Inhibition of Growth of Callus Cells by Degradation Products of Dehydroascorbic Acid

Candida pelliculosa var. acetaetherius was found to produce a β-glucosidase intracellularly. The enzyme was purified 200-fold by fractionation with ammonium sulfate and chromatography on Sephadex G-100 and DEAE Sepharose CL-6B. After polyacrylamide gel electrophoresis of the final fraction, one protein band corresponding to β-glucosidase was detected. The molecular weights determined by SDS-PAGE and by Sephacryl S-300 chromatography were 90,000 and 360,000, respectively, suggesting that the enzyme was a tetramer. The enzyme was a glycoprotein and its isoelectric point was at pH 4.9. It’s optimum pH and temperature were 6.5 and 50°C, respectively. The enzyme activity was inhibited by Zn^{2 +}, Hg^{2 +}, Cu^{2 +}, Co^{2 +}, p-chloromercuribenzoate, and glucose. Inhibition by glucose was competitive with a Ki value of 6.5 mm. Specificity studies for substrates indicated that the enzyme was specific for the p-configuration of sugars. Km values measured at 50°C were 0.5 mm for p-nitrophenyl-β-glucoside and 37 mm for cellobiose.     

Purification and characterization of an<Emphasis Type="Italic"> N</Emphasis>-acetylglucosaminidase produced by a<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> strain which controls<Emphasis Type="Italic"> Crinipellis perniciosa</Emphasis>

Isolate 1051 of Trichoderma harzianum, a mycoparasitic fungus, was found to impair development of the phytopathogen, Crinipellis perniciosa, in the field. This Trichoderma strain growing in liquid medium containing chitin produced substantial amounts of chitinases. The N-acetylglucosaminidase present in the culture-supernatant was purified to homogeneity by gel filtration and hydrophobic interaction chromatography, as demonstrated by SDS-PAGE analysis. The enzyme had a molecular mass of 36 kDa and hydrolyzed the synthetic substrate -nitrophenyl-N-acetylglucosaminide (NGlcNAc) with Michaelis–Menten kinetics. Maximal activities were determined at pH 4.0 and a temperature range of 50–60°C. K _m and V _max values for NGlcNAc hydrolysis were 8.06 moles ml^–1 and 3.36 moles ml^–1 min^–1, respectively, at pH 6.0 and 37°C. The enzyme was very sensitive to Fe³⁺, Mn²⁺ and Co²⁺ ions, but less sensitive to Zn²⁺, Al³⁺, Cu²⁺ and Ca²⁺. Glucose at a final concentration of 1 mM inhibited 65% of the original activity of the purified enzyme. Determination of the product (reducing sugar) of hydrolysis of C. perniciosa mycelium and scanning electron microscopic analysis revealed that the N-acetylglucosaminidase hydrolyses the C. perniciosa cell wall.     

Cobalt transport in nickel-resistant strains ofNeurospora crassa

Uptake of Co²⁺ by three nickel-resistant strains (Ni^R1, Ni^R2, and Ni^R3) ofNeurospora crassa that differed in resistance to Co²⁺ has been studied. Uptake was linear with Co²⁺ concentration (up to 1 mM), with time (up to 6 h), and with pH between 3 and 6. Uptake rates were in the order Ni^R2>Ni^R1>Ni^R3. In all strains, there was gradual increase in Co²⁺ uptake between 10° and 28°C, with a much sharper increase between 28° and 40°C. Metabolic inhibitors decreased Co²⁺ uptake partially in all strains, except for KF in Ni^R3. About 50–80 g Co²⁺/100 mg dry weight was surface bound. Ni²⁺, Zn²⁺, and Mn²⁺ competed with Co²⁺, the effects being strain specific. Mg²⁺ inhibited Co²⁺ uptake in all strains with preformed mycelia. In Ni^R1 and Ni^R2 only with young mycelia (40 h old) was Mg²⁺ inhibitory to Co²⁺ uptake,during growth in the presence of Co²⁺. The results suggested the presence of two transport systems for Co²⁺ in Ni^R1 and Ni^R2, only one of which was sensitive to Mg²⁺; in contrast, Ni^R3 had a single system, which was sensitive to Mg²⁺.     

Partial purification and biochemical characterization of alkaline 5'-phosphodiesterase from barley malt sprouts

An alkaline 5-phosphodiesterase (5-PDE) from barley (Hordeum distichum) malt sprouts was partially purified by thermal treatment and acetone precipitation to diminish phosphomonoesterase (PME) activity. 5-PDE was purified 40-fold to a specific activity of 30 U mg^–1 protein with a final yield of about 32%. With synthetic substrate, the enzyme had an optimum pH of 8.9, maximum activity at 70 °C over 10 min, and a Km of 0.26 mM. The partially purified enzyme was activated by 10 mM Mg²⁺ up to 168% of the original activity, while Zn²⁺, Mn²⁺ and Cu²⁺ ions, chelating agent (EDTA) and NaN₃ (1–10 mM), and 5-ribonucleotides (1–5 mM) were inhibitory. Final enzyme preparation was stable over 8 d at 4 °C), at 70 °C for up to 120 min and without loss of activity over 90 d at –18 °C.     

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.     

Characterization of a secreted cholesterol oxidase from Rhodococcus sp.GKI (CIP 105335)

Extracellular cholesterol oxidase (COX) (EC 1.1.3.6) was produced by Rhodococcus sp. GK1 cells grown in a defined mineral salt medium containing a mixture of phytosterols (sitosterol, campesterol, stigmasterol) as the sole source of carbon and energy. In the same time, the sterols acted as enzyme inducers. The medium was enriched with yeast extract in order to stimulate enzyme secretion. COX was purified from the culture supernatants by affinity-like chromatography on a column packed with kieselguhr and cholesterol. Enzyme bound onto the column was eluted with 0.05 M phosphate buffer pH 7.0 containing Triton X-100 at 0.1% (w/v). Some properties of the purified COX were determined. Its specific activity at pH 7.0 and 30 °C, was around 5.5 units mg^–1. The molecular mass of the enzyme, as estimated by SDS-PAGE, was 59 kDa. Its isoelectrofocusing point was around pH 8.9. The C-5 double bond and the alkyl chain moiety in sterol molecules were necessary for an adequate oxidation of the sterol 3-ol. Enzyme inhibition by the ions (0.1 mM): AsO₂ ^–, Ba²⁺, Co²⁺, Cd²⁺, Cu²⁺, N₃ ^–, Ni²⁺, and Pb²⁺ was negligible (around 10%). However, COX inhibition by 0.1 mM of either Zn²⁺, 2-[(ethylmercurio)-thio]benzoic acid, or Hg²⁺ was 18%, 22% and 93% respectively. Inhibition of activity by Hg²⁺ was significant, even at 1 M. The purified COX (0.1–0.15 mg ml^–1 in 0.05 M phosphate pH 7.0) was relatively heat-stable at temperatures up to 50 °C. At this temperature, the half-life of its activity was around 70 min. However, 90% of the enzyme initial activity was lost by 20 min incubation at 60 °C. The aminoacid sequence of the COX N-terminal segment was: H2N–Ala–Pro–Pro–Val–Ala–Ser–X–Arg–Tyr–X–(Phe)– (X might be 2 Cys residues).     

Purification of α-amylase from Bacillus lentus cultures

Acetone fractionation of Bacillus lentus culture filtrate yielded the highest -amylase activity and the 66.6% fraction reached 13-fold that of the crude enzyme preparation. Gel filtration and ion exchange chromatography afforded a pure -amylase (relative molecular mass, 42 000). The pure enzyme was highly active on starch and dextrin. It produced a mixture of oligosaccharides as major products of starch hydrolysis. Maximal activity was reached at 70° C and pH 6.1. Ca²⁺, Na⁺, K⁺ and Sr²⁺ ions stabilized or slightly stimulated the enzyme whereas Ag⁺, Co²⁺, Hg²⁺, Zn²⁺, Cd²⁺ and Fe³⁺ ions strongly inhibited the activity. The enzyme contained 16 amino acids, of which aspartic and glutamic acids were present in the highest proportions. Correspondence to: S. H. Omar     

Purification and characterization of a truncatedBacillus subtilis α-amylase produced byEscherichia coli

ABacillus subtilis amylase gene was inserted into a plasmid which transferred toEscherichia coli. During cloning, a 3 region encoding 171 carboxyterminal amino acids was replaced by a nucleotide sequence that encoded 33 amino acid residues not present in the indigenous protein. The transformed cells produced substantial amylolytic activity. The active protein was purified to apparent homogeneity. Its molecular mass (48 kDa), as estimated in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, was lower than the molecular mass values calculated from the derived amino acid sequences of theB. subtilis complete -amylase (57.7 kDa) and the truncated protein (54.1 kDa). This truncated enzyme form hydrolysed starch with aK _m of 3.845 mg/ml. Activity was optimal at pH 6.5 and 50°C, and the purified enzyme was stable at temperatures up to 50°C. While Hg²⁺, Fe³⁺ and Al³⁺ were effective in inhibiting the truncated enzyme Mn²⁺ and Co²⁺ considerably enhanced the activity.     

Production of thermostable α-galactosidase from thermophilic fungusHumicola sp

The thermophilic fungus,Humicola sp isolated from soil, secreted extracellular -galactosidase in a medium cotaining wheat bran extract and yeast extract. Maximum enzyme production was found in a medium containing 5% wheat bran extract as a carbon source and 0.5% beef extract as a carbon and nitrogen source. Enzyme secretion was strongly inhibited by the presence of Cu²⁺, Ni²⁺ and Hg²⁺ (1mM) in the fermentation medium. Production of enzyme under stationary conditions resulted in 10-fold higher activity than under shaking conditions. The temperature range for production of the enzyme was 37° C to 55°C, with maximum activity (5.54 U ml^–1) at 45°C. Optimum pH and temperature for enzyme activity were 5.0 and 60° C respectively. One hundred per cent of the original activity was retained after heating the enzyme at 60°C for 1 h. At 5mM Hg²⁺ strongly inhibited enzyme activity. TheK _m andV _max forp-nitrophenyl--d-galactopyranoside were 60M and 33.6 mol min^–1 mg^–1, respectively, while for raffinose those values were 10.52 mM and 1.8 mol min^–1 mg^–1, respectively.     

Pectin lyase from Aspergillus sp. CH-Y-1043

Aspergillus sp. CH-Y-1043 synthesizes pectin lyase when grown on citrus pectin at 37° C. Production is favoured by increased esterification degree of the pectin used as carbon source. This enzyme displays higher activity at pH values of 8.5–8.8 and temperatures of 40–45° C. The optimal substrate for the enzyme was highly esterified pectin and no enzymatic activity was registered on polygalacturonic acid. The activity is stimulated by, though not dependent on, divalent cations (Ca²⁺, Mg²⁺, Mn²⁺, Ba²⁺ and Co²⁺) and inhibited by Zn²⁺, and it is not sensitive to the addition of EDTA. The enzyme is very stable when exposed to pH variations: at 4° C it preserves more than 95% of its activity at pHs ranging from 2.0 to 10.0, and at 30° C stability is preserved at pHs ranging from 4.0 to 8.0. At a constant pH of 5.0, the enzyme conserves its stability at temperatures ranging from 4 to 50° C and at pH 8.0 sensitivity to temperature increased. The results on the endo-exo nature of the enzyme suggest that this is an exo-pectin lyase. Correspondence to: G. Aguilar     

Optimization of medium for extracellular nuclease formation from Rhizopus stolonifer

A strain of Rhizopus stolonifer produced a high activity of extracellular DNAase when grown on YPG (yeast extract peptone glucose) medium. The source of peptone had a marked effect on the enzyme yield and only one peptone (i.e. from Sarabhai M. Chemicals Ltd, India) supported enzyme production. Maximum enzyme activity (88 U/ml) was obtained after 4 days' growth under submerged conditions in YPG medium containing 100 M Mn²⁺, Co²⁺ or Mg²⁺, and glucose as the sole carbon source. The unpurified enzyme was optimally active at pH 7.5 and 45°C. It had a higher activity with sonicated and heat-denatured DNA than native DNA.     

Characterization of thermostable α-glucosidase from Clostridium thermohydrosulfuricum 39E

Summary Clostridium thermohydrosulfuricum 39E produced a cell-bound -glucosidase. It was partially purified 140-fold by solubilizing with Triton X-100, ammonium sulfate treatment, DEAE-Sepharose CL-6B, octyl-Sepharose and acarbose-Sepharose affinity chromatography. The optimum temperature for the action of the enzyme was at 75°C. It had a half-life of 35 min at 75°C, 110 min at 70°C and 46 h at 60°C. The enzyme was stable at pH 5.0–6.0 and had an optimum pH at 5.0–5.5. It hydrolyzed the -1,4-linkages in maltose, maltotriose, maltotetraose and maltohexaose, the rate decreasing in order of higher-sized oligosaccharides. The enzyme preparation also hydrolyzed the -1,6 linkages in isomaltose and isomaltotriose. It rapidly hydrolyzed p-nitrophenyl -d-glucoside (pNPG). The K _m values for maltose, isomaltose, panose, maltotriose, and pNPG were 1.85, 2.95, 1.72, 0.58, and 0.31 mm, respectively, at pH 5.5 and 60°C. The enzyme produced glucose from all these substrates. The enzyme preparation did not require any metal ion for activity. The -glucosidase activity was inhibited by acarbose. Offprint requests to: B. C. Saha     

Glutamine synthetase of the nitrogen-fixing alga Anabaena cylindrica

Summary High levels of glutamine synthetase, detected using both a biosynthetic assay (P _i release from ATP) and a -glutamyl transferase assay, are present in aerobically grown N₂-fixing cultures of Anabaena cylindrica. The enzyme is soluble, has a pH optimum of 6.5–7.5, with a peak at 7.1–7.2 (biosynthetic activity) or 6.9 (transferase activity), and a temperature optimum at 30°C–40°C. Partially purified preparations are stable in air at 5°C for at least 3 days. Mg²⁺, Mn²⁺, Co²⁺ and Ca²⁺ support high rates of biosynthetic activity, Zn²⁺ is less effective and Cu²⁺ and Ba²⁺ are ineffective.Enzyme activity is regulated at several levels: possibly by repression and derepression of the enzyme in response to NH₄ ⁺ level; by variation in the Mn²⁺: ATP ratio with optimum activity at a 1:1 ratio; by feed-back inhibition which may be of a cumulative type. The consensus of the evidence suggests the absence of a covalent enzyme modification of the type found in E. coli. Glutamine synthetase levels are almost twice as high on a protein basis in the heterocysts as in the vegetative cells. Apparent K _m values for whole filaments for NH₄ ⁺ and glutamate in the biosynthetic reactions are 1 mM and 2 mM respectively.     

Purification and characterization of a cellulase (CMCase) from a newly isolated thermophilic aerobic bacterium Caldibacillus cellulovorans gen. nov., sp. nov

One thermostable endoglucanase (CMCase) was purified to homogeneity from the culture supernatant of a new isolated thermophilic bacterium Caldibacillus cellulovorans. The molecular weight of the enzyme was 85.1 kDa as determined by SDS Polyacrylamide gel electrophoresis (PAGE) and 174 kDa by size-exclusion chromatography. The isoelectric point of the enzyme was at pH 4.12. The temperature for maximum activity was 80 °C, with half-lives of 32 min at 80 °C, and 2 min at 85 °C, and 83% activity remaining after 3 h at 70 °C. Thermostability of the enzyme was increased twofold by the addition of bovine serum albumin. Maximal activity was observed between pH 6.5 and 7.0. The enzyme activity was significantly inhibited by Zn²⁺, Hg²⁺, and p-chloromercuribenzenesulphonic acid. The enzyme showed high activity on carboxymethylcellulose (CMC) with much lower activity on Avicel; a low level of activity was also found against xylan. Cellobiose was the major product of hydrolysis of amorphous cellulose and CMC. Viscometric analysis indicated that the enzyme hydrolysed CMC in an exo-acting fashion. Cellotriose and cellobiose were not degraded and at least four contiguous glucosyl residues were necessary for degradation by the enzyme. The K _m and V _max of the enzyme for CMC were 3.4 mg ml^–1 and 44.7 mol min^–1 (mg protein)^–1, respectively.     

Synthesis and Biological Activities of (±)-Deoxy-abscisic Acid Isomers

Phosphodiesterase production with bis-p-nitrophenyl phosphate as a substrate by alkalophilic Bacillus No. A-40-2 increased with increasing Mn²⁺ concentration, showing maximum productivity at 10 mm. The enzyme production was negligible in the medium without Mn²⁺. The simultaneous addition of 10 mm Mn²⁺ and one of the several cations Mg²⁺, Co²⁺, Mo⁶⁺, and Pb²⁺ at suitable concentrations stimulated the enzyme production 1.8-fold at most over that with only 10 mm Mn²⁺. Inorganic phosphate hardly repressed the enzyme production. The enzyme was purified homogeneously. The purified enzyme had the optimum pH of 7.5 and was fairly stable from pH 7–11. The enzyme hydrolyzed 2′,3′-cyclic-nucleotides and 3′-nucleotides, but did not hydrolyze 3′,5′-cyclic-nucleotides or 5′-nucleotides, indicating it to be a 2′,3′-cyclic-nucleotide 2′-phosphodiesterase (EC 3.1.4.16). The enzyme had activity without metals, but Mg²⁺, Ca²⁺, Ba²⁺, and Mo⁶⁺ activated the enzyme reaction.     

Studies on d-Glucose-isomerizing Enzyme from Bacillus coagulans,Strain HN-68

A functional role of Co²⁺ and Mn²⁺ in the d-glucose- and d-xylose-isomerizing reactions by d-glucose-isomerizing enzyme obtained from the cells of Bacillus coagulans, strain HN–68 was investigated. (1) The enzyme required Co²⁺ and Mn²⁺ for d-glucose- and d-xylose-isomerizing activities, respectively. (2) The enzyme which bound the metal, Co²⁺- or Mn²⁺-enzyme, was active form. Co²⁺ was bound to the enzyme in a molar ratio of 4:1. (3) The rate of activation by metal ion varied with incubation pH. (4) The binding of substrate to the enzyme was completely independent in the presence of metal ions. (5) However, it seemed unlikely that the Co²⁺ and Mn²⁺ acted as catalyzer on the reaction. (6) The binding sites for Co²⁺ and Mn²⁺ were different from each other. (7) The experimental data obtained might be successfully explained in terms of the suitable conformational changes for d-glucose and d-xylose isomerization, which were induced in the catalytic sites of the enzyme by binding Co²⁺ and Mn²⁺, respectively.     

Purification and kinetic properties of skeletal muscle lactate dehydrogenase from the lizard Agama stellio stellio

Lactate dehydrogenase isoenzyme LDH-5 (M4) was purified to homogeneity from the skeletal muscle of lizard Agama stellio stellio as a poikilothermic animal, using colchicine-Sepharose chromatography and heat inactivation. The purified enzyme showed a single band after SDS-PAGE, corresponding to a molecular weight of 36 kD. The K _m values for pyruvate, NADH, lactate, and NAD⁺ were 0.020, 0.040, 8.1, and 0.02 mM, respectively. Pyruvate showed maximum activity at about 180 M, with a decline at higher concentrations. The enzyme was stable at 70°C for 30 min, but was rapidly inactivated at 90°C. The optimum pH for the forward reaction (pyruvate to lactate) was 7.5, and for the reverse reaction (lactate to pyruvate) was 9.2. Oxalate, glutamate, Cu²⁺, Co²⁺, Mn²⁺, and Mg²⁺ were inhibitory in both forward and reverse reactions.     

Main physicochemical features of monofunctional flavokinase from Bacillus subtilis

The main properties of a monofunctional riboflavin kinase from B. subtilis have been studied for the first time; the enzyme is responsible for a key reaction in flavin biosynthesis—the ATP-dependent phosphorylation of riboflavin with production of flavin mononucleotide. The active form of the enzyme is a monomer with molecular weight of about 26 kD with a strict specificity for reduced riboflavin. To display its maximum activity, the enzyme needs ATP and Mg²⁺. During the phosphorylation of riboflavin, Mg²⁺ could be partially replaced by ions of other bivalent metals, the efficiencies of which decreased in the series Mg²⁺ > Mn²⁺ > Zn²⁺, whereas Co²⁺ and Ca²⁺ had inhibiting effects. The flavokinase activity was maximal at pH 8.5 and 52°C. ATP could be partially replaced by other triphosphates, their donor activity decreasing in the series: ATP > dATP > CTP > UTP. The Michaelis constants for riboflavin and ATP were 0.15 and 112 M, respectively. As compared to riboflavin, a tenfold excess of its analog 7,8-dimethyl-10-(O-methylacetoxime)-isoalloxazine decreased the enzyme activity by 30%. Other analogs of riboflavin failed to markedly affect the enzyme activity.     

Regulatory effect of regucalcin on (Ca2+−Mg2+)-ATPase in rat liver plasma membranes: comparison with the activation by Mn2+ and Co2+

The effect of various metals and regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes of rat liver was investigated. Of various metals (Zn²⁺, Cu²⁺, Ni²⁺, Mn²⁺, Co²⁺ and Al³⁺; 100 M as a final concentration), Mn²⁺ and Co²⁺ increased markedly (Ca²⁺–Mg²⁺)-ATPase activity, while other metals had no effect. When Ca²⁺ was not added into enzyme reaction mixture, Mn²⁺ and Co²⁺ (25–100 M) did not significantly increase the enzyme activity, indicating that heavy metals act on Ca²⁺-stimulated phosphorylation of the enzyme. Meanwhile, regucalcin (0.25–1.0 M) caused a remarkable elevation of (Ca²⁺–Mg²⁺)-ATPase activity. This increase was not inhibited by the presence of 100 M vanadate, although the effects of Mn²⁺ and Co²⁺ (100 M) were inhibited by vanadate. Also, the inhibition of the Mn²⁺ and Co²⁺ effects by vanadate was not seen in the presence of regucalcin. Moreover, regucalcin (0.5 M) increased significantly the enzyme activity in the absence of Ca²⁺. This effect of regulcalcin was not altered by increasing concentrations of Ca²⁺ added, indicating that the regucalcin effect does not depend on Ca²⁺. The present results suggest that regucalcin activates directly (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes, and that the activation is not involved in the Ca²⁺-dependent phosphorylation of the enzyme.