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.
Promiscuity in ligand-binding: the three-dimensional structure of a Piromyces carbohydrate-binding module, CBM29-2, in complex with cello-and mannohexaose.
Charnock, S. J., Bolam, D. N., Nurizzo, D., Szabó, L., McKie, V. A., Gilbert, H. J. & Davies, G. J. (2002). Proceedings of the National Academy of Sciences, 99(22), 14077-14082.
Carbohydrate–protein recognition is central to many biological processes. Enzymes that act on polysaccharide substrates frequently contain noncatalytic domains, “carbohydrate-binding modules” (CBMs), that target the enzyme to the appropriate substrate. CBMs that recognize specific plant structural polysaccharides are often able to accommodate both the variable backbone and the side-chain decorations of heterogeneous ligands. “CBM29” modules, derived from a noncatalytic component of the Piromyces equi cellulase/hemicellulase complex, provide an example of this selective yet flexible recognition. They discriminate strongly against some polysaccharides while remaining relatively promiscuous toward both β-1,4-linked manno- and cello-oligosaccharides. This feature may reflect preferential, but flexible, targeting toward glucomannans in the plant cell wall. The three-dimensional structure of CBM29-2 and its complexes with cello- and mannohexaose reveal a β-jelly-roll topology, with an extended binding groove on the concave surface. The orientation of the aromatic residues complements the conformation of the target sugar polymer while accommodation of both manno- and gluco-configured oligo- and polysaccharides is conferred by virtue of the plasticity of the direct interactions from their axial and equatorial 2-hydroxyls, respectively. Such flexible ligand recognition targets the anaerobic fungal complex to a range of different components in the plant cell wall and thus plays a pivotal role in the highly efficient degradation of this composite structure by the microbial eukaryote.
Functional characterization and target discovery of glycoside hydrolases from the digestome of the lower termite Coptotermes gestroi.
Cairo, J. P. L. F., Leonardo, F. C., Alvarez, T. M., Ribeiro, D. A., Büchli, F., Costa-Leonardo, A. M., Carazzolle, M. F., Costa, F. F., Paes Leme, A. F., Pereira, G. A. G. & Squina, F. M. (2011). Biotechnology for Biofuels, 4(1), 50.
Background: Lignocellulosic materials have been moved towards the forefront of the biofuel industry as a sustainable resource. However, saccharification and the production of bioproducts derived from plant cell wall biomass are complex and lengthy processes. The understanding of termite gut biology and feeding strategies may improve the current state of biomass conversion technology and bioproduct production. Results: The study herein shows comprehensive functional characterization of crude body extracts from Coptotermes gestroi along with global proteomic analysis of the termite’s digestome, targeting the identification of glycoside hydrolases and accessory proteins responsible for plant biomass conversion. The crude protein extract from C. gestroi was enzymatically efficient over a broad pH range on a series of natural polysaccharides, formed by glucose-, xylose-, mannan- and/or arabinose-containing polymers, linked by various types of glycosidic bonds, as well as ramification types. Our proteomic approach successfully identified a large number of relevant polypeptides in the C. gestroi digestome. A total of 55 different proteins were identified and classified into 29 CAZy families. Based on the total number of peptides identified, the majority of components found in the C. gestroi digestome were cellulose-degrading enzymes. Xylanolytic enzymes, mannan- hydrolytic enzymes, pectinases and starch degrading and debranching enzymes were also identified. Our strategy enabled validation of liquid chromatography with tandem mass spectrometry recognized proteins, by enzymatic functional assays and by following the degradation products of specific 8-amino-1,3,6-pyrenetrisulfonic acid labeled oligosaccharides through capillary zone electrophoresis. Conclusions: Here we describe the first global study on the enzymatic repertoire involved in plant polysaccharide degradation by the lower termite C. gestroi. The biochemical characterization of whole body termite extracts evidenced their ability to cleave all types of glycosidic bonds present in plant polysaccharides. The comprehensive proteomic analysis, revealed a complete collection of hydrolytic enzymes including cellulases (GH1, GH3, GH5, GH7, GH9 and CBM 6), hemicellulases (GH2, GH10, GH11, GH16, GH43 and CBM 27) and pectinases (GH28 and GH29).
Influence of a mannan binding family 32 carbohydrate binding module on the activity of the appended mannanase.
Mizutani, K., Fernandes, V. O., Karita, S., Luís, A. S., Sakka, M., Kimura, T., Jackson, A., Zhang, X., Fontes, C. M. G. A., Gilbert, H. J. & Sakka, K. (2012). Applied and Environmental Microbiology, 78(14), 4781-4787.
In general, cellulases and hemicellulases are modular enzymes in which the catalytic domain is appended to one or more noncatalytic carbohydrate binding modules (CBMs). CBMs, by concentrating the parental enzyme at their target polysaccharide, increase the capacity of the catalytic module to bind the substrate, leading to a potentiation in catalysis. Clostridium thermocellum hypothetical protein Cthe_0821, defined here as C. thermocellum Man5A, is a modular protein comprising an N-terminal signal peptide, a family 5 glycoside hydrolase (GH5) catalytic module, a family 32 CBM (CBM32), and a C-terminal type I dockerin module. Recent proteomic studies revealed that Cthe_0821 is one of the major cellulosomal enzymes when C. thermocellum is cultured on cellulose. Here we show that the GH5 catalytic module of Cthe_0821 displays endomannanase activity. C. thermocellum Man5A hydrolyzes soluble konjac glucomannan, soluble carob galactomannan, and insoluble ivory nut mannan but does not attack the highly galactosylated mannan from guar gum, suggesting that the enzyme prefers unsubstituted β-1,4-mannoside linkages. The CBM32 of C. thermocellum Man5A displays a preference for the nonreducing ends of mannooligosaccharides, although the protein module exhibits measurable affinity for the termini of β-1,4-linked glucooligosaccharides such as cellobiose. CBM32 potentiates the activity of C. thermocellum Man5A against insoluble mannans but has no significant effect on the capacity of the enzyme to hydrolyze soluble galactomannans and glucomannans. The product profile of C. thermocellum Man5A is affected by the presence of CBM32.
Isolation and purification of thermostable β-mannanase from Paenibacillus illinoisensis ZY-08.
Lee, Y. S., Zhou, Y., Park, I. H., Chandra, M. R. G. S., Ahn, S. C. & Choi, Y. L. (2010). Journal of the Korean Society for Applied Biological Chemistry, 53(1), 1-7.
A bacterium, ZY-08 that produced extracellular mannanase, was isolated and identified as Paenibacillus illinoisensis (P. illinoisensis) on the basis of 16S rDNA phylogenetic analysis. The enzyme was purified to apparent homogeneity by ultrafiltration, DEAE-Sepharose chromatography, and Sephadex G-50 chromatography procedures. The β-mannanase was purified 3.5 fold to homogeneity with a final recovery of 22% and specificity of 9.3 U/mg protein as judged by SDS-polyacrylamide gel electrophoresis. The molecular mass was about 60 kDa. It was active at 60°C and pH 6.0 and it’s retained about 40% of activity at 70°, with a broad pH range of 3.0-6.0. The mannanase was highly specific towards glucomannan and galactomannan, but exhibited very low activity towards starch, carboxy-methyl cellulose, and birchwood xylan. Result of thin-layer chromatography analysis showed that the enzyme produced various kinds of oligosacchrides. These unique properties of the thermostable mannanase and broad substrate specificity (low activity towards cellulosic substrates) from P. illinoisensis ZY-08 make this enzyme attractive for biotechnological applications.
Model for random hydrolysis and end degradation of linear polysaccharides: Application to the thermal treatment of mannan in solution.
Nattorp, A., Graf, M., Spühler, C. & Renken, A. (1999). Industrial & Engineering Chemistry Research, 38(8), 2919-2926.
The kinetics for homogeneous hydrolysis of mannan is studied in a batch reactor at temperatures from 160 to 220°C. A formate buffer ensures a pH of 3.8−4.0, measured at 25°C. Samples are analyzed for oligosaccharides up to a degree of polymerization of 6 and also for the total amount of mannose after acid hydrolysis. A mathematical model with two reactions (1, random hydrolysis of the glucosidic bonds; 2, degradation of the reducing end of the molecule) describes accurately the time course of oligosaccharides. Optimized rate constants follow closely an Arrhenius relationship, with the degradation having a higher activation energy (140 kJ/mol) than the hydrolysis (113 kJ/mol). The mathematical model has the advantage that production of small molecules is independent of the initial chain-length distribution as long as the average initial chain length is some 5 times longer than the largest species measured. It can be applied to first-order depolymerization of other linear polymers with one link type in order to determine reaction rate constants or make predictions about molecular weight distribution on the base of known reaction rate constants.
Molecular and biochemical characterization of endo-β-mannanases from germinating coffee (Coffea Arabica) grains.
Marraccini, P., Rogers, J. W., Allard, C., André, M. L., Caillet, V., Lacoste, N., Lausamme, F. & Michaux, S. (2001). Planta, 213(2), 296-308.
The activity of endo-β-mannanase ([1→4]-β-mannan endohydrolase EC 22.214.171.124) is likely to be central to the metabolism of cell wall mannans during the germination of grains of coffee (Coffea spp.). In the present paper, we report the cloning and sequencing of two endo-β-mannanase cDNAs (manA and manB) by different strategies from Coffea arabica L. The manA cDNA was obtained by the use of oligonucleotides homologous to published sequences of other endo-β-mannanases and manB by the use of oligonucleotides deduced from a purified enzyme from coffee. ManA and B proteins share about 56% sequence homology and include highly conserved regions found in other mannan endohydrolases. Purification of the activity by chromatography followed by separation by two-dimensional electrophoresis and amino acid sequencing demonstrated the existence of at least seven isomers of the ManB form. The existence of multiple manB genes was also indicated by Southern analysis, whereas only one or two gene copies were detected for manA. Northern hybridizations with manA- and manB-specific probes showed that mRNA transcripts for both cDNAs were present at the same periods of bean germination with transcript peaks at 20 days after imbibition of water (DAI). Transcripts were not detected during grain maturation or in the other tissues such as roots, stems, flowers and leaves. The peak endo-β-mannanase activity occurred at approximately 28 DAI and was not detected in grains prior to imbibition. Activity and mRNA levels appeared to be tightly co-ordinated. Tests of substrate specificity with the purified ManB enzyme showed that activity required a minimum of five mannose units to function efficiently.
Hydrolysis of (1,4)- β-D-mannans in barley (Hordeum vulgare L.) is mediated by the concerted action of (1,4)-β-D-mannan endohydrolase and β-D-mannosidase.
Hrmova, M., Burton, R. A., Biely, P., Lahnstein, J. & Fincher, G. (2006). Biochem. J, 399, 77-90.
A family GH5 (family 5 glycoside hydrolase) (1,4)-β-D-mannan endohydrolase or β-D-mannanase (EC 126.96.36.199), designated HvMAN1, has been purified 300-fold from extracts of 10-day-old barley (Hordeum vulgare L.) seedlings using ammonium sulfate fractional precipitation, followed by ion exchange, hydrophobic interaction and size-exclusion chromatography. The purified HvMAN1 is a relatively unstable enzyme with an apparent molecular mass of 43 kDa, a pI of 7.8 and a pH optimum of 4.75. The HvMAN1 releases Man (mannose or D-mannopyranose)-containing oligosaccharides of degree of polymerization 2–6 from mannans, galactomannans and glucomannans. With locust-bean galactomannan and mannopentaitol as substrates, the enzyme has Km constants of 0.16 mg·ml-1 and 5.3 mM and Kcat constants of 12.9 and 3.9 s-1 respectively. Product analyses indicate that transglycosylation reactions occur during hydrolysis of (1,4)-β-D-manno-oligosaccharides. The complete sequence of 374 amino acid residues of the mature enzyme has been deduced from the nucleotide sequence of a near full-length cDNA, and has allowed a three-dimensional model of the HvMAN1 to be constructed. The barley HvMAN1 gene is a member of a small (1,4)-β-D-mannan endohydrolase family of at least six genes, and is transcribed at low levels in a number of organs, including the developing endosperm, but also in the basal region of young roots and in leaf tips. A second barley enzyme that participates in mannan depolymerization through its ability to hydrolyse (1,4)-β-D-manno-oligosaccharides to Man is a family GH1 β-D-mannosidase, now designated HvβMANNOS1, but previously identified as a β-D-glucosidase [Hrmova, MacGregor, Biely, Stewart and Fincher (1998) J. Biol. Chem. 273, 11134–11143], which hydrolyses 4NP (4-nitrophenyl) β-D-mannoside three times faster than 4NP β-D-glucoside, and has an action pattern typical of a (1,4)-β-D-mannan exohydrolase.
Molecular insights into substrate specificity and thermal stability of a bacterial GH5-CBM27 endo-1,4-β-D-mannanase.
dos Santos, C. R., Paiva, J. H., Meza, A. N., Cota, J., Alvarez, T. M., Ruller, R., Prade, R. A., Squina, F. M. & Murakami, M. T. (2012). Journal of Structural Biology, 177(2), 469-476.
The breakdown of β-1,4-mannoside linkages in a variety of mannan-containing polysaccharides is of great importance in industrial processes such as kraft pulp delignification, food processing and production of second-generation biofuels, which puts a premium on studies regarding the prospection and engineering of β-mannanases. In this work, a two-domain β-mannanase from Thermotoga petrophila that encompasses a GH5 catalytic domain with a C-terminal CBM27 accessory domain, was functionally and structurally characterized. Kinetic and thermal denaturation experiments showed that the CBM27 domain provided thermo-protection to the catalytic domain, while no contribution on enzymatic activity was observed. The structure of the catalytic domain determined by SIRAS revealed a canonical (α/β)8-barrel scaffold surrounded by loops and short helices that form the catalytic interface. Several structurally related ligand molecules interacting with TpMan were solved at high-resolution and resulted in a wide-range representation of the subsites forming the active-site cleft with residues W134, E198, R200, E235, H283 and W284 directly involved in glucose binding.
Structural characterization of neutral oligosaccharides by laser-enhanced in-source decay of MALDI-FTICR MS.
Yang, H., Yu, Y., Song, F. & Liu, S. (2011). Journal of The American Society for Mass Spectrometry, 22(5), 845-855.
MALDI in-source decay (ISD) technique described to date has proven to be a convenient and rapid method for sequencing purified peptides and proteins. However, the general ISD still can not produce adequate fragments for the detailed structural elucidation of oligosaccharides. In this study, an efficient and practical method termed the laser-enhanced ISD (LEISD) technique of MALDI-FTICR MS allows highly reliable and abundant fragmentation of the neutral oligosaccharides, which was attributed to the ultrahigh irradiation laser of mJ level. The yield of ISD fragmentation was evaluated under different laser powers for 7 neutral oligosaccharides using DHB as matrix. Better quality ISD spectra including fragment ions in low-mass region were obtained at higher laser power. Results from the LEISD of oligosaccharides demonstrated that a significantly better signal-to-noise ratio (S/N) and more structural information could be obtained in comparison to the conventional CID. It was also suggested that the valuable A ions derived from cross-ring cleavage of the linear oligosaccharides allowed the distinction among α(1 → 4)-, α(1 → 6)-, β(1 → 4)-, and β(1 → 3)-linked isobaric structures according to fragment types and intensities. In addition, ideal fragmentation ions observed by LEISD method facilitated the determination of the sequences and branched points of complex oligosaccharides from human milk.
Family 34 glycosyltransferase (GT34) genes and proteins in Pinus radiata (radiata pine) and Pinus taeda (loblolly pine).
Ade, C. P., Bemm, F., Dickson, J. M. J., Walter, C. & Harris, P. J. (2014). The Plant Journal, 78(2), 305-318.
Using a functional genomics approach, four candidate genes (PtGT34A, PtGT34B, PtGT34C and PtGT34D) were identified in Pinus taeda. These genes encode CAZy family GT34 glycosyltransferases that are involved in the synthesis of cell-wall xyloglucans and heteromannans. The full-length coding sequences of three orthologs (PrGT34A, B and C) were isolated from a xylem-specific cDNA library from the closely related Pinus radiata. PrGT34B is the ortholog of XXT1 and XXT2, the two main xyloglucan (1→6)-α-xylosyltransferases in Arabidopsis thaliana. PrGT34C is the ortholog of XXT5 in A. thaliana, which is also involved in the xylosylation of xyloglucans. PrGT34A is an ortholog of a galactosyltransferase from fenugreek (Trigonella foenum-graecum) that is involved in galactomannan synthesis. Truncated coding sequences of the genes were cloned into plasmid vectors and expressed in a Sf9 insect cell-culture system. The heterologous proteins were purified, and in vitro assays showed that, when incubated with UDP-xylose and cellotetraose, cellopentaose or cellohexaose, PrGT34B showed xylosyltransferase activity, and, when incubated with UDP-galactose and the same cello-oligosaccharides, PrGT34B showed some galactosyltransferase activity. The ratio of xylosyltransferase to galactosyltransferase activity was 434:1. Hydrolysis of the galactosyltransferase reaction products using galactosidases showed the linkages formed were α-linkages. Analysis of the products of PrGT34B by MALDI-TOF MS showed that up to three xylosyl residues were transferred from UDP-xylose to cellohexaose. The heterologous proteins PrGT34A and PrGT34C showed no detectable enzymatic activity.
The modular architecture of Cellvibrio japonicus mannanases in glycoside hydrolase families 5 and 26 points to differences in their role in mannan degradation.
Hogg, D., Pell, G., Dupree, P., Goubet, F., Martin-Orue, S., Armand, S. & Gilbert, H. (2003). Biochem. J, 371(3), 1027-1043.
β-1,4-Mannanases (mannanases), which hydrolyse mannans and glucomannans, are located in glycoside hydrolase families (GHs) 5 and 26. To investigate whether there are fundamental differences in the molecular architecture and biochemical properties of GH5 and GH26 mannanases, four genes encoding these enzymes were isolated from Cellvibrio japonicus and the encoded glycoside hydrolases were characterized. The four genes, man5A, man5B, man5C and man26B, encode the mannanases Man5A, Man5B, Man5C and Man26B, respectively. Man26B consists of an N-terminal signal peptide linked via an extended serine-rich region to a GH26 catalytic domain. Man5A, Man5B and Man5C contain GH5 catalytic domains and non-catalytic carbohydrate-binding modules (CBMs) belonging to families 2a, 5 and 10; Man5C in addition contains a module defined as X4 of unknown function. The family 10 and 2a CBMs bound to crystalline cellulose and ivory nut crystalline mannan, displaying very similar properties to the corresponding family 10 and 2a CBMs from Cellvibrio cellulases and xylanases. CBM5 bound weakly to these crystalline polysaccharides. The catalytic domains of Man5A, Man5B and Man26B hydrolysed galactomannan and glucomannan, but displayed no activity against crystalline mannan or cellulosic substrates. Although Man5C was less active against glucomannan and galactomannan than the other mannanases, it did attack crystalline ivory nut mannan. All the enzymes exhibited classic endo-activity producing a mixture of oligosaccharides during the initial phase of the reaction, although their mode of action against manno-oligosaccharides and glucomannan indicated differences in the topology of the respective substrate-binding sites. This report points to a different role for GH5 and GH26 mannanases from C. japonicus. We propose that as the GH5 enzymes contain CBMs that bind crystalline polysaccharides, these enzymes are likely to target mannans that are integral to the plant cell wall, while GH26 mannanases, which lack CBMs and rapidly release mannose from polysaccharides and oligosaccharides, target the storage polysaccharide galactomannan and manno-oligosaccharides.
A comparison between a yeast cell wall extract (Bio-Mos®) and palm kernel expeller as mannan-oligosac-charides sources on the performance and ileal microbial population of broiler chickens.
Navidshad, B., Liang, J. B., Jahromi, M. F., Akhlaghi, A. & Abdullah, N. (2015). Italian Journal of Animal Science, 14(1), 3452.
The present study was conducted to determine the effect of a yeast cell wall extract (Bio-Mos) and palm kernel expeller (PKE) on the performance, nutrient digestibility, and ileal bacteria population of broiler chickens. A total of 60 1-d-old male broiler chicks (Cobb 500) were fed one of the 3 isonitrogenous and isocaloric diet including a control diet, or a control diet supplemented with 2 g/kg Bio-Mos (1-42 d), and for the third group, the control diet at 1-28 d following a diet containing 200 g/kg of an enzymatically-treated PKE at 29-42 d. The weight gains of birds fed the PKE containing diet (96.17 g/d) were less than other groups (109.10 and 104.42 g/d for the Bio-Mos and control diet, respectively) (P<0.05). Dietary inclusion of PKE increased bird’s feed intake (214.45 g/d) and feed conversion ratio (FCR) (2.23) than the Bio-Mos diet (194.87 and 1.79 g/d for feed intake and FCR, respectively) (P<0.05). The PKE diet had lower digestibility coefficients for dry matter (83.37%), ash and crude protein (78.63%) than the PKE free diets (P<0.05). As a ratio of the ileal total bacteria, there were no differences in the ileal population of Lactobacilli and Enterococcus genus or Enterobacteriaceae among the experimental groups (P>0.05), but the birds fed PKE or Bio-Mos containing diets had a lower population of Escherichia colithan the control group (P<0.05). The results showed that PKE potentially has a prebiotic property for chicken; however, a 200 g/kg dietary inclusion rate of PKE is not commercially recommendable because of its negative effects on the nutrients digestibility.
A Novel β-1, 4-mannanase Isolated from Paenibacillus polymyxa KT551.
Hori, K., Kawabata, Y., Nakazawa, Y., Nishizawa, M. & Toeda, K. (2014). Food Science and Technology Research, 20(6), 1261-1265.
A β-1,4-mannanase producing bacterium was isolated from soil collected in Akita Prefecture, Japan. The bacterium was identified as Paenibacillus polymyxa KT551 and was shown to produce a novel β-1,4-mannanase. The novelty of the enzyme was established by its N-terminal amino acid sequence, molecular weight and isoelectric point. The isolated β-1,4-mannanase showed activity against mannotetraose, mannopentaose and mannohexaose to produce mannobiose, mannotriose and mannotetraose. However, the enzyme exhibited no activity against mannobiose and mannotriose. Moreover, the crude enzyme preparation of the bacterium had no or minimal β-mannosidase or α-galactosidase activity. Therefore, the enzyme preparation from P. polymyxa KT551 holds potential for the efficient production of mannooligosaccharides from natural resources of galactomannans.
Biochemical characterization of an acidophilic β-mannanase from Gloeophyllum trabeum CBS900. 73 with significant transglycosylation activity and feed digesting ability.
Wang, C., Zhang, J., Wang, Y., Niu, C., Ma, R., Wang, Y., Bai, Y., Luo, H. & Yao, B. (2016). Food Chemistry, 197, 474-481.
Acidophilic β-mannanases have been attracting much attention due to their excellent activity under extreme acidic conditions and significant industrial applications. In this study, a β-mannanase gene of glycoside hydrolase family 5, man5A, was cloned from Gloeophyllum trabeum CBS900.73, and successfully expressed in Pichia pastoris. Purified recombinant Man5A was acidophilic with a pH optimum of 2.5 and exhibited great pH adaptability and stability (>80% activity over pH 2.0-6.0 and pH 2.0-10.0, respectively). It had a high specific activity (1356 U/mg) against locust bean gum, was able to degrade galactomannan and glucomannan in a classical four-site binding mode, and catalyzed the transglycosylation of mannotetrose to mannooligosaccharides with higher degree of polymerization. Besides, it had great resistance to pepsin and trypsin and digested corn-soybean meal based diet in a comparable way with a commercial β-mannanase under the simulated gastrointestinal conditions of pigs. This acidophilic β-mannanase represents a valuable candidate for wide use in various industries, especially in the feed.
An Aspergillus nidulans GH26 endo-β-mannanase with a novel degradation pattern on highly substituted galactomannans.
von Freiesleben, P., Spodsberg, N., Blicher, T. H., Anderson, L., Jørgensen, H., Stålbrand, H., Meyer, A. S. & Krogh, K. B. (2016). Enzyme and Microbial Technology, 83, 68-77.
The activity and substrate degradation pattern of a novel Aspergillus nidulans GH26 endo-β-mannanase (AnMan26A) was investigated using two galactomannan substrates with varying amounts of galactopyranosyl residues. The AnMan26A was characterized in parallel with the GH26 endomannanase from Podospora anserina (PaMan26A) and three GH5 endomannanases from A. nidulans and Trichoderma reesei (AnMan5A, AnMan5C and TrMan5A). The initial rates and the maximal degree of enzymatically catalyzed conversion of locust bean gum and guar gum galactomannans were determined. The hydrolysis product profile at maximal degree of conversion was determined using DNA sequencer-Assisted Saccharide analysis in High throughput (DASH). This is the first reported use of this method for analyzing galactomannooligosaccharides. AnMan26A and PaMan26A were found to have a novel substrate degradation pattern on the two galactomannan substrates. On the highly substituted guar gum AnMan26A and PaMan26A reached 35-40% as their maximal degree of conversion whereas the three tested GH5 endomannanases only reached 8-10% as their maximal degree of conversion. α-Galactosyl-mannose was identified as the dominant degradation product resulting from AnMan26A and PaMan26A action on guar gum, strongly indicating that these two enzymes can accommodate galactopyranosyl residues in the -1 and in the +1 subsite. The degradation of α-64-63-di-galactosyl-mannopentaose by AnMan26A revealed accommodation of galactopyranosyl residues in the -2, -1 and +1 subsite of the enzyme. Accommodation of galactopyranosyl residues in subsites -2 and +1 has not been observed for other characterized endomannanases to date. Docking analysis of galactomannooligosaccharides in available crystal structures and homology models supported the conclusions drawn from the experimental results. This newly discovered diversity of substrate degradation patterns demonstrates an expanded functionality of fungal endomannanases, than hitherto reported.
A novel thermostable GH5_7 β-mannanase from Bacillus pumilus GBSW19 and its application in manno-oligosaccharides (MOS) production.
Zang, H., Xie, S., Wu, H., Wang, W., Shao, X., Wu, L., Rajer, F. U. & Gao, X. (2015). Enzyme and microbial technology, 78, 1-9.
A novel thermostable mannanase from a newly isolated Bacillus pumilus GBSW19 has been identified, expressed, purified and characterized. The enzyme shows a structure comprising a 28 amino acid signal peptide, a glycoside hydrolase family 5 (GH5) catalytic domain and no carbohydrate-binding module. The recombinant mannanase has molecular weight of 45 kDa with an optimal pH around 6.5 and is stable in the range from pH 5-11. Meanwhile, the optimal temperature is around 65°C, and it retains 50% relative activity at 60°C for 12 h. In addition, the purified enzyme can be activated by several ions and organic solvents and is resistant to detergents. Bpman5 can efficiently convert locus bean gum to mainly M2, M3 and M5, and hydrolyze manno-oligosaccharides with a minimum DP of 3. Further exploration of the optimum condition using HPLC to prepare oligosaccharides from locust bean gum was obtained as 10 mg/ml locust bean gum incubated with 10 U/mg enzyme at 50°C for 24 h. By using this enzyme, locust bean gum can be utilized to generate high value-added oligosaccharides with a DP of 2-6.
High-level expression and characterization of a thermophilic β-mannanase from Aspergillus niger in Pichia pastoris.
Yu, S., Li, Z., Wang, Y., Chen, W., Fu, L., Tang, W., Chen, C., Liu, Y., Zhang, X. & Ma, L. (2015). Biotechnology Letters, 37(9), 1853-1859.
Objectives: A novel, high-level expression, thermostable mannan endo-1,4-β-mannosidase is urgently needed for industrial applications. Results: The mannan endo-1,4-β-mannosidase gene (MAN) from Aspergillus niger CBS 513.88 was optimized based on the codon usage bias in Pichia pastoris and synthesized by overlapping PCR to produce MAN-P. It was expressed in P. pastoris GS115 from a constitutive expression vector pHBM-905 M. MAN-P reached 594 mg/l in shake-flasks after 192 h induction. On production in a 5 l fermenter, the yield of MAN-P reached ~3.5 mg/ml and the enzyme activity was 1612 U/ml. The enzyme exhibited a maximum activity of 3049 U/ml at 80°C and retained 60 % enzyme activity at 80°C for 2 h. The pH optimum was 4.5 and the enzyme was stable over the pH range 1.5-11. Conclusion: The thermostability of MAN-P is higher than other known fungal mannanases and the expression and thermophilic properties make MAN-P useful for industrial applications.
From native malt to pure starch-Development and characterization of a purification procedure for modified starch.
Rittenauer, M., Kolesnik, L., Gastl, M. & Becker, T. (2016). Food Hydrocolloids, 56, 50-57.
Starch characteristics influence the gelatinization process, which is an important prerequisite for the saccharification required in many industrial processes. In order to determine these characteristics in barley malt, an adapted purification procedure allowing to preserve the native starch composition and simultaneously segregating the amylolytic enzymes which were formed during the germination is indispensable. Therefore, this research aimed to develop a method based on a combination of dry milling, micro-sieving and density gradient centrifugation. The impact on the starch characteristics was evaluated for three germinated barley varieties. The purified starches showed starch contents greater than 90% and proteins contents less than 0.4%. Yields ranged from 40.3 to 48.6%, depending on the variety. Considering the starch properties, the amylose/amylopectin ratio was not modified during the purification. The circularity of the granules as well as the ratio of A- and B-type granules remained constant. The particle size distribution of A-granules was not shifted, B-granules with a specific diameter of 5-10 µm were slightly reduced in dependency of the native granule composition. The highest impact could be observed on the amylolytic enzymes, which were completely segregated regardless of their initial value. The standard deviation of repeatability was less than 5%, except for the determination of B-type particle size distribution (7%). The newly developed procedure supplements existing isolation methods of unmalted grains by enabling the purification of germinated barley in a reproducible manner, without altering the native starch properties and by providing pure starch free of amylolytic activity.
Mannan endo-1, 4-β-mannosidase from Kitasatospora sp. isolated in Indonesia and its potential for production of mannooligosaccharides from mannan polymers.
Rahmani, N., Kashiwagi, N., Lee, J., Niimi-Nakamura, S., Matsumoto, H., Kahar, P., Lisdiyanti, P., Yopi, Prasetya, B., Ogino, C. & Kondo, A. (2017). AMB Express, 7(1), 100.
Mannan endo-1,4-β-mannosidase (commonly known as β-mannanase) catalyzes a random cleavage of the β-D-1,4-mannopyranosyl linkage in mannan polymers. The enzyme has been utilized in biofuel production from lignocellulose biomass, as well as in production of mannooligosaccharides (MOS) for applications in feed and food industries. We aimed to obtain a β-mannanase, for such mannan polymer utilization, from actinomycetes strains isolated in Indonesia. Strains exhibiting high mannanase activity were screened, and one strain belonging to the genus Kitasatospora was selected. We obtained a β-mannanase from this strain, and an amino acid sequence of this Kitasatospora β-mannanase showed a 58-71% similarity with the amino acid sequences of Streptomyces β-mannanases. The Kitasatospora β-mannanase showed a significant level of activity (944 U/mg) against locust bean gum (0.5% w/v) and a potential for oligosaccharide production from various mannan polymers. The β-mannanase might be beneficial particularly in the enzymatic production of MOS for applications of mannan utilization.