Superoxide synthase and dismutase activity of plasma membranes from maize roots

Summary.  Superoxide synthase and superoxide dismutase activity have been monitored in isolated maize (Zea mays) root plasma membranes spectrophotometrically by determination of nitro-blue tetrazolium and cytochrome c reduction, respectively. Superoxide production was induced by NADH and NADPH, with similar kinetics and approaching saturation at 0.06 mM in the case of NADPH and 0.1 mM in the case of NADH, with rates of 18.6 ± 5.0 and 21.8 ± 7.2 nmol/min · mg of protein, respectively. These activities exhibited a broad pH optimum between pH 6.5 and 7.5. Diphenylene iodonium inhibited about 25% (10 μM DPI) and 40% (100 μM DPI) of this activity, imidazole inhibited about 20%, while KCN, a peroxidase inhibitor, did not show any significant inhibition. Superoxide-dismutating activity was shown to occur in the same isolates and depended on the quantity of plasma membrane protein present. Growth of plants on salicylic acid prior to membrane isolation induced a rise in the activity of both of the enzymes by 20–35%, suggesting their coordinated action. Received May 15, 2002; accepted September 30, 2002; published online May 21, 2003 RID="*"     

Redox-related peroxidative responses evoked by methyl-jasmonate in axenically cultured aeroponic sunflower (Helianthus annuus L.) seedling roots

Summary.  Methyl-jasmonate (MeJA) has been proposed to be involved in the evocation of defense reactions, as the oxidative burst in plants, substituting the elicitors or enhancing their effect. 48 h dark- and sterilely cultured (axenic) aeroponic sunflower seedling roots excised and treated with different concentrations of MeJA showed a strong and quick depression of the H⁺ efflux rate, 1.80 μM MeJA totally stopping it for approximately 90 min and then reinitiating it again at a lower rate than controls. These results were wholly similar to those obtained with nonsterilely cultured roots and have been interpreted as mainly based on H⁺ consumption for O₂ ^•− dismutation to H₂O₂. Also K⁺ influx was strongly depressed by MeJA, even transitorily reverting to K⁺ efflux. These results were consistent with those associated to the oxidative burst in plants. MeJA induced massive H₂O₂ accumulation in the middle lamella and intercellular spaces of both the root cap cells and the inside tissues of the roots. The native acidic extracellular peroxidase activity of the intact (nonexcised) seedling roots showed a sudden enhancement (by about 52%) after 5 min of MeJA addition, maintained for approximately 15 min and then decaying again to control rates. O₂ uptake by roots gave similar results. These and other results for additions of H₂O₂ or horseradish peroxidase, diphenylene iodonium, and sodium diethyldithiocarbamate trihydrate to the reaction mixture with roots were all consistent with the hypothesis that MeJA induced an oxidative burst, with the generation of H₂O₂ being necessary for peroxidase activity. Results with peroxidase activity of the apoplastic fluid were in accordance with those of the whole root. Finally, MeJA enhanced NADH oxidation and inhibited hexacyanoferrate(III) reduction by axenic roots, and diphenylene iodonium cancelled out these effects. Redox activities by CN⁻- preincubated roots were also studied. All these results are consistent with the hypothesis that MeJA enhanced the NAD(P)H oxidase of a redox chain linked to the oxidative burst, so enhancing the generation of O₂ ^•− and H₂O₂, O₂ uptake, and peroxidase activity by roots. Received July 12, 2002; accepted October 2, 2002; published online May 21, 2003 RID="*"     

Glucose-6-phosphate dehydrogenase in barley roots: kinetic properties and localisation of the isoforms

Two different isoforms of glucose-6-phosphate dehydrogenase (Glc6PDH; EC 1.1.1.49) have been partially purified from barley (Hordeum vulgare L., cv. Alfeo) roots. The procedure included an ammonium sulfate step, Q-Sepharose and Reactive Blue agarose chromatography, and led to 60-fold and 150-fold purification for the two enzymes, respectively. The Glc6PDH 1 isoform accounts for 17% of total activity of the enzyme in roots, and is very sensitive to the effects of NADP⁺/NADPH ratio and dithiothreitol; the Glc6PDH 2 isoform is less affected by reducing power and represents 83% of the total activity. The isoforms showed distinct pH optima, isoelectric points, K _m for glucose-6-phosphate and a different electrophoretic mobility. The kinetic properties for the two enzymes were affected by ATP and metabolites. Both enzymes are inhibited to different extents by ATP when magnesium is omitted from the assay mixture, whereas the addition of ATP-Mg²⁺ had no effect on Glc6PDH activities. The Glc6PDH isoforms are usually present in the plastids and cytosol of plant cells. To verify the intracellular locations of the enzymes purified from barley roots, Glc6PDH was purified from isolated barley root plastids; this isoform showed kinetic parameters coincident with those found for Glc6PDH 1, suggesting a plastid location; the enzyme purified from the soluble fraction had kinetic parameters resembling those of Glc6PDH 2, confirming that this isoform is present in the cytosol of barley roots. Received: 21 June 2000 / Accepted: 28 July 2000     

Role of extracellular peroxidase in the superoxide production by wheat root cells

Summary Extracellular peroxidase has been shown to contribute to superoxide production in wounded wheat (Triticum aestivum L. cv. Ljuba) root cells. The superoxide-synthesizing system of root cells was considerably inhibited by KCN and NaN₃ and activated by MnCl₂ and H₂O₂. Treatment of roots with salicylic acid and a range of di- and tri-carbonic acids (malic, citric, malonic, fumaric, and succinic acids) stimulated superoxide production in both root cells and extracellular solution. The H₂O₂-stimulated superoxide production in the extracellular solution was much higher when roots were preincubated with salicylic or succinic acid. Exogenous acids enhanced peroxidase activity in the extracellular solution. Pretreatment of root cells with the detergents trypsin and sodium dodecyl sulfate had similar effects on the peroxidase activity. Significant inhibition of both superoxide production and peroxidase activity by diphenylene iodonium suggests that the specificity of the latter as an inhibitor of NADPH oxidase is doubtful. Results obtained indicate that extracellular peroxidase is involved in the superoxide production in wheat root cells. The mobile form of peroxidase can be readily secreted to the apoplastic solution and serve as an emergency enzyme involved in plant wound response.Abbreviations DPI diphenylene iodonium - ECS extracellular solution - ROS reactive oxygen species - SA salicylic acid     

Induction of UDP-Glucose:Salicylic Acid Glucosyltransferase in Oat Roots

A UDP-glucose:salicylic acid 3-O-glucosyltransferase (EC 2.4.1.35) (GTase) from oat (Avena sativa L. cv Dal) root extracts was assayed in vitro using [¹⁴C]salicylic acid (SA) and an ion exchange column to separate SA from β-glucosylsalicylic acid. The GTase, present at a very low constitutive level, was inducible to 23 times the constitutive level. When excised roots were exposed to SA at pH 6.5, the specific activity of the enzyme increased within 1.5 h, peaked after 8 to 10 h, and then declined. The increase in specific activity depended on the concentration of SA in the induction medium. Among 16 phenolics and phenolic derivatives tested, GTase induction showed high specificity toward SA and acetylsalicylic acid. Specific activity of the enzyme was induced to higher levels in roots from 7-d-old seedlings than roots from younger plants. GTase activity was less inducible in basal compared with median or apical root sections. Induction of GTase activity was a result of de novo RNA and protein synthesis. Candidate peptides for the GTase were identified by comparison of two-dimensional electrophoresis gels of proteins labeled with [³⁵S]methionine during incubation of roots in the presence or the absence of SA and a gel of a partially purified GTase preparation.     

Partial purification and characterization of an ascorbate-reducible b-type cytochrome from the plasma membrane of Arabidopsis thaliana leaves

Summary.  The plant plasma membrane (PM) contains more than one b-type cytochrome. One of these proteins has a rather high redox potential (can be fully reduced by ascorbate) and is capable of transporting electrons through the PM. Four genes encoding proteins with considerable homology to the sequences of cytochrome b ₅₆₁ proteins in the animal chromaffin granule membrane have recently been identified in the genome of Arabidopsis thaliana. In order to characterize the cytochrome b ₅₆₁ located in the Arabidopsis PM, first PM vesicles were purified by aqueous polymer two-phase partitioning from the leaves of 9-week-old A. thaliana. PM proteins were solubilized by nonionic detergent, and the fully ascorbate-reducible b-type cytochrome was partially purified by anion-exchange chromatography steps. Potentiometric redox titration of the fraction, containing the fully ascorbate-reducible b-type cytochrome after the first anion-exchange chromatography step, revealed the presence of two hemes with redox potentials of 135 mV and 180 mV, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the fractions containing the fully ascorbate-reducible b-type cytochrome after the second anion-exchange chromatography step revealed the presence of a single polypeptide band at about 120 kDa. However, heat treatment (15 min, 90 °C) before electrophoresis was able to split the 120 kDa band into two bands with molecular masses of about 23 and 28 kDa. These values are lower than the apparent molecular mass for the fully ascorbate-reducible b-type cytochrome purified from Phaseolus vulgaris hypocotyls (about 52 kDa) but are in good agreement with those characteristic for the cytochrome b ₅₆₁ proteins purified from chromaffin granule membranes (about 28 kDa) and the four polypeptides predicted from the Arabidopsis genome (24–31 kDa). Received May 4, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Institute of Biophysics, BRC, Hungarian Academy of Sciences, POB 521, 6701 Szeged, Hungary.     

Identification of uronic acid oxidase in plant peroxidase preparations

Commercial plant peroxidase preparations contained a uronic acid oxidase, separable from the peroxidase activity by ion exchange chromatography. The partially purified enzyme, devoid of peroxidase, oxidized hexuronic acids, with the greatest activity for D-glucuronic acid, whereas other aldoses were not substrates. The immediate products of reaction of D-glucuronic acid with oxygen were hydrogen peroxide and a D-glucarolactone, which was a very strong inhibitor of β-glucuronidase and believed to be the 1,5-lactone. The sensitivity to sulphite inhibition suggests that the enzyme is a flavoprotein.     

Regeneration of lipophilic antioxidants by NAD(P)H:quinone oxidoreductase 1

Summary.  The aim of this work was to study the activity of NAD(P)H:(quinone acceptor) oxidoreductase 1 (EC 1.6.99.2) in the regeneration of lipophilic antioxidants, alpha-tocopherol, and reduced-coenzyme Q analogs. First, we tested whether or not two isoforms of the NAD(P)H:(quinone acceptor) oxidoreductase 1 designated as “hydrophilic” and “hydrophobic” (H. J. Prochaska and P. Talalay, Journal of Biological Chemistry 261: 1372–1378, 1986) show differential enzyme activities towards hydrophilic or hydrophobic ubiquinone homologs. By chromatography on phenyl Sepharose, we purified the two isoforms from pig liver cytosol and measured their reduction of several ubiquinone homologs of different side chain length. We also studied by electron paramagnetic resonance the effect of NAD(P)H:(quinone acceptor) oxidoreductase 1 on steady-state levels of chromanoxyl radicals generated by linoleic acid and lipooxygenase and confirmed the enzyme's ability to protect alpha-tocopherol against oxidation induced with H₂O₂-Fe²⁺. Our results demonstrated that the different hydrophobicities of the isoforms do not reflect different reactivities towards ubiquinones of different side chain length. In addition, electron paramagnetic resonance studies showed that in systems containing the reductase plus NADH, levels of chromanoxyl radicals were dramatically reduced. Morever, in the presence of oxidants, alpha-tocopherol was preserved by NAD(P)H:(quinone acceptor) oxidoreductase 1, supporting our hypothesis that regeneration of alpha-tocopherol may be one of the physiologic functions of this enzyme. Received May 20, 2002; accepted September 20, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Biología Celular, Fisiología e Inmunología, Facultad de Ciencias, Edificio Severo Ochoa, Campus de Rabanales, Universidad de Córdoba, 14014 Córdoba, Spain.     

Improved process for production of recombinant yeast-derived monomeric human G-CSF

The human granulocyte colony-stimulating factor (hG-CSF) was efficiently secreted at high levels in fed-batch cultures of recombinant Saccharomyces cerevisiae. However, the secreted recombinant hG-CSF (rhG-CSF) was shown to exist as large multimers in the culture broth due to strong hydrophobic interaction. It was hardly monomerized even by urea at high concentration. This multimer has been reported to diminish specific receptor-binding activity of hG-CSF and causes undesirable problems in the downstream process. When the rhG-CSF was secreted to extracellular broth in the presence of a non-ionic surfactant (Tween 80) in the culture media, the multimerization of the secreted rhG-CSF was efficiently prevented in the fed-batch cultures. Also, the monomer fraction and secretion efficiency of rhG-CSF were significantly increased at the higher culture pH (6.5). Without using any denaturing agents, the secreted rhG-CSF monomer was easily purified with high recovery yield and purity via a simple purification process under acidic conditions, consisting of diafiltration, cation exchange, and gel filtration chromatography. A lyophilization process devoid of intermonomer aggregation was also designed using effective stabilizing agents. Received: 2 March 1999 / Received revision: 16 April 1999 / Accepted: 23 April 1999     

Iron deficiency enhances the levels of ascorbate,glutathione, and related enzymes in sugar beet roots

Summary.  The effects of Fe deficiency stress on the levels of ascorbate and glutathione, and on the activities of the enzymes ferric chelate reductase, glutathione reductase (EC 1.6.4.2), ascorbate free-radical reductase (EC 1.6.5.4) and ascorbate peroxidase (EC 1.11.1.11), have been investigated in sugar beet (Beta vulgaris L.) roots. Plasma membrane vesicles and cytosolic fractions were isolated from the roots of the plants grown in nutrient solutions in the absence or presence of Fe for two weeks. Plants responded to Fe deficiency not only with a 20-fold increase in root ferric chelate reductase activity, but also with moderately increased levels of the general reductants ascorbate (2-fold) and glutathione (1.6-fold). The enzymes of the ascorbate-glutathione cycle in roots were also affected by Fe deficiency. Glutathione reductase activity was enhanced 1.4-fold with Fe deficiency, associated to an increased ratio of reduced to oxidized glutathione, from 3.1 to 5.2. The plasma membrane fraction from iron-deficient roots showed 1.7-fold higher ascorbate free-radical reductase activity, whereas in the cytosolic fraction the enzyme activity was not affected by Fe deficiency. The activity of the cytosolic hemoprotein ascorbate peroxidase decreased approximately by 50% with Fe deprivation. These results show that sugar beet responds to Fe deficiency with metabolic changes affecting components of the ascorbate-glutathione cycle in root cells. This suggests that the ascorbate-glutathione cycle would play certain roles in the general Fe deficiency stress responses in strategy I plants. Received November 19, 2001; accepted September 30, 2002; published online April 2, 2003 RID="*" ID="*" Correspondence and reprints: Departamento de Nutrición Vegetal, Estación Experimental de Aula Dei, CSIC, Apartado 202, 50080 Zaragoza, Spain.     

Indole-3-acetic acid oxidase and syringaldazine oxidase activities of peroxidase isozymes in soybean root nodules

Many isoperoxidases with indole-3-acetic acid oxidase (IAA) and syringaldazine oxidase activities were detected by polyacrylamide gel electrophoresis in soybean root nodules [ Glycine max (L.) Merrill, cv. Asgrow], detached at the onset of flowering. The kinetics of the two activities were studied with some of the isoperoxidases partially purified by ion exchange chromatography. IAA oxidase activity of the cationic isoforms showed a sigmoidal kinetic behaviour and a higher substrate affinity than the anionic ones, whereas typical saturation kinetics were found with an anionic fraction that contained leghemoglobins. So, nodule IAA oxidase activity may mainly be displayed by the cationic isoforms. These cationic isoperoxidases had high affinity towards syringaldazine and they also may be associated with cell wall rigidification.     

Purification and Characterization of an Extracellular Alkaline Serine Protease with Dehairing Function from <Emphasis Type="Italic">Bacillus pumilus</Emphasis>

An extracellular alkaline serine protease (called DHAP), produced by a Bacillus pumilus strain, demonstrates significant dehairing function. This protease is purified by hydrophobic interaction chromatography, ion exchange, and gel filtration. DHAP had a pI of 9.0 and a molecular weight of approximately 32,000 Dalton. It shows maximal activity at pH 10 and with a temperature of 55°C; the enzyme activity can be completely inhibited by phenylmethylsulfonyl fluoride (PMSF) and diisopropyl fluorophosphates (DFP). The first 20 amino acid residues of the purified DHAP have been determined with a sequence of AQTVPYGIPQIKAPAVHAQG. Alignment of this sequence with other alkaline protease demonstrates its high homology with protease from another B. pumilus strain. Received: 17 April 2002 / Accepted: 24 May 2002     

Degradation of sulfonated azo dyes by the purified lignin peroxidase from <Emphasis Type="Italic">Brevibacillus laterosporus</Emphasis> MTCC 2298

Lignin peroxidase (EC 1.11.1.14) was purified from the Brevibacillus laterosporus MTCC 2298 by ion exchange chromatography. The K_m value of the purified lignin peroxidase (using n-propanol as substrate) was 1.6 mM. The MW of purified enzyme determined with the help of MW-standard markers was approximately 205 kDa. Purity of the enzyme was confirmed by native polyacrylamide gel electrophoresis (PAGE) and the activity staining using a substrate L-DOPA. Sulfonated azo dyes such as Methyl orange and Blue-2B were degraded by the purified lignin peroxidase. Degradation of the dyes was confirmed by HPLC, GC-MS, and FTIR spectroscopy. The mainly elected products of Methyl orange were 4-substituted hexanoic acid (m/z = 207), 4-cyclohexenone lactone cation (m/z = 191), and 4-isopropanal-2, 5-cyclohexa-dienone (m/z = 149) and for Blue-2B were 4-(2-hexenoic acid)-2, 5-cyclohexa-diene-one (m/z = 207; M − 1 = 206) and dehydro-acetic acid derivative (m/z = 223).     

Apoplastic superoxide production and peroxidase activity by intact and excised axenically grown seedling roots of sunflower

Excised and cold-preincubated sunflower seedling roots were compared with intact non-preincubated roots to test the effect of the injury stress and cold preincubation on the oxidative burst measured as apoplastic superoxide (O (2) (.-) ) generation and exocellular peroxidase (ECPOX) activity. Preincubated excised or intact roots released into the medium apoplastic proteins with peroxidase activity. Intact and excised roots responded to methyl jasmonate by an immediate oxidative burst that could not be induced by salicylic acid; both phytohormones also induced a slight and slow O (2) (.-) generation and ECPOX activity on excised roots, when added to the cold preincubation medium. The results with cyanide, azide, SHAM (ECPOX inhibitors) and diphenylene iodonium (inhibitor of trans-plasma membrane NAD(P)H-oxidases (NOX)-respiratory burst oxidase homologue in plants (RBOH), the trans-plasmamembrane nicotinamide adenine dinucleotide phosphate oxidase) are consistent with the hypothesis that different systems may be the origin of O (2) (.-) in intact and excised roots; ECPOX was an important component of them in both, together with NOX-RBOH in intact roots, but in excised roots the last one was replaced by an oxidase sensitive to the same inhibitors as the alternative mitochondrial oxidase. According to our hypothesis, these results could be explained if the electron flux would be deviated to different interconnected plasma membrane-redox systems, with different terminal oxidases, activated by different effectors or stresses.     

Use of azo dye ligand chromatography for the partial purification of a novel extracellular peroxidase from Streptomyces viridosporus T7A

Crude peroxidase preparations from the lignocellulose-degrading actinomycete, Streptomyces viridosporus T7A, were shown to decolorize several azo dye isomers and showed a correlation of dye structure to degradability similar to that shown by fungal Mn-peroxidase, an enzyme not previously described in actinomycetes. Addition of the heme-peroxidase inhibitor KCN did not significantly change the ability of the T7A enzyme(s) to decompose the dyes. These results suggest that T7A may produce a Mn- or other peroxidase with similar substrate specificity to Mn-peroxidase. Affinity chromatography using immobilized azo dye isomers was used for purifying peroxidases from T7A. A significantly purified peroxidase preparation was obtained irrespective of the azo dye used. In comparison, concanavalin A lectin affinity chromatography showed very poor binding and resolution for T7A peroxidases. Azo dye affinity purification gave preparations sufficiently purified to allow amino acid microsequencing for two of the bound proteins. N-terminal amino acid sequences were found to share significant homology with a fungal Mn-peroxidase and actinomycete cellulases. Received: 20 May 1997 / Received revision: 17 December 1997 / Accepted: 2 January 1998     

Purification and characterization of tannase from Paecilomyces variotii: hydrolysis of tannic acid using immobilized tannase

An extracellular tannase (tannin acyl hydrolase) was isolated from Paecilomyces variotii and purified from cell-free culture filtrate using ammonium sulfate precipitation followed by ion exchange and gel filtration chromatography. Fractional precipitation of the culture filtrate with ammonium sulfate yielded 78.7% with 13.6-folds purification, and diethylaminoethyl–cellulose column chromatography and gel filtration showed 19.4-folds and 30.5-folds purifications, respectively. Molecular mass of tannase was found 149.8 kDa through native polyacrylamide gel electrophoresis (PAGE) analysis. Sodium dodecyl sulphate–PAGE revealed that the purified tannase was a monomeric enzyme with a molecular mass of 45 kDa. Temperature of 30 to 50°C and pH of 5.0 to 7.0 were optimum for tannase activity and stability. Tannase immobilized on alginate beads could hydrolyze tannic acid even after extensive reuse and retained about 85% of the initial activity. Thin layer chromatography, high performance liquid chromatography, and ¹H-nuclear magnetic resonance spectral analysis confirmed that gallic acid was formed as a byproduct during hydrolysis of tannic acid.     

Analysis of acid invertase and comparison with acid phosphatase in the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf and Kernan

Fractions of acid invertase and acid phosphatase of the ericoid mycorrhizal fungus Hymenoscyphus ericae (Read) Korf & Kernan were compared by column chromatography and polyacrylamide gel electrophoresis. Acid invertase levels were measured during the exponential phase after 14 days growth in pure culture. Most acid invertase was wall associated (50%) with 41% forming an extracellular fraction and 9% a soluble, cytoplasmic fraction. The wall-bound fraction was partially solubilized by 1 M NaCl, bulked with the extracellular fraction and separated by gel filtration into two acid invertase activity peaks. These peaks corresponded closely to two acid phosphatase activity peaks measured in the same eluates. Anion exchange chromatography under a continuous salt gradient separated the invertase and phosphatase isoforms from each other. Non-denaturing polyacrylamide gel electrophoresis demonstrated that the more active isoforms of each enzyme have different electrophoretic properties and are high mannose-type glycoproteins with a high affinity for the lectin, concanavalin A. The results are discussed in terms of the functional aspects of the two enzymes and their cytochemical localization.     

Production of ligninolytic enzymes and synthetic lignin mineralization by the bird's nest fungus Cyathus stercoreus

Production of ligninolytic enzymes and degradation of ¹⁴C-ring labeled synthetic lignin by the white-rot fungus Cyathus stercoreus ATCC 36910 were determined under a variety of conditions. The highest mineralization rate for ¹⁴C dehydrogenative polymerizates (DHP; 38% ¹⁴CO₂ after 30 days) occurred with 1 mM ammonium tartrate as nitrogen source and 1% glucose as additional carbon source, but levels of extracellular laccase and manganese peroxidase (MnP) were low. In contrast, 10 mM ammonium tartrate with 1% glucose gave low mineralization rates (10% ¹⁴CO₂ after 30 days) but higher levels of laccase and manganese peroxidase. Lignin peroxidase was not produced by C. stercoreus under any of the studied conditions. Mn(II) at 11 ppm gave a higher rate of ¹⁴C DHP mineralization than 0.3 or 40 ppm, but the highest manganese peroxidase level was obtained with Mn(II) at 40 ppm. Cultivation in aerated static flasks gave rise to higher levels of both laccase and manganese peroxidase compared to the levels in shake cultures. 3,4-Dimethoxycinnamic acid at 500 μM concentration was the most effective inducer of laccase of those tested. The purified laccase was a monomeric glycoprotein having an apparent molecular mass of 70 kDa, as determined by calibrated gel filtration chromatography. The pH optimum and isoelectric point of the purified laccase were 4.8 and 3.5, respectively. The N-terminal amino acid sequence of C. stercoreus laccase showed close homology to the N-terminal sequences determined from other basidiomycete laccases. Information on C. stercoreus, whose habitat and physiological requirements for lignin degradation differ from many other white-rot fungi, expands the possibilities for industrial application of biological systems for lignin degradation and removal in biopulping and biobleaching processes. Received: 29 January 1999 / Received revision: 5 July 1999 / Accepted: 9 July 1999     

Biosynthesis of ribosome-inactivating proteins from callus and cell suspension cultures of Mirabilis expansa (Ruiz &Pavon)

 Callus and cell suspension cultures from the little known Andean crop Mirabilis expansa were developed and maintained on Murashige and Skoog medium supplemented with 2,4-dichlorophenoxyacetic acid (1 mg/l) and kinetin (0.1 mg/l). Callus and cell suspension cultures were screened with antibodies raised against ME1 (27.5 kDa) and ME2 (27 kDa), two ribosome-inactivating proteins (RIPs) previously found in roots of M. expansa. A 29-kDa protein found in callus and cell suspensions reacted strongly with ME1 antibodies. The 29-kDa protein, named MEC, was purified to homogeneity by ammonium sulfate precipitation and cation exchange perfusion chromatography. Amino acid N-terminal sequencing revealed close homology between MEC and ME1. The MEC amino acid sequence examined was highly conserved among RIPs from widely different sources. This new RIP was immunolocalized to the cell walls of callus and cell suspension cultures. Received: 20 August 1999 / Revision received: 10 December 1999 / Accepted: 21 December 1999     

Purification and partial characterization of β-1,3-glucanase from <Emphasis Type="Italic">Chaetomium thermophilum</Emphasis>

A thermostable extracellular β-1,3-glucanase from Chaetomium thermophilum was purified to homogeneity by fractional ammonium sulfate precipitation, Pheny1-Sepharose hydrophobic interaction chromatography, ion exchange chromatography on DEAE-Sepharose and gel filtration on Sephacryl S-100. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 76.3 kDa. The enzyme exhibited optimum catalytic activity at pH 6.0 and 60 °C. It was thermostable at 50 °C, and retained 90% activity after 60 min at 60 °C. The half-life at 65 °C, 70 °C and 80 °C was 55 min, 21.5 min, and 5 min, respectively. The N-terminal amino acid sequence (8 residues) of the enzyme was HWLGDIPH. The HPLC analysis showed that the only enzymatic product formed from laminarin by the purified β-1,3-glucanase was glucose, indicating that the enzyme is an exo-β-1,3-glucanase (EC 3.2.1.58).