Cytotoxicity and Glycan-Binding Profile of a <Emphasis Type="SmallCaps">d</Emphasis>-Galactose-Binding Lectin from the Eggs of a Japanese Sea Hare (<Emphasis Type="Italic">Aplysia kurodai</Emphasis>)

A divalent cation-independent 16 kDa d-galactose binding lectin (AKL-2) was isolated from eggs of sea hare, Aplysia kurodai. The lectin recognized d-galactose and d-galacturonic acid and had a 32 kDa dimer consisting of two disulfide-bonded 16 kDa subunits. Eighteen N-terminus amino acids were identified by Edman degradation, having unique primary structure. Lectin blotting analysis with horseradish peroxidase-conjugated lectins has shown that AKL-2 was a glycoprotein with complex type oligosaccharides with N-acetyl d-glucosamine and mannose at non-reducing terminal. Two protein bands with 38 and 36 kDa in the crude extract of sea hare eggs after purification of the lectin was isolated by AKL-2-conjugated Sepharose column and elution with 0.1 M lactose containing buffer. It suggested that the lectin binds with an endogenous ligand in the eggs. AKL-2 kept extreme stability on haemagglutination activity if it was treated at pH 3 and 70 °C for 1 h. Glycan binding profile of AKL-2 by frontal affinity chromatography technology using 15 pyridylamine labeled oligosaccharides has been appeared that the lectin uniquely recognized globotriose (Galα1-4Galβ1-4Glc; Gb3) in addition to bi-antennary complex type N-linked oligosaccharides with N-acetyllactosamine. Surface plasmon resonance analysis of AKL-2 against a neo-glycoprotein, Gb3-human serum albumin showed the k _ass and k _diss values are 2.4 × 10³ M^?1 s^?1 and 3.8 × 10^?3 s^?1, respectively. AKL-2 appeared cytotoxicity against both Burkitt’s lymphoma Raji cell and erythroleukemia K562. The activity to Raji by the lectin was preferably cancelled by the co-presence of melibiose mimicing Gb3. On the other hand, K562 was cancelled effectively by lactose than melibiose. It elucidated that AKL-2 had cytotoxic ability mediated glycans structure to cultured cells.     

Deciphering the mode of action,structural and biochemical analysis of heparinase II/III (<Emphasis Type="Italic">Ps</Emphasis>PL12a) a new member of family 12 polysaccharide lyase from <Emphasis Type="Italic">Pseudopedobacter saltans</Emphasis>

Heparinases are widely used for production of clinically and therapeutically important bioactive oligosaccharides and in analyzing the polydisperse, heterogeneous, and complex structures of heparin/heparan sulfate. In the present study, the gene (1911 bp) encoding heparinase II/III of family 12 polysaccharide lyase (PsPL12a) from Pseudopedobacter saltans was cloned, expressed, and biochemically and functionally characterized. The purified enzyme PsPL12a of molecular size approximately 76 kDa exhibited maximum activity in the temperature range 45–50 °C and at pH 6.0. PsPL12a gave maximum activity at 1% (w/v) heparin under optimum conditions. The kinetic parameters, K _m and V_max, for PsPL12a were 4.6?±?0.5 mg/ml and 70?±?2 U/mg, respectively. Ten millimolars of each Mg²⁺ and Mn²⁺ ions enhanced PsPL12a activity by 80%, whereas Ni²⁺ inhibited by 75% and Co²⁺ by 10%, and EDTA completely inactivated the enzyme. Protein melting curve of PsPL12a gave a single peak at 55 °C and 10 mM Mg²⁺ ions and shifted the peak to 60 °C. The secondary structure analysis of PsPL12a by CD showed 65.12% α-helix, 11.84% β-strand, and 23.04% random coil. The degradation products of heparin by PsPL12a analyzed by ESI-MS spectra displayed peaks corresponding to heparin di-, tetra-, penta-, and hexa-saccharides revealing the endolytic mode of enzyme action. Heparinase II/III (PsPL12a) from P. saltans can be used for production of low molecular weight heparin oligosaccharides for their utilization as anticoagulants. This is the first report on heparinase cloned from P. saltans.     

A class V chitinase from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>: gene responses,enzymatic properties,and crystallographic analysis

Expression of a class V chitinase gene (At4g19810, AtChiC) in Arabidopsis thaliana was examined by quantitative real-time PCR and by analyzing microarray data available at Genevestigator. The gene expression was induced by the plant stress-related hormones abscisic acid (ABA) and jasmonic acid (JA) and by the stress resulting from the elicitor flagellin, NaCl, and osmosis. The recombinant AtChiC protein was produced in E. coli, purified, and characterized with respect to the structure and function. The recombinant AtChiC hydrolyzed N-acetylglucosamine oligomers producing dimers from the non-reducing end of the substrates. The crystal structure of AtChiC was determined by the molecular replacement method at 2.0 Å resolution. AtChiC was found to adopt an (β/α)₈ fold with a small insertion domain composed of an α-helix and a five-stranded β-sheet. From docking simulation of AtChiC with pentameric substrate, the amino acid residues responsible for substrate binding were found to be well conserved when compared with those of the class V chitinase from Nicotiana tabacum (NtChiV). All of the structural and functional properties of AtChiC are quite similar to those obtained for NtChiV, and seem to be common to class V chitinases from higher plants.     

Influence of organic solvents on the structural and thermal characteristics of silk protein from the web of <Emphasis Type="Italic">Orthaga exvinacea</Emphasis> Hampson (<Emphasis Type="Italic">Lepidoptera</Emphasis>: <Emphasis Type="Italic">Pyralidae</Emphasis>)

The silk protein from the web of Orthaga exvinacea was isolated, purified, and casted into films. This film was treated separately with methanol, acetone, ethyl acetate, and isopropyl alcohol in 50 % concentration for about 30 min. The treated films were thus dried in a desiccator and subjected to FTIR and TG-DTA analysis. The structural studies revealed that the organic solvents induce conformatory changes in the protein film, especially the most sensitive amide I (1650 cm^?1) band. This band had shifted to lower wavenumber (1633–1636 cm^?1). Furthermore, the conformatory characteristics associated with amide I band also changed from random coil to β-sheet. Generally, β-sheet contributes strength to the protein film. Among the treated films, film treated with acetone showed much thermal stability. Moreover, the film treated with methanol had shown two different temperatures of maximum degradation. It is concluded that in addition to β-sheet content, various other factors such as various processing conditions and structural organization of protein may influence the stability of the films.     

Enzyme-linked lectinosorbent assay of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

In this study, we developed a microplate sandwich analysis of Escherichia coli and Staphylococcus aureus bacterial pathogens based on the interaction of their cell wall carbohydrates with natural receptors called lectins. An immobilized lectin-cell-biotinylated lectin complex was formed in this assay. Here, we studied the binding specificity of several plant lectins to E. coli and S. aureus cells, and pairs characterized by high-affinity interactions were selected for the assay. Wheat germ agglutinin and Ricinus communis agglutinin were used to develop enzyme-linked lectinosorbent assays for E. coli and S. aureus cells with the detection limits of 4 × 10⁶ and 5 × 10⁵ cells/mL, respectively. Comparison of the enzyme-linked immonosorbent assay and the enzyme-linked lectinosorbent assay demonstrated no significant differences in detection limit values for E. coli. Due to the accessibility and universality of lectin reagents, the proposed approach is a promising tool for the control of a wide range of bacterial pathogens.     

Integrated control of tobacco black shank by combined use of riboflavin and <Emphasis Type="Italic">Bacillus subtilis</Emphasis> strain Tpb55

We investigated the effect of riboflavin on the biocontrol activity of Bacillus subtilis Tpb55 against Phytophthora nicotianae (Pn), which causes tobacco black shank. Riboflavin (0.2 mg ml^?1) significantly improved the biocontrol activity of Tpb55 (2.0 × 10⁸ cfu ml^?1). Riboflavin (0.02–0.5 mg ml^?1) alone could not significantly inhibit Pn growth. However, it enhanced the B. subtilis population, both in vitro and in tobacco roots and significantly increased the activity of defense enzymes, peroxidase, catalase, superoxide dismutase, and β-1,3-glucanase, in the roots of B. subtilis-treated tobacco seedlings. Our results indicate that riboflavin can stimulate the growth of B. subtilis Tpb55 and induce resistance to Pn in tobacco plants. These findings should boost the prospects for practical application of B. subtilis Tpb55 as a biocontrol agent against black shank of tobacco.     

Suitability of edaphic arthropods as prey for <Emphasis Type="Italic">Proctolaelaps bickleyi</Emphasis> and <Emphasis Type="Italic">Cosmolaelaps brevistilis</Emphasis> (Acari: Mesostigmata: Melicharidae,Laelapidae) under laboratory conditions

Soils are often complex habitats inhabited by a wide range of organisms, some harmful to plants and others beneficial, for example by attacking harmful organisms. Beneficial organisms include predatory mites, some of which have been commercialized for biological control of pest insects and mites. The objective of this work was to evaluate under laboratory condition the suitability of representative soil insect and mite pests, especially Aceria tulipae (Keifer), as prey to the soil-inhabiting predatory mites Proctolaelaps bickleyi (Bram) and Cosmolaelaps brevistilis (Karg). Predation, oviposition and survivorship of recently molted adult females of the predators were assessed in the dark in rearing chambers at 25 ± 1 °C and 75 ± 3% RH. Predation rate by P. bickleyi on A. tulipae was significantly higher than that by C. brevistilis (196.3 vs. 71.0 specimens/day). About 482 A. tulipae were preyed by each P. bickleyi at each day, when 500 A. tulipae were made available daily to the predator. Oviposition rate on that prey was also higher for P. bickleyi (4.2 eggs/day). For C. brevistilis, the highest level of oviposition was on Caliothrips phaseoli (Hood) (1.2 eggs/day). Survivorship was always higher for C. brevistilis (≥ 70%), given its ability to remain alive relatively long even in the absence of prey. High rates of survivorship of P. bickleyi were observed on A. tulipae, Bradysia matogrossensis (Lane) and Protorhabditis sp. Promising results were obtained for P. bickleyi on A. tulipae and even on other prey, justifying the conduction of complementary studies under field condition.     

Molecular Cloning,Expression and Characterization of Pectin Methylesterase (<Emphasis Type="Italic">Ct</Emphasis>PME) from <Emphasis Type="Italic">Clostridium thermocellum</Emphasis>

Many phytopathogenic micro-organisms such as bacteria and fungi produce pectin methylesterases (PME) during plant invasion. Plants and insects also produce PME to degrade plant cell wall. In the present study, a thermostable pectin methylesterase (CtPME) from Clostridium thermocellum belonging to family 8 carbohydrate esterase (CE8) was cloned, expressed and purified. The amino acid sequence of CtPME exhibited similarity with pectin methylesterase from Erwinia chrysanthemi with 38% identity. The gene encoding CtPME was cloned into pET28a(+) vector and expressed using Escherichia coli BL21(DE3) cells. The recombinant CtPME expressed as a soluble protein and exhibited a single band of molecular mass approximately 35.2 kDa on SDS-PAGE gels. The molecular mass, 35.5 kDa of the enzyme, was also confirmed by MALDI-TOF MS analysis. Notably, highest protein concentration (11.4 mg/mL) of CtPME was achieved in auto-induction medium, as compared with LB medium (1.5 mg/mL). CtPME showed maximum activity (18.1 U/mg) against citrus pectin with >85% methyl esterification. The optimum pH and temperature for activity of CtPME were 8.5 and 50 °C, respectively. The enzyme was stable in pH range 8.0–9.0 and thermostable between 45 and 70 °C. CtPME activity was increased by 40% by 5 mM Ca²⁺ or Mg²⁺ ions. Protein melting curve of CtPME gave a peak at 80 °C. The peak was shifted to 85 °C in the presence of 5 mM Ca²⁺ ions, and the addition of 5 mM EDTA shifted back the melting peak to 80 °C. CtPME can be potentially used in food and textile industry applications.     

Effects of Lectins on initial attachment of cariogenic <Emphasis Type="Italic">Streptococcus mutans</Emphasis>

Oral bacteria initiate biofilm formation by attaching to tooth surfaces via an interaction of a lectin-like bacterial protein with carbohydrate chains on the pellicle. This study aimed to find naturally derived lectins that inhibit the initial attachment of a cariogenic bacterial species, Streptococcus mutans (S. mutans), to carbohydrate chains in saliva in vitro. Seventy kinds of lectins were screened for candidate motifs that inhibit the attachment of S. mutans ATCC 25175 to a saliva-coated culture plate. The inhibitory effect of the lectins on attachment of the S. mutans to the plates was quantified by crystal violet staining, and the biofilm was observed under a scanning electron microscope (SEM). Surface plasmon resonance (SPR) analysis was performed to examine the binding of S. mutans to carbohydrate chains and the binding of candidate lectins to carbohydrate chains, respectively. Moreover, binding assay between the biotinylated-lectins and the saliva components was conducted to measure the lectin binding. Lectins recognizing a salivary carbohydrate chain, Galβ1-3GalNAc, inhibited the binding of S. mutans to the plate. In particular, Agaricus bisporus agglutinin (ABA) markedly inhibited the binding. This inhibition was confirmed by SEM observation. SPR analysis indicated that S. mutans strongly binds to Galβ1-3GalNAc, and ABA binds to Galβ1-3GalNAc. Finally, the biotinylated Galβ1-3GalNAc-binding lectins including ABA demonstrated marked binding to the saliva components. These results suggest that ABA lectin inhibited the attachment of S. mutans to Galβ1-3GalNAc in saliva and ABA can be useful as a potent inhibitor for initial attachment of oral bacteria and biofilm formation.     

Reproduction and development in some species of the weakly electric genus <Emphasis Type="Italic">Campylomormyrus</Emphasis> (Mormyridae,Teleostei)

Reproduction in captivity of four species of the mormyrid genus Campylomormyrus was investigated. Cyclical reproduction was provoked by changing water conductivity (C) alone: decreasing C led to gonadal recrudescence, an increase induced gonad regression. Data on the reproduction and development of three species are presented. All three species are indeterminate fractional spawners. Spawning intervals ranged from 6 to 66 days in C. rhynchophorus, 10–75 days in C. tshokwe, and 18 days in C. compressirostris (calculated values). Fecundities (eggs per fractional spawning) ranged from 70 to 1570 eggs in C. rhynchophorus, 100–1192 in C. tshokwe, and 38–246 in C. compressirostris. Spawnings/ovipositions occurred during the second half of the night; no parental care was observed; no special spawning substrates were necessary. C. compressirostris successfully spawned in breeding groups, C. rhynchophorus as pair. Agonistic behavior in the C. tshokwe pair forced us to divide the breeding tank; therefore, only ovipositions occurred. However, injection of an artificial GnRH hormone allowed us to obtain ripe eggs and sperm and to perform successful artificial reproduction. All three species produce yolky, slightly sticky eggs. Egg diameter ranges from 2.3–3.0 mm. Hatching occurred on day 3, feeding started on day 11. Transition from larval to juvenile stage occurred at around 20 mm total length (TL). At this size C. rhynchophorus developed a higher body than the two other species and differences between the species in the melanin pigmentation of the unpaired fins occurred. Between 32 and 35 mm TL the upper and lower jaws started to elongate.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> spp. increases root biomass and tropane alkaloid yields in transgenic hairy roots of <Emphasis Type="Italic">Datura</Emphasis> spp.

Transgenic hairy roots of Datura spp., established using strain A4 of Agrobacterium rhizogenes, are genetically stable and produce high levels of tropane alkaloids. To increase biomass and tropane alkaloid content of this plant tissue, four Pseudomonas strains, Pseudomonas fluorescens P64, P66, C7R12, and Pseudomonas putida PP01 were assayed as biotic elicitors on transgenic hairy roots of Datura stramonium, Datura tatula, and Datura innoxia. Alkaloids were extracted from dried biomass, and hyoscyamine and scopolamine were quantified using liquid chromatography-tandem mass spectrometry analysis. D. stramonium and D. innoxia biomass production was stimulated by all Pseudomonas spp. strains after a 5-d treatment. All strains of P. fluorescens increased hyoscyamine yields compared to untreated cultures after both 5 and 10 d of treatment. Hyoscyamine yields were highest in D. tatula cultures exposed to a 5-d treatment with C7R12 (16.633 + 0.456 mg g^?1 dry weight, a 431% increase) although the highest yield increases compared to the control were observed in D. stramonium cultures exposed to strains P64 (511% increase) and C7R12 (583% increase) for 10 d. D. innoxia showed the highest scopolamine yields after elicitation with P. fluorescens strains P64 for 5 d (0.653 + 0.021 mg g^?1 dry weight, a 265% increase) and P66 for 5 and 10 d (5 d, 0.754 + 0.0.031 mg g^?1 dry weight, a 321% increase; 10 d 0.634 + 0.046 mg g^?1 dry weight, a 277% increase). These results show that the Pseudomonas strains studied here can positively and significantly affect biomass and the yields of hyoscyamine and scopolamine from transgenic roots of the three Datura species.     

Phycoremediation potential,physiological, and biochemical response of <Emphasis Type="Italic">Amphora subtropica</Emphasis> and <Emphasis Type="Italic">Dunaliella</Emphasis> sp. to nickel pollution

Metal pollution can produce many biological effects on aquatic environments. The marine diatom Amphora subtropica and the green alga Dunaliella sp. possess a high metal absorption capacity. Nickel (Ni) removal by living cells of A. subtropica and Dunaliella sp. was tested in cultures exposed to different Ni concentrations (100, 200, 300, and 500 mg L^?1). The amount of Ni removed by the microalgae increased with the time of exposure and the initial Ni concentration in the medium. The metal, which was mainly removed by bioadsorption to Dunaliella sp. cell surfaces (93.63% of total Ni (for 500 mg Ni L^?1) and by bioaccumulation (80.82% of total Ni (for 300 mg Ni L^?1) into Amphora subtropica cells, also inhibited growth. Exposure to Ni drastically reduced the carbohydrate and protein concentrations and increased total lipids from 6.3 to 43.1 pg cell^?1, phenolics 0.092 to 0.257 mg GAE g^?1 (Fw), and carotenoid content, from 0.08 to 0.59 mg g^?1 (Fw), in A. subtropica. In Dunaliella sp., total lipids increased from 26.1 to 65.3 pg cell^?1, phenolics from 0.084 to 0.289 mg GAE g^?1 (Fw), and carotenoid content from 0.41 to 0.97 mg g^?1 (Fw). These compounds had an important role in protecting the algae against ROS generated by Ni. In order to cope with Ni stress shown by the increase of TBARS level, enzymatic (SOD, CAT, and GPx) ROS scavenging mechanisms were induced.     

Isolation,characterization and analysis of the agglutinative activity of a lectin from <Emphasis Type="Italic">Crotalaria spectabilis</Emphasis>

Lectins are proteins with ability to recognize specific carbohydrates. These are present in virtually all organisms and have increasing applications in biotechnology. Here, our aim was to purify lectins from seeds of Crotalaria spectabilis Roth and determine their agglutinative ability. In this study, 45 g of seeds were milled, their proteins were precipitated by acetone or ammonium sulfate and purified by exclusion and ion-exchange chromatography. An isolated lectin was submitted to tests for hemagglutination and inhibition of hemagglutinating activity by carbohydrates as well as tests for its response to chelating and reducing agents. Our results show that the apparent molecular weight (as determined by SDS-PAGE) of the lectin is 30 kDa, and the tests for inhibition of erythrocytes’ agglutinative activity by sugars were positive for d-galactose and N-acetyl-d-galactosamine. Data obtained with the chelating agent EDTA demonstrated the presence of divalent cations in the protein structure. However, the reducing agent 2-mercaptoethanol was unable to inhibit the protein’s bioactivity. The lectin agglutinated the blood groups A, B, AB and O, as well as bacterial lineages from the species Leptospira interrogans and Leptospira biflexa, indicating a prospective application in the diagnosis and treatment of leptospirosis.     

A review of the history of research and control of <Emphasis Type="Italic">Rhipicephalus</Emphasis> (<Emphasis Type="Italic">Boophilus</Emphasis>) <Emphasis Type="Italic">microplus</Emphasis>, babesiosis and anaplasmosis in Uruguay

The effect of five constant temperatures (16, 20, 24, 28 and 32 °C) on the development, survival and reproduction of Tetranychus cinnabarinus (Boisduval) [=?Tetranychus urticae Koch (red form)] fed on cassava leaves was examined in the laboratory at 85% relative humidity. Development time of various immature stages decreased with increasing temperature, with total egg-to-adult development time varying from 27.7 to 6.7 days. The lower thermal threshold for development was 10.8 °C and the thermal constant from egg to adult was 142.4 degree-days. Pre- and post-oviposition period and female longevity all decreased as temperature increased. The longest oviposition period was observed at 20 °C with 20.4 days. Under different temperatures, mated females laid, on average, 1.0, 2.9, 4.7, 4.7 and 4.9 eggs per day, respectively. The maximum fecundity (81.5 eggs per female) was at 28 °C and the intrinsic rate of increase (r _m ) was highest (0.25) at 32 °C. The results of this study indicate that T. cinnabarinus population could increase rapidly when cassava leaves serve as a food source. At the appropriate temperature T. cinnabarinus could seriously threaten growth of cassava.     

Hemato-Immunological Responses and Disease Resistance in Siberian Sturgeon <Emphasis Type="Italic">Acipenser baerii</Emphasis> Fed on a Supplemented Diet of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis>

A feeding trial was conducted to investigate the effects of different levels of dietary Lactobacillus plantarum on hemato-immunological parameters and resistance against Streptococcus iniae infection in juvenile Siberian sturgeon Acipenser baerii. Fish (14.6 ± 2.3 g) were fed three experimental diets prepared by supplementing a basal diet with L. plantarum at different concentrations [1 × 10⁷, 1 × 10⁸ and 1 × 10⁹ colony-forming units (cfu) g^?1] and a control (non-supplemented basal) diet for 8 weeks. Innate immune responses (immunoglobulin (Ig), alternative complement activity (ACH50) and lysozyme activity) were significantly higher in fish fed the 1 × 10⁸ and 1 × 10⁹ cfu g^?1 L. plantarum diet compared to the other groups (P < 0.05). Furthermore, fish fed on various levels of L. plantarum significantly showed higher red blood cell (RBC), hemoglobin (Hb), white blood cell (WBC) and monocyte compared to those of the control group (P < 0.05). At the end of the feeding experiment, some fish were challenged with S. iniae to quantify the level of disease resistance. The mortality after S. iniae challenge was decreased in fish fed a probiotic. These results indicated that dietary supplementation of L. plantarum improved immune response and disease resistance of Siberian sturgeon juvenile.     

Impact of elevated CO<Subscript>2</Subscript> in <Emphasis Type="Italic">Casuarina equisetifolia</Emphasis> rooted stem cuttings inoculated with <Emphasis Type="Italic">Frankia</Emphasis>

Impact of different levels of elevated CO ₂ on the activity of Frankia (Nitrogen-fixing actinomycete) in Casuarina equisetifolia rooted stem cuttings has been studied to understand the relationship between C. equisetifolia, Frankia and CO₂. The stem cuttings of C. equietifolia were collected and treated with 2000 ppm of Indole Butyric Acid (IBA) for rooting. Thus vegetative propagated rooted stem cuttings of C. equisetifolia were inoculated with Frankia and placed in the Open top chambers (OTC) with elevated CO₂ facilities. These planting stocks were maintained in the OTC for 12 months under different levels of elevated CO₂ (ambient control, 600 ppm, 900 ppm). After 12 months, the nodule numbers, bio mass, growth, and photosynthesis of C. equisetifolia rooted stem cuttings inoculated with Frankia were improved under 600 ppm of CO₂. The rooted stem cuttings of C. equisetifolia inoculated with Frankia showed a higher number of nodules under 900 ppm of CO₂ and cuttings without Frankia inoculation exhibited poor growth. Tissue Nitrogen (N) content was also higher under 900 ppm of CO₂ than ambient control and 600 ppm levels. The photosynthetic rate was higher (17.8 μ mol CO₂ m^?2 s^?1) in 900 ppm of CO₂ than in 600 ppm (13.2 μ mol CO₂ m^?2 s^?1) and ambient control (8.3 μ mol CO₂ m^?2 s^?1). This study showed that Frankia can improve growth, N fixation and photosynthesis of C. equietifolia rooted stem cuttings under extreme elevated CO₂ level conditions (900 ppm).     

Single point mutations enhance activity of <Emphasis Type="Italic">cis</Emphasis>-epoxysuccinate hydrolase

Objectives

To enhance activity of cis-epoxysuccinate hydrolase from Klebsiella sp. BK-58 for converting cis-epoxysuccinate to tartrate.

Results

By semi-saturation mutagenesis, all the mutants of the six important conserved residues almost completely lost activity. Then random mutation by error-prone PCR and high throughput screening were further performed to screen higher activity enzyme. We obtained a positive mutant F10D after screening 6000 mutations. Saturation mutagenesis on residues Phe10 showed that most of mutants exhibited higher activity than the wild-type, and the highest mutant was F10Q with activity of 812 U mg^?1 (k _cat /K _m , 9.8 ± 0.1 mM^?1 s^?1), which was 230 % higher than that of wild-type enzyme 355 U mg^?1 (k _cat /K _m , 5.3 ± 0.1 mM^?1 s^?1). However, the thermostability of the mutant F10Q slightly decreased.

Conclusions

The catalytic activity of a cis-epoxysuccinate hydrolase was efficient improved by a single mutation F10Q and Phe10 might play an important role in the catalysis.

     

Impact of Chemical,Organic and Bio-Fertilizers Application on Bell Pepper, <Emphasis Type="Italic">Capsicum annuum</Emphasis> L. and Biological Parameters of <Emphasis Type="Italic">Myzus persicae</Emphasis> (Sulzer) (Hem.: Aphididae)

Myzus persicae (Sulzer) is a polyphagous aphid that causes chlorosis, necrosis, stunting, and reduce growth rate of the host plants. In this research, the effects of Zinc sulfate and vermicompost (30%), Bacillus subtilis, Pseudomonas fluorescens, Glomus intraradices, G. intraradices × B. subtilis, and G. intraradices × P. fluorescens compared to control was investigated on the growth characters of Capsicum annuum L. and biological parameters of M. persicae. Different fertilizers caused a significant effect on growth characters of C. annuum and biological parameters of M. persicae. The highest plant growth was observed on Zinc sulfate and B. subtilis treated plants, and the lowest was on control. Increase in the amount of specific leaf area (SLA) (0.502 mm² mg^?1) was significantly higher in the B. subtilis than other fertilizer treatments. The longest (10.3 days) and the shortest (5.3 days) developmental times of M. persicae nymphs were observed on 30% vermicompost and Zinc sulfate treatments, respectively. The lowest adult longevity periods of M. persicae (11.2 and 11.3 days) were observed on G. intraradices × B. subtilis and 30% vermicompost treatments, respectively, and the longest ones (16.4 days) on Zinc sulfate. The highest rate of nymphal mortality and the lowest amount of nymphal growth index (NGI) were recorded on 30% vermicompost. The nymphs reared on Zinc sulfate treatment had the lowest rate of nymphal mortality and the highest amount of NGI. Thus, amending the soil with 30% vermicompost had a significantly negative effect on the biological parameters of M. persicae that can be used as an ecological control tactic for this pest.     

Chemical defences of native European coccinellid eggs against intraguild predation by the invasive Asian coccinellid, <Emphasis Type="Italic">Harmonia axyridis</Emphasis> (Pallas)

Harmonia axyridis (Pallas) is a coccinellid of Asian origin that has recently invaded substantial parts of Europe and is suspected to affect native coccinellid populations through intraguild predation and competition for food. Previous work has shown that two species from the Calvia genus appeared to be well protected against H. axyridis predation. To deepen our understanding on chemical protection of Calvia spp. and the predation risk by H. axyridis, we tested for susceptibility and palatability of Calvia spp. and H. axyridis eggs against predation by H. axyridis neonate larvae. Results show that eggs of C. quatuordecimguttata were mostly not eaten by H. axyridis, while eggs of the congeneric C. decemguttata were found to be largely unprotected against predation by the invasive coccinellid. We also observed that H. axyridis first instars successfully cannibalized on conspecific eggs. Removing the surface chemicals from C. quatuordecimguttata eggs resulted in significantly reduced protection from being preyed upon by H. axyridis, while applying these extracts onto C. decemguttata and H. axyridis eggs resulted in increased protection against H. axyridis larvae. The importance of surface chemicals in the interactions between H. axyridis and native coccinellids was confirmed by GC–MS analysis, showing a high diversity of hydrocarbons located on the surface of C. quatuordecimguttata eggs, i.e. more than twice as many when compared to C. decemguttata. Survival of H. axyridis larvae feeding on eggs of C. quatuordecimguttata, C. decemguttata or conspecific eggs, from which surface chemicals were removed by washing them with hexane, was not different from survival on unwashed eggs.     

<Emphasis Type="Italic">Plectosphaerella cucumerina</Emphasis> as a bioherbicide for <Emphasis Type="Italic">Cirsium arvense</Emphasis>: proof of concept

Plectosphaerella cucumerina (Lindf.) W. Gams was evaluated as a bioherbicide for Cirsium arvense L. (Scop.) using a Canadian and a New Zealand isolate. Both isolates defoliated C. arvense when applied at 10¹³ conidia ha^?1 in water volumes ranging from 250 to 6400 l ha^?1 with a rapid decline in effect with declining conidial dose. Repeat application and the addition of the adjuvant Pulse^® penetrant to the conidial suspension increased the disease severity in C. arvense. Maximum disease occurred at 20 °C with a 48 h post-application dew period. The experiments demonstrate that P. cucumerina can defoliate C. arvense under the environmental conditions of temperate pastures where the weed is problematic. The results also show that modifications to formulation and strategic application may reduce the 48 h dew period requirement and risk to non-target species respectively, supporting the conclusion that the fungus has potential as a bioherbicide for C. arvense.