate except gelatin. The high specific affinities of strain A3 for different biopolymers confirm that some representatives of the classes Cytophagia-Flavobacteria are highly adapted to growth with these compounds at µg liter-1 levels and support the hypothesis that Cytophagia-Flavobacteria play an important role in biopolymer degradation in (ultra)oligotrophic freshwater environments.
Occurrence of cellobiose residues directly linked to galacturonic acid in pectic polysaccharides.
Nunes, C., Silva, L., Fernandes, A. P., Guiné, R. P. F., Domingues, M. R. M. & Coimbra, M. A. (2012). Carbohydrate Polymers, 87(1), 620-626.
The study carried out in this work concerns the structural characterization of pectic polysaccharides from plum (Prunus domestica L.) and pear (Pyrus communis L.) cell walls and commercial pectic polysaccharides, obtained from Citrus. The α-(1 → 4)-D-galacturonic acid backbone was submitted to a selective hydrolysis with endo-polygalacturonase (EPG) and the fractions with low molecular weight (<1 kDa) obtained by size-exclusion chromatography were analysed by mass spectrometry using electrospray ionisation (ESI-MS). The ESI-MS spectra obtained revealed the presence of several [M+Na]+ ions of pectic oligosaccharides identified as belonging to different series, including oligosaccharides constituted only by galacturonic acid residues (GalAn, n = 1–5) and galacturonic acid residues substituted by pentose residues (GalA3Pentn, n = 1–2). Surprisingly, it was also observed the occurrence of galacturonic acid residues substituted by hexose residues (GalAnHexm, n = 2–4, m = 1–2). The fragmentation of the observed [M+Na]+ ions, obtained under ESI-MS/MS and MSn allowed to confirm the proposed structures constituent of these pectic oligosaccharides. Furthermore, the ESI-MSn spectra of the ions that could be identified as GalAnHexm (n = 2–4, m = 1–2) confirmed the presence of Hex or Hex2 residues linked to a GalA residue. Methylation analysis showed the presence, in all EPG treated samples, of terminally linked arabinose, terminally and 4-linked xylose, and terminally and 4-linked glucose. The occurrence of GalA substituted by Glc, and Glc-β-(1 → 4)–Glc are structural features that, as far as we know, have never been reported to occur in pectic polysaccharides.
The maltodextrin transport system and metabolism in Lactobacillus acidophilus NCFM and production of novel α‐glucosides through reverse phosphorolysis by maltose phosphorylase.
Nakai, H., Baumann, M. J., Petersen, B. O., Westphal, Y., Schols, H., Dilokpimol, A., Hachem, M. A., Lahtinen, S. J., Duus, J. Ø. & Svensson, B. (2009). FEBS Journal, 276(24), 7353-7365.
A gene cluster involved in maltodextrin transport and metabolism was identified in the genome of Lactobacillus acidophilus NCFM, which encoded a maltodextrin-binding protein, three maltodextrin ATP-binding cassette transporters and five glycosidases, all under the control of a transcriptional regulator of the LacI-GalR family. Enzymatic properties are described for recombinant maltose phosphorylase (MalP) of glycoside hydrolase family 65 (GH65), which is encoded by malP (GenBank: AAV43670.1) of this gene cluster and produced in Escherichia coli. MalP catalyses phosphorolysis of maltose with inversion of the anomeric configuration releasing β-glucose 1-phosphate (β-Glc 1-P) and glucose. The broad specificity of the aglycone binding site was demonstrated by products formed in reverse phosphorolysis using various carbohydrate acceptor substrates and β-Glc 1-P as the donor. MalP showed strong preference for monosaccharide acceptors with equatorial 3-OH and 4-OH, such as glucose and mannose, and also reacted with 2-deoxy glucosamine and 2-deoxy N-acetyl glucosamine. By contrast, none of the tested di- and trisaccharides served as acceptors. Disaccharide yields obtained from 50 mM β-Glc 1-P and 50 mM glucose, glucosamine, N-acetyl glucosamine, mannose, xylose or L-fucose were 99, 80, 53, 93, 81 and 13%, respectively. Product structures were determined by NMR and ESI-MS to be α-Glcp-(1→4)-Glcp (maltose), α-Glcp-(1→4)-GlcNp (maltosamine), α-Glcp-(1→4)-GlcNAcp (N-acetyl maltosamine), α-Glcp-(1→4)-Manp, α-Glcp-(1→4)-Xylp and α-Glcp-(1→4)-L-Fucp, the three latter being novel compounds. Modelling using L. brevis GH65 as the template and superimposition of acarbose from a complex with Thermoanaerobacterium thermosaccharolyticum GH15 glucoamylase suggested that loop 3 of MalP involved in substrate recognition blocked the binding of candidate acceptors larger than monosaccharides.
Cell-free protein synthesis of membrane (1,3)-β-D-glucan (curdlan) synthase: Co-translational insertion in liposomes and reconstitution in nanodiscs.
Periasamy, A., Shadiac, N., Amalraj, A., Garajová, S., Nagarajan, Y., Waters, S., Mertens, H. D. T. & Hrmova, M. (2013). Biochimica et Biophysica Acta (BBA)-Biomembranes, 1828(2), 743-757.
A membrane-embedded curdlan synthase (CrdS) from Agrobacterium is believed to catalyse a repetitive addition of glucosyl residues from UDP-glucose to produce the (1,3)-β-D-glucan (curdlan) polymer. We report wheat germ cell-free protein synthesis (WG-CFPS) of full-length CrdS containing a 6xHis affinity tag and either Factor Xa or Tobacco Etch Virus proteolytic sites, using a variety of hydrophobic membrane-mimicking environments. Full-length CrdS was synthesised with no variations in primary structure, following analysis of tryptic fragments by MALDI-TOF/TOF Mass Spectrometry. Preparative scale WG-CFPS in dialysis mode with Brij-58 yielded CrdS in mg/ml quantities. Analysis of structural and functional properties of CrdS during protein synthesis showed that CrdS was co-translationally inserted in DMPC liposomes during WG-CFPS, and these liposomes could be purified in a single step by density gradient floatation. Incorporated CrdS exhibited a random orientation topology. Following affinity purification of CrdS, the protein was reconstituted in nanodiscs with Escherichia coli lipids or POPC and a membrane scaffold protein MSP1E3D1. CrdS nanodiscs were characterised by small-angle X-ray scattering using synchrotron radiation and the data obtained were consistent with insertion of CrdS into bilayers. We found CrdS synthesised in the presence of the Ac-AAAAAAD surfactant peptide or co-translationally inserted in liposomes made from E. coli lipids to be catalytically competent. Conversely, CrdS synthesised with only Brij-58 was inactive. Our findings pave the way for future structural studies of this industrially important catalytic membrane protein.
Production of high-value β-1,3-glucooligosaccharides by microwave-assisted hydrothermal hydrolysis of curdlan.
Wang, D., Kim, D. H., Yoon, J. J. & Kim, K. H. (2017). Process Biochemistry, 52, 233-237.
We report the first hydrothermal hydrolysis of curdlan, a water insoluble β-1,3-glucan, to produce β-1,3-glucooligosaccharides, which are high-value materials with health-benefiting activities. In this study, hydrothermal hydrolysis was tested for the liquefaction and saccharification of curdlan. The optimal hydrothermal hydrolysis conditions were 180°C and 60 min, respectively, resulting in a high degree of liquefaction (98.4%) and low byproduct formation. Under the optimal conditions, 17.47 g/L of β-1,3-glucooligosaccharides was produced from 20 g/L of curdlan, representing a conversion yield of 87.4% (w/w). Using this process, β-1,3-glucooligosaccharides were conveniently produced in a one-step reaction without any chemicals or enzymes. This hydrothermal hydrolysis for curdlan exhibited the best performance among various hydrolysis processes reported to date. This method can be applied to large-scale production of β-1,3-glucooligosaccharides for the functional food and biopharmaceutical industries.
Characterization and comparison of polysaccharides from Lycium barbarum in China using saccharide mapping based on PACE and HPTLC.
Wu, D. T., Cheong, K. L., Deng, Y., Lin, P. C., Wei, F., Lv, X. J., Long, Z. R., Zhao, J., Ma, S. C. & Li, S. P. (2015). Carbohydrate polymers, 134, 12-19.
Water-soluble polysaccharides from 51 batches of fruits of L. barbarum (wolfberry) in China were investigated and compared using saccharide mapping, partial acid hydrolysis, single and composite enzymatic digestion, followed by polysaccharide analysis by using carbohydrate gel electrophoresis (PACE) analysis and high performance thin layer chromatography (HPTLC) analysis, respectively. Results showed that multiple PACE and HPTLC fingerprints of partial acid and enzymatic hydrolysates of polysaccharides from L. barbarum in China were similar, respectively. In addition, results indicated that β-1,3-glucosidic, α-1,4-galactosiduronic and α-1,5-arabinosidic linkages existed in polysaccharides from L. barbarum collected in China, and the similarity of polysaccharides in L. barbarum collected from different regions of China was pretty high, which are helpful for the improvement of the performance of polysaccharides from L. barbarum in functional/health foods area. Furthermore, polysaccharides from Panax notoginseng, Angelica sinensis, and Astragalus membranaceus var. mongholicus were successfully distinguished from those of L. barbarum based on their PACE fingerprints. These results were beneficial to improve the quality control of polysaccharides from L. barbarum and their products, which suggested that saccharide mapping based on PACE and HPTLC analysis could be a routine approach for quality control of polysaccharides.
The LacI family protein GlyR3 co-regulates the celC operon and manB in Clostridium thermocellum.
Choi, J., Klingeman, D. M., Brown, S. D. & Cox, C. D. (2017). Biotechnology for Biofuels, 10(1), 163.
Background: Clostridium thermocellum utilizes a wide variety of free and cellulosomal cellulases and accessory enzymes to hydrolyze polysaccharides present in complex substrates. To date only a few studies have unveiled the details by which the expression of these cellulases are regulated. Recent studies have described the auto regulation of the celC operon and determined that the celC-glyR3-licA gene cluster and nearby manB-celT gene cluster are co-transcribed as polycistronic mRNA. Results: In this paper, we demonstrate that the GlyR3 protein mediates the regulation of manB. We first identify putative GlyR3 binding sites within or just upstream of the coding regions of manB and celT. Using an electrophoretic mobility shift assay (EMSA), we determined that a higher concentration of GlyR3 is required to effectively bind to the putative manB site in comparison to the celC site. Neither the putative celT site nor random DNA significantly binds GlyR3. While laminaribiose interfered with GlyR3 binding to the celC binding site, binding to the manB site was unaffected. In the presence of laminaribiose, in vivo transcription of the celC-glyR3-licA gene cluster increases, while manB expression is repressed, compared to in the absence of laminaribiose, consistent with the results from the EMSA. An in vitro transcription assay demonstrated that GlyR3 and laminaribiose interactions were responsible for the observed patters of in vivo transcription. Conclusions: Together these results reveal a mechanism by which manB is expressed at low concentrations of GlyR3 but repressed at high concentrations. In this way, C. thermocellum is able to co-regulate both the celC and manB gene clusters in response to the availability of β-1,3-polysaccharides in its environment.
HPAEC-PAD for oligosaccharide analysis—novel insights into analyte sensitivity and response stability.
Mechelke, M., Herlet, J., Benz, J. P., Schwarz, W. H., Zverlov, V. V., Liebl, W. & Kornberger, P. (2017). Analytical and Bioanalytical Chemistry, 1-13.
The rising importance of accurately detecting oligosaccharides in biomass hydrolyzates or as ingredients in food, such as in beverages and infant milk products, demands for the availability of tools to sensitively analyze the broad range of available oligosaccharides. Over the last decades, HPAEC-PAD has been developed into one of the major technologies for this task and represents a popular alternative to state-of-the-art LC-MS oligosaccharide analysis. This work presents the first comprehensive study which gives an overview of the separation of 38 analytes as well as enzymatic hydrolyzates of six different polysaccharides focusing on oligosaccharides. The high sensitivity of the PAD comes at cost of its stability due to recession of the gold electrode. By an in-depth analysis of the sensitivity drop over time for 35 analytes, including xylo- (XOS), arabinoxylo- (AXOS), laminari- (LOS), manno- (MOS), glucomanno- (GMOS), and cellooligosaccharides (COS), we developed an analyte-specific one-phase decay model for this effect over time. Using this model resulted in significantly improved data normalization when using an internal standard. Our results thereby allow a quantification approach which takes the inevitable and analyte-specific PAD response drop into account.
Chemical characterization and immunomodulatory activity of acetylated polysaccharides from Dendrobium devonianum.
Deng, Y., Li, M., Chen, L. X., Chen, X. Q., Lu, J. H., Zhao, J. & Li, S. P. (2017). Carbohydrate Polymers, 180, 238-245.
The chain conformation, chemical characters and immunomodulatory activity of polysaccharide from Dendrobium devonianum (DDP) were investigated.Results showed that molecular weights, polydispersity index, radius of gyrations of DDP were 3.99 × 105 Da, 1.27, 74.1 nm, respectively. By applying the polymer solution theory, the exponent (v) values of
2>z 1/2 = kMwv was calculated as 0.38, which revealed that DDP existed as a globular shape in aqueous solution, and further confirmed by AFM analysis. Furthermore, the main monosaccharide compositions were Man and Glc with the ratio of 29.61:1.00. Indeed, the main glycosidic linkages were β-1,4-Manp, and substituted with acetyl groups at O-2 and O-3 position. Notably, DDP could promote the immune functions of macrophages including NO release and phagocytosis. Thus, DDP could be explored as a natural immune-stimulating agent in the health and functional food area as well as pharmaceutical industries.
Trp residue at subsite − 5 plays a critical role in the substrate binding of two protistan GH26 β-mannanases from a termite hindgut.
Hsu, Y., Koizumi, H., Otagiri, M., Moriya, S. & Arioka, M. (2018). Applied Microbiology and Biotechnology, 1-11.
Symbiotic protists in the hindgut of termites provide a novel enzymatic resource for efficient lignocellulytic degradation of plant biomass. In this study, two β-mannanases, RsMan26A and RsMan26B, from a symbiotic protist community of the lower termite, Reticulitermes speratus, were successfully expressed in the methylotrophic yeast, Pichia pastoris. Biochemical characterization experiments demonstrated that both RsMan26A and RsMan26B are endo-acting enzymes and have a very similar substrate specificity, displaying a higher catalytic efficiency to galactomannan from locust bean gum (LBG) and glucomannan than to β-1,4-mannan and highly substituted galactomannan from guar gum. Homology modeling of RsMan26A and RsMan26B revealed that each enzyme displays a long open cleft harboring a unique hydrophobic platform (Trp79) that stacks against the sugar ring at subsite - 5. The Km) values of W79A mutants of RsMan26A and RsMan26B to LBG increased by 4.8-fold and 3.6-fold, respectively, compared with those for the native enzymes, while the kcat) remained unchanged or about 40% of that of the native enzyme, resulting in the decrease in the catalytic efficiency by 4.8-fold and 9.1-fold, respectively. The kinetic values for glucomannan also showed a similar result. These results demonstrate that the Trp residue present near the subsite - 5 has an important role in the recognition of the sugar ring in the substrate.
A novel β-glucosidase from Saccharophagus degradans 2-40T for the efficient hydrolysis of laminarin from brown macroalgae.
Kim, D. H., Kim, D. H., Lee, S. H. & Kim, K. H. (2018). Biotechnology for Biofuels, 11(1), 64.
Background: Laminarin is a potential biomass feedstock for the production of glucose, which is the most preferable fermentable sugar in many microorganisms by which it can be converted to biofuels and bio-based chemicals. Also, laminarin is a good resource as functional materials because it consists of β-1,3-glucosidic linkages in its backbone and β-1,6-glucosidic linkages in its branches so that its oligosaccharides driven from laminarin have a variety of biological activities. It is industrially important to be able to produce laminarioligosaccharides as well as glucose from laminarin by a single enzyme because the enzyme cost accounts for a large part of bio-based products. In this study, we investigated the industrial applicability of Bgl1B, a unique β-glucosidase from Saccharophagus degradans 2-40T, belonging to the glycoside hydrolase family 1 (GH1) by characterizing its activity of hydrolyzing laminarin under various conditions. Results: Bgl1B was cloned and overexpressed in Escherichia coli from S. degradans 2-40T, and its enzymatic activity was characterized. Similar to most of β-glucosidases in GH1, Bgl1B was able to hydrolyze a variety of disaccharides having different β-linkages, such as laminaribiose, cellobiose, gentiobiose, lactose, and agarobiose, by cleaving β-1,3-, β-1,4-, and β-1,6-glycosidic linkages. However, Bgl1B showed the highest specific activity toward laminaribiose with a β-1,3-glycosidic linkage. In addition, it was able to hydrolyze laminarin, one of the major polysaccharides in brown macroalgae, into glucose with a conversion yield of 75% of theoretical maximum. Bgl1B also showed transglycosylation activity by producing oligosaccharides from laminarin and laminaribiose under a high mass ratio of substrate to enzyme. Furthermore, Bgl1B was found to be psychrophilic, exhibiting relative activity of 59-85% in the low-temperature range of 2-20°C. Conclusions: Bgl1B can directly hydrolyze laminarin into glucose with a high conversion yield without leaving any oligosaccharides. Bgl1B can exhibit high enzymatic activity in a broad range of low temperatures (2-20°C), which is advantageous for establishing energy-efficient bioprocesses. In addition, under high substrate to enzyme ratios, Bgl1B can produce high-value laminarioligosaccharides via its transglycosylation activity. These results show that Bgl1B can be an industrially important enzyme for the production of biofuels and bio-based chemicals from brown macroalgae.
Enzymatic properties and the gene structure of a cold-adapted laminarinase from Pseudoalteromonas species LA.
Mitsuya, D., Sugiyama, T., Zhang, S., Takeuchi, Y., Okai, M., Urano, N. & Ishida, M. (2018). Journal of Bioscience and Bioengineering, 126(2), 169-175.
We isolated a laminarin-degrading cold-adapted bacterium strain LA from coastal seawater in Sagami Bay, Japan and identified it as a Pseudoalteromonas species. We named the extracellular laminarinase LA-Lam, and purified and characterized it. LA-Lam showed high degradation activity for Laminaria digitata laminarin in the ranges of 15-50°C and pH 5.0-9.0. The major terminal products degraded from L. digitata laminarin with LA-Lam were glucose, laminaribiose, and laminaritriose. The degradation profile of laminarioligosaccharides with LA-Lam suggested that the enzyme has a high substrate binding ability toward tetrameric or larger saccharides. Our results of the gene sequence and the SDS-PAGE analyses revealed that the major part of mature LA-Lam is a catalytic domain that belongs to the GH16 family, although its precursor is composed of a signal peptide, the catalytic domain, and three-repeated unknown regions.
Effective degradation of curdlan powder by a novel endo-β-1→ 3-glucanase.
Li, K., Chen, W., Wang, W., Tan, H., Li, S. & Yin, H. (2018). Carbohydrate Polymers, 201, 122-130.
Curdlan is a water-insoluble microbial exo-polysaccharide that is hardly degraded. The gene CcGluE encoding an endo-β-1→3-glucanase consisting of 412 amino acids (44 kDa) from Cellulosimicrobium cellulans E4-5 was cloned and expressed in Escherichia coli. The recombinant CcGluE hydrolysed curdlan powder effectively. CcGluE shows high endo-β-1→3 glucanase activity and low β-1,4 and β-1,6 glucanase activities with broad substrate specificity for glucan, including curdlan, laminarin and β-1→3/1 →6-glucan, and the highest catalytic activity for curdlan. Moreover, the hydrolytic products of curdlan were glucan oligosaccharides with degrees of polymerisation of 2-13, and the main products were glucobiose and glucotriose. Degradation mode analysis indicated that CcGluE is more likely to hydrolyse glucopentaose and revealed that CcGluE was an endo-glucanase. Furthermore, upon combination with a homogenising pre-treatment method with curdlan, the degradation efficiency of CcGluE for curdlan powder was greatly improved 7.1-fold, which laid a good foundation for the utilisation of curdlan.