ABSTRACT
An operon, bglABC, that encodes two sugar permeases and a beta-glucosidase was cloned from a cellulolytic actinomycete, Thermobifida fusca, into Escherichia coli and sequenced. The bglC gene encoding an intracellular beta-glucosidase (beta-d-glucoside glucohydrolase, EC 3.2.1.21) belonging to glycosyl hydrolase family 1 was subcloned and expressed in E. coli. The purified enzyme (MW 53,407 Da; pI 4.69) hydrolyzed substrates containing both beta 1 --> 4 and beta 1 --> 2 glycosidic bonds, and was most active against cellobiose (Vmax = 29, Km = 0.34 mm), cellotriose, cellotetraose, and sophorose. The enzyme also showed aryl-beta-glucosidase activity on p-nitrophenyl-beta-d-glucopyranoside and p-nitrophenyl-beta-d-cellobioside. BglC had a pH optimum of 7.0 and a temperature optimum of 50 degrees C. The enzyme was stable at 60 degrees C, but was rapidly inactivated at 65 degrees C. BglC was inhibited by low concentrations of gluconolactone, but was insensitive to end-product inhibition by glucose and was not affected by Ca or Mg ions or EDTA. Its properties are well suited for use in a process to hydrolyze biomass cellulose to glucose.
Subject(s)
Actinomycetales/enzymology , Actinomycetales/genetics , Cellobiose/metabolism , Actinomycetales/metabolism , Amino Acid Sequence , Bacterial Proteins/analysis , Base Sequence , Cloning, Molecular , DNA Primers/chemistry , DNA, Bacterial/chemistry , DNA, Bacterial/isolation & purification , Electrophoresis, Polyacrylamide Gel , Escherichia coli/chemistry , Escherichia coli/genetics , Hydrogen-Ion Concentration , Molecular Sequence Data , Molecular Weight , Polymerase Chain Reaction , Sequence Homology, Amino Acid , Sequence Homology, Nucleic Acid , beta-GlucosidaseABSTRACT
Biosynthesis of extracellular cellulases in the cellulose-degrading actinomycete Thermobifida fusca is controlled by a transcriptional regulator, CelR, and cellobiose, which acts as an inducer interfering with the CelR-DNA interaction. We report the identification and characterization of a mutation in the celR gene that changes Ala(55) in the hinge helix of CelR to Thr. The wild-type and mutant celR genes were cloned in Escherichia coli, and their protein products were characterized. The CelR mutant protein bound DNA more weakly than the wild-type protein and formed a less stable complex with DNA in the presence of cellobiose. The results of Western analysis and gel retardation experiments suggest that CelR is produced constitutively and its DNA-binding activity is regulated through posttranslational modification.
Subject(s)
Actinomycetales/genetics , Bacterial Proteins , Cellulase/genetics , Fungal Proteins/genetics , Mutation , Repressor Proteins/genetics , Actinomycetales/metabolism , Cellobiose/metabolism , Cellulase/metabolism , Cytoplasm/metabolism , Fungal Proteins/metabolism , Gene Expression Regulation, Fungal , Promoter Regions, Genetic , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Repressor Proteins/metabolism , Transcription, GeneticABSTRACT
CelR, a protein that regulates transcription of cellulase genes in Thermomonospora fusca (Actinomycetaceae) was purified to homogeneity. A 6-kilobase NotI-SacI fragment of T. fusca DNA containing the celR gene was cloned into Esherichia coli and sequenced. The celR gene encodes a 340-residue polypeptide that is highly homologous to members of the GalR-LacI family of bacterial transcriptional regulators. CelR specifically binds to a 14-base pair inverted repeat, which has sequence similarity to the binding sites of other family members. This site is present in regions upstream of all six cellulase genes in T. fusca. The binding of CelR to the celE promoter is inhibited specifically by low concentrations of cellobiose (0.2-0.5 mM), the major end product of cellulases. The other sugars tested did not affect binding at equivalent or 50-fold higher concentrations. The results suggest that CelR may act as a repressor, and that the mechanism of induction involves a direct interaction of CelR with cellobiose.
Subject(s)
Actinomycetaceae/enzymology , Bacterial Proteins , Cellulase/genetics , Gene Expression Regulation, Enzymologic , Repressor Proteins/metabolism , Transcription, Genetic , Amino Acid Sequence , Base Sequence , Binding Sites , Cellobiose/metabolism , Cloning, Molecular , DNA, Bacterial , DNA-Binding Proteins/metabolism , Molecular Sequence Data , Promoter Regions, Genetic , Protein Binding , Repressor Proteins/genetics , Repressor Proteins/isolation & purification , Sequence Homology, Amino AcidABSTRACT
Regulation of the biosynthesis of the six cellulases comprising the cellulolytic system of the thermophilic soil bacterium Thermomonospora fusca ER1 was studied. The levels of the individual enzymes produced on different noninducing and inducing carbon sources were determined. The lowest level of cellulase synthesis (3 nM) was observed with xylose as a carbon source, and the highest level (247 to 1,670 nM for different enzymes) was found in cultures grown on microcrystalline cellulose. Endocellulases and exocellulases showed distinctly different regulation patterns. Differences in the regulation of individual enzymes appear to be determined by the specific structural organization of the upstream regulatory sequences of their genes.
Subject(s)
Actinomycetaceae/enzymology , Bacterial Proteins/biosynthesis , Cellulase/biosynthesis , Actinomycetaceae/genetics , Bacterial Proteins/genetics , Cellulase/genetics , Codon, Initiator/genetics , Xylose/metabolismABSTRACT
The cytotoxic activity of complexes of hydrolysable tannins of Chamaenerium angustifolium L. Scop. and Hippophae rhamnoides L. was compared with that of the antineoplastic drugs vinoblastin (VLB), methotrexate (MT), and 5-fluorouracil (5-FU). The tannins yield to VLB and MT in activity but resemble 5-Fu in their active concentration. They inhibited succinate oxidation by the rat liver mitochondria and their cytotoxicity was displayed in much lower concentrations than their inhibitory effect on mitochondrial respiration. It is concluded that tannins inhibit the respiratory chain at the third point of conjugation in the transfer of electrons from cytochrome c to oxygen.
Subject(s)
Antineoplastic Agents/toxicity , Mitochondria, Liver/drug effects , Tannins/toxicity , Animals , Antimetabolites, Antineoplastic/toxicity , Antineoplastic Agents/pharmacology , Antineoplastic Agents, Phytogenic/toxicity , Burkitt Lymphoma , Dose-Response Relationship, Drug , Fluorouracil/toxicity , Lymphocytes/drug effects , Male , Methotrexate/toxicity , Mitochondria, Liver/metabolism , Oxygen Consumption/drug effects , Rats , Rats, Wistar , Tannins/pharmacology , Tumor Cells, Cultured , Vinblastine/toxicityABSTRACT
Pharmacological and metabolic effects of Galleria mellonella larvae extract used in Russian folk medicine to treat cardiovascular and senile diseases were studied. It was shown that the extract possesses adaptogenic, cardiotropic, cardioprotective, and hypocoagulant properties. The extract possesses low toxicity and does not cause significant changes in biochemical parameters in the blood serum of laboratory animals. Increase in catecholamine content in the heart and aortic tissues and their decrease in adrenal glands are unfavourable effects of high doses of the extract.
Subject(s)
Adaptation, Physiological/drug effects , Heart Diseases/prevention & control , Moths/metabolism , Tissue Extracts/pharmacology , Animals , Anura , Blood Coagulation/drug effects , Epinephrine/metabolism , Glycogen/metabolism , Heart/drug effects , Heart Diseases/chemically induced , Heart Diseases/physiopathology , In Vitro Techniques , Kidney Function Tests , Muscle, Smooth, Vascular/drug effects , Muscle, Smooth, Vascular/metabolism , Myocardium/metabolism , Norepinephrine/metabolism , Organ Size/drug effects , Physical Endurance/drug effects , Rats , Strophanthins/antagonists & inhibitors , Strophanthins/toxicityABSTRACT
1. The action of the extract from larvae of the great wax moth Galleria mellonella L. used in Russian folk medicine was studied. 2. Two active components influencing the growth and morphological differentiation of cells in vitro were found. 3. The presence of these components in the extract was conditioned by consumption of honeybee products by Galleria mellonella larvae. 4. Cytotoxicity of honeybee products was studied.
Subject(s)
Bees/metabolism , Medicine, Traditional , Moths/metabolism , Tissue Extracts/pharmacology , Animals , Cells, Cultured , Humans , Larva/metabolism , Molecular WeightABSTRACT
A procedure for isolating biologically active substances from Galleria mellonella using a culture of isolated giant neurons of mollusc Lymnaea stagnalis as a test-system is described. Fractions capable of activating neurites and inhibiting aggregation of neuronal cells within a range of concentrations from 1 to 30 micrograms/ml were isolated. The fractions obtained have in their chemical composition about 10.5% N, also contain P and S. They have a carbohydrate component.
Subject(s)
Biological Products/pharmacology , Lepidoptera/analysis , Lymnaea/metabolism , Moths/analysis , Neurons/cytology , Animals , Biological Products/isolation & purification , Cell Aggregation/drug effects , Cells, Cultured , Larva/analysis , Lymnaea/drug effects , Neurons/drug effectsABSTRACT
The present study was performed to determine the chemical composition and molecular weight of channel-forming complex by fractioning the cell homogenate supernatant by different means (chromatography, gel-electrophoresis, using dissociating and denaturating agents). Availability of the channel-forming complexes in individual fractions is judged from reconstruction of channels in BLM. The Ca-channels reconstructed in BLM are formed by molecules of a protein nature (presumably, peptides). Their molecular weight is no more than 20000 (presumably, 5000). A high stability of spacial organization of channel forming molecules was observed. The channels--subunits aggregate without any change in properties of the selective filter. The thermodynamically preferred agagregates have conductivity of 200 pmho in 0.1 M KCl and seem to consist of 80 channels--subunits which are switched off and on all together.
