Intracellular precursors of endo-β-1,4-glucanase in Trichoderma reesei

Abstract Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting was employed to detect intracellular precursors of endo-β-1,4-glucanases (EGs) in Trichoderma reesei QM9414 under conditions of de novo induction by sophorose and de novo carbon catabolite derepression by lactose. Secretion of EGs was always preceded by intracellular accumulation of lower M _r precursors, which became processed to larger M _r forms immediately prior to their extracellular appearance. Treatment of the larger M _r forms with α-mannosidase converted them to forms with the same M _r as the smaller forms, whereas Endo H treatment was without effect. These results are consistent with a requirement of O -linked glycosylation for secretion of EGs by T. reesei .     

Structure and expression properties of the endo-β-1,4-glucanase A gene from the filamentous fungus Aspergillus nidulans

Endo-beta-1,4-glucanase A (EG A) of Aspergillus nidulans was purified to homogeneity, and its genomic gene (eglA) was cloned based on partial amino acid sequences of the purified enzyme and sequenced. The eglA gene comprised 1228 bp with four putative introns and encoded a polypeptide of 326 amino acids bearing high homology to the family A cellulases. The eglA promoter activity in A. nidulans was examined using the A. oryzae Taka-amylase A gene as a reporter. Expression of the reporter gene was induced by carboxymethylcellulose and cellobiose, and repressed by glucose, galactose, mannose, xylose, sorbitol, glycerol and succinate. Lactose neither induced nor repressed the expression.     

Diverse Expression of β1,4-N-Acetylgalactosaminyltransferase Gene in the Adult Mouse Brain

Abstract: Among various tissues of mouse, β1,4- N -acetylgalactosaminyltransferase (GM2/GD2 synthase) gene is expressed predominantly in the brain. Further analysis of the gene expression in the mouse CNS was performed by northern blotting and by enzyme assays using extracts from various parts of the CNS. In situ hybridization was also done to investigate the distribution of cells generating GM2/GD2 synthase. In northern blots, diverse levels of the gene expression were observed, depending on the regions examined. By in situ hybridization, pyramidal cells in the hippocampus, granular cells in dentate gyrus and cerebral cortex, Purkinje cells in cerebellum, and mitral cells in the olfactory bulb expressed high levels of the mRNA; these results corresponded to the results obtained by northern blot. Enzyme levels in these sites were accordingly high. However, enzyme levels in certain areas with low mRNA intensities, such as thalamus and pons medulla, were higher than expected from the results of northern blotting. The significance of the high gene expression in certain areas for brain function and the reason for the discrepancy between mRNA level and enzyme activity in some regions are discussed.     

Purification and characterization of thermostable β-1,3-1,4 glucanase from<Emphasis Type="Italic">Bacillus</Emphasis> sp. A8-8

In this study, the extracellular enzyme activity ofBacillus sp. A8-8 was detected on LB agar plates containing 0.5% of the following substrates: carboxymethylcellulose (CMC), xylan, cellulose, and casein, respectively. The β-1,3-1,4 glucanase produced fromBacillus sp. A8-8 was purified by ammonium sulfate and hydrophobic chromatography. The molecular size of the protein was estimated by SDS-PAGE as approximately 33 kDa. The optimum pH and temperature for the enzyme activity were 6.0 and 60°C, respectiveley. However, enzyme activity was shown over a broad range of pH values and temperatures. The purified β-1,3-1,4 glucanase retained over 70% of its original activity after incubation at 80°C for 2 h, and showed over 40% of its original activity within the pH range of 9 to 12. This suggests that β-1,3-1,4 glucanase fromBacillus sp. A8-8 is thermostable and alkalistable. In addition, β-1,3-1,4 glucanase had higher substrate specificity to lichenan than to CMC. Finally the activity of the endoglucanase was inhibited by Fe³⁺, Mg²⁺, and Mn²⁺ ions. However Co²⁺ and Ca²⁺ ions were increased its activity. These authors contributed equally to this work.     

Production of several isoforms of β-1,4-glucanase by the cyanolichen Peltigera canina

Lichen cellulase may participate in the degradation of the external substrata and/or modification of the photobiont cell wall. To promote a better understanding of the roles of cellulases in lichens, a cyanolichen was chosen because of the absence of cellulose in its symbionts. Freshly-collected thalli of Peltigera canina (L.) Wild, produce β-1,4-glucanase (EC 3.2.1.4, β-1,4-D-glucanohydrolase). This enzyme's activity was detected in the soluble and cell wall fractions and it was found to be secreted to the incubation medium when thalli were floated on water or on cellobiose. Several forms of the enzyme were detected by isoelectrofocusing. In preparative isoelectrofocusing, a single peak was obtained in each fraction, characterized by pls of 5.05, 5.25 and 4.75 in the soluble, cell wall and medium fractions, respectively. These differences were in agreement with the different pattern of bands obtained in slab-isoelectrofocusing, where the most acidic band (pl of 4.45) was present only in the soluble fraction and the band with higher pl (6.17) was more intense in the cell wall fraction. Since both symbionts in a cyanolichen lack cellulose, cellulases cannot participate in the modification of their cell wall; the presence of cellulase in Peltigera canina must therefore be related to the degradation of the tissues of the moss substratum.     

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.     

Cloning and characterization of a novel member of human β-1,4-galactosyltransferase gene family

By using the EST strategy for identifying novel members belonging to homologous gene families, a novel fulklength cDNA encoding a protein significantly homologous to UDP-Gal: N-acetylglucosamine β-1, 4-galactosyltransferase (GalT) was isolated from a human testis cDNA library. A nucleotide sequence of 2 173 bp long was determined to contain an open reading frame of 1 032 nucleotides (344 amino acids). In view of the homology to memben of the galactosyltransferase gene family and especially the closest relationship toGallus gallus GalT type I (CK I), the predicted product of the novel cDNA was designated as human β-1,4-galactosyltransferase homolog I (HumGT-H1). Its mRNA is present in different degrees in 16 tissues examined. Southern analysis of human genomic DNA revealed its locus on chromosome 3. Poject supported by the 863 High-technology Program, the National Outstanding Young Scientist Foundation and the National Natural Science Foundation of China (Grant No. 39680019).     

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.     

Early Effects of β,β'-Iminodipropionitrile on Tubulin Solubility and Neurofilament Phosphorylation in the Axon

Abstract: To elucidate the role of neurofilaments in microtubule stabilization in the axon, we studied the effects of β,β'-iminodipropionitrile (IDPN) on the solubility and transport of tubulin as well as neurofilament phosphorylation in the motor fibers of the rat sciatic nerve. IDPN is known to impair the axonal transport of neurofilaments, causing accumulation of neurofilaments in the proximal axon and segregation of neurofilaments to the peripheral axoplasm throughout the nerve. Administration of IDPN at various intervals after radioactive labeling of the spinal cord with l -[³⁵S]methionine revealed that transport inhibition occurred all along the nerve within 1–2 days. Transport of cold-insoluble tubulin, which accounts for 50% of axonal tubulin, was also affected. A significant increase in the proportion of cold-soluble tubulin was observed, reaching a maximum at 3 days after IDPN treatment and returning to the control level in the following weeks. Preceding this change in tubulin solubility, a transient decrease in the phosphorylation level of the 200-kDa neurofilament protein was detected in the ventral root using phosphorylation-dependent antibodies. These early changes agreed in timing with the onset of segregation and transport inhibition, suggesting that interaction between neurofilaments and microtubules possibly regulated by phosphorylation plays a significant role in microtubule stabilization.     

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.     

Cationic and Anionic Requirements for the Binding of 2β-Carbomethoxy-3β-(4-Fluorophenyl)[3H]tropane to the Dopamine Uptake Carrier

Abstract: The present study reports the ion dependency of 2β-carbomethoxy-3β-(4-fluorophenyl)[³H]tropane ([³H]- CFT) binding to the dopamine transporter in the rat striaturn. The results indicate that [³H]CFT binding to synaptosomal P₂ membranes requires low concentrations of Na⁺ (peak binding between 20 and 50 m M Na⁺), is stimulated by phosphate anion or l^-, but is unaffected or only slightly affected by F^-, Cl^-, Br^-, NO₃^-, or SO₄^2-, Concentrations of Na⁺ of >50 m M become inhibitory except in the presence of l^-, which shifts peak binding levels toward higher Na⁺ concentrations and also elevates the peak binding level. K⁺ strongly decreased [³H]CFT binding with a shallow inhibition curve, and Na⁺ could not overcome this effect. Saturation analysis of [³H]CFT binding revealed a single binding site changing its affinity for CFT depending on the concentration of sodium phosphate buffer (6, 10, 30, 50, 130, or 200 m M ; 1 mM plus 49 mM NaCIversus 10 m M plus 40 m M NaCI; or 1 mM plus 129 m M Nal versus 10 m M plus 120 m M Nal). No differences were observed in the density of CFT binding sites between any of the conditions examined.     

Characterization of an unglycosylated low molecular weight 1,4-β-glucan-glucanohydrolase of Trichoderma reesei

A low molecular weight endoglucanase (1,4-beta-glucan glucanohydrolase E.C.3.2.1.4) was purified to homogeneity by a two-step procedure from 7 day old culture filtrates of Trichoderma reesei. The endoglucanase was obtained by BioGel A 0.5 m gel chromatography followed by preparative PAGIF. The purified endoglucanase was homogeneous upon titration curve separation. Enzyme characteristics were: Mr 25 kDa, pI 7.5. The amino acid composition is predominantly neutral (mainly glycine). The N-terminus is arginine. The pH-optimum for this endoglucanase was 5.8 and its optimal temperature was at 52 degrees C. The activity of this endoglucanase gave a strong increase in CMC-fluidity with only a small release of reducing sugars. The endoglucanase was 0.2% of total culture medium protein content. The reducing sugars upon CMC digestion were G1-G4. The enzyme had no specificity towards crystalline cellulose (Avicel) or xylan. The endoglucanase is not a glycoprotein.     

Action of β-N-Oxalylamino-l-Alanine on Mouse Brain NADH-Dehydrogenase Activity

Abstract: β- N -Oxalylamino- l -alanine ( l -BOAA), a non-protein neuroexcitatory amino acid present in the seeds of Lathyrus sativus (chickling or grass pea), is known to produce its neurotoxic effects by overstimulation of non- N -methyl- d -aspartate receptors, especially α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors, at micromolar concentrations. It has recently been reported that l -BOAA selectively inhibits mitochondrial enzyme NADH-dehydrogenase (NADH-DH) in brain slices at subpicomolar concentrations. The present study finds that up to 4 m M concentrations of pure l -BOAA fail to inhibit NADH-DH activity in mouse brain homogenate and isolated brain mitochondria. Two known inhibitors (rotenone and 1-methyl-4-phenylpyridinium ion, MPP⁺) of this mitochondrial enzyme produced significant inhibition under identical conditions. NADH-DH inhibition was also not observed in the homogenate or mitochondria from the brains of animals systemically treated with convulsive doses of l -BOAA. Some inhibition (20–37%) of NADH-DH activity was observed in mouse brain slices incubated with 100–1,000 µ M concentrations of l -BOAA for 1 h at 37°C in an atmosphere of 95% O₂ and 5% CO₂, but the inhibition was nonselective, because the activity of another mitochondrial enzyme, succinic dehydrogenase, was similarly inhibited by l -BOAA. These results are in contrast with the report that l -BOAA inhibits mitochondrial NADH-DH selectively at subpicomolar concentrations. We suggest the observed nonselective NADH-DH inhibition in mouse brain slices treated with l -BOAA is caused by neuronal damage through an excitotoxic mechanism.     

Evaluation of β-1,3-glucanase and β-1,4-glucanase enzymes production in some Trichoderma species

Trichoderma species have become the important means of biological control for fungal diseases. This research was carried on to access the high β-1,3-glucanase and β-1,4-glucanase enzyme producer of Trichoderma species isolates using two different carbon sources for finding a method to obtain more concentrate culture filtrates. Therefore, 14 Trichoderma isolates belonging to species: Trichoderma ceramicum, T. virens, T. pseudokoningii, T. koningii, T. koningiosis, T. atroviridae, T. viridescens, T. asperellum, T. harzianum1, T. orientalis, T. harzianum2, T. brevicompactum, T. viride and T. spirale were cultured in Wiendling’s liquid medium plus 0.5% glycerol or 0.5% Phytophthora sojae-hyphe as the carbon source in shaking and non-shaking (stagnant) statuses. Enzyme activity rate and total protein were evaluated in raw, acetony and lyophilized concentrated culture filtrates and the specific enzyme activity of β-1,3-glucanase and β-1,4-glucanase were measured by milligramme glucose equivalent released per minute per milligramme total protein in culture filtrates. The results showed that using Phytophthora – hyphe in medium increased the enzyme activities as compared to glycerol at all Trichoderma species which suggested that these substrates can also act as inducer for synthesis of lytic enzymes, in addition the most enzymes activity was observed in the lyophilised concentrated culture filtrate. The most successful species in β-1,3-glucanase and β-1,4-glucanase enzymes activities were T. brevicompactum and T. virens and these species can be used for mass production of these enzymes which are supposed to be used in commercial formulation and also will be able to control P. sojae directly.     

Inhibition of Tyrosine Aminotransferase by β-N-Oxalyl-l-α,β-Diaminopropionic Acid, the Lathyrus sativus Neurotoxin

Abstract: Species differences in susceptibility are a unique feature associated with the neurotoxicity of β-N-oxalyl-l -α,β-diaminopropionic acid (l -ODAP), the Lathyrus sativus neurotoxin, and the excitotoxic mechanism proposed for its mechanism of toxicity does not account for this feature. The present study examines whether neurotoxicity of l -ODAP is the result of an interference in the metabolism of any amino acid and if it could form the basis to explain the species differences in susceptibility. Thus, Wistar rats and BALB/c (white) mice, which are normally resistant to l -ODAP, became susceptible to it following pretreatment with tyrosine (or phenylalanine), exhibiting typical neurotoxic symptoms. C57BL/6J (black) mice were, however, normally susceptible to l -ODAP without any pretreatment with tyrosine. Among the various enzymes associated with tyrosine metabolism examined, the activity of only tyrosine aminotransferase (TAT) was inhibited specifically by l -ODAP. The inhibition was noncompetitive with respect to tyrosine (K_i = 2.0 ± 0.1 mM) and uncompetitive with respect to α-ketoglutarate (K_i = 8.4 ± 1.5 mM). The inhibition of TAT was also reflected in a marked decrease in the rate of oxidation of tyrosine by liver slices, an increase in tyrosine levels of liver, and also a twofold increase in the dopa and dopamine contents of brain in l -ODAP-injected black mice. The dopa and dopamine contents in the brain of only l -ODAP-injected white mice did not show any change, whereas levels of these compounds were much higher in tyrosine-pretreated animals. Also, the radioactivity associated with tyrosine, dopa, and dopamine arising from [¹⁴C]tyrosine was twofold higher in both liver and brain of l -ODAP-treated black mice. Thus, a transient increase in tyrosine levels following the inhibition of hepatic TAT by l -ODAP and its increased availability for the enhanced synthesis of dopa and dopamine and other likely metabolites (toxic?) resulting therefrom could be the mechanism of neurotoxicity and may even underlie the species differences in susceptibility to this neurotoxin.     

Degradation and solubilization of pectin by β-galactosidases purified from avocado mesocarp

Three β-galactosidase (EC 3. 2. 1. 23) isozymes were purified from the mesocarp of ripe fruit of Persea americana Mill. cv. Lula. and their effects on pectin derived from mature-green tomato fruit were investigated. The β-galactosidases had pl values of 5. 0,5. 1 and 5. 2. and molecular weights of 41. 49 and 54 KDa. respectively. There was a partial degradation of pectin resulting in the release of monomeric galactose upon treatment with avocado β-galactosidase. This degradation resulted in increased pectin solubility and decreased apparent average molecular size as determined by microfiltration and gel permeation by high-performance liquid chromatography. The increase in solubility was due. in part. to an apparent decrease in the ability of pectin molecules to aggregate together.     

Purification and characterization of an extracellular endo-1,4-β-xylanase from Fusarium oxysporum f. sp. melonis

Abstract Fusarium oxysporum f. sp. melonis produces extracellular endo-1,4-β-xylanase and β-xylosidase when grown in shaken culture at 26°C in a mineral salts medium containing oat spelt xylan and glucose as carbon sources. Endo-1,4-β-xylanase was purified 251 times from 5-day-old culture filtrates, by Sephacryl S-200, ion exchange and gel filtration HPLC. The purified sample yielded a single band in SDS polyacrylamide gels with a molecular mass of 80 kDa on electrophoretic mobility and 83 kDa by gel filtration behavior. High activity of the endo-1,4-β-xylanase against xylan was observed between 5 and 8 pH, and between 40 and 60°C, the optimum pH and temperature being 5.0 and 50°C, respectively. Kinetic properties of the enzyme are similar to those of other fungal xylanases, showing high affinity towards oat spelt xylan with a K _m of 1 mM expressed as xylose equivalent.