Beta-glucosidase, exo-beta-glucanase and pyridoxine transglucosylase activities of rice BGlu1.
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ABSTRACT:
SUBMITTER: Opassiri R
PROVIDER: S-EPMC1224054 | biostudies-other | 2004 Apr
REPOSITORIES: biostudies-other
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Project description:BackgroundEfficient production of carbon-neutral biofuels is key to resolving global warming and exhaustion of fossil fuels. Cellulose, which is the most abundant biomass, is physically strong and biochemically stable, and these characteristics lead to difficulty of efficient saccharification of cellulosic compounds for production of fermentable glucose and other sugars.ResultsWe transformed rice with overexpressing constructs of rice genes encoding each of three classes of cellulases. The exo-glucanase overexpressing plants showed various abnormalities in leaf such as division of leaf blade, crack on leaf surface, excess lacunae in midrib structure and necrotic colour change. The overexpressing plants also showed sterility. Noticeably, these plants showed enhanced saccharification of stems after maturation. These results indicate that overexpression of the exo-glucanase gene brought about various developmental defects associated with modification of cell wall and enhanced saccharification in rice. On the other hand, endo-glucanase-overexpressing plants could not be obtained, and overexpression of β-glucosidase brought about no effect on plant growth and development.ConclusionsOur results indicate that genetic engineering of cellulosic biomass plants by overexpressing cellulase genes will be one of the approaches to confer enhanced saccharification ability for efficient production of cellulosic biofuels such as ethanol.
| S-EPMC4883724 | biostudies-literature
Project description:Fungal cell walls undergo continual remodeling that generates β-1,3-glucan fragments as products of endo-glycosyl hydrolases (GHs), which can be recognized as pathogen-associated molecular patterns (PAMPs) and trigger plant immune responses. How fungal pathogens suppress those responses is often poorly understood. Here, we study mechanisms underlying the suppression of β-1,3-glucan-triggered plant immunity by the blast fungus Magnaporthe oryzae. We show that an exo-β-1,3-glucanase of the GH17 family, named Ebg1, is important for fungal cell wall integrity and virulence of M. oryzae. Ebg1 can hydrolyze β-1,3-glucan and laminarin into glucose, thus suppressing β-1,3-glucan-triggered plant immunity. However, in addition, Ebg1 seems to act as a PAMP, independent of its hydrolase activity. This Ebg1-induced immunity appears to be dampened by the secretion of an elongation factor 1 alpha protein (EF1α), which interacts and co-localizes with Ebg1 in the apoplast. Future work is needed to understand the mechanisms behind Ebg1-induced immunity and its suppression by EF1α.
| S-EPMC10484928 | biostudies-literature
Project description:1. The maltase and glucoamylase activities of acid alpha-glucosidase purified from rabbit muscle exhibited marked differences in certain physicochemical properties. These included pH stability, inactivation by thiol-group reagents, inhibition by alphaalpha-trehalose, methyl alpha-d-glucoside, sucrose, turanose, polyols, glucono-delta-lactone and monosaccharides, pH optimum and the kinetics and pH-dependence of cation activation. 2. The results are interpreted in terms of the existence of at least two specific substrate-binding sites or sub-sites. One site is specific for the binding of maltose and probably other oligosaccharides. The second site binds polysaccharides such as glycogen. 3. The sites appear to be in close proximity, since glycogen and maltose are mutually inhibitory substrates and interact directly in transglucosylation reactions. 4. Acid alpha-glucosidase exhibited intrinsic transglucosylase activity. The enzyme catalysed glucosyl-transfer reactions from [(14)C]maltose (donor substrate) to polysaccharides (glycogen and pullulan) and to maltose itself (disproportionation). The pH optimum was 5.1, with a shoulder or secondary activity peak at pH5.4. The glucose transferred to glycogen was attached by alpha-1,4- and alpha-1,6-linkages. Three major oligosaccharide products of enzyme action on maltose (disproportionation) were detected. 5. The kinetics of enzyme action on [(14)C]maltose showed that the rate of transglucosylation increased in a sigmoidal fashion as a function of substrate concentration, approximately in parallel with a decrease in the rate of glucose release. 6. The results are interpreted to imply competitive interaction at a specific binding site between maltose and water as glucosyl acceptors. 7. The results are discussed in terms of the possible existence of multiple subgroups of glycogen-storage disease type II.
| S-EPMC1177248 | biostudies-other
Project description:During sclerotial infection of Sclerotinia sclerotiorum the mycoparasite Coniothyrium minitans penetrates through the host cell wall, which contains beta-1,3-glucan as its major component. A PCR-based strategy was used to clone a beta-1,3-glucanase-encoding gene, designated cmg1, from a cDNA library of the fungus. The nucleotide and deduced amino acid sequences of this gene showed high levels of similarity to the sequences of other fungal exo-beta-1,3-glucanase genes. The calculated molecular mass of the deduced protein (without the predicted 24-amino-acid N-terminal secretion signal peptide) was 83,346 Da, and the estimated pI was 4.73. Saccharomyces cerevisiae INVSc1 expressing the cmg1 gene secreted a approximately 100-kDa beta-1,3-glucanase enzyme (as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) into the culture medium. N-terminal sequence analysis of the purified recombinant enzyme revealed that the secreted enzyme starts at Ala-32, seven amino acids downstream from the predicted signal peptidase cleavage site. The purified recombinant glucanase inhibited in vitro mycelial growth of S. sclerotiorum by 35 and 85% at concentrations of 300 and 600 microg x ml(-1), respectively. A single copy of the cmg1 gene is present in the genome of C. minitans. Northern analyses indicated increases in the transcript levels of cmg1 due to both carbon starvation and the presence of ground sclerotia of S. sclerotiorum; only slight repression was observed in the presence of 2% glucose. Expression of cmg1 increased during parasitic interaction with S. sclerotiorum.
| S-EPMC92660 | biostudies-literature
Project description:Monolignol glucosides are storage forms of monolignols, which are polymerized to lignin to strengthen plant cell walls. The conversion of monolignol glucosides to monolignols is catalyzed by monolignol β-glucosidases. Rice Os4BGlu18 β-glucosidase catalyzes hydrolysis of the monolignol glucosides, coniferin, syringin, and p-coumaryl alcohol glucoside more efficiently than other natural substrates. To understand more clearly the basis for substrate specificity of a monolignol β-glucosidase, the structure of Os4BGlu18 was determined by X-ray crystallography. Crystals of Os4BGlu18 and its complex with δ-gluconolactone diffracted to 1.7 and 2.1 Å resolution, respectively. Two protein molecules were found in the asymmetric unit of the P212121 space group of their isomorphous crystals. The Os4BGlu18 structure exhibited the typical (β/α)8 TIM barrel of glycoside hydrolase family 1 (GH1), but the four variable loops and two disulfide bonds appeared significantly different from other known structures of GH1 β-glucosidases. Molecular docking studies of the Os4BGlu18 structure with monolignol substrate ligands placed the glycone in a similar position to the δ-gluconolactone in the complex structure and revealed the interactions between protein and ligands. Molecular docking, multiple sequence alignment, and homology modeling identified amino acid residues at the aglycone-binding site involved in substrate specificity for monolignol β-glucosides. Thus, the structural basis of substrate recognition and hydrolysis by monolignol β-glucosidases was elucidated.
| S-EPMC7817009 | biostudies-literature
Project description:beta-Glucanase present in cell-free extracts from Candida utilis was isolated and purified 562-fold by procedures that include adsorption on DEAE-Sephadex A-50 and filtration through columns of Sephadex G-50, G-100 and G-200, Bio-Gel P-10, and Concanavalin A-Sepharose 4B. The purified enzyme appeared homogeneous on polyacrylamide-gel electrophoresis and in ultracentrifugation studies (S20,w = 1.74S). The enzyme behaved as an acidic glycoprotein (pI4.1) with 68% carbohydrate and a high content of acidic amino acids. The mol.wt. was estimated to be 20000 from gel filtration and polyacrylamide-gel electrophoresis and 36000 from sedimentation experiments. Studies on the hydrolysis of different substrates showed that the enzyme is an unspecific beta-glucanase able to break down both (1 leads to 3)-eta- and (1 leads to 6)-beta-linkages by an exo-splitting mechanism. Glucono-delta-lactone, Zn2+ and Hg2+ inhibited the enzyme activity.
| S-EPMC1164153 | biostudies-other
Project description:The oomycetous, fungus-like, aquatic organism Pythium insidiosum is the causative agent of pythiosis, a life-threatening infectious disease of humans and animals living in tropical and subtropical areas of the world. Common sites of infection are the arteries, eyes, cutaneous/subcutaneous tissues, and gastrointestinal tract. Diagnosis of pythiosis is time-consuming and difficult. Radical excision of the infected organs is the main treatment for pythiosis because conventional antifungal drugs are ineffective. An immunotherapeutic vaccine prepared from P. insidiosum crude extract showed limited efficacy in the treatment of pythiosis patients. Many pythiosis patients suffer lifelong disabilities or die from an advanced infection. Recently, we identified a 74-kDa major immunodominant antigen of P. insidiosum which could be a target for development of a more effective serodiagnostic test and vaccines. Mass spectrometric analysis identified two peptides of the 74-kDa antigen (s74-1 and s74-2) which perfectly matched a putative exo-1,3-ss-glucanase (EXO1) of Phytophthora infestans. Using degenerate primers derived from these peptides, a 1.1-kb product was produced by PCR, and its sequence was found to be homologous to that of the P. infestans exo-1,3-ss-glucanase gene, EXO1. Enzyme-linked immunosorbent assays targeting the s74-1 and s74-2 synthetic peptides demonstrated that the 74-kDa antigen was highly immunoreactive with pythiosis sera but not with control sera. Phylogenetic analysis using part of the 74-kDa protein-coding sequence divided 22 Thai isolates of P. insidiosum into two clades. Further characterization of the putative P. insidiosum glucanase could lead to new diagnostic tests and to antimicrobial agents and vaccines for the prevention and management of the serious and life-threatening disease of pythiosis.
| S-EPMC2916248 | biostudies-literature
Project description:Marine bacteria residing on local red, green, and brown seaweeds were screened for exo-1,3-β-glucanase (ExoP) activity. Of the 90 bacterial species isolated from 32 seaweeds, only one, a Pseudoalteromonas sp., was found to display such activity. It was isolated from a Durvillaea sp., a brown kelp known to contain significant amounts of the storage polysaccharide laminarin (1,3-β-D-glucan with some 1,6-β branching). Four chromatographic steps were utilized to purify the enzyme (ExoP). Chymotryptic digestion provided peptide sequences for primer design and subsequent gene cloning. The exoP gene coded for 840 amino acids and was located just 50 bp downstream from a putative lichenase (endo-1,3-1,4-β-glucanase) gene, suggesting possible cotranscription of these genes. Sequence comparisons revealed ExoP to be clustered within a group of bacterial glycosidases with high similarity to a group of glycoside hydrolase (GH3) plant enzymes, of which the barley exo-1,3/1,4-β-glucanase (ExoI) is the best characterized. The major difference between the bacterial and plant proteins is an extra 200- to 220-amino-acid extension at the C terminus of the former. This additional sequence does not correlate with any known functional domain, but ExoP was not active against laminarin when this region was removed. Production of recombinant ExoP allowed substrate specificity studies to be performed. The enzyme was found to possess similar levels of exoglucanase activity against both 1,4-β linkages and 1,3-β linkages, and so ExoP is designated an exo-1,3/1,4-β-exoglucanase, the first such bacterial enzyme to be characterized. This broader specificity could allow the enzyme to assist in digesting both cell wall cellulose and cytoplasmic laminarin.
| S-EPMC2953027 | biostudies-literature
Project description:The fungus Trichoderma reesei grows on barley (Hordeum valgare L.) beta-glucan and pachyman, secreting increased quantities of exo-beta 1,3-glucanase. This enzyme is also found in commercial cellulase preparations, from which it has been partially purified. It has a mol.wt. of 700000, an isoelectric point of 4.2, is cold-stable and hydrolyses both beta 1 Leads to 3- and beta 1 Leads to 6-linkages.
| S-EPMC1162288 | biostudies-other
Project description:Pythiosis is a deadly infectious disease of humans and animals living in tropical and subtropical countries. The causative agent is the oomycete Pythium insidiosum. Treatment of pythiosis is challenging. The use of antimicrobial agents usually fails in the treatment of pythiosis. Many patients undergo surgical removal of an infected organ (i.e., eye, arm, and leg). The immunotherapeutic vaccine, prepared from the crude extract of P. insidiosum, shows limited efficacy against pythiosis. The fatal outcome occurs in patients with advanced disease. There are urgent needs for an effective therapeutic modality for pythiosis. Recently, the exo-1,3-β-glucanase (Exo1) has been identified as a conserve immunoreactive protein of P. insidiosum. Exo1 was predicted to reside at the cell membrane and hydrolyze cell wall β-glucan during cell growth. An Exo1 ortholog is absent in the human genome, making it an appealing target for drug or vaccine development. We attempted to clone and express the codon-optimized exo1 gene of P. insidiosum in E. coli. To solve the inclusion body formation, expression and purification of Exo1 were achievable in the denaturing condition using SDS- and urea-based buffers. Exo1 lacked hydrolytic activity due to the absence of proper protein folding and post-translational modifications. ELISA and Western blot analyses demonstrated the immunoreactivity of Exo1 against pythiosis sera. In conclusion, we successfully expressed and purified the immunoreactive Exo1 protein of P. insidiosum. The recombinant Exo1 can be produced at an unlimited amount and could serve as an extra protein to enhance the effectiveness of the current form of the vaccine against pythiosis.
| S-EPMC7306600 | biostudies-literature