Construction and characterization of a chimeric beta-glucosidase.
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ABSTRACT:
SUBMITTER: Singh A
PROVIDER: S-EPMC1136317 | biostudies-other | 1995 Feb
REPOSITORIES: biostudies-other
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Project description:BackgroundThe archeaon Sulfolobus solfataricus P2 encodes a thermoacidophilic cellulase which shows an extreme acid and thermal stability with a pH optimum at 1.8 and a temperature optimum at 80°C. This extraordinary enzyme could be useful for biotechnological exploitation but the expression and purification in expression hosts like E. coli is unsatisfactory due to the high aggregation tendency of the recombinant enzyme. The thermophilic cellulase CelA from Thermotoga maritima belongs to the same glycoside hydrolase family (GH12) but has a neutral pH optimum. In contrast to SSO1949 this enzyme is expressed partially soluble in E. coli.ResultsWe aimed to constructed a hybrid enzyme based on these two beta-endoglucanases which should successfully combine the advantageous properties of both cellulases, i.e. recombinant expression in E. coli, acidophily and thermophily. We constructed two hybrid proteins after bioinformatic analysis: both hybrids are expressed insoluble in E. coli, but one hybrid enzyme was successfully refolded from washed inclusion bodies.ConclusionsThe refolded active chimeric enzyme shows a temperature optimum of approximately 85°C and a pH optimum of approximately pH 3 thus retaining the advantageous properties of the Sulfolobus parent enzyme. This study suggests that the targeted construction of chimeric enzymes is an alternative to point mutational engineering efforts as long as parent enzymes with the wanted properties are available.
| S-EPMC3655044 | biostudies-literature
Project description:BACKGROUND:TH1 immune response antagonism is a desirable approach to mitigate some autoimmune and inflammatory reactions during the course of several diseases where IL-2 and IFN-gamma are two central players. Therefore, the neutralization of both cytokines could provide beneficial effects in patients suffering from autoimmune or inflammatory illnesses. RESULTS:A chimeric antagonist that can antagonize the action of TH1 immunity mediators, IFN-gamma and IL-2, was designed, engineered, expressed in E. coli, purified and evaluated for its in vitro biological activities. The TH1 antagonist molecule consists of the extracellular region for the human IFNgamma receptor chain 1 fused by a four-aminoacid linker peptide to human 60 N-terminal aminoacid residues of IL-2. The corresponding gene fragments were isolated by RT-PCR and cloned in the pTPV-1 vector. E. coli (W3110 strain) was transformed with this vector. The chimeric protein was expressed at high level as inclusion bodies. The protein was partially purified by pelleting and washing. It was then solubilized with strong denaturant and finally refolded by gel filtration. In vitro biological activity of chimera was demonstrated by inhibition of IFN-gamma-dependent HLA-DR expression in Colo 205 cells, inhibition of IFN-gamma antiproliferative effect on HEp-2 cells, and by a bidirectional effect in assays for IL-2 T-cell dependent proliferation: agonism in the absence versus inhibition in the presence of IL-2. CONCLUSION:TH1 antagonist is a chimeric protein that inhibits the in vitro biological activities of human IFN-gamma, and is a partial agonist/antagonist of human IL-2. With these attributes, the chimera has the potential to offer a new opportunity for the treatment of autoimmune and inflammatory diseases.
| S-EPMC1481661 | biostudies-literature
Project description:BackgroundIn general, biofuel production involves biomass pretreatment and enzymatic saccharification, followed by the subsequent sugar conversion to biofuel via fermentation. The crucial step in the production of biofuel from biomass is the enzymatic saccharification. Many of the commercial cellulase enzyme cocktails, such as Spezyme(®) CP (Genencor), Acellerase™ 1000 (Genencor), and Celluclast(®) 1.5L (Novozymes), are ineffectively to release free glucose from the pretreated biomass without additional β-glucosidase.ResultsIn this study, for the first time, a β-glucosidase DT-Bgl gene (1359 bp) was identified in the genome of Anoxybacillus sp. DT3-1, and cloned and heterologously expressed in Escherichia coli BL21. Phylogenetic analysis indicated that DT-Bgl belonged to glycosyl hydrolase (GH) family 1. The recombinant DT-Bgl was highly active on cello-oligosaccharides and p-nitrophenyl-β-d-glucopyranoside (pNPG). The DT-Bgl was purified using an Ni-NTA column, with molecular mass of 53 kDa using an SDS-PAGE analysis. It exhibited optimum activity at 70 °C and pH 8.5, and did not require any tested co-factors for activation. The K m and V max values for DT-Bgl were 0.22 mM and 923.7 U/mg, respectively, with pNPG as substrate. The DT-Bgl displayed high glucose tolerance, and retained 93 % activity in the presence of 10 M glucose.ConclusionsAnoxybacillus DT-Bgl is a novel thermostable β-glucosidase with low glucose inhibition, and converts long-chain cellodextrins to cellobiose, and further hydrolyse cellobiose to glucose. Results suggest that DT-Bgl could be useful in the development of a bioprocess for the efficient saccharification of lignocellulosic biomass.
| S-EPMC4994278 | biostudies-literature
Project description:Screening of gene-specific amplicons from metagenomes (S-GAM) is an efficient technique for the isolation of homologous genes from metagenomes. Using the S-GAM approach, we targeted multi-copper oxidase (MCO) genes including laccase and bilirubin oxidase (BOX) in soil and compost metagenomes, and successfully isolated novel MCO core regions. These core enzyme genes shared approximately 70% identity with that of the putative MCO from Micromonospora sp. MP36. According to the principle of S-GAM, the N- and C-terminal regions of the deduced products of the mature gene come from the known parent gene, which should be homologous and compatible with the target gene. We constructed two different MCO hybrid genes using Bacillus subtilis BOX and Micromonospora sp. MP36 MCO, to give Bs-mg-mco and Mic-mg-mco, respectively. The constructed chimeric MCO genes were fused with the maltose-binding protein (MBP) gene at the N-terminus for expression in Escherichia coli cells. We found that MBP-Mic-mg-MCO/Mic-mg-MCO possessed the characteristic properties of laccase, although MBP-Bs-mg-MCO had no activity. This novel laccase (Mic-mg-MCO) demonstrated unique substrate specificity, sufficient activity at neutral pH, and high thermal stability, which are suitable properties for its use as a laccase biocatalyst.
| S-EPMC8214651 | biostudies-literature
Project description:AimPlant materials used in the food industry contain up to five times more aromas bound to glucose (glucosides) than free, unbound aromas, making these bound aromas an unused flavouring potential. The aim of this study was to identify and purify a novel β-glucosidase from Brettanomyces yeasts that are capable of releasing bound aromas present in various food products.Methods and resultsWe screened 428 different yeast strains for β-glucosidase activity and are the first to sequence the whole genome of two Brettanomyces yeasts (Brettanomyces anomalus and Brettanomyces bruxellensis) with exceptionally high β-glucosidase activity. Heterologous expression and purification of the identified B. anomalus β-glucosidase showed that it has an optimal activity at a higher pH (5·75) and lower temperature (37°C) than commercial β-glucosidases. Adding this B. anomalus β-glucosidase to cherry beers and forest fruit milks resulted in increased amounts of benzyl alcohol, eugenol, linalool and methyl salicylate compared to Aspergillus niger and Almond glucosidase.ConclusionsThe newly identified B. anomalus β-glucosidase offers new possibilities for food bioflavouring.Significance and impact of the studyThis study is the first to sequence the B. anomalus genome and to identify the β-glucosidase-encoding genes of two Brettanomyces species, and reports a new bioflavouring enzyme.
| S-EPMC6680314 | biostudies-literature
Project description:An extracellular β-glucosidase was isolated from Proteus mirabilis VIT117 found to be growing on prawn shells. The enzyme production was found to be enhanced (14.58 U/ml) when the culture was maintained at pH 9 and provided with sorbitol as carbon source, yeast extract as nitrogen source and incubated at 37 °C for approximately 72 h. Statistical methods like Plackett-Burman and RSM were also applied here to study the effects of different combinations of growth parameters for the bacteria, where the most significant parameters were found to be inoculum size, pH, yeast extract, incubation time and sorbitol. The optimum concentrations of inoculum size, pH and yeast extract determined by RSM were 2 %, 9 and 2 %, respectively. Partial purification of the protein was done by ammonium sulfate precipitation, followed by dialysis, gel filtration chromatography and SDS-PAGE. The enzyme was found to have a molecular weight of approximately 50 kDa and was observed to be most active at 37 °C in pH 9, with a sharp decline in the enzyme activity when temperature or the pH was increased. Enzyme kinetics study was performed to understand the catalytic behavior of the enzyme and it was found that our β-glucosidase had 5.613 U/ml and 0.082 mM as V max and K m values, respectively.
| S-EPMC5047857 | biostudies-literature
Project description:The enzyme 6-phospho-β-glucosidase is an important member of the glycoside hydrolase family 1 (GH1). However, its catalytic mechanisms, especially the key residues determining substrate specificity and affinity, are poorly understood. A metagenome-derived gene sequence, encoding a novel 6-phospho-β-glucosidase designated Pbgl25-217, was isolated and characterized. The optimal conditions for enzymatic activity were 37°C and pH 7; Ca(2+), Mg(2+), and Mn(2+) stabilized the activity of Pbgl25-217, whereas Ni(2+), Fe(2+), Zn(2+), Cu(2+), and Fe(3+) inhibited its activity. The Km and Vmax of Pbgl25-217 were 4.8 mM and 1,987.0 U mg(-1), respectively. Seven conserved residues were recognized by multiple alignments and were tested by site-directed mutagenesis for their functions in substrate recognition and catalytic reaction. The results suggest that residues S427, Lys435, and Tyr437 act as "gatekeepers" in a phosphate-binding loop and play important roles in phosphate recognition. This functional identification may provide insights into the specificity of 6-phospho-β-glycosidases in GH1 and be useful for designing further directed evolution.
| S-EPMC3623223 | biostudies-literature
Project description:Bacillus licheniformis CGMCC 2876, an aerobic spore-forming bacterium, produces a polysaccharide bioflocculant that is biodegradable and harmless. The present study determined that β-glucosidase played a negative role in bioflocculant synthesis. The gene encoding β-glucosidase was cloned and expressed in Escherichia coli BL21. This gene consists of 1437 bp and encodes 478 amino acid residues. The recombinant β-glucosidase (Bgl.bli1) was purified and showed a molecular mass of 53.4 kDa by SDS-PAGE. The expression and reaction conditions of Bgl.bli1 were optimized; the activity of β-glucosidase reached a maximum at 45.44 U/mL. Glucose clearly inhibited the activity of β-glucosidase. The purified recombinant Bgl.bli1 hydrolysed polysaccharide bioflocculant in vitro and synergised with other cellulases. The ability of Bgl.bli1 to hydrolyse polysaccharide bioflocculant was the reason for the decrease in flocculating activity and indicated the utility of this enzyme for diverse industrial processes.
| S-EPMC5673865 | biostudies-literature
Project description:Analysis of a 2.4-kb cDNA of the cellulose-binding extracellular beta-glucosidase (CBGL) from Phanerochaete chrysosporium suggested that CBGL is organized into two domains, an N-terminal cellulose-binding domain and a C-terminal catalytic domain. Genomic sequence analysis suggested that cbgl is encoded by 30 exons. Southern analysis of DNA from homokaryotic cultures indicated that CBGL is encoded by two alleles, cbgl-1 and cbgl-2, of a single gene.
| S-EPMC106459 | biostudies-literature
Project description:BackgroundCold-active enzymes, sourced from cold-adapted organisms, are characterized by high catalytic efficiencies at low temperatures compared with their mesophilic counterparts, which have poor activity. This property makes them advantageous for biotechnology applications as it: (i) saves energy costs, (ii) shortens the times for processes operated at low temperatures, (iii) protects thermosensitive substrates or products of the enzymatic reaction, (iv) prevents undesired chemical transformations, and (v) prevents the loss of volatile compounds.ResultsA bglMKg gene that encodes a monomeric cold-active glycoside hydrolase family 1 enzyme with an apparent molecular mass of 50 kDa was isolated by the functional screening of a marine metagenomic library. The BglMKg enzyme was expressed in E. coli, purified by FPLC and characterized. The recombinant BglMKg could effectively hydrolyze various chromogenic substrates and β-linked oligosaccharides, and had remarkably high β-galactosidase, β-glucosidase and β-fucosidase activities. Because of the lack of information about the usefulness of β-fucosidases in industry, further characterization of the enzymatic properties of BglMKg was only carried out with substrates specific for β-glucosidase or β-galactosidase. The BglMKg had maximal β-galactosidase and β-glucosidase activities at approximately 40°C and 45°C, respectively. The optimum pH for β-galactosidase activity was 6.5, whereas the optimum pH for β-glucosidase activity was 7.5. In general, the enzyme was stable below 30°C and from pHs 6.0 to 8.0. The results of the kinetic studies revealed that BglMKg more efficiently hydrolyzed β-glucosidase substrates than β-galactosidase ones.ConclusionsBglMKg is a small, monomeric, cold-active β-glucosidase with additional enzymatic activities. It was efficiently expressed in E. coli indicating that BglMKg might be a candidate for industrial applications.
| S-EPMC3605331 | biostudies-literature