Structural basis for inhibition of xyloglucan-specific endo-β-1,4-glucanase (XEG) by XEG-protein inhibitor

Microorganisms such as plant pathogens secrete glycoside hydrolases (GHs) to digest the polysaccharide chains of plant cell walls. The degradation of cell walls by these enzymes is a crucial step for nutrition and invasion. To protect the cell wall from these enzymes, plants secrete glycoside hydrolase inhibitor proteins (GHIPs). Xyloglucan-specific endo-β-1,4-glucanase (XEG), a member of GH family 12 (GH12), could be a great threat to many plants because xyloglucan is a major component of the cell wall in most plants. Understanding the inhibition mechanism of XEG by GHIP is therefore of great importance in the field of plant defense, but to date the mechanism and specificity of GHIPs remain unclear. We have determined the crystal structure of XEG in complex with extracellular dermal glycoprotein (EDGP), a carrot GHIP that inhibits XEG. The structure reveals that the conserved arginines of EDGP intrude into the active site of XEG and interact with the catalytic glutamates of the enzyme. We have also determined the crystal structure of the XEG-xyloglucan complex. These structures show that EDGP closely mimics the XEG-xyloglucan interaction. Although EDGP shares structural similarity to a wheat GHIP (Triticum aestivum xylanase inhibitor-IA (TAXI-IA)) that inhibits GH11 family xylanases, the arrangement of GH and GHIP in the XEG-EDGP complex is distinct from that in the xylanase-TAXI-IA complex. Our findings imply that plants have evolved structures of GHIPs to inhibit different GH family members that attack their cell walls.     

Purification and properties of an extracellular endo-1,4-β-glucanase fromLenzites trabea

An extracellular endo-1,4--glucanase (EC 3.2.1.4) has been isolated and purified from the culture solution of the basidiomyceteLenzites trabea grown on glucose and cellulose. Besides-glucosidase activity (EC 3.2.1.21) no evidence for C₁-activity (EC 3.2.1.91) in the culture solution was found.The endoglucanase has been purified in a four-step procedure including chromatography on Sepharose 6-B and DEAE-Sephadex A-50, adsorption on hydroxylapatite and gel filtration on Bio-Gel P-100. The enzyme showed maximum activity at pH 4.4 and 70°C. A molecular weight of 29000 Daltons was estimated by calibration on Bio-Gel P-100. The enzyme hydrolyses carboxymethyl cellulose (CMC) as well as xylan.List of Abbreviations CMC carboxymethyl cellulose - D.S. degree of substitution - D.P. degree of polymerisation - MW molecular weight     

Purification and properties of the endo-1,4-β-glucanase III from Ruminococcus albus

Endoglucanase III (EGIII) was purified from Ruminococcus albus culture supernatant. An enzyme having a molecular weight of 53,000 was stabilized by mercaptoethanol and inhibited by sulfhydryl group-blocking reagents, and exhibited its highest CMC-degrading activity of pH 5.7 and 55°C. The enzyme hydrolyzed cellobiose (G2) and cellotriose (G3) only negligibly, but significantly hydrolyzed cellotetraose (G4), cellopentaose (G5) and cellohexaose (G6). The major hydrolysis reactions conducted by the enzyme were G4→2G2, G5→G2+G3, G6→G2+G4 and G6→2G3. The V_max values of these reactions increased remarkably while the K_m values decreased significantly with an increase in degree of polymerization of the substrate.     

Production and properties of carboxymethylcellulase (endo-1,4-β-glucanase) fromCurvularia lunata

An extracellular carboxymethylcellulase (endo-1,4--glucanase) fromCurvularia lunata, grown at 30°C with an initial pH of 6.0, had optimal activity at pH 4.8 and 50°C. The enzyme was unstable above 50°C. The enzyme had aK _m for carboxymethylcellulose of 0.97 g/l and aV _max of 5.4 IU/ml.     

Crystal structure of basic 7S globulin, a xyloglucan-specific endo-β-1,4-glucanase inhibitor protein-like protein from soybean lacking inhibitory activity against endo-β-glucanase

β-Linked glucans such as cellulose and xyloglucan are important components of the cell walls of most dicotyledonous plants. These β-linked glucans are constantly exposed to degradation by various endo-β-glucanases from pathogenic bacteria and fungi. To protect the cell wall from degradation by such enzymes, plants secrete proteinaceous endo-β-glucanases inhibitors, such as xyloglucan-specific endo-β-1,4-glucanase inhibitor protein (XEGIP) in tomato. XEGIPs typically inhibit xyloglucanase, a member of the glycoside hydrolase (GH)12 family. XEGIPs are also found in legumes, including soybean and lupin. To date, tomato XEGIP has been well studied, whereas XEGIPs from legumes are less well understood. Here, we determined the crystal structure of basic 7S globulin (Bg7S), a XEGIP from soybean, which represents the first three-dimensional structure of XEGIP. Bg7S formed a tetramer with pseudo-222 symmetry. Analytical centrifugation and size exclusion chromatography experiments revealed that the assembly of Bg7S in solution depended on pH. The structure of Bg7S was similar to that of a xylanase inhibitor protein from wheat (Tritinum aestivum xylanase inhibitor) that inhibits GH11 xylanase. Surprisingly, Bg7S lacked inhibitory activity against not only GH11 but also GH12 enzymes. In addition, we found that XEGIPs from azukibean, yardlongbean and mungbean also had no impact on the activity of either GH12 or GH11 enzymes, indicating that legume XEGIPs generally do not inhibit these enzymes. We reveal the structural basis of why legume XEGIPs lack this inhibitory activity. This study will provide significant clues for understanding the physiological role of Bg7S.     

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-β-1,4-glucanase (GH12) from Aspergillus niveus

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-β-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60°C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan β-1,3 or β-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in β-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3°C and 81.3°C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60°C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.     

Purification and characterization of a thermostable endo-β-1,4-glucanase from a novel strain of Penicillium purpurogenum

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium purpurogenum KJS506 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. P. purpurogenum produced one of the highest levels of EG (5.6 U mg-protein^?1) with rice straw and corn steep powder as carbon and nitrogen sources, respectively. The extracellular EG was purified to homogeneity by sequential chromatography of P. purpurogenum culture supernatants on a DEAE sepharose column, a gel filtration column, and then on a Mono Q column with fast protein liquid chromatography. The purified EG was a monomeric protein with a molecular weight of 37 kDa and showed broad substrate specificity with maximum activity towards lichenan. P. purpurogenum EG showed t_1/2 value of 2 h at 70 °C and catalytic efficiency of 118 ml mg^?1 s^?1, one of the highest levels seen for EG-producing microorganisms. Although EGs have been reported elsewhere, the high catalytic efficiency and thermostability distinguish P. purpurogenum EG.     

Cloning and functional characterization of a novel endo-β-1,4-glucanase gene from a soil-derived metagenomic library

A metagenomic library consisting of 3,024 bacterial artificial chromosome clones was prepared in Escherichia coli DH10B with high-molecular-weight DNA extracted from red soil in South China. A novel cellulase gene with an open reading frame of 1,332 bp, cel5G, encoding an endo-β-1,4-glucanase was cloned using an activity-based screen. The deduced enzyme, Cel5G, belongs to the glycosyl hydrolase family 5 and shares <39% identity with endoglucanases in the GenBank database. cel5G was expressed in E. coli BL21, and the recombinant enzyme Cel5G was purified to homogeneity for enzymatic analysis. Cel5G hydrolyzed a wide range of β-1,4-, β-1,3/β-1,4-, or β-1,3/β-1,6-linked polysaccharides, amorphous cellulose, filter paper, and microcrystalline cellulose. Its highest activity was in 50 mM citrate buffer, pH 4.8, at 50°C. Cel5G is stable over a wide range of pH values (from 2.0 to 10.6) and is thermally stable under 60°C. It is highly tolerant and active in high salt concentrations and is stable in the presence of pepsin and pancreatin. The K _m and V _max values of Cel5G for carboxymethyl cellulose are 19.92 mg/ml and 1,941 μmol min⁻¹ mg⁻¹, respectively. These characteristics indicate that Cel5G has potential for industrial use.     

Characterization of a novel endo-β-1,4-glucanase from Armillaria gemina and its application in biomass hydrolysis

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Armillaria gemina KJS114 based on its morphology and internal transcribed spacer rDNA gene sequence. A. gemina EG (AgEG) was purified using a single-step purification by gel filtration. The relative molecular mass of AgEG by sodium dodecyl sulfate polyacrylamide gel electrophoresis was 65 kDa and by size exclusion chromatography was 66 kDa, indicating that the enzyme is a monomer in solution. The pH and temperature optima for hydrolysis were 5.0 and 60 °C, respectively. Purified AgEG had the highest catalytic efficiency with carboxymethylcellulose (k _cat/K _m?=?3,590 mg mL^?1 s^?1) unlike that reported for any fungal EG, highlighting the significance of the current study. The amino acid sequence of AgEG showed homology with hydrolases from the glycoside hydrolase family 61. The addition of AgEG to a Populus nigra hydrolysate reaction containing a commercial cellulase mixture (Celluclast 1.5L and Novozyme 188) showed a stimulatory effect on reducing sugar production. AgEG is a good candidate for applications that convert lignocellulosic biomass to biofuels and chemicals.     

Functional characterization and oligomerization of a recombinant xyloglucan-specific endo-β-1,4-glucanase (GH12) from Aspergillus niveus

Xyloglucan is a major structural polysaccharide of the primary (growing) cell wall of higher plants. It consists of a cellulosic backbone (beta-1,4-linked glucosyl residues) that is frequently substituted with side chains. This report describes Aspergillus nidulans strain A773 recombinant secretion of a dimeric xyloglucan-specific endo-β-1,4-glucanohydrolase (XegA) cloned from Aspergillus niveus. The ORF of the A. niveus xegA gene is comprised of 714 nucleotides, and encodes a 238 amino acid protein with a calculated molecular weight of 23.5 kDa and isoelectric point of 4.38. The optimal pH and temperature were 6.0 and 60 °C, respectively. XegA generated a xyloglucan-oligosaccharides (XGOs) pattern similar to that observed for cellulases from family GH12, i.e., demonstrating that its mode of action includes hydrolysis of the glycosidic linkages between glucosyl residues that are not branched with xylose. In contrast to commercial lichenase, mixed linkage beta-glucan (lichenan) was not digested by XegA, indicating that the enzyme did not cleave glucan β-1,3 or β-1,6 bonds. The far-UV CD spectrum of the purified enzyme indicated a protein rich in β-sheet structures as expected for GH12 xyloglucanases. Thermal unfolding studies displayed two transitions with mid-point temperatures of 51.3 °C and 81.3 °C respectively, and dynamic light scattering studies indicated that the first transition involves a change in oligomeric state from a dimeric to a monomeric form. Since the enzyme is a predominantly a monomer at 60 °C, the enzymatic assays demonstrated that XegA is more active in its monomeric state.     

Characterization of endo-β-1,4-glucanase from a novel strain of Penicillium pinophilum KMJ601

A novel endo-β-1,4-glucanase (EG)-producing strain was isolated and identified as Penicillium pinophilum KMJ601 based on its morphology and internal transcribed spacer (ITS) rDNA gene sequence. When rice straw and corn steep powder were used as carbon and nitrogen sources, respectively, the maximal EG activity of 5.0 U mg protein⁻¹, one of the highest levels among EG-producing microorganisms, was observed. The optimum temperature and pH for EG production were 28°C and 5.0, respectively. The increased production of EG by P. pinophilum in culture at 28°C was confirmed by two-dimensional electrophoresis followed by MS/MS sequencing of the partial peptide. A partial EG gene (eng5) was amplified by degenerate polymerase chain reaction (PCR) based on the peptide sequence. A full-length eng5 was cloned by genome-walking PCR, and P. pinophilum EG was identified as a member of glycoside hydrolase family 5. The present results should contribute to improved industrial production of EG by P. pinophilum KMJ601.     

Crystal structure of hyperthermophilic endo-β-1,4-glucanase: implications for catalytic mechanism and thermostability

Endo-β-1,4-glucanase from thermophilic Fervidobacterium nodosum Rt17-B1 (FnCel5A), a new member of glycosyl hydrolase family 5, is highly thermostable and exhibits the highest activity on carboxymethylcellulose among the reported homologues. To understand the structural basis for the thermostability and catalytic mechanism, we report here the crystal structures of FnCel5A and the complex with glucose at atomic resolution. FnCel5A exhibited a (β/α)(8)-barrel structure typical of clan GH-A of the glycoside hydrolase families with a large and deep catalytic pocket located in the C-terminal end of the β-strands that may permit substrate access. A comparison of the structure of FnCel5A with related structures from thermopile Clostridium thermocellum, mesophile Clostridium cellulolyticum, and psychrophile Pseudoalteromonas haloplanktis showed significant differences in intramolecular interactions (salt bridges and hydrogen bonds) that may account for the difference in their thermostabilities. The substrate complex structure in combination with a mutagenesis analysis of the catalytic residues implicates a distinctive catalytic module Glu(167)-His(226)-Glu(283), which suggests that the histidine may function as an intermediate for the electron transfer network between the typical Glu-Glu catalytic module. Further investigation suggested that the aromatic residues Trp(61), Trp(204), Phe(231), and Trp(240) as well as polar residues Asn(51), His(127), Tyr(228), and His(235) in the active site not only participated in substrate binding but also provided a unique microenvironment suitable for catalysis. These results provide substantial insight into the unique characteristics of FnCel5A for catalysis and adaptation to extreme temperature.     

Transfer ofBacillus subtilis endo-β-1,4-glucanase gene intoZymomonas anaerobia

Summary The endo--1,4-glucanase gene ofBacillus subtilis origin cloned previously in a plasmid pBS1 was subcloned in a new plasmid pSCR815, and with the new plasmidZymomonas anaerobia was transformed. TheBacillus glucanase gene expressed in theZymomonas cells with efficiency much lower than inEscherichia coli.     

Endogenous production of endo-β-1,4-glucanase by decapod crustaceans

The potential ability to produce cellulase enzymes endogenously was examined in decapods crustaceans including the herbivorous gecarcinid land crabs Gecarcoidea natalis and Discoplax hirtipes, the amphibious freshwater crab Austrothelphusa transversa, the terrestrial hermit crab, Coenobita variabilis the parastacid crayfish Euastacus, and the crayfish Cherax destructor. The midgut gland of both G. natalis and D. hirtipes contained substantial total cellulase activities and activities of the cellulase enzymes endo-β-1,4-glucanase and β-glucosidase. With the exception of total cellulase and β-glucosidase from D. hirtipes, the enzyme activities within the midgut gland were higher than those within the digestive juice. Hence, the enzyme activities appear to reside predominantly within midgut gland, providing indirect evidence for endogenous synthesis of cellulase enzymes by this tissue. A 900 bp cDNA fragment encoding a portion of the endo-β-1,4-glucanase amino acid sequence was amplified by RT-PCR using RNA isolated from the midgut gland of C. destructor, Euastacus, A. transversa and C. variabilis. This provided direct evidence for the endogenous production of endo-β-1,4-glucanase. The 900 bp fragment was also amplified from genomic DNA isolated from the skeletal muscle of G. natalis and D. hirtipes, clearly indicating that the gene encoding endo-β-1,4-glucanase is also present in these two species. As this group of evolutionary diverse crustacean species possesses and expresses the endo-β-1,4-glucanase gene it is likely that decapod crustaceans generally produce cellulases endogenously and are able to digest cellulose.     

Purification and properties of endo-1,4-β-xylanase from Trichosporon cutaneum

Summary The endoxylanase (1,4-D-xylan xylanohydrolase, EC 3.2.1.8) was purified 3,7 fold from the culture filtrate of the yeast Trichosporon cutaneum grown on oathusk xylan. The final enzyme preparation gave a single protein band on disc gel electrophoresis and has a molecular weight of approx. 45000. The enzyme has a pH optimum of 5.0 and a temperatur optimum of 50°C. Patterns of hydrolysis demonstrate that this xylanase is an endo-splitting enzyme able to break down xylans at random giving xylobiose, xylotriose and xylose as the main end-products. Since the enzyme seems not to be capable of liberating L-arabinose from arabino-xylan branched arabinose-containing xylooligosaccharides are formed, too. This enzyme contains carbohydrates in a noncovalent manner, indicating that this extracellular xylanase, is not a glycoprotein.     

A novel salt-tolerant endo-β-1,4-glucanase Cel5A in Vibrio sp. G21 isolated from mangrove soil

Although cellulases have been isolated from various microorganisms, no functional cellulase gene has been reported in the Vibrio genus until now. In this report, a novel endo-β-1,4-glucanase gene, cel5A, 1,362 bp in length, was cloned from a newly isolated bacterium, Vibrio sp. G21. The deduced protein of cel5A contains a catalytic domain of glycosyl hydrolase family 5 (GH5), followed by a cellulose binding domain (CBM2). The GH5 domain shows the highest sequence similarity (69%) to the bifunctional beta 1,4-endoglucanase/cellobiohydrolase from Teredinibacter turnerae T7902. The mature Cel5A enzyme was overexpressed in Escherichia coli and purified to homogeneity. The optimal pH and temperature of the recombinant enzyme were determined to be 6.5–7.5 and 50°C, respectively. Cel5A was stable over a wide range of pH and retained more than 90% of total activity even after treatment in pH 5.5–10.5 for 1 h, indicating high alkali resistance. Moreover, the enzyme was activated after pretreatment with mild alkali, a novel characteristic that has not been previously reported in other cellulases. Cel5A also showed a high level of salt tolerance. Its activity rose to 1.6-fold in 0.5 M NaCl and remained elevated even in 4 M NaCl. Further experimentation demonstrated that the thermostability of Cel5A was improved in 0.4 M NaCl. In addition, Cel5A showed specific activity towards β-1,4-linkage of amorphous region of lignocellulose, and the main final hydrolysis product of carboxymethylcellulose sodium and cellooligosaccharides was cellobiose. As an alkali-activated and salt-tolerant enzyme, Cel5A is an ideal candidate for further research and industrial applications.     

Purification, biochemical characterization, and structure of recombinant endo-1,4-β-xylanase XylE

The gene xylE encoding endo-1,4-β-xylanase from the 10th family of glycosyl hydrolases produced by the mycelial fungus Penicillium canescens has been expressed under the control of the strong promoter of the bgaS gene encoding β-galactosidase from P. canescens. As a result, a strain-producer of endoxylanase XylE was developed. The recombinant enzyme was isolated and purified to homogeneity with specific activity of 50 U/mg. The physicochemical and biochemical properties of the endoxylanase were studied. The maximal enzymatic activity was observed at pH 6.0 and 70°C. Endoxylanase XylE was shown to be a highly thermostable enzyme with half-inactivation period τ_1/2 of 7 h at 60°C. The kinetic parameters were 0.52 mg/ml (K _m) and 75 μmol/min per mg (V _max) using birch xylan as the substrate. Crystals of endoxylonase XylE were obtained, and the 3D structure was solved at 1.47 ? resolution. The 3D structure of an endo-1,4-β-xylanase from the 10th family containing carbohydrate and unique cyclic structure located at the C-terminus of the polypeptide chain was obtained for the first time.     

Production of endo-1,4-β-glucanase and xylanase by Trichoderma reesei immobilized on polyurethane foam

Summary Endo-1,4--glucanase and xylanase were produced by Trichoderma reesei immobilized on polyurethane foam using lactose as the main carbon source. The most porous carrier was found to be the best of those tested. The nitrogen source and KH₂PO₄ concentration of the production medium had a marked effect on culture pH during the course of fermentation and, consequently, on xylanase activity. An increase in lactose concentration from 7 to 27 g/l resulted in an increase in endoglucanase activity (max. 730 U/ml), xylanase activity (max. 3350 U/ml) and filter paper activity (max. 3.0 FPU/ml).