Beta-1,4-glucanase-like protein from the cyanobacterium Synechocystis PCC6803 is a beta-1,3-1,4-glucanase and functions in salt stress tolerance.
ABSTRACT: In the present study, we characterized the gene (Cyanobase accession number slr0897) designated Ssglc encoding a beta-1,4-glucanase-like protein (SsGlc) from Synechocystis PCC6803. The deduced amino acid sequence for Ssglc showed a high degree of similarity to sequences of GH (glycoside hydrolase) family 9 beta-1,4-glucanases (cellulases) from various sources. Surprisingly, the recombinant protein obtained from the Escherichia coli expression system was able to hydrolyse barley beta-glucan and lichenan (beta-1,3-1,4-glucan), but not cellulose (beta-1,4-glucan), curdlan (beta-1,3-glucan), or laminarin (beta-1,3-1,6-glucan). A 1H-NMR analysis of the enzymatic products revealed that the enzyme hydrolyses the beta-1,4-glycosidic linkage of barley beta-glucan through an inverting mechanism. The data indicated that SsGlc was a novel type of GH9 glucanase which could specifically hydrolyse the beta-1,3-1,4-linkage of glucan. The growth of mutant Synechocystis cells in which the Ssglc gene was disrupted by a kanamycin-resistance cartridge gene was almost the same as that of the wild-type cells under continuous light (40 micromol of photons/m2 per s), a 12 h light (40 micromol of photons/m2 per s)/12 h dark cycle, cold stress (4 degrees C), and high light stress (200 micromol of photons/m2 per s). However, under salt stress (300-450 mM NaCl), growth of the Ssglc-disrupted mutant cells was significantly inhibited as compared with that of the wild-type cells. The Ssglc-disrupted mutant cells showed a decreased rate of O2 consumption and NaHCO3-dependent O2 evolution as compared with the wild-type cells under salt stress. Under osmotic stress (100-400 mM sorbitol), there was no difference in growth between the wild-type and the Ssglc-disrupted mutant cells. These results suggest that SsGlc functions in salt stress tolerance in Synechocystis PCC6803.
Project description:Xylanases are utilized in a variety of industries for the breakdown of plant materials. Most native and engineered bifunctional/multifunctional xylanases have separate catalytic domains within the same polypeptide chain. Here we report a new bifunctional xylanase (XynBE18) produced by Paenibacillus sp. E18 with xylanase and beta-1,3-1,4-glucanase activities derived from the same active center by substrate competition assays and site-directed mutagenesis of xylanase catalytic Glu residues (E129A and E236A). The gene consists of 981 bp, encodes 327 amino acids, and comprises only one catalytic domain that is highly homologous to the glycoside hydrolase family 10 xylanase catalytic domain. Recombinant XynBE18 purified from Escherichia coli BL21(DE3) showed specificity toward oat spelt xylan and birchwood xylan and beta-1,3-1,4-glucan (barley beta-glucan and lichenin). Homology modeling and molecular dynamic simulation were used to explore structure differences between XynBE18 and the monofunctional xylanase XynE2, which has enzymatic properties similar to those of XynBE18 but does not hydrolyze beta-1,3-1,4-glucan. The cleft containing the active site of XynBE18 is larger than that of XynE2, suggesting that XynBE18 is able to bind larger substrates such as barley beta-glucan and lichenin. Further molecular docking studies revealed that XynBE18 can accommodate xylan and beta-1,3-1,4-glucan, but XynE2 is only accessible to xylan. These results indicate a previously unidentified structure-function relationship for substrate specificities among family 10 xylanases.
Project description:Streptococcus bovis JB1 was found to produce a 25-kDa extracellular enzyme active against beta-(1,3-1,4)-glucans. A gene was isolated encoding a specific beta-(1,3-1,4)-glucanase that corresponds to this size and belongs to glycoside hydrolase family 16. A 4- to 10-fold increase in supernatant beta-glucanase activity was obtained when the cloned beta-glucanase gene was reintroduced into S. bovis JB1 by use of constructs based on the plasmid vector pTRW10 or pIL253. The beta-(1,3-1,4)-glucanase gene was also expressed upon introduction of the pTRW10 construct pTRWL1R into Lactococcus lactis IL2661 and Enterococcus faecalis JH2-SS, although extracellular activity was 8- to 50-fold lower than that in S. bovis JB1. The beta-(1,3-1,4)-glucanase purified from the culture supernatant of S. bovis JB1 carrying pTRWL1R showed a K(m) of 2.8 mg per ml and a Vmax of 338 mumol of glucose equivalents per min per mg of protein with barley beta-glucan as the substrate. The S. bovis beta-(1,3-1,4)-glucanase may contribute to the ability of this bacterium to utilize starch by degrading structural polysaccharides present in endosperm cell walls.
Project description:?-1,3:1,4-Glucan is a major cell wall component accumulating in endosperm and young tissues in grasses. The mixed linkage glucan is a linear polysaccharide mainly consisting of cellotriosyl and cellotetraosyl units linked through single ?-1,3-glucosidic linkages, but it also contains minor structures such as cellobiosyl units. In this study, we examined the action of an endo-?-1,3(4)-glucanase from Trichoderma sp. on a minor structure in barley ?-1,3:1,4-glucan. To find the minor structure on which the endo-?-1,3(4)-glucanase acts, we prepared oligosaccharides from barley ?-1,3:1,4-glucan by endo-?-1,4-glucanase digestion followed by purification by gel permeation and paper chromatography. The endo-?-1,3(4)-glucanase appeared to hydrolyze an oligosaccharide with degree of polymerization 5, designated C5-b. Based on matrix-assisted laser desorption/ionization (MALDI) time-of-flight (ToF)/ToF-mass spectrometry (MS)/MS analysis, C5-b was identified as ?-Glc-1,3-?-Glc-1,4-?-Glc-1,3-?-Glc-1,4-Glc including a cellobiosyl unit. The results indicate that a type of endo-?-1,3(4)-glucanase acts on the cellobiosyl units of barley ?-1,3:1,4-glucan in an endo-manner.
Project description:A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 184.108.40.206) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.
Project description:Background:The Bacillus subtilis endo-?-1,4-glucanase (BsCel5A) hydrolyzes ?-1,3-1,4-linked glucan, and the enzyme includes a family 3 carbohydrate-binding module (CBM3) that binds ?-1,4-linked glucan. Methods:Here we investigate the BsCel5A ?-1,3-1,4 glucanase activity after exchanging the CBM3 domain for the family 11 CBM from Ruminiclostridium thermocellum celH (RtCBM11) having ?-1,3-1,4 glucan affinity. Results:The BsCel5A-RtCBM11 presents a 50.4% increase in Vmax, a 10% reduction in K0.5, and a 2.1-fold increase in catalytic efficiency. Enzyme mobility and binding to barley ?-1,3-1,4 glucan and pre-treated sugarcane bagasse were investigated using Electron Paramagnetic Resonance (EPR) with Site-Directed Spin Labeling (SDSL) of the binding site regions of the CBM3 and RtCBM11 domains in the BsCel5A-CBM3 and BsCel5A-RtCBM11, respectively. Although higher mobility than the RtCBM11 was shown, no interaction of the spin-labeled CBM3 with ?-1,3-1,4 glucan was observed. In contrast, a Ka value of 0.22 mg/mL was estimated from titration of the BsCel5A-RtCBM11 with ?-1,3-1,4 glucan. Enzyme binding as inferred from altered EPR spectra of the BsCel5A-RtCBM11 was observed only after xylan or lignin extraction from sugarcane bagasse. Binding to xylan- or lignin-free lignocellulose was correlated with a 4.5- to 5-fold increase in total reducing sugar release as compared to the milled intact sugarcane bagasse, suggesting that xylan impedes enzyme access to the ?-1,3-1,4 glucan. Conclusions:These results show that the non-specific binding of the BsCel5A-RtCBM11 to the lignin component of the cell wall is minimal, and represent the first reported use of EPR to directly study the interaction of glycoside hydrolyse enzymes with natural insoluble substrates.
Project description:The three-dimensional structure of the hybrid Bacillus 1,3-1,4-beta-glucanase (beta-glucanase; 1,3-1,4-beta-D-glucan 4-glucanohydrolase, lichenase, EC 220.127.116.11) designated H(A16-M) was determined by x-ray crystallography at a resolution of 2.0 A and refined to an R value of 16.4% using stereochemical restraints. The protein molecule consists mainly of two seven-stranded antiparallel beta-pleated sheets arranged atop each other to form a compact, sandwich-like structure. A channel crossing one side of the protein molecule accommodates an inhibitor, 3,4-epoxybutyl beta-D-cellobioside, which binds covalently to the side chain of Glu-105, as seen in a crystal structure analysis at 2.8-A resolution of the protein-inhibitor complex (R = 16.8%). That Glu-105 may be indispensible for enzyme catalysis by H(A16-M) is suggested by site-directed mutagenesis of this residue, which inevitably leads to an inactive enzyme.
Project description:Here we report the cloning of the Pa_3_10940 gene from the coprophilic fungus Podospora anserina, which encodes a C-terminal family 1 carbohydrate binding module (CBM1) linked to a domain of unknown function. The function of the gene was investigated by expression of the full-length protein and a truncated derivative without the CBM1 domain in the yeast Pichia pastoris. Using a library of polysaccharides of different origins, we demonstrated that the full-length enzyme displays activity toward a broad range of ?-glucan polysaccharides, including laminarin, curdlan, pachyman, lichenan, pustulan, and cellulosic derivatives. Analysis of the products released from polysaccharides revealed that this ?-glucanase is an exo-acting enzyme on ?-(1,3)- and ?-(1,6)-linked glucan substrates and an endo-acting enzyme on ?-(1,4)-linked glucan substrates. Hydrolysis of short ?-(1,3), ?-(1,4), and ?-(1,3)/?-(1,4) gluco-oligosaccharides confirmed this striking feature and revealed that the enzyme performs in an exo-type mode on the nonreducing end of gluco-oligosaccharides. Excision of the CBM1 domain resulted in an inactive enzyme on all substrates tested. To our knowledge, this is the first report of an enzyme that displays bifunctional exo-?-(1,3)/(1,6) and endo-?-(1,4) activities toward beta-glucans and therefore cannot readily be assigned to existing Enzyme Commission groups. The amino acid sequence has high sequence identity to hypothetical proteins within the fungal taxa and thus defines a new family of glycoside hydrolases, the GH131 family.
Project description:The low nutritional value of barley for poultry is because of the absence of an intestinal enzyme for efficient depolymerization of (1, 3-1,4)-beta-glucan, the major polysaccharide of the endosperm cell walls. This leads to high viscosity in the intestine, limited nutrient uptake, decreased growth rate, and unhygienic sticky droppings adhering to chickens and floors of the production cages. Consequently, the 7.5 billion broiler chickens produced annually in the United States are primarily raised on corn-soybean diets. Here we show that addition to normal barley of 6.2% transgenic malt containing a thermotolerant (1,3-1,4)-beta-glucanase (4.28 microg.g(-1) soluble protein) provides a weight gain equivalent to corn diets. The number of birds with adhering sticky droppings is drastically reduced. Intestines and excrements of chickens fed the barley control diet contained large amounts of soluble (1,3-1,4)-beta-glucan, which was reduced by 75 and 50%, respectively, by adding transgenic malt to the diet. The amount of active recombinant enzyme in the small intestine corresponded to that present in the feed, whereas an 11-fold concentration of the enzyme was observed in the ceca, and a 7.5-fold concentration occurred in the excrement. Glycosylation of the beta-glucanase isolated from the ceca testified to its origin from the transgenic barley. Analysis of the data from this trial demonstrates the possibility of introducing individual recombinant enzymes into various parts of the gastrointestinal tract of chickens with transgenic malt and thereby the possibility of evaluating their effect on the metabolism of a given ingredient targeted by the enzyme.
Project description:β-1,3-Glucanase is considered as a useful enzymatic tool for β-1,3-glucan degradation to produce (1→3)-linked β-glucan oligosaccharides with pharmacological activity properties. To validly isolate β-1,3-glucanase-producing microorganisms, the soil of Wolfiporia extensa, considered an environment rich in β-1,3-glucan-degrading microorganisms, was subjected to high throughput sequencing. The results demonstrated that the genera Streptomyces (1.90%) and Arthrobacter (0.78%) belonging to the order Actinomycetales (8.64%) in the phylum Actinobacteria (18.64%) were observed in soil for P. cocos cultivation (FTL?). Actinomycetes were considered as the candidates for isolation of glucan-degrading microorganisms. Out of 58 isolates, only 11 exhibited β-1,3-glucan-degrading activity. The isolate SYBCQL belonging to the genus Kitasatospora with β-1,3-glucan-degrading activity was found and reported for the first time and the isolate SYBC17 displayed the highest yield (1.02 U/mg) among the isolates. To check the β-1,3-glucanase contribution to β-1,3-glucan-degrading activity, two genes, 17-W and 17-Q, encoding β-1,3-glucanase in SYBC17 and one gene QLK1 in SYBCQL were cloned and expressed for verification at the molecular level. Our findings collectively showed that the isolates able to secrete β-1,3-glucanase could be obtained with the assistance of high-throughput sequencing and genes expression analysis. These methods provided technical support for isolating β-1,3-glucanase-producing microorganisms.
Project description:A novel ?-(1,3)-glucanase gene designated lamC, cloned from Corallococcus sp. strain EGB, contains a fascin-like module and a glycoside hydrolase family 16 (GH16) catalytic module. LamC displays broad hydrolytic activity toward various polysaccharides. Analysis of the hydrolytic products revealed that LamC is an exo-acting enzyme on ?-(1,3)(1,3)- and ?-(1,6)-linked glucan substrates and an endo-acting enzyme on ?-(1,4)-linked glucan and xylan substrates. Site-directed mutagenesis of conserved catalytic Glu residues (E304A and E309A) demonstrated that these activities were derived from the same active site. Excision of the fascin-like module resulted in decreased activity toward ?-(1,3)(1,3)-linked glucans. The carbohydrate-binding assay showed that the fascin-like module was a novel ?-(1,3)-linked glucan-binding module. The functional characterization of the fascin-like module and catalytic module will help us better understand these enzymes and modules.IMPORTANCE In this report of a bacterial ?-(1,3)(1,3)-glucanase containing a fascin-like module, we reveal the ?-(1,3)(1,3)-glucan-binding function of the fascin-like module present in the N terminus of LamC. LamC displays exo-?-(1,3)/(1,6)-glucanase and endo-?-(1,4)-glucanase/xylanase activities with a single catalytic domain. Thus, LamC was identified as a novel member of the GH16 family.