Dataset Information

Re-interpreting the role of endo-beta-mannanases as mannan endotransglycosylase/hydrolases in the plant cell wall.

ABSTRACT: BACKGROUND: Mannans are hemicellulosic polysaccharides in the plant primary cell wall with two major physiological roles: as storage polysaccharides that provide energy for the growing seedling; and as structural components of the hemicellulose-cellulose network with a similar function to xyloglucans. Endo-beta-mannanases are hydrolytic enzymes that cleave the mannan backbone. They are active during seed germination and during processes of growth or senescence. The recent discovery that endo-beta-mannanase LeMAN4a from ripe tomato fruit also has mannan transglycosylase activity requires the role of endo-beta-mannanases to be reinterpreted. AIMS: In this review, the role of endo-beta-mannanases as mannan endotransglycosylase/hydrolases (MTHs) in remodelling the plant cell wall is considered by analogy to the role of xyloglucan endotransglucosylase/hydrolases (XTHs). The current understanding of the reaction mechanism of these enzymes, their three-dimensional protein structure, their substrates and their genes are reported. FUTURE OUTLOOK: There are likely to be more endohydrolases within the plant cell wall that can carry out hydrolysis and transglycosylation reactions. The challenge will be to demonstrate that the transglycosylation activities shown in vitro also exist in vivo and to validate a role for transglycosylation reactions during the growth and development of the plant cell wall.

SUBMITTER: Schroder R

PROVIDER: S-EPMC2710900 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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Re-interpreting the role of endo-beta-mannanases as mannan endotransglycosylase/hydrolases in the plant cell wall.

Schröder Roswitha R Atkinson Ross G RG Redgwell Robert J RJ

Annals of botany 20090519 2

<h4>Background</h4>Mannans are hemicellulosic polysaccharides in the plant primary cell wall with two major physiological roles: as storage polysaccharides that provide energy for the growing seedling; and as structural components of the hemicellulose-cellulose network with a similar function to xyloglucans. Endo-beta-mannanases are hydrolytic enzymes that cleave the mannan backbone. They are active during seed germination and during processes of growth or senescence. The recent discovery that e ...[more]

PMID: 19454593

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Project description:The genome sequence of the hyperthermophilic bacterium Thermotoga maritima encodes a number of glycosyl hydrolases. Many of these enzymes have been shown in vitro to degrade specific glycosides that presumably serve as carbon and energy sources for the organism. However, because of the broad substrate specificity of many glycosyl hydrolases, it is difficult to determine the physiological substrate preferences for specific enzymes from biochemical information. In this study, T. maritima was grown on a range of polysaccharides, including barley beta-glucan, carboxymethyl cellulose, carob galactomannan, konjac glucomannan, and potato starch. In all cases, significant growth was observed, and cell densities reached 10(9) cells/ml. Northern blot analyses revealed different substrate-dependent expression patterns for genes encoding the various endo-acting beta-glycosidases; these patterns ranged from strong expression to no expression under the conditions tested. For example, cel74 (TM0305), a gene encoding a putative beta-specific endoglucananse, was strongly expressed on all substrates tested, including starch, while no evidence of expression was observed on any substrate for lam16 (TM0024), xyl10A (TM0061), xyl10B (TM0070), and cel12A (TM1524), which are genes that encode a laminarinase, two xylanases, and an endoglucanase, respectively. The cel12B (TM1525) gene, which encodes an endoglucanase, was expressed only on carboxymethyl cellulose. An extracellular mannanase encoded by man5 (TM1227) was expressed on carob galactomannan and konjac glucomannan and to a lesser extent on carboxymethyl cellulose. An unexpected result was the finding that the cel5A (TM1751) and cel5B (TM1752) genes, which encode putative intracellular beta-specific endoglucanases, were induced only when T. maritima was grown on konjac glucomannan. To investigate the biochemical basis of this finding, the recombinant forms of Man5 (M(r), 76,900) and Cel5A (M(r), 37,400) were expressed in Escherichia coli and characterized. Man5, a T. maritima extracellular enzyme, had a melting temperature of 99 degrees C and an optimun temperature of 90 degrees C, compared to 90 and 80 degrees C, respectively, for the intracellular enzyme Cel5A. While Man5 hydrolyzed both galactomannan and glucomannan, no activity was detected on glucans or xylans. Cel5A, however, not only hydrolyzed barley beta-glucan, carboxymethyl cellulose, xyloglucan, and lichenin but also had activity comparable to that of Man5 on galactomannan and higher activity than Man5 on glucomannan. The biochemical characteristics of Cel5A, the fact that Cel5A was induced only when T. maritima was grown on glucomannan, and the intracellular localization of Cel5A suggest that the physiological role of this enzyme includes hydrolysis of glucomannan oligosaccharides that are transported following initial hydrolysis by extracellular glycosidases, such as Man5.

Project description:1. Cell-free extracts of all plants tested contained a novel enzyme activity (xyloglucan endotransglycosylase, XET) able to transfer a high-Mr portion from a donor xyloglucan to a suitable acceptor such as a xyloglucan-derived nonasaccharide (Glc4Xyl3GalFuc; XG9). 2. A simple assay for the enzyme, using [3H]XG9 and based on the ability of the [3H]polysaccharide product to bind to filter paper, is described. 3. The enzyme was highly specific for xyloglucan as the glycosyl donor, and showed negligible transglycosylation of other polysaccharides, including CM-cellulose. 4. The Km for XG9 was 50 microM; certain other 3H-labelled xyloglucan oligosaccharides also acted as acceptors, and certain non-radioactive xyloglucan oligosaccharides competed with [3H]XG9 as acceptor; the minimum acceptor structure was deduced to be: [formula: see text] 5. The pH optimum was approx. 5.5 and the enzyme was less than half as active at pH 7.0. The enzyme was slightly activated by Ca2+, Mg2+, Mn2+, spermidine, ascorbate and 2-mercaptoethanol, and inhibited by Ag+, Hg2+, Zn2+ and La3+. 6. XET activity was essentially completely extracted by aqueous solutions of low ionic strength; Triton X-100, Ca2+, La3+, and Li+ did not enhance extraction. Negligible activity was left in the unextractable (cell-wall-rich) residue. 7. The enzyme differed from the major cellulases (EC 3.2.1.4) of pea in: (a) susceptibility to inhibition by cello-oligosaccharides, (b) polysaccharide substrate specificity, (c) inducibility by auxin, (d) requirement for salt in the extraction buffer and (e) activation by 2-mercaptoethanol. XET is therefore concluded to be a new enzyme activity (xyloglucan: xyloglucan xyloglucanotransferase; EC 2.4.1.-). 8. XET was detected in extracts of the growing portions of dicotyledons, monocotyledons (graminaceous and liliaceous) and bryophytes. 9. The activity was positively correlated with growth rate in different zones of the pea stem. 10. We propose that XET is responsible for cutting and rejoining intermicrofibrillar xyloglucan chains and that it thus causes the wall-loosening required for plant cell expansion.

Project description:Mannans are key components of lignocellulose present in the hemicellulosic fraction of plant primary cell walls. Mannan endo-1,4-beta-mannosidases (1,4-beta-D-mannanases) catalyze the random hydrolysis of beta-1,4-mannosidic linkages in the main chain of beta-mannans. Biodegradation of beta-mannans by the action of thermostable mannan endo-1,4-beta-mannosidase offers significant technical advantages in biotechnological industrial applications, i.e. delignification of kraft pulps or the pretreatment of lignocellulosic biomass rich in mannan for the production of second generation biofuels, as well as for applications in oil and gas well stimulation, extraction of vegetable oils and coffee beans, and the production of value-added products such as prebiotic manno-oligosaccharides (MOS).A gene encoding mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannan mannanohydrolase (E.C. 3.2.1.78), commonly termed beta-mannanase, from Aspergillus niger BK01, which belongs to glycosyl hydrolase family 5 (GH5), was cloned and successfully expressed heterologously (up to 243 microg of active recombinant protein per mL) in Pichia pastoris. The enzyme was secreted by P. pastoris and could be collected from the culture supernatant. The purified enzyme appeared glycosylated as a single band on SDS-PAGE with a molecular mass of approximately 53 kDa. The recombinant beta-mannanase is highly thermostable with a half-life time of approximately 56 h at 70 degrees C and pH 4.0. The optimal temperature (10-min assay) and pH value for activity are 80 degrees C and pH 4.5, respectively. The enzyme is not only active towards structurally different mannans but also exhibits low activity towards birchwood xylan. Apparent Km values of the enzyme for konjac glucomannan (low viscosity), locust bean gum galactomannan, carob galactomannan (low viscosity), and 1,4-beta-D-mannan (from carob) are 0.6 mg mL-1, 2.0 mg mL-1, 2.2 mg mL-1 and 1.5 mg mL-1, respectively, while the kcat values for these substrates are 215 s-1, 330 s-1, 292 s-1 and 148 s-1, respectively. Judged from the specificity constants kcat/Km, glucomannan is the preferred substrate of the A. niger beta -mannanase. Analysis by thin layer chromatography showed that the main product from enzymatic hydrolysis of locust bean gum is mannobiose, with only low amounts of mannotriose and higher manno-oligosaccharides formed.This study is the first report on the cloning and expression of a thermostable mannan endo-1,4-beta-mannosidase from A. niger in Pichia pastoris. The efficient expression and ease of purification will significantly decrease the production costs of this enzyme. Taking advantage of its acidic pH optimum and high thermostability, this recombinant beta-mannanase will be valuable in various biotechnological applications.

Project description:BackgroundMannans are one of the key polymers in hemicellulose, a major component of lignocellulose. The Mannan endo-1,4-beta-mannosidase or 1,4-beta-D-mannanase (EC 3.2.1.78), commonly named beta-mannanase, is an enzyme that can catalyze random hydrolysis of beta-1,4-mannosidic linkages in the main chain of mannans, glucomannans and galactomannans. The enzyme has found a number of applications in different industries, including food, feed, pharmaceutical, pulp/paper industries, as well as gas well stimulation and pretreatment of lignocellulosic biomass for the production of second generation biofuel. Bacillus licheniformis is a Gram-positive endospore-forming microorganism that is generally non-pathogenic and has been used extensively for large-scale industrial production of various enzymes; however, there has been no previous report on the cloning and expression of mannan endo-1,4-beta-mannosidase gene (manB) from B. licheniformis.ResultsThe mannan endo-1,4-beta-mannosidase gene (manB), commonly known as beta-mannanase, from Bacillus licheniformis strain DSM13 was cloned and overexpressed in Escherichia coli. The enzyme can be harvested from the cell lysate, periplasmic extract, or culture supernatant when using the pFLAG expression system. A total activity of approximately 50,000 units could be obtained from 1-l shake flask cultures. The recombinant enzyme was 6 x His-tagged at its C-terminus, and could be purified by one-step immobilized metal affinity chromatography (IMAC) to apparent homogeneity. The specific activity of the purified enzyme when using locust bean gum as substrate was 1672 +/- 96 units/mg. The optimal pH of the enzyme was between pH 6.0 - 7.0; whereas the optimal temperature was at 50 - 60 degrees C. The recombinant beta-mannanase was stable within pH 5 - 12 after incubation for 30 min at 50 degrees C, and within pH 6 - 9 after incubation at 50 degrees C for 24 h. The enzyme was stable at temperatures up to 50 degrees C with a half-life time of activity (tau1/2) of approximately 80 h at 50 degrees C and pH 6.0. Analysis of hydrolytic products by thin layer chromatography revealed that the main products from the bioconversion of locus bean gum and mannan were various manno-oligosaccharide products (M2 - M6) and mannose.ConclusionOur study demonstrates an efficient expression and secretion system for the production of a relatively thermo- and alkali-stable recombinant beta-mannanase from B. licheniformis strain DSM13, suitable for various biotechnological applications.

Dataset Information

Re-interpreting the role of endo-beta-mannanases as mannan endotransglycosylase/hydrolases in the plant cell wall.

Publications

Re-interpreting the role of endo-beta-mannanases as mannan endotransglycosylase/hydrolases in the plant cell wall.

Similar Datasets

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