Antibacterial potential of γ-linolenic acid from Fischerella sp. colonizing Neem tree bark

Summary Pharmaceutically important γ-linolenic acid (GLA) was produced (4.1 mg g⁻¹ dry wt) by laboratory grown cyanobacterium Fischerella sp. colonizing Neem (Azadirachta indica) tree bark. GLA isolated from the test cyanobacterium was active against Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25992, Salmonella typhi (local strain), Pseudomonas aeruginosa ATCC 27853 and Enterobacter aerogenes MTCC 2822. The overproduction of GLA was also monitored by altering phosphate and nitrate levels in the nutrient medium. A doubling in phosphate concentration (58 μM) increased GLA level up to 12% over that of control cells while half of this phosphate level reduced GLA synthesis by 8%. In contrast, elevated nitrate concentrations (5 and 10 mM) stimulated biomass yield but not GLA, as the levels approximated to the nitrate-lacking control. The antibacterial potential of GLA from Fischerella sp. grown at varying P or N levels was at variance as evidenced by the diameter of inhibition zones against S. aureus. This variation in inhibition zones reflected differing levels of GLA as ascertained quantitatively by HPLC.     

Isolation and identification of a new antibacterial entity from the Antarctic cyanobacterium Nostoc CCC 537

The present study was aimed at the isolation, purification and structural elucidation of an antibacterial entity/lead molecule from the Antarctic cyanobacterium Nostoc CCC 537. A methanolic extract of the cyanobacterium was bioassayed with Enterobacter aerogenes as a target. The extract was purified by TLC, and the most active band was subjected to HPLC. The fraction (retention time 15.7 min) designated as the active principle was antibacterial towards Gram positive Mycobacterium tuberculosis H37Rv, Staphylococcus aureus ATCC 25923, Gram negative Salmonella typhi MTCC 3216, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25992, Enterobacter aerogenes MTCC 2822 and multi-drug resistant strains of Escherichia coli GS 2003/01, 02, 03. Based on UV, IR, ¹H NMR, EIMS, and ESIMS data, the structure of the active principle is proposed as 4-[(5-carboxy-2-hydroxy)-benzyl]-1,10-dihydroxy-3,4,7,11,11-pentamethyloctahydrocyclopenta<a>naphthalene (Mr 428, Mp 243–249°C). This intracellular biomolecule is similar to anthraquinone and indane derivatives of a diterpenoid. The rate of production of the active principle currently corresponds to 1.70 mg g⁻¹ biomass dry weight. The inherent property of Nostoc sp. to synthesise niche-specific biomolecules/lead molecules may be exploited for future drug development.     

Functional response of Anodonta anatina feeding on a green alga and four strains of cyanobacteria, differing in shape, size and toxicity

We studied the functional response of the freshwater unionid bivalve Anodonta anatina, feeding on five phytoplankton strains differing in food quality: the small green alga Scenedesmus obliquus, a toxic and a non-toxic strain of the filamentous cyanobacterium Planktothrix agardhii and a toxic and a non-toxic strain of the coccoid cyanobacterium Microcystis aeruginosa. On S. obliquus, A. anatina had a type II functional response with a maximum mass-specific ingestion rate (IR_max) of 5.24 mg C g DW⁻¹ h⁻¹ and a maximum mass-specific clearance rate (CR_max) of 492 (±38) ml g DW⁻¹ h⁻¹, the highest values for all the phytoplankton strains that were investigated. On toxic and non-toxic P. agardhii filaments, A. anatina also had a type II functional response, but IR_max and CR_max were considerably lower (IR_max 1.90 and 1.56 mg C g DW⁻¹ h⁻¹; CR_max 387 (±97) and 429 (±71) ml g DW⁻¹ h⁻¹, respectively) than on S. obliquus. Toxicity of P. agardhii had no effect on the filtration rate of the mussels. On the non-toxic M. aeruginosa (small coccoid cells), we also observed a type II functional response, although a type I functional response fitted almost as good to these data. For the colonial and toxic M. aeruginosa, a type I functional response fitted best to the data: IR increased linearly with food concentration and CR remained constant. CR_max and IR_max values for the (colonial) toxic M. aeruginosa (383 (±40) ml g DW⁻¹ h⁻¹; 3.7 mg C g DW⁻¹ h⁻¹) demonstrated that A. anatina filtered and ingested this cyanobacterium as good as the other cyanobacterial strains. However, on the non-toxic M. aeruginosa we observed the lowest CR_max of all phytoplankters (246 (±23) ml g DW⁻¹ h⁻¹, whereas IR_max was similar to that on toxic M. aeruginosa. The high maximum ingestion rates on S. obliquus and M. aeruginosa indicate a short handling time of these phytoplankton species. The high clearance rates on S. obliquus, toxic M. aeruginosa and P. agardhii reflect a high effort of the mussels to filter these particles out of the water column at low concentrations. The low clearance rates on non-toxic M. aeruginosa may be explained by the small size and coccoid form of this cyanobacterium, which may have impaired A. anatina to efficiently capture the cells. Although A. anatina had relatively high maximum clearance rates on non-toxic and toxic P. agardhii, this cyanobacterium does not seem to be a good food source, because of the observed high rates of pseudofaeces production and hence low ingestion rates.     

Endocrinological investigation into the reproductive cycles of two sympatric skate species, Malacoraja senta and Amblyraja radiata, in the western Gulf of Maine

The smooth skate, Malacoraja senta, and thorny skate, Amblyraja radiata, are two commercially exploited batoids found within the Gulf of Maine. During the past five years, we conducted a large study to accurately describe important biological life history parameters previously lacking for these species. As part of that project, the current study reports our findings on the hormonal profiles associated with the reproductive cycles of M. senta and A. radiata. Blood samples were obtained from mature M. senta and A. radiata of both sexes from all months of the year, and plasma testosterone (T), estradiol (E₂) and progesterone (P₄) concentrations were determined using radioimmunoassay (RIA). In female M. senta and A. radiata, monthly T concentrations ranged from 4,522 pg ml⁻¹ to 1,373 pg ml⁻¹ and 31,940 pg ml⁻¹ to 14,428 pg ml⁻¹, E₂ concentrations from 831 pg ml⁻¹ to 60 pg ml⁻¹ and 8,515 pg ml⁻¹ to 2,902 pg ml⁻¹, and P₄ concentrations from 3,027 pg ml⁻¹ to 20 pg ml⁻¹ and 3,264 pg ml⁻¹ to 331 pg ml⁻¹, respectively. No statistical differences were detected between any months for any hormone. Estradiol concentrations were not correlated with ovary weight, shell gland weight, or diameter of the largest follicles in either species. Monthly T concentrations in male M. senta and A. radiata ranged from 23,146 to 12,660 pg ml⁻¹ and from 57,500pg ml⁻¹ to 24,737 pg ml⁻¹, while E₂ concentrations ranged from 7.5 pg ml⁻¹ to undetectable and 103 to 30 pg ml⁻¹, respectively. No statistical differences were observed between months for either steroid. Testosterone concentrations were weakly correlated with testes weight and percent of stage VI spermatocysts in A. radiata, however, no correlation was detected between T and stage VI spermatocysts in M. senta. Collectively, these data support the previous conclusion that M. senta and A. radiata of both sexes are capable of reproducing year round in the western Gulf of Maine.     

The mmgA gene from Bacillus subtilis encodes a degradative acetoacetyl-CoA thiolase

Early in sporulation, the mother cell compartment of Bacillus subtilis transcribes the mother cell metabolic gene (mmg) operon. The gene mmgA was assigned by other workers using sequence homology as an acetyl-CoA acetyltransferase [E.C. 2.3.1.9]. The gene was overexpressed in Escherichia coli, and the protein was purified by Ni²⁺-affinity chromatography. However, the expected MmgA-catalyzed biosynthesis of acetoacetyl-CoA from acetyl-CoA was undetectable by a standard UV assay, HPLC, and mass spectrometry. These methods indicated a preference for the reverse degradative thiolytic reaction, with a k _cat of 80 s⁻¹, and a K _m of 70 and 50 μM for CoA and acetoacetyl-CoA, respectively.     

Comparison of microbial hosts and expression systems for mammalian CYP1A1 catalysis

Mammalian cytochrome P450 enzymes are of special interest as biocatalysts for fine chemical and drug metabolite synthesis. In this study, the potential of different recombinant microorganisms expressing rat and human cyp1a1 genes is evaluated for such applications. The maximum specific activity for 7-ethoxyresorufin O-deethylation and gene expression levels were used as parameters to judge biocatalyst performance. Under comparable conditions, E. coli is shown to be superior over the use of S. cerevisiae and P. putida as hosts for biocatalysis. Of all tested E. coli strains, E. coli DH5α and E. coli JM101 harboring rat CYP1A1 showed the highest activities (0.43 and 0.42 U g_CDW⁻¹, respectively). Detection of active CYP1A1 in cell-free E. coli extracts was found to be difficult and only for E. coli DH5α, expression levels could be determined (41 nmol g_CDW⁻¹). The presented results show that efficient expression of mammalian cyp1a1 genes in recombinant microorganisms is troublesome and host-dependent and that enhancing expression levels is crucial in order to obtain more efficient biocatalysts. Specific activities currently obtained are not sufficient yet for fine chemical production, but are sufficient for preparative-scale drug metabolite synthesis.     

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.     

Larvicidal activities against agricultural pests of transgenic Escherichia coli expressing combinations of four genes from Bacillus thuringiensis

The genes cry1Ac and cry1Ca from Bacillus thuringiensis subsps. kurstaki HD-73 and aizawai 4J4, respectively, encoding δ-endotoxins against lepidopteran larvae were isolated, cloned and expressed in Escherichia coli, with and without cyt1Aa (encoding cytolytic protein) and p20 (accessory protein) from subsp. israelensis. Nine combinations of the genes under control of an early T7, P _A1 inducible promoter, produced the encoding proteins. Toxicities were examined against larvae of three major agricultural pests: Pectinophora gossypiella, Helicoverpa armigera and Spodoptera littoralis. The clones expressing cyt1Aa, with or without p20, were not toxic. The clone expressing cry1Ac (pBt-1A) was the most toxic to P. gossypiella (LC₅₀ of 0.27 × 10⁸ cells g⁻¹). Clone pBt-1CA expressing cry1Ca and cry1Ac displayed the highest toxicity (LC₅₀ of 0.12 × 10⁸ cells ml⁻¹) against S. littoralis. Clone pBt-1CARCy expressing all four genes (cry1Ca, cry1Ac, p20, cyt1Aa) in tandem exhibited the highest toxicity to H. armigera (LC₅₀ of 0.16 × 10⁸ cells ml⁻¹). Cyt1Aa failed to raise the toxicity of these Cry toxins against P. gossypiella and S. littoralis but significantly enhanced toxicity against H. armigera. Two additional clones expressing either cry1Ac or cry1Ca under tandem promoters, P _A1 and P _psbA (constitutive), displayed significantly higher toxicities (7.5- to 140-fold) than their counterparts with P _A1 alone, reducing the LC₅₀ values to below 10⁷ cells ml⁻¹. Vadim Khasdan and Maria Sapojnik are contributed equally to this work.     

Bioactive metabolites from Alternaria brassicicola ML-P08, an endophytic fungus residing in Malus halliana

A total of 48 strains were isolated from the normal tissues of Malus halliana and the EtOAc extracts of their cultures were subjected to primary antimicrobial screening against four test bacteria and three fungi. As a result, 22 strains exhibited antimicrobial activity against at least one test microbe. Among them, Alternaria brassicicola ML-P08 showing strong activity (MICs: 0.31–2.50 mg/ml) was selected for further investigation on its secondary metabolites. Bioassay-guided fractionation of the EtOAc extract of its liquid culture afforded seven compounds, which were identified as alternariol (1), alternariol 9-methyl ether (2), altechromone A (3), herbarin A (4), cerevisterol (5), 3β,5α-dihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6) and 3β-hydroxy-(22E,24R)-ergosta-5,8,22-trien-7-one (7), respectively, by spectral means (MS, IR, ¹H- and ¹³C-NMR). In vitro antimicrobial assay showed that compound 3 was substantially active against Bacillus subtilis, Escherichia coli, Pseudomonas fluorescens and Candida albicans with the MICs of 3.9, 3.9, 1.8, and 3.9 μg/ml, respectively. Compound 4 also showed pronounced antifungal activity against Trichophyton rubrum and C. albicans with MICs of both 15.6 μg/ml. In addition, compound 1 exhibited strong xanthine oxidase inhibitory activity with the IC₅₀ of 15.5 μM, comparable to that of positive control, allopurinol (IC₅₀: 10.7 μM).     

Bioaugmentation of the decolorization rate of acid red GR by genetically engineered microorganism <Emphasis Type="Italic">Escherichia coli</Emphasis> JM109 (pGEX-AZR)

The study showed that the genetically engineered microorganism (GEM) bioaugment successfully the dye wastewater biotreatment systems to enhance acid red GR (ARGR) removal. Escherichia coli JM109 (pGEX-AZR) was the GEM with higher azoreductase activity. The kinetics of the ARGR decolorization by the E. coli JM109 (pGEX-AZR) agreed with Andrews model. The kinetic parameters, r _dye,max, K _s and K _i , were found to be 42.45 mg g⁻¹ h⁻¹, 584.93 mg L⁻¹ and 556.89 mg L⁻¹, respectively. The E. coli JM109 (pGEX-AZR) was tested in anaerobic sequencing batch reactors (AnSBR) in order to enhance the ARGR decolorization. The decolorization rate of ARGR was affected by the amount of E. coli JM109 (pGEX-AZR) inoculation and the best amount of inoculation was 10%. The continuous operations of the four bioreactors with different E. coli JM109 (pGEX-AZR) immobilization supports showed that the E. coli JM109 (pGEX-AZR) could bioaugment decolorization in AnSBRs with suspended and immobilized on macroporous foam carriers. For 42 days continuous operation in the AnSBRs, both the tolerance to ARGR concentration shock and the decolorization rate in these two bioaugmented AnSBRs are higher than those of the other two systems, control system and bioaugmented AnSBRs system with the sodium-alginate immobilized cells, the decolorization rate reached 90%. Changes in microbial community were detected by ribosomal intergenic spacer analysis (RISA) and amplified ribosomal DNA restriction analysis (ARDRA), which revealed that the introduced E. coli JM109 (pGEX-AZR) was persistent in the augmented systems and maintained higher metabolic activity.     

Fumigant toxicity of cassia bark and cassia and cinnamon oil compounds to <Emphasis Type="Italic">Dermatophagoides </Emphasis><Emphasis Type="Italic">farinae </Emphasis>and <Emphasis Type="Italic">Dermatophagoides pteronyssinus</Emphasis> (Acari: Pyroglyphidae)

The toxicity to adults of the American house dust mite, Dermatophagoides farinae, and the European house dust mite, Dermatophagoides pteronyssinus, of cassia bark and cassia and cinnamon oil compounds was examined using residual contact and vapour-phase toxicity bioassays. Results were compared with those of the currently used acaricides: benzyl benzoate and dibutyl phthalate. The acaricidal principles of cassia bark were identified as (E)-cinnamaldehyde and salicylaldehyde. In fabric-circle residual contact bioassays with adult D. farinae, salicylaldehyde (17.3 mg/m²) and (E)-cinnamaldehyde (25.8 mg/m²) were 2.5 and 1.7 times more toxic than benzyl benzoate (43.7 mg/m²), respectively, based on 24-h LD₅₀ values. The acaricidal activity was more pronounced in benzaldehyde, menthol, α-terpineol, and thymol (70.8–234.3 mg/m²) than in dibutyl phthalate (281.0 mg/m²). Against adult D. pteronyssinus, salicylaldehyde (17.3 mg/m²) and (E)-cinnamaldehyde (19.3 mg/m²) were 2.4- and 2.2-fold more active than benzyl benzoate (41.9 mg/m²). The toxicity of benzaldehyde, menthol, α-terpineol, and thymol (75.3–179.2 mg/m²) was higher than that of dibutyl phthalate (285.1 mg/m²). In vapour-phase toxicity tests with adult D. farinae, the test compounds described were much more effective in closed—but not in open—containers, indicating that the effect of these compounds was largely a result of action in the vapour phase.     

Citric acid production from sucrose using a recombinant strain of the yeast Yarrowia lipolytica

The yeast Yarrowia lipolytica is able to secrete high amounts of several organic acids under conditions of growth limitation and carbon source excess. Here we report the production of citric acid (CA) in a fed-batch cultivation process on sucrose using the recombinant Y. lipolytica strain H222-S4(p67ICL1) T5, harbouring the invertase encoding ScSUC2 gene of Saccharomyces cerevisiae under the inducible XPR2 promoter control and multiple ICL1 copies (10–15). The pH-dependent expression of invertase was low at pH 5.0 and was identified as limiting factor of the CA-production bioprocess. The invertase expression was sufficiently enhanced at pH 6.0–6.8 and resulted in production of 127–140 g l⁻¹ CA with a yield Y _CA of 0.75–0.82 g g⁻¹, whereas at pH 5.0, 87 g l ⁻¹ with a yield Y _CA of 0.51 gg⁻¹ were produced. The CA-productivity Q _CA increased from 0.40 g l ⁻¹ h⁻¹ at pH 5.0 up to 0.73 g l ⁻¹ h⁻¹ at pH 6.8. Accumulation of glucose and fructose at high invertase expression level at pH 6.8 indicated a limitation of CA production by sugar uptake. The strain H222-S4(p67ICL1) T5 also exhibited a gene–dose-dependent high isocitrate lyase expression resulting in strong reduction (<5%) of isocitric acid, a by-product during CA production.     

A highly thermostable trehalase from the thermophilic bacterium <Emphasis Type="Italic">Rhodothermus marinus</Emphasis>

Trehalases play a central role in the metabolism of trehalose and can be found in a wide variety of organisms. A periplasmic trehalase (α,α-trehalose glucohydrolase, EC 3.2.1.28) from the thermophilic bacterium Rhodothermus marinus was purified and the respective encoding gene was identified, cloned and overexpressed in Escherichia coli. The recombinant trehalase is a monomeric protein with a molecular mass of 59 kDa. Maximum activity was observed at 88°C and pH 6.5. The recombinant trehalase exhibited a K _m of 0.16 mM and a V _max of 81 μmol of trehalose (min)⁻¹ (mg of protein)⁻¹ at the optimal temperature for growth of R. marinus (65°C) and pH 6.5. The enzyme was highly specific for trehalose and was inhibited by glucose with a K _i of 7 mM. This is the most thermostable trehalase ever characterized. Moreover, this is the first report on the identification and characterization of a trehalase from a thermophilic bacterium.     

Cloning and characterization of a novel <Emphasis Type="SmallCaps">l</Emphasis>-arabinose isomerase from <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>

Based on analysis of the genome sequence of Bacillus licheniformis ATCC 14580, an isomerase-encoding gene (araA) was proposed as an l-arabinose isomerase (L-AI). The identified araA gene was cloned from B. licheniformis and overexpressed in Escherichia coli. DNA sequence analysis revealed an open reading frame of 1,422 bp, capable of encoding a polypeptide of 474 amino acid residues with a calculated isoelectric point of pH 4.8 and a molecular mass of 53,500 Da. The gene was overexpressed in E. coli, and the protein was purified as an active soluble form using Ni–NTA chromatography. The molecular mass of the purified enzyme was estimated to be ~53 kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and 113 kDa by gel filtration chromatography, suggesting that the enzyme is a homodimer. The enzyme required a divalent metal ion, either Mn²⁺or Co²⁺, for enzymatic activity. The enzyme had an optimal pH and temperature of 7.5 and 50°C, respectively, with a k _cat of 12,455 min⁻¹ and a k _cat/K _m of 34 min⁻¹ mM⁻¹ for l-arabinose, respectively. Although L-AIs have been characterized from several other sources, B. licheniformis L-AI is distinguished from other L-AIs by its wide pH range, high substrate specificity, and catalytic efficiency for l-arabinose, making B. licheniformis L-AI the ideal choice for industrial applications, including enzymatic synthesis of l-ribulose. This work describes one of the most catalytically efficient L-AIs characterized thus far.     

Heterologous expression of cDNAs encoding monodehydroascorbate reductases from the moss, <Emphasis Type="Italic">Physcomitrella patens</Emphasis> and characterization of the expressed enzymes

Monodehydroascorbate reductase (MDHAR; EC 1.6.5.4) catalyses the reduction of the monodehydroascorbate (MDHA) radical to ascorbate, using NADH or NADPH as an electron donor, and is believed to be involved in maintaining the reactive oxygen scavenging capability of plant cells. This key enzyme in the ascorbate-glutathione cycle has been studied here in the moss Physcomitrella patens, which is tolerant to a range of abiotic stresses and is increasingly used as a model plant. In the present study, three cDNAs encoding different MDHAR isoforms of 47 kDa were identified in P. patens, and found to exhibit enzymic characteristics similar to MDHARs in vascular plants despite low-sequence identity and a distant evolutionary relationship between the species. The three cDNAs for the P. patens MDHAR enzymes were expressed in Escherichia coli and the active enzymes were purified and characterized. Each recombinant protein displayed an absorbance spectrum typical of flavoenzymes and contained a single non-covalently bound FAD coenzyme molecule. The K _m and k _cat values for the heterologously expressed PpMDHAR enzymes ranged from 8 to 18 μM and 120–130 s⁻¹, respectively, using NADH as the electron donor. The K _m values were at least an order of magnitude higher for NADPH. The K _m values for the MDHA radical were ∼0.5–1.0 μM for each of the purified enzymes, and further kinetic analyses indicated that PpMDHARs follow a ‘ping–pong’ kinetic mechanism. In contrast to previously published data, site-directed mutagenesis indicated that the conserved cysteine residue is not directly involved in the reduction of MDHA.     

Functional identification of sll1383 from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp PCC 6803 as L-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning,expression and characterization

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K _m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V _max (∼0.033 μmol Pi/min/mg protein) was low. Li⁺ was found to be an inhibitor (IC₅₀ 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na⁺, K⁺ NH₄⁺) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg²⁺ dependent, which can be substituted partially by Mn²⁺. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.     

Improvement of succinate production by overexpression of a cyanobacterial carbonic anhydrase in Escherichia coli

Succinate fermentation was investigated in Escherichia coli strains overexpressing cyanobacterium Anabaena sp. 7120 ecaA gene encoding carbonic anhydrase (CA). In strain BL21 (DE3) bearing ecaA, the activity of CA was 21.8 U mg⁻¹ protein, whereas non-detectable CA activity was observed in the control strain. Meanwhile, the activity of phosphoenolpyruvate carboxylase (PEPC) increased from 0.2 U mg⁻¹ protein to 1.13 U mg⁻¹ protein. The recombinant bearing ecaA reached a succinate yield of 0.39 mol mol⁻¹ glucose at the end of the fermentation. It was 2.1-fold higher than that of control strain which was just 0.19 mol mol⁻¹ glucose. EcaA gene was also introduced into E. coli DC1515, which was deficient in glucose phosphotransferase, lactate dehydrogenase and pyruvate:formate lyase. Succinate yield can be further increased to 1.26 mol mol⁻¹ glucose. It could be concluded that the enhancement of the supply of HCO₃⁻ in vivo by ecaA overexpression is an effective strategy for the improvement of succinate production in E. coli.     

3-Methylglutaconyl-CoA hydratase from Acinetobacter sp

Acinetobacter strain IVS-B aerobically grows on isovalerate as sole carbon and energy source. Isovalerate is metabolised via isovaleryl-CoA, an intermediate of the oxidative (S)-leucine degradation pathway. A 3-methylglutaconyl-CoA hydratase (EC 4.2.1.18) was purified 65-fold to apparent homogeneity from cell-free extracts of isovalerate-grown cells of Acinetobacter strain IVS-B. The enzyme was found to be a homotetramer (115.2 kDa) composed of four identical subunits of 28.8 kDa not containing any cofactors. The enzyme was shown to catalyse the hydration of (E)-glutaconyl-CoA (k _cat=18 s⁻¹, K _m=40 μM) and the dehydration of (S)-3-hydroxyglutaryl-CoA (k _cat=13 s⁻¹, K _m=52 μM), albeit with somewhat lower catalytic efficiencies as compared to the 3-methyl derivatives, 3-methylglutaconyl-CoA (k _cat=138 s⁻¹, K _m=14 μM) and (S)-3-hydroxy-3-methylglutaryl-CoA (k _cat=60 s⁻¹, K _m=36 μM). Thus, the mechanistically simple syn-addition of water to the (E)-isomer of 3-methylglutaconyl-CoA of the leucine degradative pathway leading to the common intermediate (S)-3-hydroxy-3-methylglutaryl-CoA was assigned as the major physiological role to this enzyme. The amino acid sequence of 3-methylglutaconyl-CoA hydratase from Acinetobacter sp. was found to be related to over 100 prokaryotic enoyl-CoA hydratases (up to 50% identity), possibly all being 3-methylglutaconyl-CoA hydratases.An erratum to this article can be found at     

Molecular characterization of the recombinant iron-containing alcohol dehydrogenase from the hyperthermophilic Archaeon, Thermococcus strain ES1

The gene encoding a thermostable iron-containing alcohol dehydrogenase from Thermococcus Strain ES1 (ES1 ADH) was cloned, sequenced and expressed in Escherichia coli. The recombinant and native ES1 ADHs were purified using multistep column chromatography under anaerobic conditions. Both enzymes appeared to be homotetramers with a subunit size of 45 ± 1 kDa as revealed by SDS-PAGE, which was close to the calculated value (44.8 kDa). The recombinant ADH contained 1.0 ± 0.1 g-atom iron per subunit. Both enzymes were sensitive to oxygen with a half-life upon exposure to air of about 4 min. The recombinant enzyme exhibited a specific activity of 105 ± 2 U mg⁻¹, which was very similar to that of the native enzyme (110 ± 3 U mg⁻¹). The optimal pH-values for both enzymes for ethanol oxidation and acetaldehyde reduction were 10.4 and 7.0, respectively. Both enzymes also showed similar temperature-dependent activities, and catalyzed the oxidation of primary alcohols, but there was no activity towards methanol and secondary alcohols. Kinetic parameters of the enzymes showed lower K _m-values for acetaldehyde and NADPH and higher K _m-values for ethanol and NADP⁺. It is concluded that the gene encoding ES1 ADH was expressed successfully in E. coli. This is the first report of a fully active recombinant version of an iron-containing ADH from a hyperthermophile.