Cloning and characterization of β-galactoside and β-glucoside hydrolysing enzymes of Thermotoga maritima

Abstract A gene library of the hyperthermophilic bacterium Thermotoga maritima strain MSB8 was constructed in Escherichia coli . Two non-related T. maritima chromosomal DNA fragments were physically characterized. They conferred the synthesis of thermostable X-Gal (5-bromo-4-chloro-3-indolyl-β- d -galactopyranoside)-hydrolysing activity upon the host organism. The biochemical properties of the recombinant enzymes indicated that genes for a β-galactosidase (BgaA) and a broad-specificity β-glucosidase (Bg1A) had been isolated. The genes were desiignted bgaA and bglA , respectively. According to analytical size exclusion chromatography data, BgaA and BglA had native molecular masses of approximately 240 kDa and 95 kDa, respectively. Both enzymes apparently have dimeric subunit structure. An additional β-glucosidase (designated BglB) activity, clearly distinct from BglA in terms of substrate specificity, could be detected in a crude extract of T. maritima .     

Purification and properties of a β-glucosidase from Penicillium oxalicum autolysates

A beta-glucosidase from the medium of an autolyzed culture of Penicillium oxalicum has been purified by tannic acid precipitation, sephacryl S-200, DEAE-Biogel, CM-Biogel and Mono Q successively. The purification process produced a homogeneous band in the SDS-PAGE that correspond to a Mr of 133,500. The enzyme had a pl of 4, and the active optima were found at pH 5.5 and 55 degrees C. The enzyme hydrolyzed different substrates showing maximum affinity against p-nitrophenyl-beta-D-glucoside with a Km value of 0.37 mM. The beta-glucosidase was inhibited by Glucono-D-lactone but not by glucose in the concentration range of 1 to 10 mM. The enzyme was adsorbed by Concanavalin-A-Sepharose.     

Physical characterization of isozymes of endo-β-1,4-glucanase and β-1,4-glucosidase from Aspergillus species

Electrophoretic data revealed the presence of various isozymes of endoglucanase and beta-glucosidase, the number of which varied from one to three in various species of the genus Aspergillus. pH 5.0 was optimum for all the isozymes whereas metal ion treatment showed complete inhibition of almost all the isozymes by Hg2+ and partial inhibition by Ca2+ and Co2+ of isozymes of both the enzymes. An alteration in the electrophoretic mobility of isozymes of beta-glucosidase was also noticed in some species with Hg2+ treatment.     

Induction studies showing evidence of the similarities between an inducible intracellular and extracellular β-d-glucosidase produced by a species of Monilia

Abstract A Monilia sp. produced an inducible intracellular β- d -glucosidase (IG-2) which is the nascent form of the extracellular enzyme (EG-1) prior to its secretion into the extracellular medium. The other intracellular β- d -glucosidase (IG-1) produced was a constitutive enzyme. Highest yields of the inducible β- d -glucosidase resulted when Monilia sp. was grown on insoluble cellulose. Cellobiose and d -glucose appeared to repress β- d -glucosidase formation at high substrate levels and synthesis occurred only once the levels of these sugars in the medium were nearly depleted.     

β-Glucosidase and β-Galactosidase in Primary Cultures of Rat Astrocytes: Comparison to the Brain Enzymes

In primary astrocyte cultures beta-glucosidase (EC 3.2.1.21) and beta-galactosidase (EC 3.2.1.23) showed pH optima and Km values identical to rat brain enzymes, using methylumbelliferyl glycosides and labeled gluco- and galactocerebrosides as substrates. The activities of both glycosidases increased in culture up to 3-4 weeks. In rat brain only galactosidase increased; glucosidase activity declined between 12-20 days after birth. The specific activities were two- to sixfold higher in astrocyte cultures than in rat brain. These activities were not due to uptake of enzymes from the growth medium. Secretion of beta-galactosidase, but not beta-glucosidase nor acid phosphatase could be demonstrated. These results support the suggestion of a degradative function for astrocytes in the brain.     

β-Glucosidase from the cellulolytic system of Alternaria alternata autolyzed cultures

Abstract A β-glucosidase from centrifugated autolyzed cultures of Alternaria alternata has been purified 71 times by Sephadex G-200, CM-Biogel A and DEAE-Biogel A successively. The enzyme is a glycoprotein with 16% sugar and a M _r of 160 000, formed by two subunits of 60 000 and 80 000. The enzyme has optimum pH of 5 units and optimum reaction temperature of 50°C, being stable in a pH range of 3–8 and 0 to 60°C. The enzyme hydrolyzes different substrates showing maximum affinity and maximum hydrolysis velocity on cellobiose. The β-glucosidase is inhibited by gluconolactone but not by 10 mM glucose.     

Influence of L-sorbose on the location of β-glucosidase in Trichoderma pseudokoningii

Abstract The effect of l -sorbose on growth, morphology, cell wall composition and β-glucosidase location has been examined with Trichoderma pseudokoningii . Sorbose-grown cultures exhibited a longer lag phase, a tendency to more frequent hyphal branching and showed a decreased cell wall content of β-1,3-glucan. In sorbose-containing cultures, a significant higher portion of total β-glucosidase was present in the culture fluid, whereas in sorbose-lacking control cultures the major part of activity was associated with the cell walls. The results support the previous hypothesis (Kubicek, C.P. (1982) Arch. Microbiol. 132, 349–354) that β-1.3-glucan is involved in cell wall binding of β-glucosidase in Trichoderma pseudokoningii .     

Selection and study of a Candida molischiana mutant derepressed for β-glucosidase production

Abstract A mutant strain of Candida molischiana was selected. Analysis of the exocellular activity of Candida molischiana 35M5N grown on different carbon sources revealed that the biosynthesis of β-glucosidase is derepressed in this yeast strain. The strain is not a hyper-producer mutant. There were no observed differences in the endocellular and parietal activities of the wild and mutant strains. However, the mutant strain produced 35-fold more enzyme than the wild-type in the culture medium with glucose as carbon source. When glucose was used as carbon source, the mutant strain produced 90% more exocellular enzyme than when cellobiose was used as the carbon source.     

Effect of β-glucanases on Penicillium oxalicum cell wall fractions

The polysaccharidic effect of a purified 1,3- β -glucanase, a purified β -glucosidase, and of partially purified endo-1,3- β -glucanase from autolysed Penicillium oxalicum cultures on cell wall isolate fractions from the same fungus were studied.
Fractionation of 5-day-old cell wall gave rise to a series of fractions that were identified using infrared spectrophotometry. The fractions used were: F₁, an α -glucan; F₃, a β -glucan; F₄, a chitin-glucan; and F_4b, a β -glucan. The fractions were incubated with each of the enzymes and with a mixture of equal parts of the three enzymes and the products of the enzymatic hydrolysis were analyzed after 96 h incubation.
The enzymes were found to degrade fraction F_4b ( β -glucan); the greatest degree of hydrolysis was reached when the three enzymes were used together, suggesting the need for synergic action by these enzymes in the cell wall degradation process.     

Selection and study of mutants of Dekkera intermedia and Candida wickerhamii derepressed for β-glucosidase production

Abstract Mutants of Candida wickerhamii and Dekkera intermedia , derepressed for β-glucosidase biosynthesis, were isolated. These mutants were also shown to hyperproduce this enzyme. In anaerobic culture, the C. wickerhamii mutant still hyperproduced β-glucosidase and was derepressed. Glucose-cellobiose anaerobic fermentation by this strain was thus improved. On the other hand, the D. intermedia mutant did not show any difference from the wild-type strain in anaerobic culture.     

Bleomycin-induced β-lactamase overexpression in Escherichia coli carrying a bleomycin-resistance gene from Streptomyces verticillus and its application to screen bleomycin analogues

Abstract A bleomycin-resistance gene, designated blmA , has been cloned from bleomycin-producing Streptomyces verticillus by Sugiyama et al. (Gene 151 (1994) 11–16). The present study shows that Escherichia coli harboring the blmA -carrying pUC plasmid overproduced β-lactamase, encoded by an ampicillin-resistance gene on the plasmid, when cultured in the presence of bleomycin, which suggests that bleomycin may act as an inducer (or an activator) for the expression of the specific gene in the presence of blmA . We constructed a vector, designated pMAB50, which senses bleomycin and produces a pigment, using blmA and a Streptomyces tyrosinase gene located under the control of β-lactamase promoter: E. coli harboring pMAB50 produced the melanin pigment in the presence of bleomycin-type antibiotics, suggesting that the transformed E. coli can be employed as a reporter organism to screen bleomycin analogues.     

A constitutive, plasma-membrane bound β-glucosidase in Trichoderma reesei

Abstract Plasma membranes of Trichoderma reesei QM 9414, isolated from protoplasts by means of the concanavalin A procedure, contained β-glucosidase activity, which appeared constitutively upon growth on glucose. The enzyme had a pH optimum around 6, and was active on p -nitrophenyl-β- d -glucoside, cellobiose and sophorose ( K _m 0.7, 3.9 and 3.1 mM, respectively). Glucose was only weakly inhibitory ( K _i 7 mM). Treatment of the plasma membranes with Triton X-100, Tween 80 or digitonin solubilized more than 60% of the membrane-bound β-glucosidase activity. The enzyme so solubilized exhibited an M _r of 70 000 ± 5000 and an isoelectric point at pH 8.2 ± 0.3.     

β,β''-Iminodipropionitrile Impairs Retrograde Axonal Transport

beta,beta'-Iminodipropionitrile (IDPN), a neurotoxin, causes redistribution of neurofilaments in axons followed by the development of proximal axonal swellings and, in chronic intoxication, a distal decrease in axonal caliber. The latter changes are caused by a selective impairment in the slow anterograde axonal transport of neurofilament proteins. To assess the role of retrograde axonal transport in IDPN toxicity, we used [3H]N-succinimidyl propionate ([3H]NSP) to label covalently endogenous axonal proteins in sciatic nerve of the rat and measured the accumulation of radioactively labeled proteins in the cell bodies of motor and sensory neurons over time. IDPN was injected intraneurally 6 h or intraperitoneally 1 day before subepineurial injection of [3H]NSP into the sciatic nerve, and the animals were killed 1, 2, and 7 days after [3H]NSP injection. Neurotoxicity was assessed by electron microscopic observation of the nerves of similarly treated animals. Both intraneural and intraperitoneal injection of IDPN caused an acute reduction in the amount of labeled proteins transported back to the cell bodies. The early appearance of these changes suggests that alterations in retrograde transport may play a role in the production of the neuropathic changes.     

The occurrence of β-galactosidase and β-phosphogalactosidase in Lactobacillus casei strains

Abstract Several strains of Lactobacillus casei of different origins were compared and it was observed that lactose metabolism varied from one strain to the other. Certain strains contained a β-galactosidase, others a β-phosphogalactosidase and others contain both. It was shown that the activities present in these last strains are catalyzed by two proteins differing in their electrophoretic mobilities and M _r values. Genetic divergence of the studied strains is considered.     

Cloning and nucleotide sequence of the bglA gene from Erwinia herbicola and expression of β-glucosidase activity in Escherichia coli

Abstract Genomic DNA fragments encoding β-glucosidase activity from the wild-type strain WD4 of Erwinia herbicola were cloned into Escherichia coli . Two clones containing a common fragment encoded a polypeptide of 58000 Da. Cloned β-glucosidase, expressed in E. coli , showed activity against natural β-glucoside sugars except for cellobiose. An open reading frame of 1442 bp termed bglA was identified by nucleotide sequencing and it coded for a protein of 480 amino acids ( M _r 53896) which showed significant homology with β-glucosidases from glycosyl hydrolase family 1.     

Cloning and expression of a β-1,4-endoglucanase gene from Cellulomonas sp. CelB7 in Escherichia coli; purification and characterization of the recombinant enzyme

Abstract A gene library of a newly isolated Cellulomonas sp. strain was constructed in Escherichia coli and clones were screened for endoglucanase activity using dye-labelled carboxymethylcellulose. Seventeen clones were isolated that carried DNA inserts coding for endoglucanase enzymes. Of the 17 clones, one carrying the gene cegA , was further characterized. The recombinant endoglucanase was purified by FPLC. The endoglucanase was active against carboxymethylcellulose, lichenin and also degraded crystalline cellulose and birchwood xylan. The molecular mass of the enzyme (36 kDa), and its pH (7.4) and temperature (35 °C) optima were determined.     

Purification and characterization of two extracellular β-glucosidases from Aspergillus nidulans

We have characterized the toxic and adhesive properties of Escherichia coli strains producing the second type of cytotoxic necrotizing factor (CNF2) and belonging to the classic enteropathogenic serogroup O55. Bovine CNF2 strains of serotype O55:H4 express P fimbriae as do pyelonephritic Escherichia coli that cause infections in humans. In contrast, strains of serotype O55:H21 which produce CNF2 from bovine origin possess the Vir surface antigen. One human strain of serotype O55:H- was positive for production of CNF2, but was negative for the two adhesive factors and for mannose-resistant haemagglutination.     

Phosphorylation-Dependent Immunoreactivity of Neurofilaments Increases During Axonal Maturation and β,β''-Iminodipropionitrile Intoxication

The immunoreactivity of the high-molecular-weight neurofilament (NF) subunit toward antibodies that react with phosphorylation-related epitopes was determined at different anatomic sites in the PNS of rats during normal maturation and after intoxication with beta,beta'-iminodipropionitrile (IDPN). A maturational increase in the relative binding of phosphorylation-dependent antibodies compared to phosphorylation-inhibited antibodies occurred from age 3 to 12 weeks. An increase in phosphorylation-related immunoreactivity with increasing distance from the cell bodies was present in ventral and dorsal roots at all ages. The degree of phosphorylation-related immunoreactivity was greater for centrally directed axons in the dorsal roots of the L5 ganglion than for peripherally directed axons. IDPN, a toxin that impairs NF transport, caused a marked increase in reactivity toward the phosphorylation-dependent antibody. NFs from IDPN-treated rats also bound less of an antibody that is normally phosphorylation independent and this inhibition of binding was sensitive to phosphatase digestion. In each instance, greater degrees of phosphorylation-dependent immunoreactivity correlate with conditions known to exhibit slower net rates of axonal transport of NF proteins.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Caffeine Stimulates Amyloid β-Peptide Release from β-Amyloid Precursor Protein-Transfected HEK293 Cells

Abstract: Extracellular amyloid β-peptide (Aβ) deposition is a pathological feature of Alzheimer's disease and the aging brain. Intracellular Aβ accumulation is observed in the human muscle disease, inclusion body myositis. Aβ has been reported to be toxic to neurons through disruption of normal calcium homeostasis. The pathogenic role of Aβ in inclusion body myositis is not as clear. Elevation of intracellular calcium following application of calcium ionophore increases the generation of Aβ from its precursor protein (βAPP). A receptor-based mechanism for the increase in Aβ production has not been reported to our knowledge. Here, we use caffeine to stimulate ryanodine receptor (RYR)-regulated intracellular calcium release channels and show that internal calcium stores also participate in the genesis of Aβ. In cultured HEK293 cells transfected with βAPP cDNA, caffeine (5–10 m M ) significantly increased the release of Aβ fourfold compared with control. These actions of caffeine were saturable, modulated by ryanodine, and inhibited by the RYR antagonists ruthenium red and procaine. The calcium reuptake inhibitors thapsigargin and cyclopiazonic acid potentiated caffeine-stimulated Aβ release. NH₄Cl and monensin, agents that alter acidic gradients in intracellular vesicles, abolished both the caffeine and ionophore effects. Immunocytochemical studies showed some correspondence between the distribution patterns of RYR and cellular βAPP immunoreactivities. The relevance of these findings to Alzheimer's disease and inclusion body myositis is discussed.