F420H2 oxidase (FprA) from Methanobrevibacter arboriphilus, a coenzyme F420-dependent enzyme involved in O2 detoxification

Cell suspensions of Methanobrevibacter arboriphilus catalyzed the reduction of O₂ with H₂ at a maximal specific rate of 0.4 U (mol/min) per mg protein with an apparent K _m for O₂ of 30 M. The reaction was not inhibited by cyanide. The oxidase activity was traced back to a coenzyme F₄₂₀-dependent enzyme that was purified to apparent homogeneity and that catalyzed the oxidation of 2 F₄₂₀H₂ with 1 O₂ to 2 F₄₂₀ and 2 H₂O. The apparent K _m for F₄₂₀ was 30 M and that for O₂ was 2 M with a V _max of 240 U/mg at 37°C and pH 7.6, the pH optimum of the oxidase. The enzyme did not use NADH or NADPH as electron donor or H₂O₂ as electron acceptor and was not inhibited by cyanide. The 45-kDa protein, whose gene was cloned and sequenced, contained 1 FMN per mol and harbored a binuclear iron center as indicated by the sequence motif H–X–E–X–D–X₆₂–H–X₁₈–D–X₆₀–H. Sequence comparisons revealed that the F₄₂₀H₂ oxidase from M. arboriphilus is phylogenetically closely related to FprA from Methanothermobacter marburgensis (71% sequence identity), a 45-kDa flavoprotein of hitherto unknown function, and to A-type flavoproteins from bacteria (30–40%), which all have dioxygen reductase activity. With heterologously produced FprA from M. marburgensis it is shown that this protein is also a highly efficient F₄₂₀H₂ oxidase and that it contains 1 FMN and 2 iron atoms. The presence of F₄₂₀H₂ oxidase in methanogenic archaea may explain why some methanogens, e.g., the Methanobrevibacter species in the termite hindgut, cannot only tolerate but thrive under microoxic conditions.Dedicated to Hans Schlegel on the occasion of his 80th birthday.     

Purification and partial characterization ofd-(−)-lactate dehydrogenase fromLactobacillus helveticus CNRZ 32

Summary d-(–)-Lactate dehydrogenase (LDH) was purified to homogeneity from a cell-free extract ofLactobacillus helveticus CNRZ 32. The native enzyme was determined to have a molecular weight of 152 000 and consisted of four identical subunits of 38 000. This enzyme was NAD dependent fructose 1,6-diphosphate (FDP) and ATP independent. It was most active on pyruvate followed by -hydroxypyruvate as substrates. TheK _m values for pyruvate andd-(–)-lactate were 0.64 and 68.42 mM respectively, indicating that the enzyme has a higher affinity for pyruvate. The enzyme activity was completely inhibited byp-chloromercuribenzoate (1 mM) and partially by iodoacetate, suggesting the involvement of the sulfhydryl group (-SH) in catalysis. Optima for activity by the purified enzyme were pH 4.0 and 50–60°C. Limited inhibition ofd-(–)-LDH was observed with several divalent cations. Additionally, HgCl₂ was observed to strongly inhibit enzyme activity. The purified enzyme was not affected by dithiothreitol or any of the metal chelating agents examined.     

Biochemical characterisation of extracellular phytase (myo-inositol hexakisphosphate phosphohydrolase) from a hyper-producing strain of Aspergillus niger van Teighem

Aspergillus niger van Teighem, isolated in our laboratory from samples of rotten wood logs, produced extracellular phytase having a high specific activity of 22,592 units (mg protein)^–1 . The enzyme was purified to near homogeneity using ion-exchange and gel-filtration chromatography. The molecular properties of the purified enzyme suggested the native phytase to be oligomeric, with a molecular weight of 353 kDa, the monomer being 66 kDa. The purified enzyme exhibited maximum activity at pH 2.5 and 52–55°C. The enzyme retained 97% activity after a 24-h incubation at 55°C in the presence of 10 mM glycine, while 87% activity was retained when no thermoprotectant was added. Phytase activity was not affected by most metal ions, inhibitors and organic solvents. Non-ionic and cationic detergents (0.1–5%) stabilise the enzyme, while the anionic detergent (SDS), even at a 0.1% level, severely inhibited enzyme activity. The chaotropic agents guanidinium hydrochloride, urea, and potassium iodide (0.5–8 M), significantly affected phytase activity. The maximum hydrolysis rate (V_max) and apparent Michaelis-Menten constant (K_m) were 1,074 IU/mL and 606 M, respectively, with a catalytic turnover number of 3×10⁵ s^–1 and catalytic efficiency of 3.69×10⁸ M^–1 s^–1.     

Isolation and characterization of α-amylase derived from starchgrownClostridium acetobutylicum ATCC 824

Summary An extracellular -amylase was purified to homogeneity from the culture supernatant ofClostridium acetobutylicum ATCC 824 grown in synthetic medium containing starch by using a combination of ammonium sulfate fractionation, anion exchange chromatography and HPLC-gel filtration. The molecular weight of the 160-fold purified -amylase was determined by SDS-PAGE to be 61 kDa. HPLC analysis of end-products of enzyme activity on various substrates indicated that the enzyme acted specifically in an endo-fashion on the -1,4-glucosidic linkages. Enzyme activity was optimal over a pH range of 4.5–5.0 and temperature of 55°C, but was rapidly inactivated at higher temperatures. Addition of calcium chloride (2–5 mM) increased -amylase activity by ca. 20%, while the addition of 19 g ml^–1 of acarbose (a differential inhibitor of amylases) resulted in 50% inhibition. TheV _max andK _m of -amylase were 2.17 mg min^–1 and 3.28 mg ml^–1 on amylose, and 1.67 mg min^–1 and 1.73 mg ml^–1 on soluble starch, respectively.     

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.     

Cultivation conditions and properties of extracellular crude lipase from the psychrotrophic fungus Penicillium chrysogenum 9′

Among 97 fungal strains isolated from soil collected in the arctic tundra (Spitsbergen), Penicillium chrysogenum 9 was found to be the best lipase producer. The maximum lipase activity was 68 units mL^–1 culture medium on the fifth day of incubation at pH 6.0 and 20°C. Therefore, P. chrysogenum 9 was classified as a psychrotrophic microorganism. The non-specific extracellular lipase showed a maximum activity at 30°C and pH 5.0 for natural oils or at pH 7.0 for synthetic substrates. Tributyrin was found to be the best substrate for lipase, among those tested. The K_m and V_max were calculated to be 2.33 mM and 22.1 units mL^–1, respectively, with tributyrin as substrate. The enzyme was inhibited more by EDTA than by phenylmethylsulfonyl fluoride and was reactivated by Ca²⁺. The P. chrysogenum 9 lipase was very stable in the presence of hexane and 1,4-dioxane at a concentration of 50%, whereas it was unstable in presence of xylene.     

Modelling continuous culture of Rhodospirillum rubrum in photobioreactor under light limited conditions

Rhodospirillum rubrum was grown continuously and photoheterotrophically under light limitation using a cylindrical photobioreactor in which the steady state biomass concentration was varied between 0.4 to 4 kg m^–3 at a constant radiant incident flux of 100 W m^–2. Kinetic and stoichiometric models for the growth are proposed. The biomass productivities, acetate consumption rate and the CO₂ production rate can be quantitatively predicted to a high level of accuracy by the proposed model calculations. Nomenclature: C _X, biomass concentration (kg m^–3) D, dilution rate (h^–1) Ea, mean mass absorption coefficient (m² kg^–1) I , total available radiant light energy (W m^–2) K, half saturation constant for light (W m^–2) R _W, boundary radius defining the working illuminated volume (m) r _X, local biomass volumetric rate (kg m^–3 h^–1) <r _X>, mean volumetric growth rate (kg m^–3 h^–1) V _W, illuminated working volume in the PBR (m^–3). Greek letters: , working illuminated fraction (–) _M, maximum quantum yield (–) bar, mean energetic yield (kg J^–1).     

Purification of lipase from Cunninghamella verticillata and optimization of enzyme activity using response surface methodology

The fungus Cunninghamella verticillata was selected from isolates of oil-mill waste as a potent lipase producer as determined by the Rhodamine-B plate method. The lipase was purified from C. verticillata by ammonium sulphate fractionation, ion exchange chromatography and gel filtration. The purified enzyme was formed from a monomeric protein with molecular masses of 49 and 42 kDa by SDS–PAGE and gel filtration, respectively. The optimum pH at 40 °C was 7.5 and the optimum temperature at pH 7.5 was 40 °C. The enzyme was stable between a pH range of 7.5 and 9.0 at 30 °C for 24 h. The enzyme activity was strongly inhibited by AgNO₃, NiCl₂, HgCl₂, CdCl₂ and EDTA. However, the presence of Ca²⁺, Mn²⁺ and Ba²⁺ ions enhanced the activity of the enzyme. The activity of purified lipase with respect to pH, temperature and salt concentration was optimized using a Box–Behnken design experiment. A polynomial regression model used in analysing this data, showed a significant lack of fitness. Therefore, quadratic terms were incorporated in the regression model through variables. Maximum lipase activity (100%) was observed with 2 mM CaCl₂, (pH 7.5) at a temperature of 40 °C. Regression co-efficient correlation was calculated as 0.9956.     

A novel lipase/chaperone pair from<Emphasis Type="Italic"> Ralstonia</Emphasis> sp. M1: analysis of the folding interaction and evidence for gene loss in<Emphasis Type="Italic"> R. solanacearum</Emphasis>

A microbial strain (referred to as M1) that produces an extracellular lipase was isolated from a soil sample in Vietnam, and identified as a Ralstonia species by partial sequencing of its 16S rDNA. A genomic library was constructed from Pst I fragments, and a colony showing lipase activity was selected for further analysis. Sequencing of the 4.7-kb insert in this clone (named M1-72) revealed one incomplete and three complete ORFs, predicted to encode a partial hypothetical glutaminyl tRNA synthetase (304 aa), a hypothetical transmembrane protein (500 aa), a lipase (328 aa) and a lipase chaperone (352 aa), respectively. Alignment of the insert sequence with the corresponding region of the genome of R. solanacearum GMI1000 (GenBank Accession No. AL646081) confirmed the presence in the latter of the genes for the hypothetical transmembrane protein and glutaminyl tRNA synthetase, which exhibited 89–91% identity to their counterparts in M1. However, R. solanacearum GMI1000 lacks the complete lipase-encoding gene and the major part of the chaperone-encoding gene, creating a so-called black hole. The deduced amino acid sequences of the products of the lipase gene lipA and chaperone gene lipB from strain M1 shared 49.3–60.3% and 23.9–32.7% identity, respectively, with those of the Burkholderia lipase/chaperone subfamily I.2. lipB is located downstream of lipA, and separated from it by only 9 bp, and each gene has a putative ribosome binding site. The mature lipase LipA, a His-tagged derivative (LipAhis), the tagged full-length chaperone LipBhis and a truncated form (LipBhis) lacking the 56 N-terminal residues were expressed in Escherichia coli BL21. LipA, LipAhis and LipBhis could be expressed at high levels (70, 15 and 12 mg/g wet cells, respectively) and were easily purified. However, LipBhis was expressed at a much lower level which precluded purification. The specific activity of purified LipAhis, expressed on its own, was very low (<52 U/mg). However, after co-incubation with the purified LipBhis in vitro, the specific activity of the enzyme was markedly enhanced, indicating that the chaperone facilitated correct folding of the enzyme. A lipase:chaperone ratio of 1:10 was found to be optimal, yielding an enzyme preparation with a specific activity of 650 U/mg.Communicated by H. Ikeda     

Identification of enzymes involved in indole-3-acetic acid degradation

Strains of Bradyrhizobium japonicum with the ability to catabolize indole-3-acetic acid (IAA) and strains of B. japonicum, Rhizobium loti, and Rhizobium galegae, unable to catabolize IAA, were analyzed for enzymes involved in the pathway for IAA degradation. Two enzymes having isatin as substrate were detected. An isatin amidohydrolase catalyzing the hydrolysis of isatin into isatinic acid was found in some B. japonicum strains and in two Rhizobium species, R loti and R. galegae. The enzyme was inducible (4–5-fold) by its substrate, isatin, and the partially purified enzyme from R. loti showed an apparent K_M of 11 M for isatin. A NADPH-dependent isatin reductase was measured in extracts from a strain of B. japonicum lacking the isatin amidohydrolase. The structure of the reaction product, dioxindole was verified by NMR spectroscopy. Isatin reductase activity was also detected in extracts of dry pea seeds, and present in at least two isoforms. A low K_M of 10 M for isatin was found with a partially purified preparation of the pea enzyme. The presence of such an enzyme activity in pea indicates dioxindole and isatin as possible intermediates in IAA degradation in pea.     

Purification and some properties of superoxide dismutase from<Emphasis Type="Italic"> Deinococcus radiophilus</Emphasis>, the UV-resistant bacterium

The superoxide dismutase (SOD, EC 1.15.1.1) of Deinococcus radiophilus, a bacterium extraordinarily resistant to UV, ionizing radiations, and oxidative stress, was purified 1,920-fold with a 58% recovery yield from the cell-free extract of stationary cells by steps of ammonium sulfate fractionation and Superdex G-75 gel-filtration chromatography. A specific activity of the purified enzyme preparation was ca. 31,300 U mg^–1 protein. D. radiophilus SOD is Mn/FeSOD, judging by metal analysis and its insensitivity to cyanide and a partial sensitivity to H₂O₂. The molecular weights of the purified enzyme estimated by gel chromatography and polyacrylamide gel electrophoresis are 51.5±1 and 47.1±5 kDa, respectively. The SOD seems to be a homodimeric protein with a molecular mass of 26±0.5 kDa per monomer. The purified native SOD showed very acidic pI of ca. 3.8. The enzyme was stable at pH 5.0–11.0, but quite unstable below pH 5.0. SOD was thermostable up to 40°C, but a linear reduction in activity above 50°C. Inhibition of the purified SOD activity by -naphthoquinone-4-sulfonic acid, -diazobenzene sulfonic acid, and iodine suggests that lysine, histidine, and tyrosine residues are important for the enzyme activity. The N-terminal peptide sequence of D. radiophilus Mn/FeSOD (MAFELPQLPYAYDALEPHIDA(>D) is strikingly similar to those of D. radiodurans MnSOD and Aerobacter aerogenes FeSOD.Communicated by G. Antranikian     

Purification and properties of an extra cellular xylanase enzyme ofClostridium strain SAIV

An extracellular xylanase enzyme fraction A from a mesophilicClostridium strain SAIV was purified by ammonium sulfate precipitation, Sephadex G-50 gel filtration and DEAE-Sephadex A-50 ion exchange. The xylanase exhibited a molecular weight of 30,000 and it was stable upto 55° C with an optimum temperature of 50° C. It was most stable between pH 5–7, with an optimum pH of around 6. The Km value was 7.0 mg·xylan ml^-1 and V_max was 36 mol·xylose liberated mg^-1 min^-1. Carboxymethyl cellulose, filter paper cellulose and 4-p-nitrophenyl -D-xylopyranoside were not hydrolysed. The specific activity of xylanase fraction A (9.8 U mg^-1) is 2–10 fold higher than the specific activity of xylanase in other mesophilic, xylanolytic, obligate anaerobic bacteria. A minor fraction of xylanase activity designated as xylanase B was also obtained supporting the view that the multiplicity of xylanases is common in microorganisms.     

Purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of horn fly, Haematobia irritans irritans (Diptera: Muscidae)

This work describes the purification and characterization of a trypsin-like enzyme with fibrinolytic activity present in the abdomen of Haematobia irritans irritans (Diptera: Muscidae). The enzyme was purified using a one-step process, consisting of affinity chromatography on SBTI-Sepharose. The purified protease showed one major active proteinase band on reverse zymography with 0.15% gelatin, corresponding to a molecular mass of 25.5 kDa, with maximum activity at pH 9.0. The purified trypsin-like enzyme preferentially hydrolyzed synthetic substrates with arginine residue at the P1 position. The K _m values determined for three different substrates were 1.88 × 10^–4, 1.28 × 10^–4, and 1.40 × 10^–4 M for H--benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S2222), dl-Ile-Pro-Arg-p-nitroanilide (S2288), and DL-Phe-Pip-Arg-p-nitroanilide (S2238), respectively. The enzyme was strongly inhibited by typical serine proteinase inhibitors such as SBTI (soybean trypsin inhibitor, K _i = 0.19 nM) and BuXI (Bauhinia ungulata factor Xa inhibitor, K _i = 0.48 nM), and less inhibited by LDTI (leech-derived tryptase inhibitor, K _i = 1.5 nM) and its variants LDTI 2T and 5T (0.8 and 1.5 nM, respectively). The most effective inhibitor for this protease was r-aprotinin (r-BPTI) with a K _i value of 39 pM. Synthetic serine protease inhibitors presented only weak inhibition, e.g., benzamidine with K _i = 3.0 × 10^–4 M and phenylmethylsulfonyl fluoride (PMSF) showed traces of inhibition. The purified trypsin-like enzyme also digested natural substrates such as fibrinogen and fibrin net. The protease showed higher activity against fibrinogen and fibrin than did bovine trypsin. These data suggest that the proteolytic enzyme of H. irritans irritans is more specific to proteins from blood than are the vertebrate digestive enzymes. This enzyme's characteristics may be an adaptation resulting from the feeding behavior of this hematophagous insect.     

Purification and Characterization of S-Adenosyl-L-Methionine Nicotinic Acid-N-Methyltransferase from Leaves of Glycine max

Trigonelline (TRG), which act as a cell cycle regulator and a compatible solute in response to salinity and water-stress, is the N-methyl conjugate of nicotinic acid the formation of which is catalyzed by S-adenosyl-L-methionine nicotinic acid-N-methyltransferase. The enzyme was purified 2650-fold from soybean (Glycine max L.) leaves with a recovery of 4 %. The purification procedure included ammonium sulfate (45 – 60 %) precipitation, linear gradient DEAE-Sepharose chromatography, adenosine-agarose affinity chromatography, hydroxyapatite chromatography and gel filtration (Sephacryl-S-200). The purified enzyme preparation showed a major band with a molecular mass of 41.5 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis that is related to the enzyme activity. The native enzyme had a molecular mass of about 85 kDa as estimated by gel filtration. The K_m values for S-adenosyl-L-methionine and nicotinic acid were 31 and 12.5 M, respectively. The purified enzyme showed optimum activity at pH 6.5 and temperature of 40 – 45 °C. High concentration of dithiothreitol (10 mM) and glycerol (20 %) stabilize the enzyme during purification and storage. Hg²⁺ strongly inhibits enzyme activity.     

Enzyme production in continuous cultivation by the thermophilic fungus, Thermomyces lanuginosus

The production of extracellular enzymes by the thermophilic fungus Thermomyces lanuginosus was studied in chemostat cultures at a dilution rate of 0.08 h^–1 in relation to variation in the ammonium concentration in the feed medium. Under steady state conditions, three growth regimes were recognised and the production of several extracellular enzymes from T. lanuginosus was recorded under different nutrient limitations ranging from nitrogen limitation to carbon/energy limitation. The range and the production of carbohydrate hydrolysing enzymes and lipase increased from Regime I (NH₄Cl 600 mg l^–1) to Regime III (NH₄CI 1200 mg l^–1), whereas production of protease was highest in Regime II (600 mg l^–1 < NH₄Cl <1200 mg l^–1).     

Purification and characterization of the complex I from the respiratory chain of Rhodothermus marinus

The rotenone sensitive NADH: menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50°C and pH 8.1, and the enzyme presents a K _M of 9 M for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 ± 3.5 iron atoms and 3.7 menaquinone per FMN. At least five iron—sulfur centers are observed by EPR spectroscopy: two [2Fe–2S]^2+/1+ and three [4Fe–4S]^2+/1+ centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.     

Pyrimidine biosynthesis in Serratia marcescens: Polypeptide interactions of three nonsequential enzymes

Orotidine-5-monophosphate pyrophosphorylase (OMPppase, E.C. 2.4.2.10) and orotidylate decarboxylase (OMPdecase, E.C. 4.1.1.23) were purified from Serratia marcescens HY. These enzymes required physical association for maximal catalytic activities and formed a fragile complex with dihydroorotase (DHOase, E.C. 3.5.2.3.). OMPppase reversibly lost 50% of its activity upon separation from DHOase. The kinetic characteristics of OMPppase were modified by this separation. In the presence of DHOase, the K _ms for PRPP and orotate were stoichiometric: 2.3×10^–6 m and 2.6×10^–6 m, respectively. Following separation, the K _ms were significantly different: 1.3 × 10^–6 m for PRPP and 4.1×10^–6 m for orotate. OMPppase and OMPdecase could be reversibly separated by acrylamide gel electrophoresis, but the separation was accompanied by a loss of catalytic efficiency for both enzymes. DHOase readily associated into multiple molecular forms and could not be purified. The DHOase-OMPppase-OMPdecase interactions demonstrate that a weakly aggregated, multifunctional enzyme complex participates in the biosynthesis of pyrimidine nucleotides in S. marcescens. This unique association of nonsequential biosynthetic enzymes may represent a larger complex which provides a channeling or regulatory unit.This work was supported by grants from the National Science Foundation (NSF GB 5811) and the Office of Naval Research (Nonr 4413). One of us (J.W.) was a National Science Foundation Graduate Fellow.     

Influence of iron(III) and pyoverdine on extracellular proteinase and lipase production by Pseudomonas fluorescens B52

Factors associated with the production of extracellular lipase and proteinase by Pseudomonas fluorescens B52 during the late-log, early-stationary phase of grown were examined. Active lipase production by resting cell suspensions was observed when cells were harvested during the log phase (A₆₀₀ of 0.3–0.9) Resting suspensions of younger cells (A₆₀₀<0.1) synthesized lipase after a significant lag. Addition of cells of the proteinase-and lipasedeficient mutant P. fluorescens RM14 to B52 cells at low density resulted in stimulation of lipase and proteinase production. Similar results were found using cell-free culture fluid of RM14. Gel filtration on Biogel P2 revealed that the stimulatory factor co-chromatographed with the iron(III) siderophore, pyoverdine. Partially purified pyoverdine stimulated enzyme synthesis at a concentration of 6 M while having no effect on activity of preformed enzyme. Production of pyoverdine and extracellular enzymes was also stimulated by transferrin, a strong iron(III) binding protein. Growth of B52 in deferrated media was limited to 27% of that found with untreated media. Maximum pyoverdine, proteinase and lipase synthesis was obtained at a final iron(III) concentration of 5.75 M. Growth was maximal in 8.75 M iron(III) while synthesis of pyoverdine, proteinase and lipase was reduced to 3.6, 6.6 and 30% respectively in 23.75 M iron(III). Lipase activity in cell-free culture fluid was slightly inhibited by the addition of up to 400 M iron(III) while proteinase activity was unaffected. In dilute cell suspensions, lipase synthesis was more sensitive to iron(III) than was proteinase (50% inhibition at 1.6 M and a maximum of 40% inhibition at 5.0 M, respectively). In the case of lipase, added pyoverdine was able to partially protect enzyme production from the effects of iron(III). The results are consistent with a role for iron(III) in the regulation of extracellular lipase and proteinase synthesis by P. fluorescens.Contribution No. 677 from the Food Research Centre     

Purification of a soluble casein kinase II from Dictyostelium discoideum lacking the β subunit: regulation during proliferation and differentiation

A type II casein kinase has been purified from the soluble fraction of Dictyostelium discoideum vegetative cells. The enzyme has been purified 370 fold and behaves catalytically as casein kinase type II, in the sense that it utilizes GTP as well as ATP as phosphoryl donors, it is inhibited by low heparin concentrations and phosphorylates a specific peptide for CK II. It is a tetramer of 38 kDa-subunits with catalytic activity and ability to autophosphorylate in vitro. The comparison of this activity with the nuclear enzyme previously purified from the same organism indicates that both have the same molecular structure. Both enzymes have antigenic determinants in common with casein kinase II from bovine thymus, suggesting a high degree of conservation during evolution. Studies on the activity of this enzyme during early differentiation, and in the transition from quiescence to proliferation shows an increase in specific activity suggesting a crucial role for the enzyme in this organism. (Mol Cell Biochem 118: 49–60, 1992)Abbreviations CK Casein Kinase - TLCK N--p Tosyl Lysil-Chloromethylketone - SDS Sodium Dodecyl Sulphate - PAGE Polyacrylamide Gel Electrophoresis - R₃E₃TE₃ Arg-Arg-Arg-Glu-Glu-Glu-Thr-Glu-Glu-Glu - TCA Trichloroacetic Acid     

Purification and characterization of hyperthermotolerant leucine aminopeptidase from Geobacillus thermoleovorans 47b

A thermophilic bacterium, which we designated as Geobacillus thermoleovorans 47b was isolated from a hot spring in Beppu, Oita Prefecture, Japan, on the basis of its ability to grow on bitter peptides as a sole carbon and nitrogen source. The cell-free extract from G. thermoleovorans 47b contained leucine aminopeptidase (LAP; EC 3.4.11.10), which was purified 164-fold to homogeneity in seven steps, using ammonium sulfate fractionation followed by the column chromatography using DEAE-Toyopearl, hydroxyapatite, MonoQ and Superdex 200 PC gel filtration, followed again by MonoQ and hydroxyapatite. The enzyme was a single polypeptide with a molecular mass of 42,977.2 Da, as determined by matrix-assisted laser desorption ionization and time-of-flight mass spectrometry, and was found to be thermostable at 90°C for up to 1 h. Its optimal pH and temperature were observed to be 7.6–7.8 and 60°C, respectively, and it had high activity towards the substrates Leu-p-nitroanilide (p-NA)(100%), Arg-p-NA (56.3%) and LeuGlyGly (486%). The K_m and V_max values for Leu-p-NA and LeuGlyGly were 0.658 mM and 25.0 mM and 236.2 mol min^–1 mg^–1 protein and 1,149 mol min^–1 mg^–1 protein, respectively. The turnover rate (k_cat) and catalytic efficiency (k_cat/ K_m) for Leu-p-NA and LeuGlyGly were 10,179 s^–1 and 49,543 s^–1 and 15,470 mM^–1 s^–1 and 1981.7 mM^–1 s^–1, respectively. The enzyme was strongly inhibited by EDTA, 1,10-phenanthroline, dithiothreitol, -mercaptoethanol, iodoacetate and bestatin; and its apoenzyme was found to be reactivated by Co²⁺ .