Comparison of the effects of cereal and legume proteinaceous seed extracts on α-amylase activity and development of the Sunn pest

The Sunn pest, Eurygaster integriceps Puton (Hemiptera: Scutelleridae), is a significant limiting factor in the production of wheat and barley in many areas of the world. In the current study, the effect of semi-purified proteinaceous extracts of seeds on digestive enzymes, and the growth and development of the Sunn pest were studied. The results showed that the purified α-amylase inhibitor from Triticum aestivum (type І) and rice semi-purified seed extract did not significantly affect the Sunn pest α-amylase activity. However, bean and cowpea seed extracts significantly affected α-amylase activity in vitro. For example, the bean seed extract at concentrations of 0.125 and 2.0 mg · mL^− 1 inhibited α-amylase activity of the pest by 15% and 45%, respectively, while the cowpea seed extract, at the same concentrations, inhibited α-amylase activity of the pest by 9% and 40%, respectively. Further, incorporation of the seed extracts into the insect diet showed that the rice seed extract did not affect insect development time, while bean and cowpea seed extracts at high concentrations (e.g., 3.0%) significantly affected nymphal development time and survivability (P > 0.05). These results show that semi-purified seed extracts affect α-amylase activity, developmental time, and survivability but not the adult weight of the Sunn pest.     

Digestive amylase of a primitive animal,the scorpion: Purification and biochemical characterization

Scorpion, one of the most ancient invertebrates was chosen, as a model of a primitive animal, to purify and characterize an amylase located in the hepatopancreas. The scorpion digestive amylase (SDA) was purified. Pure SDA was obtained after heat treatment followed by ammonium sulfate fractionation and three steps of chromatography. The pure amylase is not glycosylated and has a molecular mass of 59,101 Da determined by MALDI-TOF MS analysis. The maximal amylase activity was measured at pH 7.0 and 50 °C, in the presence of Ca²⁺ and using potato starch as substrate. The enzyme was able to hydrolyze also, glycogen and amylose. The 23 NH₂-terminal amino acid SDA residues were sequenced. The sequence obtained is similar to those of mammalian and avian pancreatic amylases. Nevertheless, polyclonal antibodies directed against SDA failed to recognize classical digestive amylases like the porcine pancreatic one.     

Entomotoxic properties of Dioclea violacea lectin and its effects on digestive enzymes of Anagasta kuehniella (Lepidoptera)

Entomotoxic plant lectins have been extensively studied in the past two decades, yet the exact mechanisms underlying their toxic effects remain unknown. This study investigated the effects of Dioclea violacea lectin (DVL) on larval development in Anagasta kuehniella. Chronic exposure of larvae (from neonates to the fourth instar) demonstrated that DVL interfered with larval growth, retarding development and decreasing larval mass without affecting survival. DVL decreased trypsin-like, chymotrypsin-like, and α-amylase activities and proved resistant to proteolysis by midgut proteases up to 24 h. Shorter exposures to dietary DVL had no effect on midgut enzyme activity. Feeding fourth-instar larvae with fluorescently-labeled DVL revealed lectin binding to the peritrophic membrane.     

Digestive proteolytic activity in larvae and adults of Bactrocera oleae Gmelin (Diptera: Tephritidae)

Digestive proteolytic activity in larvae and adults of Bactrocera oleae was studied using specific substrates and inhibitors. The optimal pH for general proteolytic activity was 4 and 10 for soluble and membrane-bound fractions of larvae, and 9 for the soluble fraction of adults. The highest activities of general proteases were revealed at temperatures of 25 °C and 45 °C for both the soluble and membrane-bound fractions of larvae as well as the soluble fraction of adults. Determination of the specific protease activities demonstrated the presence of serine and cysteine proteases in addition to two exopeptidases in the larvae and adults. However, trypsin-like protease, chymotrypsin-like protease, and two exopeptidases of larvae, and chymotrypsin-like protease as well as cathepsin L of adults had no activity in the soluble fraction. The presence of specific proteases was verified by using specific inhibitors such as PMSF, TLCK, TPCK, E-64, EDTA, phenanthroline, and DTT. Finally, feeding of B. oleae larvae on different olive varieties revealed the highest trypsin-like protease, chymotrypsin-like protease, elastase, cathepsin B, cathepsin L, and cathepsin D on Amigdalifolia, Coratina, Baladi, Mari, Conservalia, Baladi, and Arbequina, respectively. These results showed digestive proteolytic activities in B. oleae for the first time, and could be the basic knowledge required for finding a control procedure to decrease the damage of this destructive pest around the world.     

Psychrophilic α-amylase from Aeromonas veronii NS07 isolated from farm soils

Among 120 isolates examined in this study, three isolates were selected for amylase production on starch agar plates following incubation at 10 °C. Identification by 16SrRNA on selected bacterium disclosed the highest similarity for protean regions of this gene as Aeromonas veronii NS07. A 63 kDa psychrophilic amylase enzyme from NS07 strain was purified by two-steps chromatography. The enzyme had the highest specific activity at pH 4 and was active at the range of temperatures from 0 to 50 °C, although the optimum temperature for enzyme activity was found at 10 °C. Analysis of the N-terminal amino acid sequencing disclosed 20 amino acids from purified amylase which had no similarity with other known α-amylases, indicating that the presented enzyme was novel. Amylase activity was enhanced in relation to optimum activity with the presence of sodium sulphate (161%), MnCl₂ (298%), CaCl₂ (175%), FeCl₂ (182%), MgCl₂ (237%), ZnCl₂ (169%), NiCl₂ (139%), NaCl (158%), each at 5 mM, while EDTA, phenylmethane sulphonylfluoride (PMSF) (3 mM), urea (8 M) and SDS (1%) inhibited the enzyme up to 5%, 2%, 80% and 18%, respectively. NS07 strain seems to be suitable as biocatalyst for practical use in liquefaction of starch at low temperatures, detergent and textile industries.     

Application of experimental designs to optimize medium composition for production of thermostable lipase/esterase by Geobacillus thermodenitrificans AZ1

Thirty two morphologically different bacterial were isolated from different soil samples and screened for their ability to produce lipolytic enzymes. Among all isolates, the isolate coded AZ1 was selected due to its high potency to produce lipase at elevated temperature up to 65 °C. Phylogenetic analysis based on 16SrDNA sequence revealed its close relationship to Geobacillus thermodenitrificans. The effect of ten culture variable on lipase production was evaluated by implementing Plackett–Burman statistical design. d-sucrose, peptone and soy bean flour were the most significant variables affecting lipase production. A pre-optimized medium based on this experiment yielded an enzyme activity of 260 U min^?1 ml^?1. For further optimization, a fourteen trials’ multi-factorial Box–Behnken experimental design was applied to find out the optimum level of each of the significant variables. The tested variables, namely: d-sucrose (X₁); peptone (X₂) and soy bean flour (X₃) were examined, each at three different levels coded ?1, 0, +1. The optimal levels of the three components were founded to be (g/L): d-sucrose, 6.56; peptone, 6.35; and soy bean flour, 6.92, with a predicted activity of approximately 610 U min^?1 ml^?1. According to the results of the Plackett–Burman and Box–Behnken designs the following medium composition is expected to be optimum (g/L): d-sucrose 6.56, peptone 6.35, soy bean flour 6.92, CaCl₂ 0.02, Y.E. 2.5, K₂HPO₄ 1.0, MgSO₄.7H₂O 0.2 and Fe₂ (SO₄)₃ 0.02; pH, 8; cultivation temperature 55 °C and incubation time 24 h, the enzyme activity measured in the medium was approximately 593 U min^?1 ml^?1.     

Optimization and immobilization of amylase obtained from halotolerant bacteria isolated from solar salterns

The objective of the present study was to isolate halotolerant bacteria from the sediment sample collected from Marakanam Solar Salterns, Tamil Nadu, India using NaCl supplemented media and screened for amylase production. Among the 22 isolates recovered, two strains that had immense potential were selected for amylase production and designated as P1 and P2. The phylogenetic analysis revealed that P1 and P2 have highest homology with Pontibacillus chungwhensis (99%) and Bacillus barbaricus (100%). Their amylase activity was optimized to obtain high yield under various temperature, pH and NaCl concentration. P1 and P2 strain showed respective, amylase activity maximum at 35 °C and 40 °C; pH 7.0 and 8.0; 1.5 M and 1.0 M NaCl concentration. Further under optimized conditions, the amylase activity of P1 strain (49.6 U mL^?1) was higher than P2 strain. Therefore, the amylase enzyme isolated from P. chungwhensis P1 was immobilized in sodium alginate beads. Compared to the free enzyme form (49.6 U mL^?1), the immobilized enzyme showed higher amylase activity as 90.3 U mL^?1. The enzyme was further purified partially and the molecular mass was determined as 40 kDa by SDS–PAGE. Thus, high activity of amylase even under increased NaCl concentration would render immense benefits in food processing industries.     

Synthesis,biological evaluation and molecular docking of N-phenyl thiosemicarbazones as urease inhibitors

Urease is an important enzyme which breaks urea into ammonia and carbon dioxide during metabolic processes. However, an elevated activity of urease causes various complications of clinical importance. The inhibition of urease activity with small molecules as inhibitors is an effective strategy for therapeutic intervention. Herein, we have synthesized a series of 19 benzofurane linked N-phenyl semithiocarbazones (3a–3s). All the compounds were screened for enzyme inhibitor activity against Jack bean urease. The synthesized N-phenyl thiosemicarbazones had varying activity levels with IC₅₀ values between 0.077 ± 0.001 and 24.04 ± 0.14 μM compared to standard inhibitor, thiourea (IC₅₀ = 21 ± 0.11 μM). The activities of these compounds may be due to their close resemblance of thiourea. A docking study with Jack bean urease (PDB ID: 4H9M) revealed possible binding modes of N-phenyl thiosemicarbazones.     

Purification and biochemical characterization of a novel SDS and surfactant stable,raw starch digesting,and halophilic α-amylase from a moderately halophilic bacterium,Nesterenkonia sp. strain F

An extracellular halophilic α-amylase from Nesterenkonia sp. strain F was purified to homogeneity by 80% ethanol precipitation, Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography, with a 10.8-fold increase in specific activity. The molecular mass of the amylase was estimated to be 100 kDa and 106 kDa by SDS–PAGE and gel filtration chromatography, respectively. The enzyme showed maximal activity at pH 7.5 and 45 °C. The amylase was active in a wide range of salt concentrations (0–4 M) with its maximum activity at 0.5 M NaCl or 1 M KCl and was stable at the salts concentrations between 1 M and 4 M. Fe³⁺, Cu²⁺, Zn²⁺ and Al³⁺ strongly inhibited the enzyme, whereas Ca²⁺ stimulated the amylase activity. The α-amylase was inhibited by EDTA, but was not inhibited by PMSF and β-mercaptoethanol. The enzyme showed remarkable stability towards 0.5% SDS and sarcosyl, and 2% each of Triton X-100, Tween 80 and Tween 20. K_m value of the amylase for soluble starch was 4.5 mg/ml. The amylase hydrolyzed 38% of raw wheat starch and 20% of corn starch in a period of 48 h. The major products of soluble starch hydrolysis were maltose, maltotriose and maltotetraose, indicating an α-amylase activity.     

α-Amylase (AmyP) of glycoside hydrolase subfamily GH13_37 is resistant to various toxic compounds

The α-amylase (AmyP) from a marine metagenomic library shows very low sequence similarity with characterized α-amylases and belongs to a new glycoside hydrolase subfamily GH13_37. This amylase retained above 87% residual activity in the presence of metal ions (concentrations <10 mM) tested except Hg²⁺ and was strongly stimulated by 5 mM Cu²⁺. AmyP was active over a wide range of salt concentration (0–3 M) with the optimal concentration at 1 M. The enzyme exhibited 119, 106, 108, 42 and 31% of its activity the presence of 2% Tween 20, Tween 40, Triton X-100, SDS and CTAB, respectively, showing excellent resistance. Oxidizing agents (H₂O₂ and NaClO₃) not strongly inactivated the enzyme. DTT was found to greatly enhance the activity (to 198% of original activity), while 2-mercaptoethanol had no significant effect on the enzyme. Moreover, AmyP retained considerable activity in both hydrophobic solvents and hydrophilic solvents, and n-octanol even increased the amylase activity to 113%. Compared to other α-amylases capable of resisting toxic compounds, AmyP was the first α-amylase with such broad spectrum resistance.     

Larvicidal activities of the stem bark extract and rotenoids of Millettia usaramensis subspecies usaramensis on Aedes aegypti L. (Diptera: Culicidae)

The dichloromethane/methanol (1:1) extract of the stem bark of Millettia usaramensis subspecies usaramensis was tested for its larvicidal activity against the 4th instar Aedes aegypti larvae and demonstrated activity with LC₅₀ value of 50.8 ± 0.06 μg/mL at 48 h. Compounds isolated from the extract were also tested for their larvicidal activities, and the rotenoid usararotenoid-A (LC₅₀ 4.3 ± 0.8 μg/mL at 48 h) was identified as the most active principle. This compound appears to be the first rotenoid having a trans-B/C ring junction and methylenedioxy group at C-2/C-3 with high larvicidal activity. Related rotenoids with the same configuration at the B/C-ring junction did not show significant activity at 100 μg/mL.     

First proof of bismuth oxide nanoparticles as efficient radiosensitisers on highly radioresistant cancer cells

This study provides the first proof of the novel application of bismuth oxide as a radiosensitiser. It was shown that on the highly radioresistant 9L gliosarcoma cell line, bismuth oxide nanoparticles sensitise to both kilovoltage (kVp) or megavoltage (MV) X-rays radiation. 9L cells were exposed to a concentration of 50 μg.mL⁻¹ of nanoparticle before irradiation at 125 kVp and 10 MV. Sensitisation enhancement ratios of 1.48 and 1.25 for 125 kVp and 10 MV were obtained in vitro, respectively. The radiation enhancement of the nanoparticles is postulated to be a combination of the high Z nature of the bismuth (Z = 83), and the surface chemistry. Monte Carlo simulations were performed to elucidate the physical interactions between the incident radiation and the nanoparticle. The results of this work show that Bi₂O₃ nanoparticles increase the radiosensitivity of 9L gliosarcoma tumour cells for both kVp and MV energies. Monte Carlo simulations demonstrate the advantage of a platelet morphology.     

Chemical composition and larvicidal activity of Piper capense essential oil against the malaria vector,Anopheles gambiae

Hydro-distilled essential oil from Kenyan Piper capense (Piperaceae) was analysed by gas chromatography mass spectrometry (GC–MS) and evaluated for larvicidal activity against the malaria vector, Anopheles gambiae. The oil consisted mainly of sesquiterpene hydrocarbons which accounted for 43.9% of the oil. The major sesquiterpenes were δ-cadinene (16.82%), β-bisabolene (5.65%), and bicyclogermacrene (3.30%). The oil also had appreciable amounts of monoterpene hydrocarbons (30.64%), including β-pinene (7.24%) and α-phellandrene (4.76%), and arylpropanoids (8.64%), including myristicin (4.26%). The oil showed larvicidal activity against third instar larvae of A. gambiae, with LC₅₀ and LC₉₀ values of 34.9 and 85.0 ppm, respectively. Most of the larvae died within the first few hours. The high larvicidal activity of this oil was indicated by the fact that over 80% mortality was observed at a concentration of 100 ppm after 24 h. These results compared favourably with the commercial larvicide pylarvex® which had LC₅₀ and LC₉₀ values of 3.7 and 7.8 ppm, respectively. Application of this oil or of products derived from it to larval habitats may lead to promising results in malaria and mosquito management programmes.     

Forming nanoparticles of α-amylase and embedding them into solid surfaces

In the current work nanoparticles (NPs) of α-amylase were generated in an aqueous solution using high-intensity ultrasound, and were subsequently immobilized on polyethylene (PE) films, or polycarbonate (PC) plates, or on microscope glass slides. The α-amylase NPs coated on the solid surfaces have been characterized by ESEM, TEM, FTIR, XPS and AFM. The substrates immobilized with α-amylase were used for hydrolyzing soluble potato starch to maltose. The amount of enzyme introduced in the substrates, leaching properties, and the catalytic activity of the immobilized enzyme were compared. The catalytic activity of the amylase deposited on the three solid surfaces was compared to that of the same amount of free enzyme at different pHs and temperatures. α-Amylase coated on PE showed the best catalytic activity in all the examined parameters when compared to native amylase, especially at high temperatures. When immobilized on glass, α-amylase showed better activity than the native enzyme over all pH and temperature values studied. However, the immobilization on PC did not improve the enzyme activity at any pH and any temperature compared to the free amylase. The kinetic parameters, K_m and V_max were also calculated. The amylase coated PE showed the most favorable kinetic parameters (K_m = 5 g L⁻¹ and V_max = 5E−07 mol mL⁻¹ min⁻¹). In contrast, the anchored enzyme-PC exhibited unfavorable kinetic parameters (K_m = 16 g L⁻¹, V_max = 4.2E−07 mol mL⁻¹ min⁻¹). The corresponding values for amylase-glass were K_m = 7 g L⁻¹, V_max = 1.8E−07 mol mL⁻¹ min⁻¹, relative to those obtained for the free enzyme (K_m = 6.6 g L⁻¹, V_max = 3.3E−07 mol mL⁻¹ min⁻¹).     

An inhibitor from Lupinus bogotensis seeds effective against aspartic proteases from Hypothenemus hampei

The coffee berry borer, Hypothenemus hampei (Ferrari), is one of the most devastating coffee pests (Coffea arabica L.) worldwide. Digestion in the midgut of H. hampei is facilitated by aspartic proteases. This is the first report of an aspartic protease inhibitor from Lupinus bogotensis. The L. bogotensis aspartic protease inhibitor (LbAPI) exhibited a molecular mass of 12.84 kDa, as determined by MALDI-TOF, and consists of a single polypeptide chain with an isoelectric point of 4.5. In thermal activity experiments, stability was retained at pH 2.5 after heating the protein at 70 °C for 30 min, but was unstable at 100 °C. The protein was also stable over a broad range of pH, from 2 to 11, at 30 °C. In in vitro assays, LbAPI was highly effective against aspartic proteases from H. hampei guts with a half maximal inhibitory concentration (IC₅₀) of 2.9 μg. LbAPI inhibits pepsin in a stoichiometric ratio of 1:1. LbAPI inhibition of pepsin was competitive, with a K_i of 3.1 μM, using hemoglobin as substrate. Its amino-terminal sequence had 76% homology with the seed storage proteins vicilin and β-conglutin. The homology of LbAPI to vicilins from Lupinus albus L. suggests that they may also serve as storage proteins in the seed. LbAPI could be a promising tool to make genetically modified coffee with resistance to H. hampei.     

Host suitability and fitness-related parameters of Campoletis sonorensis (Hymenoptera: Ichneumonidae) as a parasitoid of the cabbage looper,Trichoplusia ni (Lepidoptera: Noctuidae)

The seven age-classes of Trichoplusia ni (Hübner) larvae evaluated in this study as hosts of Campoletis sonorensis indicates that early 2nd larval instar (3–5 day-old larvae) of T. ni represents the most suitable host stage for the development of the larval endoparasitoid C. sonorensis. The higher suitability of early 2nd larval instar of T. ni resulted in more parasitised larvae, a higher rate of successful parasitoid emergence, a higher rate of female progeny, and a lower rate of immature parasitoid mortality. The fitness gain of C. sonorensis on late 1st larval instar (2 day-old larvae) and late 2nd larval instar –early 3rd instars (6–8 day-old larvae) stages of T. ni is negatively affected by the trade-offs between the different physiological and behavioral characteristics influencing their suitability as hosts of C. sonorensis.     

Effect of the interaction between Anticarsia gemmatalis multiple nucleopolyhedrovirus and Epinotia aporema granulovirus,on A. gemmatalis (Lepidoptera: Noctuidae) larvae

The bean shoot borer Epinotia aporema Wals. (Lepidoptera: Tortricidae) and the velvet bean caterpillar Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) are key pests of soybean and other legume crops in South America. They are often found simultaneously in certain regions. A. gemmatalis nucleopolyhedrovirus (AgMNPV) is widely used to control A. gemmatalis. More recently, E. aporema granulovirus (EpapGV) has been characterized and evaluated as a bioinsecticide for E. aporema. In order to increase its potential use and to design optimized strategies for the management of lepidopteran pests, we evaluated the interaction between EpapGV and AgMNPV on third instar A. gemmatalis larvae. Larvae fed with 50 AgMNPV OBs/larva showed an increase in the mortality rates (from 42% to 81%) and a decrease in the median survival time (from 7.7 days to 5.7 days) when these OBs were mixed with 6000 EpapGV OBs/larva. When 300 AgMNPV OBs/larva were used alone or in combination with EpapGV OBs no changes in biological parameters were observed. No mortality was detected in A. gemmatalis larvae treated with EpapGV alone. In larvae fed with the viral mixtures, only AgMNPV DNA was detected by PCR. A. gemmatalis peritrophic membranes (PMs) examined by SDS–PAGE and scanning electron microscopy showed signs of damage. Notably, we found the presence of spheroidal bodies associated with damaged areas in the PMs of larvae fed with EpapGV but not in those that were given AgMNPV alone. These results show that EpapGV increases the viral potency of AgMNPV, and thus the insecticidal efficiency, suggesting that the use of formulations including both viruses might be a valuable tool for pest management.     

Assessment of oral virulence against Spodoptera litura,acquired by a previously non-pathogenic Metarhizium anisopliae isolate,following integration of a midgut-specific insecticidal toxin

All entomopathogenic fungi infect insects by direct penetration through the cuticle rather than per os through the gut. Genetic transformation can confer fungi with per os virulence. However, unless the recipient isolate is nonpathogenic to the target insect, mortality caused by a transgenic isolate cannot be attributed solely to oral virulence due to the potential for some simultaneous cuticular infection. Here, a Metarhizium anisopliae wild-type isolate (MaWT) nonpathogenic to Spodoptera litura was genetically engineered to provide a transformed isolate (MaVipT31) expressing the insect midgut-specific toxin Vip3Aa1. Toxin expression was confirmed in MaVipT31 hyphae and conidia using Western blotting. Mortality, leaf consumption and body weight of S. litura larvae (instars I–IV) exposed to a range of concentrations of MaWT conidia were not significantly different to controls although the number of conidia ingested by surviving larvae during the bioassay ranged from 2.3 × 10⁵ (instar I) to 8.1 × 10⁶ (instar IV). In contrast, consumption of MaVipT31 conidia caused high mortalities, reduced leaf consumption rates and decreased body weights in all instars evaluated, demonstrating that oral virulence had been acquired by MaVipT31. Larval mortalities were much more dependent on the number of MaVipT31 conidia ingested than the duration of time spent feeding on conidia-treated leaves (r²: 0.83–0.94 for instars I–IV). LC₅₀ and LT₅₀ trends for MaVipT31 estimated by time-concentration-mortality modeling analyses differed greatly amongst the instars. For 50% kill to be achieved, instar I larvae required 3, 4 and 5 days feeding on the leaves bearing 103, 28 and 8 conidia/mm² respectively; instar IV larvae required 6, 7 and 8 days feeding on leaves bearing 1760, 730 and 410 conidia/mm² respectively. Our results provide a deeper insight into the high oral virulence acquired by an engineered isolate and highlight its great potential for biological control.     

Synthesis and molecular docking study of some 5,6-dichloro-2-cyclopropyl-1H-benzimidazole derivatives bearing triazole,oxadiazole, and imine functionalities as potent inhibitors of urease

A new series of benzimidazole compounds including hydrazinecarbothioamide, 1,2,4-triazole, 1,3,4-oxadiazole and imine function were synthesized starting from 5,6-dichloro-2-cyclopropyl-1H-benzimidazole. All of the benzimidazole derivatives exhibited good urease inhibitor activity. Compound 6a proved to be the most potent showing an enzyme inhibitory activity with an IC₅₀ = 0.06 µM. Molecular docking studies were also conducted on enzyme extracted from Jack bean urease to identify the binding mode of the newly synthesized compounds.