Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.21 (beta-glucosidase)
3,280 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Using molecular genetic techniques, a fusion protein has been produced which contains the cellulose-binding domain (CBD) of an exoglucanase (Cex) from Cellulomonas fimi fused to a beta-glucosidase (Abg) from Agrobacterium sp. The CBD functions as an affinity tag for the simultaneous purification and immobilization of the enzyme on cellulose. Binding to cellulose was stable for prolonged periods at temperatures from 4 degrees C to at least 50 degrees C, at ionic strengths from 10 mM to greater than 1 M, and at pH values below 8. The fusion protein can be desorbed from cellulose with distilled water or at pH greater than 8. Immobilized enzyme columns of the fusion protein bound to cotton fibers exhibited stable beta-glucosidase activity for at least 10 days of continuous operation at temperatures up to 37 degrees C. At higher temperatures, the bound enzyme lost activity. The thermal stability of the fusion protein was greatly improved by immobilization. Immobilization did not alter the pH stability. Except for its ability to bind to cellulose, the properties of the fusion protein were virtually the same as those of the native enzyme.
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PMID:Enzyme immobilization using a cellulose-binding domain: properties of a beta-glucosidase fusion protein. 136 28

The endoglucanase CenA and the exoglucanase Cex from Cellulomonas fimi each contain a discrete cellulose-binding domain (CBD), at the amino-terminus or carboxyl-terminus respectively. The gene fragment encoding the CBD can be fused to the gene of a protein of interest. Using this approach hybrid proteins can be engineered which bind reversibly to cellulose and exhibit the biological activity of the protein partner. Alkaline phosphatase (PhoA) from Escherichia coli, and a beta-glucosidase (Abg) from an Agrobacterium sp. are dimeric proteins. The fusion polypeptides CenA-PhoA and Abg-CBC(Cex) are sensitive to proteolysis at the junctions between the fusion partners. Proteolysis results in a mixture of homo- and heterodimers; these bind to cellulose if one or both of the monomers carry a CBD, e.g. CenA-PhoA/CenA-PhoA and CenA-PhoA/PhoA. CBD fusion polypeptides could be used in this way to purify polypeptides which associate with the fusion partner.
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PMID:Cellulose-binding domains: potential for purification of complex proteins. 140 57

A fusion protein, Sta-CBDCex, which comprises streptavidin with a cellulose-binding domain (CBDCex) fused to its C terminus, was produced in the cytoplasm of Escherichia coli, where it formed inclusion bodies. Renatured Sta-CBDCex, recovered from the inclusion bodies, adsorbed to Avicel, a microcrystalline cellulose. The cellulose-bound Sta-CBDCex in turn bound biotinylated alkaline phosphatase or biotinylated beta-glucosidase. The immobilized beta-glucosidase remained fully active during 2 weeks of continuous column operation at 50 degrees C.
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PMID:A streptavidin-cellulose-binding domain fusion protein that binds biotinylated proteins to cellulose. 776 88

Since Saccharomyces cerevisiae lacks the cellulase complexes that hydrolyze cellulosic materials, which are abundant in the world, two types of hydrolytic enzymes involved in the degradation of cellulosic materials to glucose were genetically co-immobilized on its cell surface for direct utilization of cellulosic materials, one of the final goals of our studies. The genes encoding FI-carboxymethylcellulase (CMCase) and beta-glucosidase from the fungus Aspergillus aculeatus were individually fused with the gene encoding the C-terminal half (320 amino acid residues from the C terminus) of yeast alpha-agglutinin and introduced into S. cerevisiae. The delivery of CMCase and beta-glucosidase to the cell surface was carried out by the secretion signal sequence of the native signal sequence of CMCase and by the secretion signal sequence of glucoamylase from Rhizopus oryzae for beta-glucosidase, respectively. The genes were expressed by the glyceraldehyde-3-phosphate dehydrogenase promoter from S. cerevisiae. The CMCase and beta-glucosidase activities were detected in the cell pellet fraction, not in the culture supernatant. The display of CMCase and beta-glucosidase proteins on the cell surface was confirmed by immunofluorescence microscopy. The cells displaying these cellulases could grow on cellobiose or water-soluble cellooligosaccharides as the sole carbon source. The degradation and assimilation of cellooligosaccharides were confirmed by thin-layer chromatography. This result showed that the cell surface-engineered yeast with these enzymes can be endowed with the ability to assimilate cellooligosaccharides. This is the first step in the assimilation of cellulosic materials by S. cerevisiae expressing heterologous cellulase genes.
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PMID:Assimilation of cellooligosaccharides by a cell surface-engineered yeast expressing beta-glucosidase and carboxymethylcellulase from aspergillus aculeatus 983 74

Oat beta-glucosidase (EC 3.2.1.21) exists in two isomeric forms of homomultimer (type I) and heteromultimer (type II), which are comprised of two 60 kDa monomers of As-Glu1 and As-Glu2. The cDNA of As-Glu2 was cloned in this study, whereas As-Glu1 was previously cloned as As-P60. The As-Glu2 cDNA encodes a plastid-directing transit peptide of 57 amino acid residues and a mature protein of 521 amino acid residues. The amino acid sequence of As-Glu2 is highly homologous to that of As-Glu1, except for their C-terminal portions. When the two cDNAs of the mature proteins were expressed as T7.Tag-fused proteins in Escherichia coli, they produced soluble and enzymatically active T7.Tag-As-Glu1 and T7.Tag-As-Glu2 proteins. The T7.Tag-As-Glu1 was assembled into a donut-shaped hexamer ring which was in turn stacked in integer numbers to form long fibrillar homomultimers of different lengths with a molecular mass of up to several million daltons. On the other hand, the T7.Tag-As-Glu2 primarily formed a dimer rather than a multimer. When both cDNAs of As-Glu1 and As-Glu2 were co-expressed as T7.Tag-fused mature proteins, they were also assembled into a hexamer ring comprised of the two monomers in a 1:1 stoichiometry. The heteromeric hexamer was stacked in smaller numbers to form the heteromultimer of T7. Tag-As-Glu1 and -As-Glu2. The results indicate that the As-Glu1 monomer plays a crucial role in the formation of both the As-Glu1 homomultimer and the As-Glu1 and As-Glu2 heteromultimer. We describe here a unique structure for the oat beta-glucosidase fibrillar multimer that is formed by stacking the hexamer rings composed of As-Glu1 and/or As-Glu2.
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PMID:Formation of fibrillar multimers of oat beta-glucosidase isoenzymes is mediated by the As-Glu1 monomer. 1106 78

A genetic system has been exploited to immobilize proteins in their active and functional forms on the cell surface of yeast, Saccharomyces cerevisiae. DNAs encoding proteins with a secretion signal peptide were fused with the genes encoding yeast agglutinins, a- and alpha-type proteins involved in mating. The fusion gene was introduced into S. cerevisiae and expressed under the control of several promoters. Appearance of the fused proteins expressed on the cell surface was demonstrated biochemically and by immunofluorescence and immunoelectron microscopy techniques. Alpha-galactosidase from Cyamopsis tetragonoloba seeds, peptide libraries including scFv and variable regions of the T cell receptor from mammalian cells have been successfully immobilized on the yeast cell wall in the active form. Recently, surface-engineered yeasts have been constructed by immobilizing the enzymes and a functional protein, for example, green fluorescent protein (GFP) from Aequorea victoria. The yeasts were termed 'arming yeasts' with biocatalysts or functional proteins. Such arming cells displaying glucoamylase from Rhizopus oryzae and alpha-amylase from Bacillus stearothermophilus, or carboxymethylcellulase and beta-glucosidase from Aspergillus acleatus, could assimilate starch or cellooligosaccharides as the sole carbon source, although S. cerevisiae cannot intrinsically assimilate these substrates. GFP-arming cells can emit green fluorescence from the cell surface in response to the environmental conditions. The approach described in this review will enable us to endow living cells, including yeast cells, with novel additional abilities and to open new dimensions in the field of biotechnology.
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PMID:Genetic immobilization of proteins on the yeast cell surface. 1453 13

A cell surface engineering system of yeast Saccharomyces cerevisiae has been established and novel yeasts armed by biocatalysts (enzymes-glucoamylase, alpha-amylase, CM-cellulase, beta-glucosidase, and lipase), termed "arming yeasts", were constructed. The gene encoding Rhizopus oryzae glucoamylase with its secretion signal peptide was fused with the gene encoding the C-terminal half of yeast alpha-agglutinin and expressed in S. cerevisiae. Glucoamylase was shown to be displayed on the cell surface in its active form and anchored covalently to the cell wall. S. cerevisiae itself is unable to utilize starch, while the surface-engineered yeast could grow on starch as the sole carbon source. For further improvement of the ability to directly ferment starchy materials by the cell surface-engineered yeast, engineered yeasts displaying two amylolytic enzymes on the cell surface were constructed. The gene encoding R. oryzae glucoamylase with its own secretion signal peptide and a truncated fragment of the alpha-amylase gene from Bacillus stearothermophilus with the prepro secretion signal sequence of the yeast alpha-factor were fused with the gene encoding the C-terminal half of the yeast alpha-agglutinin. The surface-engineered yeast co-displaying glucoamylase and alpha-amylase by the integration of their genes into the chromosomes could grow faster on starch as the sole carbon source than the engineered cells displaying only glucoamylase. The system was further applied to the construction of a novel cellulose-utilizing yeast by displaying cellulolytic enzymes in their active form on the cell surface of S. cerevisiae. Engineered yeasts co-displaying FI-carboxymethylcellulase (CM-cellulase), one of the endo-type cellulases, and beta-glucosidase from Aspergillus aculeatus on their cell surface were also constructed. The yeasts displaying these cellulases were given the ability to assimilate cellooligosaccharide, suggesting the possibility that the assimilation of cellulosic materials may be carried out by S. cerevisiae displaying heterologous cellulase proteins on the cell surface. The system has also been used for the cell surface display of R. oryzae lipase (ROL). Linker peptides (spacers) consisting of the Gly/Ser repeat sequence were inserted at the C-terminal portion of ROL to enhance the lipase activity. The insertion of an appropriate length of a linker peptide as a spacer is effective in the display of ROL, having the active region at the C-terminal portion, on the cell surface. Thus, cell surface engineering will be capable of conferring novel additional abilities upon living cells and will herald a new era in the field of biotechnology.
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PMID:Cell surface engineering of yeast: construction of arming yeast with biocatalyst. 1623 31

The beta-glucosidase gene of maize (ZmGLU1) was suggested to hydrolyze cytokinin-conjugate and release free cytokinin during plant growth and development. A clone containing the upstream region of ZmGLU1 was isolated and sequenced from a maize genomic library. The full-length ZmGLU1 promoter and a series of its 5' deletions were fused to the beta-glucuronidase (GUS) reporter gene and transferred into tobacco. The GUS activity of transgenic plants was assayed at various developmental stages. The results showed that ZmGLU1 promoter-driven GUS gene had the highest expression level in the roots and that the expression of GUS gene declined during seed maturation and down to the lowest level in mature seeds. The ZmGLU1 promoter-driven GUS expression increased during seed germination, reaching a peak on day 11. The results also showed that this promoter could be inhibited by 6-BA, trans-zeatin, and NAA, but was not affected by GA(3), ABA, SA, cold, salt, drought, and submergence treatments. The histochemical staining revealed that GUS activity was located in vigorous cell division zones with dominant staining associated with vascular tissues. Deletion analysis showed that the promoter contained a putative leaf-specific and stem-specific negative regulative element and two putative enhancers.
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PMID:Isolation of a maize beta-glucosidase gene promoter and characterization of its activity in transgenic tobacco. 1677 Jun 27

The microplasmodia of Physarum polycephalum express three types of beta-glucosidases: secretory enzyme, a soluble cytoplasmic enzyme and a membrane-bound enzyme. We are interested in the physiological role of three enzymes. We report the sequence of cDNA for membrane beta-glucosidase 1, which consists of 3825 nucleotides that includes an open reading frame encoding 1248 amino acids. The molecular weight of membrane beta-glucosidase 1 was calculated to be 131,843 based on the predicted amino acid composition. Glycosyl hydrolase family 3 N-terminal and C-terminal domains were found within the N-terminal half of the membrane beta-glucosidase 1 sequence and were highly homologous with the primary structures of fungal beta-glucosidases. Notably, the C-terminal half of membrane beta-glucosidase 1 contains two calx-beta motifs, which are known to be Ca(2+) binding domains in the Drosophila Na(+)/Ca(2+) exchanger; an RGD sequence, which is known to be a cell attachment sequence; and a transmembrane region. In this way, Physarum membrane beta-glucosidase 1 differs from all previously identified family 3 beta-glucosidases. In addition to cDNA for membrane beta-glucosidase 1, two other distinctly different mRNAs were also isolated. Two sequences were largely identical to cDNA for membrane beta-glucosidase 1, but included a long insert sequence having a stop codon, leading to truncation of their products, which could account for other beta-glucosidase forms occurred in Physarum poycephalum. Thus, the membrane beta-glucosidase is a new type family 3 enzyme fused with the Calx-beta domain. We propose that Calx-beta domain may modulate the beta-glucosidase activity in response to changes in the Ca(2+) concentration.
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PMID:A cDNA cloned from Physarum polycephalum encodes new type of family 3 beta-glucosidase that is a fusion protein containing a calx-beta motif. 1691 64

Enzyme engineering was performed to link the beta-glucosidase enzyme (BGL1) from Saccharomycopsis fibuligera to the cellulose-binding domain (CBD2) of Trichoderma reesei cellobiohydrolase (CBHII) to investigate the effect of a fungal CBD on the enzymatic characteristics of this non-cellulolytic yeast enzyme. Recombinant enzymes were constructed with single and double copies of CBD2 fused at the N-terminus of BGL1 to mimic the two-domain organization displayed by cellulolytic enzymes in nature. The engineered S. fibuligera beta-glucosidases were expressed in Saccharomyces cerevisiae under the control of phosphoglycerate-kinase-1 promoter (PGK1 ( P )) and terminator (PGK1 ( T )) and yeast mating pheromone alpha-factor secretion signal (MFalpha1 ( S )). The secreted enzymes were purified and characterized using a range of cellulosic and non-cellulosic substrates to illustrate the effect of the CBD on their enzymatic activity. The results indicated that the recombinant enzymes of BGL1 displayed a 2-4-fold increase in their hydrolytic activity toward cellulosic substrates like avicel, amorphous cellulose, bacterial microcrystalline cellulose, and carboxy methyl cellulose in comparison with the native enzyme. The organization of the CBD in these recombinant enzymes also resulted in enhanced substrate affinity, molecular flexibility and synergistic activity, thereby improving the ability of the enzymes to act on and hydrolyze cellulosic substrates, as characterized by adsorption, kinetics, thermal stability, and scanning electron microscopic analyses.
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PMID:Effect of the cellulose-binding domain on the catalytic activity of a beta-glucosidase from Saccharomycopsis fibuligera. 1733 92


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