Flufenerim, a novel insecticide acting on diverse insect pests: biological mode of action and biochemical aspects

A new chemical compound was tested for its insecticidal activity against several major insect pests. The compound, called "flufenerim", has a core pyrimidine structure and an unknown mode of action and showed potent activity against the sweet potato whitefly Bemisia tabaci (Gennadius), the green peach aphid Myzus persicae (Sulzer), and the African cotton leafworm Spodoptera littoralis (Boisduval); however, it did not show any activity against two thrips species: western flower thrips Frankliniella occidentalis (Pergande) and tobacco thrips Thrips tabaci (Lindeman). The compound was relatively potent against the three tested pests and caused mortality rates that reached up to 100% at concentrations under 10 mg of active ingredient (ai) L(-1). The action of the compound was very fast, and mortality was observed within 48 h after exposure of the insects to treated leaves. A unique characteristic of this compound is its very short residual activity, which approximates to 4 days after application under laboratory conditions and to 2 days under outdoor conditions for both B. tabaci and S. littoralis. Although this new compound's mode of action is yet unknown, its rapid and potent action against sap-sucking pests suggests that it acts on a very important target site in the insect body and possibly could be applied very close to harvesting.     

Purification, cloning, and immunological characterization of arginine kinase, a novel allergen of Octopus fangsiao

Arginine kinase (AK) is an important enzyme participating in energy metabolism in invertebrates, but, to date, there have been no reports that AK from octopus is an allergen. In this study, octopus AK was purified, and its molecular biological, immunological, and physicochemical characterizations were analyzed. The results showed that octopus AK was purified and confirmed by mass spectrometry for the first time, and its molecular mass was 38 kDa. The full-length gene sequence of octopus AK encompassed 1209 bp and was predicted to encode a protein with 348 amino acid residues. The homology of octopus AK and crustacean AK was about 54%, but the similarity between their three-dimensional structures was high. Octopus AK could react with mouse anti-shrimp AK and rabbit anti-crab AK polyclonal antibody singly. Octopus AK could also react with specific IgE of the sera from octopus-allergic patients effectively, whereas crab AK could inhibit the reaction between them. Finally, the IgE-binding activity of octopus AK could be reduced in the processes of thermal or acid-alkali treatment. In summary, AK was identified as a novel allergen in octopus, which had a sensitizing ability similar to that of crustacean AK. This is significant in allergy diagnosis and the treatment of octopus-allergic disorders.     

Molecular cloning, recombinant expression, and immunological characterization of a novel allergen from tartary buckwheat

Buckwheat is generally regarded as a nutritionally rich food source. However, earlier studies prove that it also causes allergies to subjects. Allergenic proteins with a strong IgE-binding activity have been identified in common buckwheat (CB) and a 24 kDa allergen (rTBa) in tartary buckwheat (TB). The objective of this research was to clone and express a novel allergen in tartary buckwheat and to evaluate its structure and immunological activity. The 1773 bp full-length cDNA was amplified and cloned from the total RNA of TB by polymerase chain reaction (PCR) and rapid amplification of cDNA ends (RACE) methods. Its nucleotide sequence had high similarity with legume-like 13S storage protein mRNA in CB. The deduced amino acid sequence included a putative signal peptide and 18 fragments as its epitope sites. The predicted full-length TB allergen sequence was found to have two domains, and the recombinant protein reacted with sera from patients with positive IgE binding to buckwheat and had a lower binding ability than the recombinant TBa and recombinant TBb (C- and N-terminal amino acid sequence of TBt codes for protein). This fact suggests that full-length TB allergen may hydrolyze to two domains in vivo, decreasing the IgE-binding ability.     

Isolation and characterization of a novel tannase from a metagenomic library

A novel gene (designated as tan410) encoding tannase was isolated from a cotton field metagenomic library by functional screening. Sequence analysis revealed that tan410 encoded a protein of 521 amino acids. SDS-PAGE and gel filtration chromatography analysis of purified tannase suggested that Tan410 was a monomeric enzyme with a molecular mass of 55 kDa. The optimum temperature and pH of Tan410 were 30 °C and 6.4. The activity was enhanced by addition of Ca(2+), Mg(2+) and Cd(2+). In addition, Tan410 was stable in the presence of 4 M NaCl. Chlorogenic acid, rosmarinic acid, ethyl ferulate, tannic acid, epicatechin gallate and epigallocathchin gallate were efficiently hydrolyzed by recombinant tannase. All of these excellent properties make Tan410 an interesting enzyme for biotechnological application.     

Directed evolution and characterization of a novel D-pantonohydrolase from Fusarium moniliforme

D-Pantonohydrolase has attracted increasing attention as a biocatalyst for stereospecific production of D-pantoic acid. The Fusarium moniliforme D-pantonohydrolase was selected for directed evolution through error-prone Polymerase Chain Reaction (PCR) combined with DNA shuffling for improved activity and pH stability using a convenient two-step high-throughput screening method based on the product formation and pH indicator. After three sequential error-prone PCRs and two rounds of DNA shuffling followed by screening, about 60 positive mutants were produced and a best mutant, Mut H-1287, with improved activity and pH stability was obtained. As compared to wild-type D-pantonohydrolase, Mut H-1287 showed a 10.5-fold higher specific activity; moreover, it could retain 85% of its original activity after incubation under low pH. Gene analysis indicated that the Mut H-1287 had D63H, K118Q, and V241I substitutions. The wild-type and evolved D-pantonohydrolase (Mut H-1287) was purified in three steps. The activities and characteristics of purified wild-type and evolved D-pantonohydrolase were also studied and compared.     

Preparation and characterization of the inclusion complex of chlorpyrifos in cyclodextrins to improve insecticide formulations

The chemical control of crops by organophosphate insecticide treatment is usually limited because the insecticides do not maintain their efficiency for long periods for several reasons, including environmental conditions or rapid degradation of the active ingredient. Chlorpyrifos is an organophosphate insecticide used worldwide to control a variety of soil insects and arthropods in a wide range of crops. It is easily soluble in organic solvents but shows poor water solubility. The inclusion of chrorpyrifos in cyclodextrins (CDs) improves its water solubility, bioavailability, and insecticidal activity and helps prevent overdosing, leading to more cost-effective and more environmentally friendly agricultural practices. Solubility studies of chlorpyrifos in the presence of different types of CDs show G2-beta-CDs to be the most effective CDs in the complexation process, giving 1:2 complexes, with complexation constant (Kc) values of 12.34 +/- 3.1 M(-1) for K1 and 3895 +/- 183 M(-1) for K2. These complexation constant values were corroborated by applying a fluorimetric method.     

Purification and characterization of a novel extracellular tripeptidyl peptidase from Rhizopus oligosporus

A novel extracellular tripeptidyl peptidase (TPP) was homogenously purified from the culture supernatant of Rhizopus oligosporus by sequential fast protein liquid chromatography. The purified enzyme was a 136.5 kDa dimer composed of identical subunits. The effects of inhibitors and metal ions indicated that TPP is a metallo- and serine protease. TPP was activated by divalent cations, such as Co(2+) and Mn(2+), and completely inhibited by Cu(2+). Enzyme activity was optimal at pH 7.0 and 45 °C with a specific activity of 281.9 units/mg for the substrate Ala-Ala-Phe-pNA. The purified enzyme catalyzed cleavage of various synthetic tripeptides but not when proline occupied the P1 position. Purified TPP cleaved the pentapeptide Ala-Ala-Phe-Tyr-Tyr and tripeptide Ala-Ala-Phe, confirming the TPP activity of the enzyme.     

Molecular cloning, purification, and biochemical characterization of a novel pyrethroid-hydrolyzing esterase from Klebsiella sp. strain ZD112

The gene encoding pyrethroid-hydrolyzing esterase (EstP) from Klebsiella sp. strain ZD112 was cloned into Escherichia coli and sequenced. A sequence analysis of the DNA responsible for the estP gene revealed an open reading frame of 1914 bp encoding for a protein of 637 amino acid residues. No similarities were found by a database homology search using the nucleotide and deduced amino acid sequences of the esterases and lipases. EstP was heterologously expressed in E. coli and purified. The molecular mass of the native enzyme was approximately 73 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of EstP indicated molecular masses of 73 and 73.5 kDa, respectively, suggesting that EstP is a monomer. The purified EstP not only degraded many pyrethroid pesticides and the organophosphorus insecticide malathion, but also hydrolyzed rho-nitrophenyl esters of various fatty acids, indicating that EstP is an esterase with broad substrates. The K(m) for trans- and cis-permethrin and k(cat)/K(m) values indicate that EstP hydrolyzes both these substrates with higher efficiency than the carboxylesterases from resistant insects and mammals. The catalytic activity of EstP was strongly inhibited by Hg2+, Ag+, and rho-chloromercuribenzoate, whereas a less pronounced effect (3-8% inhibition) was observed in the presence of divalent cations, the chelating agent EDTA, and phenanthroline.     

Purification and characterization of a novel pyrethroid hydrolase from Aspergillus niger ZD11

The pyrethroid pesticides residues on foods and environmental contamination are a public safety concern. Pretreatment with pyrethroid hydrolase has the potential to alleviate the conditions. For this purpose, a fungus capable of using pyrethroid pesticides as a sole carbon source was isolated from the soil and characterized as Aspergillus niger ZD11. A novel pyrethroid hydrolase from cell extract was purified 41.5-fold to apparent homogeneity with 12.6% overall recovery. It is a monomeric structure with a molecular mass of 56 kDa, a pI of 5.4, and the enzyme activity was optimal at 45 degrees C and pH 6.5. The activities were strongly inhibited by Hg(2+), Ag(+), and rho-chloromercuribenzoate, whereas less pronounced effects (5-10% inhibition) were observed in the presence of the remaining divalent cations, the chelating agent EDTA and phenanthroline. The purified enzyme hydrolyzed various insecticides with similar carboxylester. trans-Permethrin is the preferred substrate.     

Isolation,purification, and biochemical characterization of a novel water soluble protein from Inca peanut (Plukenetia volubilis L.)

A water soluble storage albumin from Inca peanut (IPA) accounted for approximately 25% (w/w) of defatted seed flour weight, representing 31% of the total seed protein. IPA is a 3S storage protein composed of two glycosylated polypeptides, with estimated molecular weights (MW) of 32800 and 34800 Da, respectively. IPA has an estimated sugar content of 4.8% +/- 0.92% (n = 6). IPA is a basic protein (pI of approximately 9.4) and contains all of the essential amino acids in adequate amounts when compared to the FAO/WHO recommended pattern for a human adult. The tryptophan content of IPA is unusually high (44 mg/g of protein), whereas the phenylalanine content is low (9 mg/g of protein). IPA is a highly digestible protein in vitro.     

Enantiomeric separation and toxicity of an organophosporus insecticide, pyraclofos

Despite the fact that the biological processes of chiral pesticides are enantioselective, knowledge of the toxicities of pyraclofos due to enantiospecificity is scarce. In this study, the optical isomers of pyraclofos were separated and their toxicities to butyrylcholinesterase (BChE) and Daphnia magna were assessed. Baseline resolution of the enantiomers was obtained on both Chiralcel OD and Chiralpak AD columns. The effect of the mobile phase composition and column temperature were then discussed. The resolved enantiomers were characterized by their optical rotation and circular dichroism signs. The anti-BChE tests demonstrated that (-)-pyraclofos was about 15 times more potent than its (+)-form. However, acute aquatic assays suggested that (+)-pyraclofos was about 6 times more toxic than its antipode. Moreover, the joint toxicity of pyraclofos enantiomers to D. magna was found to be an additive effect. These results demonstrated that the overall toxicity of pyraclofos should be assessed using the individual enantiomers.     

Isolation and characterization of a novel Arabidopsis thatiana mutant that requires a high concentration of boron

We isolated a novel Arabidopsis thaliana mutant line that requires high levels of boron (B) for normal growth. Line 8–21 was identified from ethyl methanesulfonate-mutagenized M2 population. When grown in medium containing 3 μm or less B in the form of boric acid, the fresh weights of aerial portions of the mutant were about a half of those of the wild type, but in that containing 300 μm B, the growth appeared normal. The mutant plants did not shown any difference in root growth from the wild-type plants in the range of B concentrations tested. When grown with 30 μm B, the B concentration in shoots of the line 8–21 was similar to that in the wild-type, suggesting that the mutant could not utilize B efficiently. Line 8–21 was not allelic to bor1-1 (Noguchi et al., Plant Physiol., 115, 901–906, 1997). A significant portion of F2 plants from the crosses between the wild-type and the mutant grew poorly on a low B media, suggesting segregation of the mutation.     

Influence of a pyrethroid insecticide on soil fungi

The selective effect of the pyrethroid insecticide (Polytrin) on soil fungi was studied at three doses when incorporated in soil or in agar or liquid medium. Polytrin induced an inhibitory effect on soil fungi after 2, 5 and 40 days when applied at certain levels and a stimulatory effect after 20 days of treatment with the high dose. When incorporated in the agar medium, Polytrin decreased the total counts of fungi, particularly, Aspergillus and Penicillium at the high dose (10.0 ppm). In liquid medium, it showed no significant effect except in case of A. terreus which was significantly enhanced by the three doses.     

Algal degradation of a known endocrine disrupting insecticide, alpha-endosulfan, and its metabolite, endosulfan sulfate, in liquid medium and soil

The role of algae in the persistence, transformation, and bioremediation of two endocrine disrupting chemicals, alpha-endosulfan (a cyclodiene insecticide) and its oxidation product endosulfan sulfate, in soil (incubated under light or in darkness) and a liquid medium was examined. Incubation of soil under light dramatically decreased the persistence of alpha-endosulfan and enhanced its transformation to endosulfan sulfate, over that of dark-incubated soil samples, under both nonflooded and flooded conditions. This enhanced degradation of soil-applied alpha-endosulfan was associated with profuse growth of indigenous phototrophic organisms such as algae in soil incubated under light. Inoculation of soil with green algae, Chlorococcum sp. or Scenedesmus sp., further enhanced the degradation of alpha-endosulfan. The role of algae in alpha-endosulfan degradation was convincingly demonstrated when these algae degraded alpha-endosulfan to endosulfan sulfate, the major metabolite, and endosulfan ether, a minor metabolite, in a defined liquid medium. When a high density of the algal inoculum was used, both metabolites appeared to undergo further degradation as evident from their accumulation only in small amounts and the appearance of an endosulfan-derived aldehyde. Interestingly, beta-endosulfan was detected during degradation of alpha-endosulfan by high density algal cultures. These algae were also capable of degrading endosulfan sulfate but to a lesser extent than alpha-endosulfan. Evidence suggested that both alpha-endosulfan and endosulfan sulfate were immediately sorbed by the algae from the medium, which then effected their degradation. Biosorption, coupled with their biotransformation ability, especially at a high inoculum density, makes algae effective candidates for remediation of alpha-endosulfan-polluted environments.     

Biotransformation of an organochlorine insecticide,endosulfan, by Anabaena species

This study assesses the role of the blue-green algal species present in the soil in the dissipation of endosulfan and its metabolites in the soil environment. Two Anabaena species, Anabaena sp. PCC 7120 and Anabaena flos-aquae, were used in this study. Anabaena sp. PCC 7120 produced three principal biotransformation compounds, chiefly endosulfan diol (endodiol), and minor amounts of endosulfan hydroxyether and endosulfan lactone. Trace amounts of endosulfan sulfate were detected. In comparison, the biotransformation of endosulfan by Anabaena flos-aquae yielded mainly endodiol with minor amounts of endosulfan sulfate. An unknown compound was produced up to 70% from endosulfan spiked in the medium inoculated by A. flos-aquae after 8 days of incubation. Therefore, the endosulfan fate was dependent on the species. Within 1 day of incubation, two Anabaena species produced low amounts of beta-endosulfan after application of alpha-endosulfan. These results suggest the presence of isomerase in the Anabaena species. Further studies using a fermentor to control the medium pH at 7.2 to minimize chemical hydrolysis of endosulfan revealed a major production of endodiol with minor amounts of endosulfan sulfate and the unknown compound. These results showed that the production of the unknown compound might be dependent on the alkaline pH in the medium and that the production of endodiol by A. flos-aquae might be biologically controlled. This study showed that two algal species could contribute in the detoxification pathways of endosulfan in the soil environment.     

Molecular cloning and characterization of oryzacystatin-III, a novel member of phytocystatin in rice (Oryza sativa L. japonica)

On the basis of cDNA sequences, we found that the calli of rice encodes an amino acid sequence that shares 56% and 89% identity, respectively, with oryzacystatin-I and oryzacystatin-II. This sequence differs from that of oryzacystatin-II in the N-terminal region (Gln(7)-Ala(19) in the oryzacystatin-III numbering), and this region contained a glycine residue (Gly(14)), which is evolutionarily conserved in the cystatin superfamily. We named this novel protein oryzacystatin-III. Nucleotide sequencing of the 5'-flanking region of the oryzacystatin-III gene showed that it is highly homologous to the oryzacystatin-II gene but distinct from the oryzacystatin-II locus. Oryzacystatin-III inhibited papain, ficin, and human cathepsin B. The inhibition constants for papain and ficin differ from those of oryzacystatin-I and -II, and cathepsin B activity is affected only by oryzacystatin-III, showing differences in the interaction of these inhibitors with enzymes. These data suggest that the above three inhibitors may play unique physiological roles in the regulations of rice cysteine proteinases.     

Biotransformation of 3-methyl-4-nitrophenol, a main product of the insecticide fenitrothion, by Aspergillus niger

Biotransformation of the environmental pollutant 3-methyl-4-nitrophenol (MNP), a newly characterized estrogenic chemical, and the primary breakdown product of the heavily used insecticide fenitrothion was investigated using a common soil fungus. In 96 h, daily culture sacrifice, extraction, and analysis showed that the filamentous fungus, Aspergillus niger VKM F-1119, removed more than 85% of the MNP present in solution (original concentration = 25 mg/L), mostly through biodegradation. Additionally, in 16-day time-course studies, A. niger was capable of biotransformation of MNP at concentrations as high as 70 mg/L. Gas chromatography mass spectroscopy (MS) analyses of culture fluid extracts indicated the formation of four metabolites: 2-methyl-1,4-benzenediol, 4-amino-3-methylphenol, and two singly hydroxylated derivatives of MNP. Culture scale up and metabolite analysis by liquid chromatography MS resulted in the confirmation of the original metabolites plus the detection of an azo derivative metabolite that has not been previously reported before during MNP biodegradation by any micro-organisms.