Purification and characterization of recombinant spider silk expressed in Escherichia coli

A partial cDNA clone, from the 3′ end of the dragline silk gene was isolated from Nephila clavipes major ampullate glands. This clone contains a 1.7-kb insert, consisting of a repetitive coding region of 1.4-kb and a 0.3-kb nonrepetitive coding region; 1.5-kb of the 1.7-kb fragment was cloned into Escherichia coli and a␣43-kDa recombinant silk protein was expressed. Characterization of the purified protein by Western blot, amino acid composition analysis, and matrix-assisted laser desorption ionization/time-of-flight mass spectrometry confirms it to be spider dragline silk. Received: 7 April 1997 / Received revision: 24 July 1997 / Accepted: 25 August 1997     

Molecular cloning and nucleotide sequence of the pyruvate kinase gene of an actinomycete Microbispora thermodiastatica

The gene for the thermostable pyruvate kinase of Microbispora thermodiastatica IFO 14046, a moderate thermophilic actinomycete, was cloned in Escherichia coli. This gene consists of an open reading frame of 1422 nucleotides and encodes a protein of 474 amino acids with molecular mass of 50 805 Da. The open reading frame was confirmed as the pyruvate kinase gene by comparison with the N-terminal amino acid sequence of the purified pyruvate kinase from M. thermodiastatica. Received: 19 May 1997 / Received last revision: 22 September 1997 / Accepted: 14 October 1997     

Microbial oxidative metabolism of diclofenac: production of 4′-hydroxydiclofenac using Epiccocum nigrum IMI354292

The 4′-hydroxylated metabolite of diclofenac was produced by biocatalysis for probing specific human drug-metabolising enzymes (CYP2C9). An initial screen of 11 microorganisms was carried out (50 ml scale) to identify the organism best suited to the regioselective conversion of diclofenac to its 4′-hydroxylated metabolite. From this screen, the fungus Epicoccum nigrum IMI354292 was selected as the most suitable microorganism. Scale-up was carried out in a 30-l fermenter to which 2 g diclofenac was added. After 48 h, 50% of the diclofenac had been converted to it 4′-hydroxylated metabolite. The broth was then extracted with ethyl acetate and purified by chromatography and crystallisation. This yielded 0.3 g 4′-hydroxydiclofenac with a purity of at least 99%. The 4′-hydroxydiclofenac produced by E. nigrum was characterised by HPLC, mass spectrometry and NMR. Received: 28 July 1997 / Received revision: 8 December 1997 / Accepted: 14 December 1997     

Purification and properties of an extremely thermostable NADP+-specific glutamate dehydrogenase from Archaeoglobus fulgidus

NADP⁺-specific glutamate dehydrogenase (EC 1.4.1.4) was purified to homogeneity from the extremely thermophilic, strictly anaerobic, sulfate-reducing archaeon Archaeoglobus fulgidus strain 7324. The native enzyme (263 kDa) is composed of subunits of mol. mass 46 kDa, suggesting a hexameric structure. The temperature optimum for enzyme activity was > 95° C. The enzyme was highly thermostable, having a half-life of 140 min at 100° C. Potassium phosphate, KCl, and NaCl enhanced the thermal stability and increased the rate of activity three- to fourfold. The N-terminal 26-amino-acid sequence showed a high degree of similarity to glutamate dehydrogenases from Pyrococcus spp. and Thermococcus spp. Received: 25 March 1997 / Accepted: 11 July 1997     

Transgenic Brassica carinata as a vehicle for the production of recombinant proteins in seeds

Hirudin, a blood anticoagulant protein from leeches, and β-glucuronidase were produced in Brassica carinata Braun (Ethiopian mustard) seeds using oleosin as a carrier. Cotyledonary petioles were infected with Agrobacterium strains containing oleosin-glucuronidase (pCGNOBPGUS-A) or oleosin-hirudin (pCGN-OBHIRT) constructs. Polymerase chain reaction and neomycin phosphotransferase II enzyme assays confirmed the presence of the fusion genes in plants regenerating under selection. The fusion polypeptides were correctly expressed and targeted to the oil-bodies of the seeds with high fidelity (ca. 90%). Recombinant protein was purified from all other cellular protein by a simple flotation process and cleaved from oil-bodies using the endoprotease, Factor Xa. Hirudin activity was measured using a colorimetric thrombin inhibition assay and an activity in the range of 0.2–0.4 antithrombin units per milligram of oil-body protein was detected. B. carinata offers an attractive alternative for the production of recombinant proteins using oleosin technology. Received: 20 March 1997 / Revision received: 5 June 1997 / Accepted: 30 July 1997     

Effect of solvent adaptation on the antibiotic resistance in Pseudomonas putida S12

The effect of the adaptation to toluene on the␣resistance to different antibiotics was investigated in the␣solvent-resistant strain Pseudomonas putida S12. We␣followed the process of the solvent adaptation of P.␣putida S12 by cultivating the strain in the presence␣of␣increasing concentrations of toluene and studied␣the correlation of this gradual adaptation to the resistance towards antibiotics. It was shown that the tolerance to various chemically and structurally unrelated antibiotics, with different targets in the cell, increased during this gradual adaptation. The survival of P. putida S12 in the presence of antibiotics like tetracycline, nigericin, polymyxin B, piperacillin or chloramphenicol increased 30- to and 1000-fold after adaptation to 600 mg/l toluene. However, cells grown in the absence of any solvents lost their adaptation to toluene even when grown in the presence of antibiotics. Results are discussed in terms of the physico-chemical properties of membranes as affected by the observed cis/trans isomerization of unsaturated fatty acids, as well as in terms of the active efflux of molecules from the cytoplasmic membrane. Received: 9 May 1997 / Received revision: 4 July 1997 / Accepted: 4 July 1997     

4-Hydroxybenzoate 3-geranyltransferase from Lithospermum erythrorhizon: purification of a plant membrane-bound prenyltransferase

Geranyldiphosphate:4-hydroxybenzoate 3-geranyltransferase is a regulatory enzyme in the biosynthesis of shikonin, a phytoalexin and pharmaceutical produced by cell cultures of Lithospermum erythrorhizon Sieb. et Zucc.. In Linsmaier-Skoog medium, the activity of this enzyme could be enhanced more than 200-fold by addition of methyl jasmonate, and this culture material was used for the solubilization and purification of the enzyme. Of various detergents examined, digitonin was the most suitable for the solubilization of the enzyme. The solubilized enzyme was purified 800-fold by chromatography over diethylaminoethyl (DEAE)-Sephacel, Heparin-Sepharose, Reactive Green 19-Agarose, and Cholic Acid-Agarose. The purified enzyme required magnesium ions as cofactor and was highly specific for geranyldiphosphate (GPP) and 4-hydroxybenzoate (4HB) as substrates. The K _m values for 4HB and GPP were calculated by the method of Lineweaver and Burk as 18.4 μM and 13.8 μM, respectively. Received: 2 July 1997 / Accepted: 14 October 1997     

Optimal conditions for bioconversion of ferulic acid into vanillic acid by Pseudomonas fluorescens BF13 cells

Pseudomonas fluorescens BF13 is especially capable of promoting the formation of vanillic acid during ferulic acid degradation. We studied the possibility of enhancing the formation of this intermediary metabolite by using suspensions of cells at high density. The bioconversion of ferulic into vanillic acid was affected by several parameters, such as the concentration of the biomass, the amount of ferulic acid that was treated, the carbon source on which the biomass was grown. The optimal yield of vanillic acid was obtained with 6 mg/ml cells pre-grown on p-coumaric acid and 2 mg/ml ferulic acid. Under these conditions the bioconversion rate was 95% in 5 h. Therefore BF13 strain represents a valid biocatalyst for the preparative synthesis of vanillic acid. Received: 1 July 1997 / Received revision: 28 October 1997 / Accepted: 16 November 1997     

Development of an efficient Agrobacterium-mediated transformation system for Brassica carinata

Shoot organogenesis and plant regeneration were readily achieved from cotyledonary petioles and hypocotyls of Brassica carinata. These explants were used for Agrobacterium-mediated transformation. A construct containing the selectable marker genes, neomycin phosphotransferase II, phosphinothricin acetyl transferase and the reporter gene β-glucuronidase, under the control of a tandem 35S promoter, was used for transformation. Although transformation was achieved with both cotyledonary petioles and hypocotyls, cotyledonary petioles responded best, with 30–50% of the explants producing GUS-positive shoots after selection on 25 mg/l kanamycin. Direct selection on L-phosphinothricin also produced resistant shoots but at a lower frequency (1–2%). Received: 9 April 1997 / Revision received: 3 July 1997 / Accepted: 30 July 1997     

Effects of phenolic compounds on the growth and the fatty acid composition of Lactobacillus plantarum

The effects of different phenolic compound concentrations on the fatty acid composition of Lactobacillus plantarum isolated from traditional home-made olive brines were determined. Increasing amounts of caffeic and ferulic acids induced a gradual increase in the amounts of myristic, palmitoleic, stearic and 9,10-methylenehexadecanoic (C17Δ, where Δ represents the cyclopropane group) acid with a concomitant decrease of lactobacillic acid (C₁₉Δ). On the other hand, the addition of tannins induced an increase in the C₁₉Δ level at the expense of vaccenic acid content. The presence of acidic phenols and tannins also affected bacterial growth, inducing the most obvious effect with tannin at 1 g l⁻¹. Received: 1 July 1997 / Received revision: 9 September 1997 / Accepted: 15 September 1997     

Biotransformation of isoquinoline, phenanthridine, phthalazine, quinazoline, and quinoxaline by Streptomyces viridosporus

Streptomyces viridosporus T7A (ATCC␣39115), during growth in tryptone/yeast extract broth, cometabolized five heterocyclic nitrogen-containing compounds. The metabolites produced from the azaarenes were identified by high-performance liquid chromatography, UV/visible absorption spectroscopy, and mass spectrometry. Isoquinoline was transformed to 1(2H)-isoquinolinone (14%), phenanthridine to 6(5H)-phenanthridinone (25%), phthalazine to 1(2H)-phthalazinone (46%), quinazoline to 2,4(1H,3H)-quinazolinedione (4%), and quinoxaline to 2(1H)-quinoxalinone (8%) and 1-methyl-2(1H)-quinoxalinone (12%). Received: 29 July 1997 / Received revision: 19 November 1997 / Accepted: 29 November 1997     

Anthracyclines: isolation of overproducing strains by the selection and genetic recombination of putative regulatory mutants of Streptomycespeucetius var. caesius

In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium at 6 days of fermentation. Received: 14 April 1997 / Received revision: 19 June 1997 / Accepted: 4 July 1997     

Autoregulation of the partition genes of the mini-F plasmid and the intracellular localization of their products in Escherichia coli

The sopAB operon and the sopC sequence, which acts as a centromere, are essential for stable maintenance of the mini-F plasmid. Immunoprecipitation experiments with purified SopA and SopB proteins have demonstrated that these proteins interact in vitro. Expression studies using the lacZ gene as a reporter revealed that the sopAB operon is repressed by the cooperative action of SopA and SopB. Using immunofluorescence microscopy, we found discrete fluorescent foci of SopA and SopB in cells that produce both SopA and SopB in the presence of the sopC DNA segment, but not in the absence of sopC, suggesting the SopA-SopB complex binds to sopC segments. SopA was exclusively found to colocalize with nucleoids in cells that produced only SopA, while, in the absence of SopA, SopB was distributed in the cytosolic spaces. Received: 14 July 1997 / Accepted: 3 October 1997     

Modelling the kinetics of growth of Acetobacter aceti in discontinuous culture: influence of the temperature of operation

Acetic acid fermentation is the biochemical process by which, under strict conditions of aerobiosis, Acetobacter aceti oxidises the ethanol contained in alcoholic substrates into acetic acid. This paper studies the effect of temperature on the specific growth rate of the microorganisms (μ _C), in particular, the mathematical modelling of this process, with the aim of developing previous studies of the mathematical relationships between μ _C of A. aceti and the concentrations of substrate (ethanol), product (acetic acid) and dissolved oxygen. Until now this relationship has not been widely studied, and only a few studies have looked at the influence of temperature on growth kinetics of this bacteria. We have developed an extensive experimental system, to determine precisely the influence of temperature on the maximum specific growth rate. Received: 15 July 1997 / Received revision: 7 October 1997 / Accepted: 19 October 1997     

Alginate promotes production of various enzymes by Catharanthus roseus cells

When 1.0 g/l of alginate was added to a Catharanthus roseus L. cell culture, many proteins were released from the cells as detected by SDS polyacrylamide gel electrophoresis. In particular, production and/or release of 5′-phosphodiesterase (5′-PDase), catalase and chitinase by C. roseus L. cells were promoted by the addition of alginate. The promotive effects of alginate on 5′-PDase production were observed for various C. roseus cell lines and similar results were obtained when different alginates with various mannuronate/guluronate ratios and viscosities were used. In contrast, agar, agarose, and chitosan did not promote 5′-PDase production. The promotion of 5′-PDase production was not due to cell mutation, the alginate acted rather as a kind of elicitor. During 82 subcultures (577 days) in Murashige and Skoog medium containing 1.0 g/l of alginate, production and release of 5′-PDase by C. roseus L. cells were promoted without inhibition of cell growth. Received: 27 February 1997 / Revision received: 10 July 1997 / Accepted: 20 July 1997     

Gene cluster for creatinine degradation in Arthrobacter sp. TE1826

The genes encoding creatininase (CrnA; 258 residues) and creatinase (CreA; 411 residues) from Arthrobacter sp. TE1826 were cloned and sequenced. The genes form a cluster with the sarcosine oxidase gene (soxA) and its regulator gene (soxR), which were cloned previously. The deduced amino acid sequences of CrnA and CreA show 35.9% and 63.1% identity, respectively to the corresponding Pseudomonas enzymes. CrnA and CreA were purified from the recombinant strains and characterized. Other open reading frames (creB and crnB), encoding proteins similar to several transporters, were found downstream of creA and crnA, respectively. Received: 15 July 1997 / Accepted: 20 October 1997     

A Sandwich Enzyme-Linked Immunosorbent Assay for the Bacillus sphaericus Binary Toxin

Bacillus sphaericus (Bs) binary toxin was purified from recombinant E. coli DH5α harboring the recombinant plasmid pAR5, which carries a 3.6-kb DNA fragment of Bs 1593M encoding mosquito larvicidal activity. The binary toxin preparation, designated BsEcAg, contained mainly 51- and 42-kDa toxin proteins and was toxic to 50% of Culex quinquefasciatus larvae at a concentration of 9.22 ng toxin protein/ml. This preparation was used to raise antibodies in sheep and mice. The sandwich ELISA used sheep antitoxin antibody as primary antibody (coating antibody), mouse antitoxin antibody as second antibody, and goat antimouse antibody as an alkaline phosphatase-conjugated detecting antibody. The assay sensitivity was 200 ng/ml for both BsEcAg and binary toxin antigen (BsAg) from Bs 2362 cells. There is a significant correlation between toxin level determined by ELISA and bioassay. This procedure has also been used to monitor toxin levels in batch fermentations of Bs 2362. Received: 2 July 1997 / Accepted: 12 August 1997     

Changes in intrinsic fluorescence during the production of viable but nonculturable Escherichia coli

The potential of intrinsic fluorescence spectroscopy to detect and differentiate viable but nonculturable bacteria in the presence of culturable bacteria was explored. Escherichia coli cells, starved for 210 days in nutrient-free normal saline, show new fluorescence emissions near 400 and 440 nm, and reduced emission near 340 nm. Received 7 July 1997/ Accepted in revised form 26 November 1997     

Purification of ornithine carbamoyltransferase from kidney bean (Phaseolus vulgaris L.) leaves and comparison of the properties of the enzyme from canavanine-containing and -deficient plants

Kidney bean (Phaseolus vulgaris L.) ornithine carbamoyltransferase (OCT; EC 2.1.3.3) was purified to homogeneity from leaf homogenates in a single-step procedure, using δ-N-(phosphonoacetyl)-l-ornithine-Sepharose 6B affinity chromatography. The 8540-fold-purified OCT exhibited a specific activity of 526 micromoles citrulline per minute per milligram of protein at 35 °C and pH 8.0. The enzyme represents approximately 0.01% of the total soluble protein in the leaf. The molecular mass of the native enzyme was approximately 109 kDa as estimated by Sephacryl S-200 gel filtration chromatography. The purified protein ran as a single band of molecular mass 36 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and at a single isoelectric point of 6.6 when subjected to denaturing isoelectric focusing. These results suggest that the enzyme is a trimer of identical subunits. Among the tested amino acids, l-cysteine and S-carbamoyl-l-cysteine were the most effective inhibitors of the enzyme. The OCT of kidney bean showed a very low activity towards canaline. The OCTs of canavanine-deficient plants have very low canaline-dependent activities, but the OCTs of canavanine-containing plants showed high canaline-dependent activities. It was assumed that the substrate specificity of this enzyme determines the canavanine synthetic activity of the urea cycle. Received: 22 July 1997 / Accepted: 4 December 1997