Purification and chemical properties of two 1,3;1,4-beta-glucan endohydrolases from germinating barley

Two 1,3;1,4-beta-glucan endohydrolases have been purified from extracts of germinating barley by ammonium sulphate precipitation, ion-exchange and gel filtration chromatography. Both enzymes are monomeric, basic proteins. Enzyme I has a molecular weight of 28000 and an isoelectric point of 8.5, while enzyme II has a molecular weight of 33000 and an isoelectric point greater than 10. Enzyme II is a glycoprotein containing 3.6% carbohydrate, of which three residues are probable N-acetylglucosamine, but enzyme I contains only traces of associated carbohydrate. The amino acid compositions of the two 1,3;1,4-beta-glucan endohydrolases are similar and the cross-reactivity of antibodies raised against the purified enzymes suggests that they share common antigenic determinants.     

Purification and characterization of a low molecular weight 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414

A low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) (1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) has been isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by a two-step procedure of gel filtration and ion-exchange chromatography. The isolated enzyme appeared homogeneous upon polyacrylamide gel electrophoresis at pH 2.9, isoelectric focusing in a polyacrylamide gel slab, sedimentation equilibrium analysis and chromatography of the reduced and alkylated enzyme on a column of Sepharose 6B in 6 M guanidine - HCl. A molecular weight was calculated at approx. 20 000 and the isoelectric point was determined at pH 7.52. The purified enzyme was not a carbohydrate-containing protein.     

A novel cellulase gene from the mulberry longicorn beetle, Apriona germari: gene structure, expression, and enzymatic activity

We have previously cloned a cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Ag-EGase I) belonging to glycoside hydrolase family (GHF) 45 from the mulberry longicorn beetle, Apriona germari. We report here the gene structure, expression and enzyme activity of an additional celluase (Ag-EGase II) from A. germari and also described the gene structure of Ag-EGase I. The Ag-EGase II gene spans 1033 bp and consisted of two introns and three exons coding for 239 amino acid residues. The 2713-bp-long genomic DNA of Ag-EGase I also consisted of two introns and three exons. The Ag-EGase II showed 61% protein sequence identity to Ag-EGase I and 51% to another beetle, Phaedon cochleariae, cellulase, belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase II. The Ag-EGase II has 14 conserved cysteine residues and three putative N-glycosylation sites. Northern blot analysis confirmed midgut-specific expression of Ag-EGase II, suggesting that the midgut is the prime site for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase II was expressed as a 36-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase II was approximately 812 U/mg of recombinant Ag-EGase II. The enzymatic properties of the purified recombinant Ag-EGase II showed the highest activity at 50 °C and pH 6.0, and were stable at 60 °C at least for 10 min.     

Catalytic properties and mode of action of three endo-beta-glucanases from Talaromyces emersonii on soluble beta-1,4- and beta-1,3;1,4-linked glucans

In this paper, we present the first detailed analysis of the modes of action of three purified, thermostable endo-beta-D-glucanases (EG V-VII) against a range of soluble beta-linked glucans. Studies indicated that EG V-VII, purified to homogeneity from a new source, the thermophilic fungus Talaromyces emersonii, are strict beta-glucanases that exhibit maximum activity against mixed-link 1,3;1,4-beta-D-glucans. Time-course hydrolysis studies of 1,4-beta-D-glucan (carboxymethylcellulose; CMC), 1,3;1,4-beta-D-glucan from barley (BBG) and lichenan confirmed the endo-acting nature of EG V-VII and verified preference for 1,3;1,4-beta-D-glucan substrates. The results suggest that EG VI and EG VII belong to EC 3.2.1.6, as both enzymes also exhibit activity against 1,3-beta-glucan (laminaran), in contrast to EG V. Although cellobiose, cellotriose and glucose were the main glucooligosaccharide products released, the range and relative amount of each product was dependent on the particular enzyme, substrate and reaction time. Kinetic constants (Km, Vmax, kcat and kcat/Km) determined for EG V-VII with BBG as substrate yielded similar Km and Vmax values for EG V and EG VI. EG VII exhibited highest affinity for BBG (Km value of 9.1 mg ml(-1)) and the highest catalytic efficiency (kcat/Km of 12.63 s(-1) mg(-1) ml).     

Solubilization of beta-glucan synthases from the membranes of cultured ryegrass endosperm cells.

beta-Glucan synthases were solubilized by treating membrane preparations from suspension-cultured ryegrass (lolium multiflorum) endosperm cells with detergents. Of the seven detergents tested only digitonin and octyl glucoside dissociated active synthases from the membranes. The digitonin-solubilized enzymes produced 1,4-beta-glucans and 1,3:1,4-beta-glucans, whereas the digitonin-insoluble enzymes produced, in addition, 1,3-beta-glucans. Chromatography of the digitonin-solubilized beta-glucan synthases on DEAE-Sepharose resulted in their partial purification. The octyl glucoside-solubilized enzymes produced more 1,3-beta-glucans than did the membrane-bound preparations. These results suggest that the 1,3-beta-glucan synthase is a separate enzyme and is not involved in 1,3:1,4-beta-glucan synthesis. Digitonin not only dissociated synthases from the membranes, but also stimulated synthase activity. This effect may be related to the inhibition by digitonin of glucosyl transfer from UDP-glucose to form steryl glucosides.     

Characterization of Potent Fibrinolytic Enzymes in Earthworm,Lumbricus rubellus

Stable and potent fibrinolytic enzymes (six homogeneous proteins) were purified to homogeneity from extracts of the lyophilized powder of an earthworm, Lumbricus rubellus. The molecular weight of each enzyme estimated by SDS–polyacrylamide gel electrophoresis was different from those by gel filtration chromatography in the six purified proteins. The exact molecular weight of each enzyme (F-III-2, F-III-1, F-II, F-I-2, F-I-l, and F-I-0) measured by ionspray MS analysis was 29, 662, 29, 667, 24, 664, 24, 220, 24, 196, and 23,013, respectively. The isoelectric point (pI) of each enzyme was 3.40, 3.60, 4.20, 4.00, 4.30, and 4.85, respectively. The enzymes were single polypeptide chains. They had a very strong fibrinolytic activity and the maximum reactivity for chromogenic substrates from pH 9-11. The enzymes, acidic proteins that had abundant asparagine and aspartic acid, and low lysine in their amino acid composition, did not contain component sugars. The enzymes were stable at from pH 1-11 and up to 60°C. Studies on substrate specificity and inhibition indicated that these enzymes were alkaline trypsin-like serine proteases. N-Terminal amino acid sequences of the enzymes had local similarities to those of trypsin-like enzymes such as elastase and coagulation factor IX. From the results of amino acid sequence, amino acid composition analyses and immunological analyses, it was suggested that these six enzyme proteins were derived as isozyme(s) from at least four different genes.     

New perspectives on the endo-beta-glucanases of glycosyl hydrolase Family 17

Isozymes of glycosyl hydrolase Family 17 hydrolyze 1,3-beta-glucan polysaccharides found in the cell wall matrix of plants and fungi, enabling these plant enzymes to serve diverse roles in plant defense and plant development. Fourteen genes from Family 17 have been characterized in the genome of rice. A sequence dendrogram analysis divided these genes into four subfamilies. The recombinant GNS1 enzyme from subfamily B had 1,3;1,4-beta-glucanase activity, suggesting a role for this isozyme in plant development.     

Molecular modeling of family GH16 glycoside hydrolases: potential roles for xyloglucan transglucosylases/hydrolases in cell wall modification in the poaceae

Family GH16 glycoside hydrolases can be assigned to five subgroups according to their substrate specificities, including xyloglucan transglucosylases/hydrolases (XTHs), (1,3)-beta-galactanases, (1,4)-beta-galactanases/kappa-carrageenases, "nonspecific" (1,3/1,3;1,4)-beta-D-glucan endohydrolases, and (1,3;1,4)-beta-D-glucan endohydrolases. A structured family GH16 glycoside hydrolase database has been constructed (http://www.ghdb.uni-stuttgart.de) and provides multiple sequence alignments with functionally annotated amino acid residues and phylogenetic trees. The database has been used for homology modeling of seven glycoside hydrolases from the GH16 family with various substrate specificities, based on structural coordinates for (1,3;1,4)-beta-D-glucan endohydrolases and a kappa-carrageenase. In combination with multiple sequence alignments, the models predict the three-dimensional (3D) dispositions of amino acid residues in the substrate-binding and catalytic sites of XTHs and (1,3/1,3;1,4)-beta-d-glucan endohydrolases; there is no structural information available in the databases for the latter group of enzymes. Models of the XTHs, compared with the recently determined structure of a Populus tremulos x tremuloides XTH, reveal similarities with the active sites of family GH11 (1,4)-beta-D-xylan endohydrolases. From a biological viewpoint, the classification, molecular modeling and a new 3D structure of the P. tremulos x tremuloides XTH establish structural and evolutionary connections between XTHs, (1,3;1,4)-beta-D-glucan endohydrolases and xylan endohydrolases. These findings raise the possibility that XTHs from higher plants could be active not only on cell wall xyloglucans, but also on (1,3;1,4)-beta-D-glucans and arabinoxylans, which are major components of walls in grasses. A role for XTHs in (1,3;1,4)-beta-D-glucan and arabinoxylan modification would be consistent with the apparent overrepresentation of XTH sequences in cereal expressed sequence tags databases.     

A stress-induced rice (Oryza sativa L.) beta-glucosidase represents a new subfamily of glycosyl hydrolase family 5 containing a fascin-like domain

GH5BG, the cDNA for a stress-induced GH5 (glycosyl hydrolase family 5) beta-glucosidase, was cloned from rice (Oryza sativa L.) seedlings. The GH5BG cDNA encodes a 510-amino-acid precursor protein that comprises 19 amino acids of prepeptide and 491 amino acids of mature protein. The protein was predicted to be extracellular. The mature protein is a member of a plant-specific subgroup of the GH5 exoglucanase subfamily that contains two major domains, a beta-1,3-exoglucanase-like domain and a fascin-like domain that is not commonly found in plant enzymes. The GH5BG mRNA is highly expressed in the shoot during germination and in leaf sheaths of mature plants. The GH5BG was up-regulated in response to salt stress, submergence stress, methyl jasmonate and abscisic acid in rice seedlings. A GUS (glucuronidase) reporter tagged at the C-terminus of GH5BG was found to be secreted to the apoplast when expressed in onion (Allium cepa) cells. A thioredoxin fusion protein produced from the GH5BG cDNA in Escherichia coli hydrolysed various pNP (p-nitrophenyl) glycosides, including beta-D-glucoside, alpha-L-arabinoside, beta-D-fucoside, beta-D-galactoside, beta-D-xyloside and beta-D-cellobioside, as well as beta-(1,4)-linked glucose oligosaccharides and beta-(1,3)-linked disaccharide (laminaribiose). The catalytic efficiency (kcat/K(m)) for hydrolysis of beta-(1,4)-linked oligosaccharides by the enzyme remained constant as the DP (degree of polymerization) increased from 3 to 5. This substrate specificity is significantly different from fungal GH5 exoglucanases, such as the exo-beta-(1,3)-glucanase of the yeast Candida albicans, which may correlate with a marked reduction in a loop that makes up the active-site wall in the Candida enzyme.     

Metazoan cellulase genes from termites: intron/exon structures and sites of expression.

Endogenous endo-beta-1,4-glucanase (EGase, EC 3.2.1.4) cDNAs were cloned from representatives of the termite families Termitidae and Rhinotermitidae. These EGases are all composed of 448 amino acids and belong to glycosyl hydrolase family 9 (GHF9), sharing high levels of identity (40-52%) with selected bacterial, mycetozoan and plant EGases. Like most plant EGases, they consist of a single catalytic domain, lacking the ancillary domains found in most microbial cellulases. Using a PCR-based strategy, the entire sequence of the coding region of NtEG, a gene putatively encoding an EGase from Nasutitermes takasagoensis (Termitidae), was determined. NtEG consists of 10 exons interrupted by 9 introns and contains typical eukaryotic promoter elements. Genomic fragments of EGase genes from Reticulitermes speratus (Rhinotermitidae) were also sequenced. In situ hybridization of N. takasagoensis guts with an antisense NtEG RNA probe demonstrated that expression occurs in the midgut, which contrasts to EGase expression being detected only in the salivary glands of R. speratus. NtEG, when expressed in Escherichia coli, was shown to have in vitro activity against carboxymethylcellulose.     

Glycosylphosphatidylinositol-anchored glucanosyltransferases play an active role in the biosynthesis of the fungal cell wall

A novel 1,3-beta-glucanosyltransferase isolated from the cell wall of Aspergillus fumigatus was recently characterized. This enzyme splits internally a 1,3-beta-glucan molecule and transfers the newly generated reducing end to the non-reducing end of another 1, 3-beta-glucan molecule forming a 1,3-beta linkage, resulting in the elongation of 1,3-beta-glucan chains. The GEL1 gene encoding this enzyme was cloned and sequenced. The predicted amino acid sequence of Gel1p was homologous to several yeast protein families encoded by GAS of Saccharomyces cerevisiae, PHR of Candida albicans, and EPD of Candida maltosa. Although the expression of these genes is required for correct morphogenesis in yeast, the biochemical function of the encoded proteins was unknown. The biochemical assays performed on purified recombinant Gas1p, Phr1p, and Phr2p showed that these proteins have a 1,3-beta-glucanosyltransferase activity similar to that of Gel1p. Biochemical data and sequence analysis have shown that Gel1p is attached to the membrane through a glycosylphosphatidylinositol in a similar manner as the yeast homologous proteins. The activity has been also detected in membrane preparations, showing that this 1,3-beta-glucanosyltransferase is indeed active in vivo. Our results show that transglycosidases anchored to the plasma membrane via glycosylphosphatidylinositols can play an active role in fungal cell wall synthesis.     

Expression of novel β-glucanase Cel12A from Stachybotrys atra in bacterial and fungal hosts

β-glucanase Cel12A from Stachybotrys atra has been cloned and expressed in Aspergillus niger. The purified enzyme showed high activity of β-1,3-1,4-mixed glucans, was also active on carboxymethylcellulose (CMC), while it did not hydrolyze crystalline cellulose or β-1,3 glucans as laminarin. Cel12A showed a marked substrate preference for β-1,3-1,4 glucans, showing maximum activity on barley β-glucans (27.69 U mg(-1)) while the activity on CMC was much lower (0.51 U mg(-1)). Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focussing (IEF), and zymography showed the recombinant enzyme has apparent molecular weight of 24 kDa and a pI of 8.2. Optimal temperature and pH for enzyme activity were 50°C and pH 6.5. Thin layer chromatography analysis showed that major hydrolysis products from barley β-glucan and lichean were 3-O-β-cellotriosyl-D-glucose and 3-O-β-cellobiosyl-D-glucose, while glucose and cellobiose were released in smaller amounts. The amino acid sequence deduced from cel12A revealed that it is a single domain enzyme belonging to the GH12 family, a family that contains several endoglucanases with substrate preference for β-1,3-1,4 glucans. We believe that S. atra Cel12A should be considered as a lichenase-like or nontypical endoglucanase.     

Identification of an endo-beta-1,4-D-xylanase from Magnaporthe grisea by gene knockout analysis, purification, and heterologous expression

Magnaporthe grisea, a destructive ascomycetous pathogen of rice, secretes cell wall-degrading enzymes into a culture medium containing purified rice cell walls as the sole carbon source. From M. grisea grown under the culture conditions described here, we have identified an expressed sequenced tag, XYL-6, a gene that is also expressed in M. grisea-infected rice leaves 24 h postinoculation with conidia. This gene encodes a protein about 65% similar to endo-beta-1,4-D-glycanases within glycoside hydrolase family GH10. A M. grisea knockout mutant for XYL-6 was created, and it was shown to be as virulent as the parent strain in infecting the rice host. The proteins secreted by the parent strain and by the xyl-6Delta mutant were each fractionated by liquid chromatography, and the collected fractions were assayed for endo-beta-1,4-D-glucanase or endo-beta-1,4-D-xylanase activities. Two protein-containing peaks with endo-beta-1,4-D-xylanase activity secreted by the parent strain are not detectable in the column eluant of the proteins secreted by the mutant. The two endoxylanases (XYL-6alpha and XYL-6beta) from the parent were each purified to homogeneity. N-terminal amino acid sequencing indicated that XYL-6alpha is a fragment of XYL-6beta and that XYL-6beta is identical to the deduced protein sequence encoded by the XYL-6 gene. Finally, XYL-6 was introduced into Pichia pastoris for heterologous expression, which resulted in the purification of a fusion protein, XYL-6H, from the Pichia pastoris culture filtrate. XYL-6H is active in cleaving arabinoxylan. These experiments unequivocally established that the XYL-6 gene encodes a secreted endo-beta-1,4-D-xylanase.     

N-glycosylation is necessary for enzymatic activity of a beetle (Apriona germari) cellulase

We previously reported that the beta-1,4-endoglucanase (EGase) belonging to glycoside hydrolase family 45 cloned from the mulberry longicorn beetle, Apriona germari (Ag-EGase I), is composed of 237 amino acid residues and has a potential N-glycosylation site at 97-100 amino acid residues (NSTF). We here describe the N-glycosylation and its role for enzymatic activity of the Ag-EGase I. The N-glycosylation of Ag-EGase I was revealed by the treatment of tunicamycin to the recombinant virus-infected insect Sf9 cells and by endoglycosidase F to the purified recombinant Ag-EGase I, demonstrating that the carbohydrate moieties are not necessary for secretion but essential for Ag-EGase I enzyme activity. To further elucidate the functional role of the N-glycosylation in Ag-EGase I, we have assayed the cellulase enzyme activity in Thr99Gln mutant. Lack of N-glycosylation in Ag-EGase I showed no substantial enzyme activity. This result demonstrates that N-glycosylation at site 97-100 amino acid residues (NSTF) is essential for enzyme activity.     

Functional metagenomics unveils a multifunctional glycosyl hydrolase from the family 43 catalysing the breakdown of plant polymers in the calf rumen

Microbial communities from cow rumen are known for their ability to degrade diverse plant polymers at high rates. In this work, we identified 15 hydrolases through an activity-centred metagenome analysis of a fibre-adherent microbial community from dairy cow rumen. Among them, 7 glycosyl hydrolases (GHs) and 1 feruloyl esterase were successfully cloned, expressed, purified and characterised. The most striking result was a protein of GH family 43 (GHF43), hereinafter designated as R_09-02, which had characteristics very distinct from the other proteins in this family with mono-functional β-xylosidase, α-xylanase, α-L-arabinase and α-L-arabinofuranosidase activities. R_09-02 is the first multifunctional enzyme to exhibit β-1,4 xylosidase, α-1,5 arabinofur(pyr)anosidase, β-1,4 lactase, α-1,6 raffinase, α-1,6 stachyase, β-galactosidase and α-1,4 glucosidase activities. The R_09-02 protein appears to originate from the chromosome of a member of Clostridia, a class of phylum Firmicutes, members of which are highly abundant in ruminal environment. The evolution of R_09-02 is suggested to be driven from the xylose- and arabinose-specific activities, typical for GHF43 members, toward a broader specificity to the glucose- and galactose-containing components of lignocellulose. The apparent capability of enzymes from the GHF43 family to utilise xylose-, arabinose-, glucose- and galactose-containing oligosaccharides has thus far been neglected by, or could not be predicted from, genome and metagenome sequencing data analyses. Taking into account the abundance of GHF43-encoding gene sequences in the rumen (up to 7% of all GH-genes) and the multifunctional phenotype herein described, our findings suggest that the ecological role of this GH family in the digestion of ligno-cellulosic matter should be significantly reconsidered.     

Molecular cloning, expression, and enzymatic activity of a novel endogenous cellulase from the mulberry longicorn beetle, Apriona germari

A novel endogenous beta-1,4-endoglucanase (Ag-EGase III) gene belonging to the glycoside hydrolase family (GHF) 5 was cloned from the mulberry longicorn beetle, Apriona germari. The Ag-EGase III gene spans 1061 bp and consists of a single exon coding for 325 amino acid residues. The Ag-EGase III showed 89% protein sequence identity to another beetle, Psacothea hilaris, cellulase belonging to GHF 5. The Ag-EGase III has the potential proton donor and nucleophile amino acids conserved in GHF 5 and two putative N-glycosylation sites. Northern blot and Western blot analyses showed that Ag-EGases were expressed in the gut; Ag-EGase III and Ag-EGase I were expressed in three gut regions, and no Ag-EGase II was found in hindgut, indicating that the foregut and midgut are the prime sites for cellulase synthesis in A. germari larvae. The cDNA encoding Ag-EGase III was expressed as a 47-kDa polypeptide in baculovirus-infected insect Sf9 cells and the enzyme activity of the purified recombinant Ag-EGase III was approximately 1037 U per mg of recombinant Ag-EGase III. The enzymatic property of the purified recombinant Ag-EGase III showed the highest activity at 55 degrees C and pH 6.0, and was stable at 60 degrees C at least for 10 min. In addition, the N-glycosylation of Ag-EGase III was revealed by treatment with tunicamycin of recombinant virus-infected insect Sf9 cells and with endoglycosidase F of purified recombinant Ag-EGase III, demonstrating that the carbohydrate moieties are not necessary for enzyme activity.     

Purification and characterization of a beta-1,3-glucan binding protein from plasma of the crayfish Pacifastacus leniusculus

The plasma of the crayfish Pacifastacus leniusculus contains a protein which is able to bind to laminarin (a soluble beta-1,3-glucan) and which has been isolated by two independent methods, affinity precipitation with a beta-1,3-glucan or immunoaffinity chromatography. The purified beta-1,3-glucan binding protein was homogenous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. It is a monomeric glycoprotein with a molecular mass of approximately 100,000 Da and an isoelectric point of approximately 5.0. Amino acid analysis showed a very high similarity with the amino acid composition of beta-1,3-glucan binding proteins recently purified from two insects, the cockroach Blaberus craniifer and the silkworm Bombyx mori. The N-terminal amino acid sequence was determined to be: H2N-Asp-Ala-Gly-X-Ala-Ser-Leu-Val-Thr-Asn-Phe-Asn-Ser-Ala-Lys-Leu-X-X-Ly s--- Using monospecific rabbit polyclonal antibodies, the presence of this protein has also been shown within the blood cells. The purified beta-1,3-glucan binding protein did not show any peptidase or phenoloxidase activity but was able to enhance the activation of hemocyte-derived peptidase and prophenoloxidase only in the presence of the beta-1,3-glucan, laminarin, whereas mannan, dextran (alpha-glucan), or cellulose (beta-1,4-glucan) incubated with the beta-1,3-glucan binding protein had no effect on these enzyme activities. The beta-1,3-glucan binding protein could only be affinity-precipitated from crayfish plasma by the beta-1,3-glucans laminarin or curdlan (an insoluble beta-1,3-glucan), while mannan or dextran did not bind to the beta-1,3-glucan binding protein. No hemagglutinating activity of the purified beta-1,3-glucan binding protein could be detected.