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|Stability:||> 10 years under recommended storage conditions|
|Source:||Ivory nut seeds|
|Monosaccharides (%):||Mannose = 99|
|Main Chain Glycosidic Linkage:||β-1,4|
|Substrate For (Enzyme):||endo-1,4-β-Mannanase|
High purity Mannan (Ivory Nut) for use in research, biochemical enzyme assays and in vitro diagnostic analysis.
Treated with sodium borohydride to lower reducing sugar levels. Traces of arabinose and xylose.
Zhang, Q. & Mischnick, P. (2018). Macromolecular Chemistry and Physics, In Press.
Konjak glucomannan (KGM) is methylated with NaOH/MeI in water in the presence of borate and as a reference without this additive. With increasing equiv. of borate increasing suppression of 2- and 3-O-methylation of mannosyl residues (M) is observed, while glucosyl units (G) are mainly affected at O-6, but to much lower extent. Raising the temperature and/or addition of acetone as a co-solvent enhance reactivity, but at the cost of M/G regioselectivity. O-Methyl konjac glucomannans (M-KGM) with an average degree of substitution (DS) up to 0.8 and a DS ratio for G and M up to 2 are obtained. From liquid chromatrography–electrospray ionization–mass spectrometry (LC-ESI-MS) of the oligosaccharides obtained from M-KGM, it is concluded that borate-mediated transient protection might also depend on the location of M within KGM. Comparison with random and block models supports random distribution of M and G in KGM. Thermogravimetric analysis shows higher decomposition temperature of M-KGMs with increasing DS.Hide Abstract
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.Hide Abstract
Morrill, J., Månberger, A., Rosengren, A., Naidjonoka, P., von Freiesleben, P., Krogh, K. B., Nylander, T., Karlsson, E. N., Adlercreutz, P. & Stålbrand, H. (2018). Applied Microbiology and Biotechnology, 102, 5149-5163.
β-Mannanases catalyze the conversion and modification of β-mannans and may, in addition to hydrolysis, also be capable of transglycosylation which can result in enzymatic synthesis of novel glycoconjugates. Using alcohols as glycosyl acceptors (alcoholysis), β-mannanases can potentially be used to synthesize alkyl glycosides, biodegradable surfactants, from renewable β-mannans. In this paper, we investigate the synthesis of alkyl mannooligosides using glycoside hydrolase family 5 β-mannanases from the fungi Trichoderma reesei (TrMan5A and TrMan5A-R171K) and Aspergillus nidulans (AnMan5C). To evaluate β-mannanase alcoholysis capacity, a novel mass spectrometry-based method was developed that allows for relative comparison of the formation of alcoholysis products using different enzymes or reaction conditions. Differences in alcoholysis capacity and potential secondary hydrolysis of alkyl mannooligosides were observed when comparing alcoholysis catalyzed by the three β-mannanases using methanol or 1-hexanol as acceptor. Among the three β-mannanases studied, TrMan5A was the most efficient in producing hexyl mannooligosides with 1-hexanol as acceptor. Hexyl mannooligosides were synthesized using TrMan5A and purified using high-performance liquid chromatography. The data suggests a high selectivity of TrMan5A for 1- hexanol as acceptor over water. The synthesized hexyl mannooligosides were structurally characterized using nuclear magnetic resonance, with results in agreement with their predicted β-conformation. The surfactant properties of the synthesized hexyl mannooligosides were evaluated using tensiometry, showing that they have similar micelle-forming properties as commercially available hexyl glucosides. The present paper demonstrates the possibility of using β-mannanases for alkyl glycoside synthesis and increases the potential utilization of renewable β-mannans.Hide Abstract
Zhang, Q. & Mischnick, P. (2017). Macromolecular Chemistry and Physics, 218(17), 1700119.
Methylation in water with NaOH/MeI is applied to study the influence of the stereochemistry on relative reactivities of D-mannosyl (M) compared to D-glucosyl (G) units in konjac glucomannan (KGM). The pH is kept constant at 13.6 over the course of the reaction and aliquots are removed after various time intervals. Methyl distribution in G and M residues is determined after perethylation, hydrolysis, and conversion to O-ethyl-O-methyl-alditol acetates. The order of relative rate constants determined for the O-methyl Konjac glucomannans (M-KGMs) in degree of substitution (DS) range 0.3–0.8 is G-k6 > M-k6 > G-k2 ≈ M-k2 > M-k3 > G-k3. Oligosaccharides obtained by partial hydrolysis after full protection of M-KGM with MeI-d3 are labeled with m-amino-benzoic acid and measured by liquid chromatography–electrospray ionization–mass spectrometry. DS/DP profiles are in full agreement with random distribution of methyl groups. Thermal properties of M-KGMs are analyzed by differential scanning calorimetry and thermogravimetric analysis. Decomposition temperature increases with DS, while the temperature of an endothermic change decreases.Hide Abstract
Kim, D. Y., Lee, S. H., Lee, M. J., Cho, H. Y., Lee, J. S., Rhee, Y. H., Shin, D. H., Son K. H. & Park, H. Y. (2017). International Journal of Biological Macromolecules, 106, 620-628.
The gene (1488-bp) encoding a novel GH10 endo-β-1,4-xylanase (XylM) consisting of an N-terminal catalytic GH10 domain and a C-terminal ricin- type β-trefoil lectin domain-like (RICIN) domain was identified from Luteimicrobium xylanilyticum HY-24. The GH10 domain of XylM was 72% identical to that of Micromonospora lupine endo-β-1,4-xylanase and the RICIN domain was 67% identical to that of Actinospica robiniae hypothetical protein. The recombinant enzyme (rXylM: 49 kDa) exhibited maximum activity toward beechwood xylan at 65°C and pH 6.0, while the optimum temperature and pH of its C-terminal truncated mutant (rXylM△RICIN: 35 kDa) were 45°C and 5.0, respectively. After pre- incubation of 1 h at 60°C, rXylM retained over 80% of its initial activity, but the thermostability of rXylM△RICIN was sharply decreased at temperatures exceeding 40°C. The specific activity (254.1 U mg-1) of rXylM toward oat spelts xylan was 3.4-fold higher than that (74.8 U mg-1) of rXylM△RICIN when the same substrate was used. rXylM displayed superior binding capacities to lignin and insoluble polysaccharides compared to rXylM△RICIN. Enzymatic hydrolysis of β-1,4-D-xylooligosaccharides (X3-X6) and birchwood xylan yielded X3 as the major product. The results suggest that the RICIN domain in XylM might play an important role in substrate-binding and biocatalysis.Hide Abstract
Gruben, B. S., Mäkelä, M. R., Kowalczyk, J. E., Zhou, M., Benoit-Gelber, I. & De Vries, R. P. (2017). BMC genomics, 18(1), 900.
Background: The Aspergillus niger genome contains a large repertoire of genes encoding carbohydrate active enzymes (CAZymes) that are targeted to plant polysaccharide degradation enabling A. niger to grow on a wide range of plant biomass substrates. Which genes need to be activated in certain environmental conditions depends on the composition of the available substrate. Previous studies have demonstrated the involvement of a number of transcriptional regulators in plant biomass degradation and have identified sets of target genes for each regulator. In this study, a broad transcriptional analysis was performed of the A. niger genes encoding (putative) plant polysaccharide degrading enzymes. Microarray data focusing on the initial response of A. niger to the presence of plant biomass related carbon sources were analyzed of a wild-type strain N402 that was grown on a large range of carbon sources and of the regulatory mutant strains δxlnR, δaraR, δamyR, δrhaR and δgalX that were grown on their specific inducing compounds. Results: The cluster analysis of the expression data revealed several groups of co-regulated genes, which goes beyond the traditionally described co-regulated gene sets. Additional putative target genes of the selected regulators were identified, based on their expression profile. Notably, in several cases the expression profile puts questions on the function assignment of uncharacterized genes that was based on homology searches, highlighting the need for more extensive biochemical studies into the substrate specificity of enzymes encoded by these non-characterized genes. The data also revealed sets of genes that were upregulated in the regulatory mutants, suggesting interaction between the regulatory systems and a therefore even more complex overall regulatory network than has been reported so far. Conclusions: Expression profiling on a large number of substrates provides better insight in the complex regulatory systems that drive the conversion of plant biomass by fungi. In addition, the data provides additional evidence in favor of and against the similarity-based functions assigned to uncharacterized genes.Hide Abstract
Cheng, L., Duan, S., Feng, X., Zheng, K., Yang, Q. & Liu, Z. (2016). BioMed Research International, 2016, In Press.
β-mannanase has shown compelling biological functions because of its regulatory roles in metabolism, inflammation, and oxidation. This study separated and purified the β-mannanase from Bacillus subtilis BE-91, which is a powerful hemicellulose-degrading bacterium using a “two-step” method comprising ultrafiltration and gel chromatography. The purified β-mannanase (about 28.2 kDa) showed high specific activity (79, 859.2 IU/mg). The optimum temperature and pH were 65°C and 6.0, respectively. Moreover, the enzyme was highly stable at temperatures up to 70°C and pH 4.5-7.0. The β-mannanase activity was significantly enhanced in the presence of Mn2+, Cu2+, Zn2+, Ca2+, Mg2+, and Al3+, and strongly inhibited by Ba2+, and Pb2+. Km and Vmax values for locust bean gum were 7.14 mg/mL and 107.5 µmol/min/mL versus 1.749 mg/mL and 33.45 µ mol/min/mL for Konjac glucomannan, respectively. Therefore, β-mannanase purified by this work shows stability at high temperatures and in weakly acidic or neutral environments. Based on such data, the β-mannanase will have potential applications as a dietary supplement in treatment of inflammatory processes.Hide Abstract
Park, Y. B., Kafle, K., Lee, C. M., Cosgrove, D. J., & Kim, S. H. (2015). Cellulose, 22(6), 3531-3540.
In nature, algae produce cellulose I where all glucan chains are aligned parallel. However, the presence of cellulose II with anti-parallel glucan chains has been reported for certain Derbesia (Chlorophyceae algae) cell walls; if this is true, it would mean a new biological process for synthesizing cellulose that has not yet been recognized. To answer this question, we examined cellulose structure in Derbesia cell walls, intact as well as treated with cellulose isolation procedures, using sum-frequency-generation spectroscopy, infrared (IR) spectroscopy and X-ray diffraction (XRD). Derbesia walls contain large amounts of mannan and small amounts of crystalline cellulose. Evidence for cellulose II in the intact cell walls was not found, whereas cellulose II in the trifluoroacetic acid (TFA) treated cell wall samples were detected by IR and XRD. A control experiment conducted with ball-milled Avicel cellulose samples showed that cellulose II structure could be formed as a result of TFA treatment and drying of amorphous cellulose. These data suggest that the cellulose II structure detected in the TFA-treated Derbesia gametophyte wall samples is most likely due to reorganization of amorphous cellulose during the sample preparation. Our results contradict the previous report of cellulose II in native alga cell walls. Even if the crystalline cellulose II exists in intact Derbesia gametophyte cell walls, its amount would be very small (below the detection limit) and thus biologically insignificant.Hide Abstract
Kiemle, S. N., Zhang, X., Esker, A. R., Toriz, G., Gatenholm, P. & Cosgrove, D. J. (2014). Biomacromolecules, 15 (5), 1727-1736.
(1,3)(1,4)-β-D-Glucan (mixed-linkage glucan or MLG), a characteristic hemicellulose in primary cell walls of grasses, was investigated to determine both its role in cell walls and its interaction with cellulose and other cell wall polysaccharides in vitro. Binding isotherms showed that MLG adsorption onto microcrystalline cellulose is slow, irreversible, and temperature-dependent. Measurements using quartz crystal microbalance with dissipation monitoring showed that MLG adsorbed irreversibly onto amorphous regenerated cellulose, forming a thick hydrogel. Oligosaccharide profiling using endo-(1,3)(1,4)-β-glucanase indicated that there was no difference in the frequency and distribution of (1,3) and (1,4) links in bound and unbound MLG. The binding of MLG to cellulose was reduced if the cellulose samples were first treated with certain cell wall polysaccharides, such as xyloglucan and glucuronoarabinoxylan. The tethering function of MLG in cell walls was tested by applying endo-(1,3)(1,4)-β-glucanase to wall samples in a constant force extensometer. Cell wall extension was not induced, which indicates that enzyme-accessible MLG does not tether cellulose fibrils into a load-bearing network.Hide Abstract
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.Hide Abstract
Westphal, Y., Schols, H. A., Voragen, A. G. J. & Gruppen, H. (2010). Journal of Chromatography A, 1217(5), 689-695.
Separation and characterization of complex mixtures of oligosaccharides is quite difficult and, depending on elution conditions, structural information is often lost. Therefore, the use of a porous-graphitized-carbon (PGC)-HPLC-ELSD-MSn-method as analytical tool for the analysis of oligosaccharides derived from plant cell wall polysaccharides has been investigated. It is demonstrated that PGC-HPLC can be widely used for neutral and acidic oligosaccharides derived from cell wall polysaccharides. Furthermore, it is a non-modifying technique that enables the characterization of cell wall oligosaccharides carrying, e.g. acetyl groups and methylesters. Neutral oligosaccharides are separated based on their size as well as on their type of linkage and resulting 3D-structure. Series of the planar β-(1,4)-xylo- and β-(1,4)-gluco-oligosaccharides are retained much more by the PGC material than the series of β-(1,4)-galacto-, β-(1,4)-manno- and α-(1,4)-gluco-oligosaccharides. Charged oligomers such as α-(1,4)-galacturonic acid oligosaccharides are strongly retained and are eluted only after addition of trifluoroacetic acid depending on their net charge. Online-MS-coupling using a 1:1 splitter enables quantitative detection of ELSD as well as simple identification of many oligosaccharides, even when separation of oligosaccharides within a complex mixture is not complete. Consequently, PGC-HPLC-separation in combination with MS-detection gives a powerful tool to identify a wide range of neutral and acidic oligosaccharides derived from various cell wall polysaccharides.Hide Abstract
Marcus, S. E., Blake, A. W., Benians, T. A. S, Lee, K. J., Poyser, C., Donaldson, L., Leroux, O., Rogowski, A., Petersen, H. L., Boraston, A., Gilbert, H. J., Willats, W. G. T. & Paul Knox, J. (2010). The Plant Journal, 64(2), 191-203.
How the diverse polysaccharides present in plant cell walls are assembled and interlinked into functional composites is not known in detail. Here, using two novel monoclonal antibodies and a carbohydrate-binding module directed against the mannan group of hemicellulose cell wall polysaccharides, we show that molecular recognition of mannan polysaccharides present in intact cell walls is severely restricted. In secondary cell walls, mannan esterification can prevent probe recognition of epitopes/ligands, and detection of mannans in primary cell walls can be effectively blocked by the presence of pectic homogalacturonan. Masking by pectic homogalacturonan is shown to be a widespread phenomenon in parenchyma systems, and masked mannan was found to be a feature of cell wall regions at pit fields. Direct fluorescence imaging using a mannan-specific carbohydrate-binding module and sequential enzyme treatments with an endo-β-mannanase confirmed the presence of cryptic epitopes and that the masking of primary cell wall mannan by pectin is a potential mechanism for controlling cell wall micro-environments.Hide Abstract
Gunnarsson, L. C., Karlsson, E. N., Albrekt, A. -S., Andersson, M., Holst, O. & Ohlin, M. (2004). Protein Engineering, Design & Selection, 17(3), 213-221.
The growing field of biotechnology is in constant need of binding proteins with novel properties. Not just binding specificities and affinities but also structural stability and productivity are important characteristics for the purpose of large‐scale applications. In order to find such molecules, libraries are created by diversifying naturally occurring binding proteins, which in those cases serve as scaffolds. In this study, we investigated the use of a thermostable carbohydrate binding module, CBM4‐2, from a xylanase found in Rhodothermus marinus, as a diversity‐carrying scaffold. A combinatorial library was created by introducing restricted variation at 12 positions in the carbohydrate binding site of the CBM4‐2. Despite the small size of the library (1.6×106 clones), variants specific towards different carbohydrate polymers (birchwood xylan, Avicel and ivory nut mannan) as well as a glycoprotein (human IgG4) were successfully selected for, using the phage display method. Investigated clones showed a high productivity (on average 69 mg of purified protein/l shake flask culture) when produced in Escherichia coli and they were all stable molecules displaying a high melting transition temperature (75.7 ± 5.3°C). All our results demonstrate that the CBM4‐2 molecule is a suitable scaffold for creating variants useful in different biotechnological applications.Hide Abstract
Bolam, D. N., Xie, H., Pell, G., Hogg, D., Galbraith, G., Henrissat, B. & Gilbert, H. J. (2004). Journal of Biological Chemistry, 279(22), 22953-22963.
The hydrolysis of the plant cell wall by microbial glycoside hydrolases and esterases is the primary mechanism by which stored organic carbon is utilized in the biosphere, and thus these enzymes are of considerable biological and industrial importance. Plant cell wall-degrading enzymes in general display a modular architecture comprising catalytic and non-catalytic modules. The X4 modules in glycoside hydrolases represent a large family of non-catalytic modules whose function is unknown. Here we show that the X4 modules from a Cellvibrio japonicus mannanase (Man5C) and arabinofuranosidase (Abf62A) bind to polysaccharides, and thus these proteins comprise a new family of carbohydrate-binding modules (CBMs), designated CBM35. The Man5C-CBM35 binds to galactomannan, insoluble amorphous mannan, glucomannan, and manno-oligosaccharides but does not interact with crystalline mannan, cellulose, cello-oligosaccharides, or other polysaccharides derived from the plant cell wall. Man5C-CBM35 also potentiates mannanase activity against insoluble amorphous mannan. Abf62A-CBM35 interacts with unsubstituted oat-spelt xylan but not substituted forms of the hemicellulose or xylo-oligosaccharides, and requires calcium for binding. This is in sharp contrast to other xylan-binding CBMs, which interact in a calcium-independent manner with both xylo-oligosaccharides and decorated xylans.Hide Abstract
Xu, B., Hägglund, P., Stålbrand, H. & Janson, J. C. (2002). Journal of Biotechnology, 92(3), 267-277.
Two variants of an endo-β-1,4-mannanase from the digestive tract of blue mussel, Mytilus edulis, were purified by a combination of immobilized metal ion affinity chromatography, size exclusion chromatography in the absence and presence of guanidine hydrochloride and ion exchange chromatography. The purified enzymes were characterized with regard to enzymatic properties, molecular weight, isoelectric point, amino acid composition and N-terminal sequence. They are monomeric proteins with molecular masses of 39 216 and 39 265 Da, respectively, as measured by MALDI-TOF mass spectrometry. The isoelectric points of both enzymes were estimated to be around 7.8, however slightly different, by isoelectric focusing in polyacrylamide gel. The enzymes are stable from pH 4.0 to 9.0 and have their maximum activities at a pH about 5.2. The optimum temperature of both enzymes is around 50–55°C. Their stability decreases rapidly when going from 40 to 50°C. The N-terminal sequences (12 residues) were identical for the two variants. They can be completely renatured after denaturation in 6 M guanidine hydrochloride. The enzymes readily degrade the galactomannans from locust bean gum and ivory nut mannan but show no cross-specificity for xylan and carboxymethyl cellulose. There is no binding ability observed towards cellulose and mannan.Hide Abstract
Sabini, E., Wilson, K. S., Siika‐aho, M., Boisset, C. & Chanzy, H. (2000). European Journal of Biochemistry, 267(8), 2340-2344.
The enzymatic degradation of single crystals of mannan I with the catalytic core domain of a β-mannanase (EC 18.104.22.168 or Man5A) from Trichoderma reesei was investigated by transmission electron microscopy and electron diffraction. The enzyme attack took place at the edge of the crystals and progressed towards their centres. Quite remarkably the crystalline integrity of the crystals was preserved almost to the end of the digestion process. This behaviour is consistent with an endo-mechanism, where the enzyme interacts with the accessible mannan chains located at the crystal periphery and cleaves one mannan molecule at a time. The endo mode of digestion of the crystals was confirmed by an analysis of the soluble degradation products.Hide Abstract
Sachslehner, A. & Haltrich, D. (1999). FEMS Microbiology Letters, 177(1), 47-55.
The phytopathogenic fungus Sclerotium (Athelia) rolfsii forms one major endo-β-1,4-D-mannanase (EC 22.214.171.124) under non-induced and derepressed conditions, i.e. after depletion of glucose which was used as the only carbohydrate substrate for its cultivation. This mannanase was purified to electrophoretic homogeneity by ammonium sulfate precipitation, hydrophobic interaction chromatography, anion exchange chromatography and gel filtration. The enzyme is a glycoprotein with a molecular mass of 46.5±2 kDa (SDS-PAGE), an isoelectric point of 2.75, and a pH optimum of 3.0–3.5. The enzyme is especially stable in the acidic region with an exceptional half-life of activity of 41 days at pH 4.5 and 50°C. It exerts activity on β-1,4-mannan from ivory nut, which is hydrolyzed mainly to mannobiose and mannotriose, as well as on glucomannan, galactomannan, galactoglucomannan, and mannooligosaccharides not smaller than mannotetraose. The main end-products mannotriose and to a lesser extent mannobiose inhibit its activity moderately.Hide Abstract
Braithwaite, K. L., Black, G. W., Hazlewood, G. P., Ali, B. R. S. & Gilbert, H. J. (1995). Biochem. J, 305, 1005-1010.
Pseudomonas fluorescens subsp. cellulosa when cultured in the presence of carob galactomannan degraded the polysaccharide. To isolate gene(s) from P. fluorescens subsp. cellulosa encoding endo-β-1,4-mannanase (mannanase) activity, a genomic library of Pseudomonas DNA, constructed in lambda ZAPII, was screened for mannanase-expressing clones using the dye-labelled substrate, azo-carob galactomannan. The nucleotide sequence of the pseudomonad insert from a mannanase-positive clone revealed a single open reading frame of 1257 bp encoding a protein of Mr 46,938. The deduced N-terminal sequence of the putative polypeptide conformed to a typical prokaryotic signal peptide. Truncated derivatives of the mannanase, lacking 54 and 16 residues from the N- and C-terminus respectively of the mature form of the enzyme, did not exhibit catalytic activity. Inspection of the primary structure of the mannanase did not reveal any obvious linker sequences or protein motifs characteristic of the non-catalytic domains located in other Pseudomonas plant cell wall hydrolases. These data indicate that the mannanase is non-modulator, comprising a single catalytic domain. Comparison of the mannanase sequence with those in the SWISSPROT database revealed greatest sequence homology with the mannanase from Bacillus sp. Thus the Pseudomonas enzyme belongs to glycosyl hydrolase Family 26, a family containing mannanases and endoglucanases. Analysis of the substrate specificity of the mannanase showed that the enzyme hydrolysed mannan and galactomannan, but displayed little activity towards other polysaccharides located in the plant cell wall. The enzyme had a pH optimum of approx. 7.0, was resistant to proteolysis and had an Mr of 46,000 when expressed by Escherichia coli.Hide Abstract