Purification and characterization of naringinase from a newly isolated strain of <Emphasis Type="Italic">Aspergillus niger</Emphasis> 1344 for the transformation of flavonoids

Summary An extracellular naringinase (an enzyme complex consisting of α-L-rhamnosidase and β-D-glucosidase activity, EC 3.2.1.40) that hydrolyses naringin (a trihydroxy flavonoid) for the production of rhamnose and glucose was purified from the culture filtrate of Aspergillus niger 1344. The enzyme was purified 38-fold by ammonium sulphate precipitation, ion exchange and gel filtration chromatography with an overall recovery of 19% with a specific activity of 867 units per mg of protein. The molecular mass of the purified enzyme was estimated to be about 168 kDa by gel filtration chromatography on a Sephadex G-200 column and the molecular mass of the subunits was estimated to be 85 kDa by sodium dodecyl sulphate-Polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme had an optimum pH of 4.0 and temperature of 50 °C, respectively. The naringinase was stable at 37 °C for 72 h, whereas at 40 °C the enzyme showed 50% inactivation after 96 h of incubation. Hg²⁺, SDS, p-chloromercuribenzoate, Cu²⁺ and Mn²⁺ completely inhibited the enzyme activity at a concentration of 2.5–10 mM, whereas, Ca²⁺, Co²⁺ and Mg²⁺ showed very little inactivation even at high concentrations (10–100 mM). The enzyme activity was strongly inhibited by rhamnose, the end product of naringin hydrolysis. The enzyme activity was accelerated by Mg²⁺ and remained stable for one year after storage at −20 °C. The purified enzyme preparation successfully hydrolysed naringin and rutin, but not hesperidin.     

Characterization of a naringinase from Aspergillus oryzae 11250 and its application in the debitterization of orange juice

Naringinase plays a rather important role in reducing the bitterness of juice by hydrolyzing naringin. A novel extracellular naringinase was purified from Aspergillus oryzae 11250 cultured in the presence of orange peel. A 26.78-fold purification rate was achieved by salt-induced precipitation, followed by anion-exchange and gel filtration chromatography with 32% recovery and specific activity of 2194.62 units per mg protein (U/mg). The optimum pH and temperature for naringinase activity were 5.0 and 45 °C, respectively. This enzyme was stable at 30 °C for 5 h. The K_m and V_max of naringinase toward naringin determined by Lineweaver-Burk method were 1.60 ± 0.13 mM and 126.21 ± 5.52 μmol/(min mg), respectively. The enzyme activity was inhibited completely by Ag⁺ at 10 mM. Naringinase is capable of hydrolyzing naringin, neohesperidin, and some other glycosides. A supplement of 6 U/mL of this naringinase in citrus juice sufficiently removed naringin to relieve the bitterness of citrus juice. These properties make the enzyme an ideal candidate for commercial application in the debitterization of orange juice.     

Purification and properties of extracellular phytase from Bacillus sp. KHU-10

Bacillus species producing a thermostable phytase was isolated from soil, boiled rice, and mezu (Korean traditinal koji). The activity of phytase increased markedly at the late stationary phase. An extracellular phytase from Bacillus sp. KHU-10 was purified to homogeneity by acetone precipitation and DEAE-Sepharose and phenyl-Sepharose column chromatographies. Its molecular weight was estimated to be 46 kDa on gel filtration and 44 kDa on SDS-polyacrylamide gel elctrophoresis. Its optimum pH and temperature for phytase activity were pH 6.5-8.5 and 40°C without 10 mM CaCl₂ and pH 6.0-9.5 and 60°C with 10 mM CaCl₂. About 50% of its original activity remained after incubation at 80°C or 10 min in the presence of 10 mM CaCl₂. The enzyme activity was fairly stable from pH 6.5 to 10.0. The enzyme had an isoelectric point of 6.8. As for substrate specificity, it was very specific for sodium phytate and showed no activity on other phosphate esters. The K _m value for sodium phytate was 50 M. Its activity was inhibited by EDTA and metal ions such as Ba²⁺, Cd²⁺, Co²⁺, Cr³⁺, Cu²⁺, Hg²⁺, and Mn²⁺ ions.     

Purification and characterization of a high molecular weight endoxylanase from the solid-state culture of an alkali-tolerant Aspergillus fumigatus MKU1

Summary A high molecular weight endoxylanase (XylF2) from the solid state culture of Aspergillus fumigatus MKU1 was purified to homogeneity by a combination of tube gel electrophoresis and electroelution methods. The purity was demonstrated by SDS-PAGE and the molecular mass of the XylF2 was found to be 66 kDa. The optimal pH and temperature for activity were 5.0 and 90 °C, respectively. The apparent K _m and V _max values of XylF2 with oat spelt xylan as substrate were 1.6 mg/ml and 3.25 mmol/min/mg protein respectively. The enzyme showed high activity towards oat spelt xylan while negligible activity was observed on carboxymethylcellulose. The activity of XylF2 was strongly inhibited by Hg²⁺, Ni²⁺, Zn²⁺, SDS and N-bromosuccinimide and stimulated by l-cysteine and iodoacetamide. The hydrolysis of oat spelt xylan by XylF2 released only xylo-oligosaccharides.     

Purification and properties of a cyanide-degrading enzyme from Burkholderia cepacia strain C-3

A cyanide-hydrolysing enzyme from Burkholderia cepacia strain C-3 isolated from soil was purified to electrophoretic homogeneity by ammonium sulphate precipitation and column chromatography on HiTrap Q (DEAE-agarose) and phenyl-Sepharose HP. The enzyme was purified 48-fold with a 0.8% yield and a final specific activity of 26.8 u/mg protein. The purified enzyme was observed as a single polypeptide band of molecular mass 38 kDa during both denaturing and non-denaturing gel electrophoresis. Enzymatic activity was optimal at pH 8.0–8.5 and at 30–35 °C. Activity was stimulated by Mo²⁺, Sn²⁺, and Zn²⁺, and inhibited by Al³⁺, Co²⁺, Cu²⁺ and Hg²⁺. The enzyme was specific for cyanide and thiocyanate with formate and ammonia as the main products from KCN degradation. Its K _m and V _max values were 1.4 mM and 15.2 u/mg protein, respectively. Apparent substrate inhibition occurred at cyanide concentrations greater than 2 mM.     

Solid‐state fermentation for the production of debittering enzyme naringinase using Aspergillus niger MTCC 1344

Aspergillus niger produced high levels of naringinase using easily available, inexpensive industrial waste residues such as rice bran, wheat bran, sugar cane bagasse, citrus peel, and press mud in solid‐state fermentation (SSF). Among these, rice bran was found to be the best substrate. Naringinase production was highest after 96 h of incubation at 27°C and at a substrate‐to‐moisture ratio of 1:1 w/v. Supplementation of the medium with 10% naringin caused maximum induction. An inoculum age of 72 h and an inoculum level of 15% resulted in maximum production of naringinase. Enzyme production was stimulated by the addition of nutrients such as naringin and peptone. Thus, A. niger produced a very high level of naringinase within a short time in solid‐state fermentation using inexpensive agro‐residues, a level that is much higher than reported for any other microbes.     

A novel highly acidic β-mannanase from the acidophilic fungus Bispora sp. MEY-1: gene cloning and overexpression in Pichia pastoris

Using degenerate polymerase chain reaction (PCR) and thermal asymmetric interlaced PCR, a 1,347-bp full-length complementary DNA fragment encompassing the gene man5A, which encodes a 429-amino acid β-mannanase with a calculated mass of 46.8 kDa, was cloned from acidophilic Bispora sp. MEY-1. The deduced amino acid sequence (catalytic domain) displayed highest identity (54.1%) with the Emericella nidulans endo-β-1,4-d-mannanase, a member of the glycoside hydrolase family 5. Recombinant MAN5A was overexpressed in Pichia pastoris, and its activity in the culture medium reached 500 U ml⁻¹. The enzyme was acidophilic, with highest activity at pH 1.0–1.5, lower than any known mannanases, and optimal temperature for activity was 65°C. MAN5A had good pH adaptability, excellent thermal and pH stability, and high resistance to both pepsin and trypsin. The specific activity, K _m, and V _max for locust bean gum substrate was 3,373 U mg⁻¹, 1.56 mg ml⁻¹, and 6,587.6 μmol min⁻¹ mg⁻¹, respectively. The enzymatic activity was not significantly affected by ions such as Ca²⁺, Cr³⁺, Co²⁺, Zn²⁺, Na⁺, K⁺, and Mg²⁺ and enhanced by Ni²⁺, Fe³⁺, Mn²⁺ and Ag⁺. These favorable properties make MAN5A a potential candidate for use in various industrial applications.     

Peroxidase from Bacillus sp. VUS and its role in the decolorization of textile dyes

Peroxidase was purified by an ion exchange chromatography followed by gel filtration chromatography from dye degrading Bacillus sp. strain VUS. The optimum pH and temperature of the enzyme activity was 3.0 and 65°C, respectively. This enzyme showed more activity with n-propanol than other substrates tested viz. xylidine, 3-(3,4-dihydroxy phenyl) Lalanine (L-DOPA), hydroxyquinone, ethanol, indole, and veratrole. Km value of the enzyme was 0.076 mM towards n-propanol under standard assay conditions. Peroxidase was more active in presence of the metal ions like Li²⁺, Co²⁺, K²⁺, Zn²⁺, and Cu²⁺ where as it showed less activity in the presence of Ca²⁺ and Mn²⁺. Inhibitors like ethylenediamine tetraacetic acid (EDTA), glutamine, and phenylalanine inhibited the enzyme partially, while sodium azide (NaN₃) completely. The crude as well as the purified peroxidase was able to decolourize different industrial dyes. This enzyme decolourized various textile dyes and enhanced percent decolourization in the presence of redox mediators. Aniline was the most effective redox mediator than other mediators tested. Gas chromatography-Mass spectrometry (GC-MS) confirmed the formation of 7-Acetylamino-4-hydroxy-naphthalene-2-sulphonic acid as the final product of Reactive Orange 16 indicating asymmetric cleavage of the dye.     

α-Rhamnosidase and β-glucosidase expressed by naringinase immobilized on new ionic liquid sol-gel matrices: Activity and stability studies

Novel ionic liquid (IL) sol-gel materials development, for enzyme immobilization, was the goal of this work. The deglycosylation of natural glycosides were performed with α-l-rhamnosidase and β-d-glucosidase activities expressed by naringinase. To attain that goal ILs with different structures were incorporated in TMOS/Glycerol sol-gel matrices and used on naringinase immobilization.The most striking feature of ILs incorporation on TMOS/Glycerol matrices was the positive impact on the enzyme activity and stability, which were evaluated in fifty consecutive runs. The efficiency of α-rhamnosidase expressed by naringinase TMOS/Glycerol@ILs matrices increased with cation hydrophobicity as follows: [OMIM] > [BMIM] > [EMIM] > [C₂OHMIM] > [BIM] and [OMIM] ≈ [E₂-MPy] ? [E₃-MPy]. Regarding the imidazolium family, the hydrophobic nature of the cation resulted in higher α-rhamnosidase efficiencies: [BMIM]BF₄ ? [C₂OHMIM]BF₄ ? [BIM]BF₄. Small differences in the IL cation structure resulted in important differences in the enzyme activity and stability, namely [E₃-MPy] and [E₂-MPy] allowed an impressive difference in the α-rhamnosidase activity and stability of almost 150%. The hydrophobic nature of the anion influenced positively α-rhamnosidase activity and stability. In the BMIM series the more hydrophobic anions (PF₆⁻, BF₄⁻ and Tf₂N⁻) led to higher activities than TFA. SEM analysis showed that the matrices are shaped lens with a film structure which varies within the lens, depending on the presence and the nature of the IL.The kinetics parameters, using naringin and prunin as substrates, were evaluated with free and naringinase encapsulated, respectively on TMOS/Glycerol@[OMIM][Tf₂N] and TMOS/Glycerol@[C₂OHMIM][PF₆] and on TMOS/Glycerol. An improved stability and efficiency of α-l-rhamnosidase and β-glucosidase expressed by encapsulated naringinase on TMOS/Glycerol@[OMIM][Tf₂N] and TMOS/Glycerol@[C₂OHMIM][PF₆] were achieved. In addition to these advantageous, with ILs as sol-gel templates, environmental friendly processes can be implemented.     

PRODUCTION OF NARINGINASE FROM A NEW SOIL ISOLATE,Bacillus methylotrophicus: ISOLATION,OPTIMIZATION AND SCALE-UP STUDIES

Five strains of naringin-degrading bacteria were isolated and found to be positive for extracellular naringinase activity. The one that showed highest activity in the selective medium was identified by 16S rRNA analysis as Bacillus methylotrophicus. The best combination of carbon–nitrogen source was determined by employing two-level full factorial analyses, comprising 24 experiments in shake flasks. Sucrose–yeast extract showed significant increase in naringinase activity (7.46 U/L) compared to the basal medium. Naringinase production was found to be inducible and naringin was found to be the best inducer among naringin, naringenin, hesperidin, and L-rhamnose. Inoculum size of 2% (v/v) and age of 48 hr favored naringinase and biomass production. Highest naringinase activity of 8 U/L was observed at the initial medium pH of 6. Response surface modeling was applied based on central composite design to determine the effects of three independent variables (sucrose, yeast extract, and naringin) and their mutual interactions. In total, 20 experiments were conducted and a statistical model was developed, which predicted naringinase production of 10.61 U/L. Subsequently, verification experiments were conducted and validity of the model was verified. Bioreactor studies conducted with the optimized medium showed an enzyme production of 12.05 U/L within 34 hr of fermentation.     

Production,partial purification and characterization of α-<Emphasis Type="SmallCaps">l</Emphasis>-rhamnosidase from <Emphasis Type="Italic">Penicillium ulaiense</Emphasis>

Penicillium ulaiense is a post-harvest pathogenic fungus that attacks citrus fruits. The objective of this work was to study this microorganism as an α-l-rhamnosidase producer and to characterize it from P. ulaiense. The enzyme under study is used for different applications in food and beverage industries. α-l-Rhamnosidase was produced in a stirred-batch reactor using rhamnose as the main carbon source. The kinetic parameters for the growth of the fungi and for the enzyme production were calculated from the experimental values. A method for partial purification, including (NH₄)₂SO₄ precipitation, incubation at pH 12 and DEAE-sepharose chromatography yielded an enzyme with very low β-glucosidase activity. The pH and temperature optima were 5.0 and 60°C, respectively. The Michaelis–Menten constants for the hydrolysis of p-nitrophenyl-α-l-rhamnoside were V _max = 26 ± 4 IU ml⁻¹ and K _m  = 11 ± 2 mM. The enzyme showed good thermostability up to 60°C and good operational stability in white wine. Co²⁺ affected positively the activity; EDTA, Mn²⁺, Mg²⁺, dithiotreitol and Cu²⁺ reduced the activity by different amounts, and Hg²⁺ completely inhibited the enzyme. The enzyme showed more activity on p-nitrophenyl-α-l-rhamnoside than on naringin. According to these results, this enzyme has potential for use in the food and pharmacy industries since P. ulaiense does not produce mycotoxins.     

Purification and properties of oxaloacetate decarboxylase fromCorynebacterium glutamicum

Oxaloacetate (OAA) decarboxylase (E.C. 4.1.1.3) was isolated fromCorynebacterium glutamicum. In five steps the enzyme was purified 300-fold to apparent homogeneity. The molecular mass estimated by gel filtration was 118 ± 6 kDa. SDS-PAGE showed a single subunit of 31.7 KDa, indicating an ₄ subunit structure for the native enzyme. The enzyme catalyzed the decarboxylation of OAA to pyruvate and CO₂, but no other -ketoacids were used as substrate. The cation Mn²⁺ was required for full activity, but could be substituted by Mg²⁺, Co²⁺, Ni²⁺ and Ca²⁺. Monovalent ions like Na⁺, K⁺ or NH ₄ ⁺ were not required for activity. The enzyme was inhibited by Cu²⁺, Zn²⁺, ADP, coenzyme A and succinate. Avidin did not inhibit the enzyme activity, indicating that biotin is not involved in decarboxylation of OAA. Analysis of the kinetic properties revealed a K _m for OAA of 2.1 mM and a K _m of 1.2 mM for Mn²⁺. The V _max was 158 µmol of OAA converted per min per mg of protein, which corresponds to an apparent k _cat of 311 s^–1.Abbreviations OAA oxaloacetate - LDH lactate dehydrogenase     

Production and characterization of extracellular protease of mutant Aspergillus niger AB100 grown on fish scale

Fish scale, the chief waste material of fish processing industries was processed and tested for production of extracellular protease by mutant Aspergillus niger AB₁₀₀. Protease production by A. niger AB₁₀₀ was greatly enhanced in presence of processed fish scale powder. Where as among the three complex nutrients tested, soya bean meal shows maximum stimulatory effect over protease production (2,776 μmol/ml/min) when used in combination with glucose (5% w/v) and urea (2.5% w/v). The protease was optimally active at pH 7.0, retaining more than 60% of its activity in the pH range of 5–9. The enzyme was found to be most active at 50°C and stable at 30°C for 1 h. Purification of enzyme by CM-Cellulose and SDS-PAGE resulted in about 26-fold increase in the specific activity of the enzyme with a molecular weight of 30.9 kDa. HPLC study shows the purity of the enzyme as 75.92%. By the activating effect of divalent cations (Fe²⁺, Zn²⁺, Mn²⁺, Ca²⁺and Mg²⁺) and inhibiting effect of chelating agent (EDTA) and Hg²⁺, the enzyme was found to be a metalloprotease.     

An alkaline protease from Bacillus circulans BM15, newly isolated from a mangrove station: characterization and application in laundry detergent formulations

An investigation on the properties of an alkaline protease secreted by Bacillus circulans BM15 strain isolated from a mangrove sediment sample was carried out in order to characterize the enzyme and to test its potency as a detergent additive. The protease was purified to apparent homogeneity by ammonium sulphate precipitation and was a 30-kDa protease as shown by SDS-PAGE and its proteolytic activity was detected by casein zymography. It had optimum activity at pH 7, was stable at alkaline pH range (7 to 11), had optimum temperature of activity 40°C and was stable up to a temperature of 55°C after incubation for one hour. Hg²⁺, Zn²⁺, Co²⁺, and Cu²⁺completely inhibited the enzyme activity, while Ca²⁺, Mg²⁺, K⁺ and Fe³⁺ were enhancing the same. The serine protease inhibitor PMSF and metal chelator EDTA inhibited the activity of this protease while the classic metalloprotease inhibitor 1, 10 phenanthroline did not show inhibition. The enzyme was stable in SDS, Triton-X-100 and H₂ O₂ as well as in various commercial detergents after incubation for one hour. The extracellular production of the enzyme, the pH and temperature stability and stability in presence of oxidants, surfactants and commercial detergents suggest its possible use as a detergent additive.     

Expression,purification and characterization of pectate lyase A from <Emphasis Type="Italic">Aspergillus nidulans</Emphasis> in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Pectate lyase A (PelA) of Aspergillus nidulans was successfully expressed in Escherichia coli and effectively purified using a Ni²⁺-nitrilotriacetate-agarose column. Enzyme activity of the recombinant PelA could reach 360 U ml⁻¹ medium. The expressed PelA exhibited its optimum level of activity over the range of pH 7.5–10 at 50°C. Mn²⁺, Ca²⁺, Fe²⁺, Mg²⁺ and Fe³⁺ ions stimulated the pectate lyase activity, but Cu²⁺ and Zn²⁺ inhibited it. The recombinant PelA had a V _max of 77 μmol min⁻¹ mg⁻¹ and an apparent K _m of 0.50 mg ml⁻¹ for polygalacturonic acid. Low-esterified pectin was the optimum substrate for the PelA, whereas higher-esterified pectin was hardly cleaved by it. PelA efficiently macerated mung bean hypocotyls and potato tuber tissues into single cells.     

Properties of L-arabinose isomerase from Escherichia coli as biocatalyst for tagatose production

L-arabinose isomerase (EC 5.3.1.4) mediates the isomerization of D-galactose into D-tagatose as well as the conversion of L-arabinose into L-ribulose. To investigate the properties of L-arabinose isomerase as a biocatalyst for the conversion of galactose to tagatose, the L-arabinose isomerase of Escherichia coli was characterized. The substrate specificity for L-arabinose was 166-fold higher than that for D-galactose. The optimal pH and temperature for the galactose isomerization reaction were 8.0 and 30 °C, respectively. The enzyme activity was stable for 1 h at temperatures below 35 °C and within a pH range of 8–10. The Michaelis constant, K _m, for galactose was 1480 mM, which is 25-fold higher than that for arabinose. The addition of Fe²⁺ and Mn²⁺ ions enhanced the conversion of galactose to tagatose, whereas the addition of Cu²⁺, Zn²⁺, Hg²⁺, and Fe³⁺ ions inhibited the reaction completely. In the presence of 1 mM Fe²⁺ ions, the K _m for galactose was found to be 300 mM.     

De novo synthesis,constitutive expression of <Emphasis Type="Italic">Aspergillus sulphureus</Emphasis> β-xylanase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis> and partial enzymic characterization

The endo-β-1, 4-xylanase gene xynA from Aspergillus sulphureus, encoded a lack-of-signal peptide protein of 184 amino acids, was de novo synthesized by splicing overlap extension polymerase chain reaction according to Pichia pastoris protein’s codon bias. The synthetic DNA, composed of 572 nucleotides, was ligated into the downstream sequence of an α-mating factor in a constitutive expression vector pGAPzαA and electrotransformed into the P. pastoris X-33 strain. The transformed yeast screened by Zeocin was able to constitutively secrete the xylanase in yeast–peptone–dextrose liquid medium. The heterogenous DNA was stabilized in the strain by 20-times passage culture. The recombinant enzyme was expressed with a yield of 120 units/mL under the flask culture at 28°C for 3 days. The enzyme showed optimal activity at 50°C and pH 2.4–3.4. Residual activity of the raw recombinant xylanase was not less than 70% when fermentation broth was directly heated at 80°C for 30 min. However, the dialyzed xylanase supernatant completely lost the catalytic activity after being heated at 60°C for 30 min. The recombinant xylanase showed no obvious activity alteration by being pretreated with Na₂HPO₄-citric acid buffer of pH 2.4 for 2 h. The xylanase also showed resistance to certain metal ions (Na⁺, Mg²⁺, Ca²⁺, K⁺, Ba²⁺, Zn²⁺, Fe²⁺, and Mn²⁺) and EDTA. These biochemical characteristics suggest that the recombinant xylanase has a prospective application in feed industry as an additive.     

Effects of Metal Ions on the Conformation and Activity of Acutolysin D from Agkistrodon Acutus Venom

Acutolysin D, isolated from the venom of Agkistrodon acutus, possesses marked haemorrhagic and proteolytic activities. The molecular weight and the absorption coefficients (A ^1% ₂₈₀) of acutolyisn D have been determined to be 47,850 ± 8 amu and 9.3 by mass spectrometer and UV spectrum, respectively. The effects of metal ions on the conformation and activity of acutolysin D have been studied by following fluorescence, circular dichroism and biological activity measurements. Acutolysin D contains two Ca²⁺-binding sites and two Zn²⁺-binding sites determined by atomic absorption spectrophotometer. Zn²⁺ is essential for the enzyme activities of acutolysin D, however, the presence of 1 mM Zn²⁺ significantly decreases its caseinolytic activity and intrinsic fluorescence intensity at pH 9.0 due to Zn(OH)₂ precipitate formation. Ca²⁺ is important for the structural integrity of acutolysin D, and the presence of 1 mM Ca²⁺ markedly enhances its caseinolytic activity. Interestingly, the caseinolytic activity which is inhibited partly by Cu²⁺, Co²⁺, Mn²⁺ or Tb³⁺ and inhibited completely by Cd²⁺, is enhanced by Mg²⁺. The fluorescence intensity of the protein decreases in the presence of Cu²⁺, Co²⁺, Cd²⁺ or Mn²⁺, but neither for Ca²⁺, Mg²⁺ nor for Tb³⁺. Zn²⁺, Ca²⁺, Mg²⁺, Cu²⁺, Mn²⁺, Co²⁺ and Tb³⁺ have slight effects on its secondary structure contents. In addition, Cd²⁺ causes a marked increase of antiparallel β-sheet content from 45.5% to 60.2%.     

Al3+-Ca2+ interactions in aluminum rhizotoxicity

Several mineral rhizotoxicities, including those induced by Al³⁺, H⁺, and Na⁺, can be relieved by elevated Ca²⁺ in the rooting medium. This leads to the hypothesis that the toxic cations displace Ca²⁺ from transport channels or surface ligands that must be occupied by Ca²⁺ in order for root elongation to occur. In this study with wheat (Triticum aestivum L.) seedlings, we have determined, in the case of Al³⁺, that (i) Ca²⁺, Mg²⁺, and Sr²⁺ are equally ameliorative, (ii) that root elongation does not increase as Ca²⁺ replaces Mg²⁺ or Sr²⁺ in the rooting media, and (iii) that rhizotoxicity is a function solely of Al³⁺ activity at the root-cell membrane surface as computed by a Gouy-Chapman-Stern model. The rhizotoxicity was indifferent to the computed membrane-surface Ca²⁺ activity. The rhizotoxicity induced by high levels of tris(ethylenediamine)cobaltic ion (TEC³⁺), in contrast to Al³⁺, was specifically relieved by Ca²⁺ at the membrane surface. The rhizotoxicity induced by H⁺ exhibited a weak specific response to Ca²⁺ at the membrane surface. We conclude that the Ca²⁺-displacement hypothesis fails in the case of Al³⁺ rhizotoxicity and that amelioration by cations (including monovalent cations) occurs because of decreased membrane-surface negativity and the consequent decrease in the membrane-surface activity of Al³⁺. However, TEC³⁺, but not Al³⁺, may be toxic because it inhibits Ca²⁺ uptake. The nature of the specific H⁺-Ca²⁺ interaction is uncertain.Abbreviations {Al³⁺ }₀ chemical activity of Al³⁺ at the root-cell membrane surface - {Al³⁺ }_E chemical activity of Al³⁺ in the external rooting medium - E₀ electrical potential at the root-cell membrane surface - HXM²⁺ hexamethonium ion - TEC³⁺ tris(ethylenediamine)cobaltic ion     

Improved expression of a soluble single chain antibody fusion protein containing tumor necrosis factor in <Emphasis Type="Italic">Escherichia coli</Emphasis>

An epoxide hydrolase gene of about 0.8 kb was cloned from Rhodococcus opacus ML-0004, and the open reading frame (ORF) sequence predicted a protein of 253 amino acids with a molecular mass of about 28 kDa. An expression plasmid carrying the gene under the control of the tac promotor was introduced into Escherichia coli, and the epoxide hydrolase gene was successfully expressed in the recombinant strains. Some characteristics of purified recombinant epoxide hydrolase were also studied. Epoxide hydrolase showed a high stereospecificity for l(+)-tartaric acid, but not for d(+)-tartaric acid. The epoxide hydrolase activity could be assayed at the pH ranging from 3.5 to 10.0, and its maximum activity was obtained between pH 7.0 and 7.5. The enzyme was sensitive to heat, decreasing slowly between 30°C and 40°C, and significantly at 45°C. The enzyme activity was activated by Ca²⁺ and Fe²⁺, while strongly inhibited by Ag⁺ and Hg⁺, and slightly inhibited by Cu²⁺, Zn²⁺, Ba²⁺, Ni⁺, EDTA–Na₂ and fumarate.