苏云金芽孢杆菌毒素Cry1Aa、Cry2Aa、Cry3Aa和Cry4Aa结构的计算机对比分析

运用生物信息学软件对苏云金芽孢杆菌毒素Cry1Aa、Cry2Aa、Cry3Aa和Cry4Aa的 序列和基本参数、二级结构、三级结构、跨膜区和表面电势进行了预测比较。它们在一级 结构上有较大差异,但二级结构和跨膜区相似,三级结构中各毒素的结构域Ⅰ之间基本相 似,结构域Ⅱ之间差异较大,其中Cry2Aa为差异最大成员。4种毒素的表面电势分布不同。 毒素之间的结构相似性和差异性与其作用机理和杀虫特异性有关。     

Identification of a Bacillus thuringiensis Cry11Ba toxin-binding aminopeptidase from the mosquito, Anopheles quadrimaculatus

Background  

Aminopeptidase N (APN) type proteins isolated from several species of lepidopteran insects have been implicated as Bacillus thuringiensis (Bt) toxin-binding proteins (receptors) for Cry toxins. We examined brush border membrane vesicle (BBMV) proteins from the mosquito Anopheles quadrimaculatus to determine if APNs from this organism would bind mosquitocidal Cry toxins that are active to it.     

Synergism of the Bacillus thuringiensis Cry1, Cry2, and Vip3 Proteins in Spodoptera frugiperda Control

The polyphagous caterpillar, Spodoptera frugiperda, has been controlled with either chemical insecticides or transgenic plants such as Bt maize that expresses the cry and/or vip genes of the Bacillus thuringiensis (Bt) bacterium. Despite the efficiency of Bt toxins in lepidopteran control, populations resistant to Bt plants have emerged in different locations around the world. Thus, understanding how combined proteins interact against pests can assist resistance control and management. This work demonstrated the toxicity of Cry1Ab, Cry1Ac, Cry1Ca, Cry1Ea, Cry2Aa, Cry2Ab, Vip3Aa, and Vip3Ca in single and combined assays against S. frugiperda neonatal larvae. All protein mixtures had synergistic action in the control of the larvae. The Vip3Aa + Cry1Ab mixture had the highest toxicity, sequentially followed by Vip3Aa + Cry2Ab, Cry1Ab + Cry2Ab + Vip3Aa, Cry1Ea + Cry1Ca, Cry1Ab + Cry2Ab, Vip3Ca + Cry1Ea, and Vip3Ca + Cry1Ca. Cry1Ab, Cry1Ac, Cry2Ab, and Vip3Aa bound to more than one site on the brush border membrane vesicles (BBMV) of S. frugiperda. The Cry1Ab and Cry1Ac proteins share binding site, while Cry1Ab does not share binding site with the Cry2Aa and Cry2Ab proteins. The Vip3Aa protein does not share receptors with the tested Cry1 and Cry2. The results suggest that combination these tested proteins may increase toxicity against S. frugiperda neonates.

     

Nanobody-based electrochemical immunoassay for Bacillus thuringiensis Cry1Ab toxin by detecting the enzymatic formation of polyaniline

We describe an electrochemical immunoassay for the Cry1Ab toxin that is produced by Bacillus thuringiensis. It is making use of a nanobody (a heavy-chain only antibody) that was selected from an immune phage displayed library. A biotinylated primary nanobody and a HRP-conjugated secondary nanobody were applied in a sandwich immunoassay where horseradish peroxidase (HRP) is used to produce polyaniline (PANI) from aniline. PANI can be easily detected by differential pulse voltammetry at a working voltage as low as 40 mV (vs. Ag/AgCl) which makes the assay fairly selective. This immunoassay for Cry1Ab has an analytical range from 0.1 to 1000 ng∙mL^-1 and a 0.07 ng∙mL^-1 lower limit of detection. The average recoveries of the toxin from spiked samples are in the range from 102 to 114 %, with a relative standard deviation of <7.5 %. The results demonstrated that the assay represented an attractive alternative to existing immunoassays in enabling affordable, sensitive, robust and specific determination of this toxin.

Nanobodies specific to Cry1Ab toxin were isolated from an immunized camel. A biotinylated primary nanobody and a HRP-conjugated secondary nanobody were applied in a sandwich immunoassay with horseradish peroxidase being used to produce polyaniline, which can be easily detected by differential pulse voltammetry.

     

Deciphering the Selectivity of Bombyx mori Pheromone Binding Protein for Bombykol over Bombykal: A Theoretical Approach

In this article we report calculations dedicated to estimate the selectivity of the Bombyx mori pheromone binding protein towards the two closely related pheromonal components Bombykol and Bombykal. The selectivity is quantified by the binding free‐energy difference, obtained either by the thermodynamic integration or by the MM‐GBSA approach. In the latter, the selectivity is decomposed on a per‐residue basis, which identifies the residues considered crucial for the selectivity of the protein for Bombykol over Bombykal. A discussion on the role of Bombyx mori pheromone binding protein is provided on the basis of these results.     

Effect of gamma irradiation on nutritional components and Cry1Ab protein in the transgenic rice with a synthetic cry1Ab gene from Bacillus thuringiensis

The effects of gamma irradiation on the transgenic rice containing a synthetic cry1Ab gene from Bacillus thuringiensis were investigated. There was almost no difference in the content of the major nutritional components, i.e. crude protein, crude lipid, eight essential amino acids and total ash between the irradiated grains and the non-irradiated transgenic rice. However, the amounts of Cry1Ab protein and apparent amylose in the irradiated transgenic rice were reduced significantly by the doses higher than 200 Gy. In vivo observation showed that Cry1Ab protein contents also decreased in the fresh leaf tissues of survival seedlings after irradiation with 200 Gy or higher doses and showed inhibition of seedling growth. The results indicate that gamma irradiation might improve the quality of transgenic rice due to removal of the toxic Cry1Ab protein.     

Determination of 1-deoxynojirimycin in the Larvae of the Silkworm,Bombyx mori,by High-Performance Liquid Chromatography

1-Deoxynojirimycin, known to be one of the most potent α-glycosidase inhibitors, has been used as an antihyperglycemia agent. In this study, a rapid method for the determination of 1-deoxynojirimycin in the larvae of the silkworm, Bombyx mori was established, using high-performance liquid chromatography equipped with fluorescence detection after derivatization with 9-fluorenylmethyl chloroformate. The 1-deoxynojirimycin was efficiently extracted from the larvae using 0.05 mol/L hydrochloric acid, vortexing for 15 s at room temperature, followed by sonication for 30 min. The linear dynamic range extended from 0.7 to 28 µg/mL (r = 0.9999). The average recovery of the 1-deoxynojirimycin was 104% with a relative standard deviation of 3.30%. The limit of detection was 0.05 µg/mL at a signal-to-noise ratio of 3. There were high concentrations of 1-deoxynojirimycin in the larvae, reaching 0.50% of total body mass in the gluttonous stage of the third instar and significant variation in the fifth instar, suggests that the larvae may be a valuable source of 1-deoxynojirimycin for the treatment of carbohydrate-mediated disorders.     

The study on phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis: synthesis of homogeneous substrates, substrate specificity and other properties.

The properties of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis were studied in detail. The enzyme was extremely thermostable in 0.1% bovine serum albumin and retained 73% of its activity at 100 degrees C for 10 min, while it was labile in the absence of albumin. The enzymatic activity was inhibited by HgCl2 or p-chloromercuriphenylsulfonic acid and restored by dithiothreitol. The kinetic parameters (Km and Vmax) of PI-PLC were determined for the mixed micelle of yeast phosphatidylinositol (PI)/Triton X-100 or sodium deoxycholate. Four PIs having different acyl chains: dilauroylphosphatidylinositol (DLPI), dimyristoylphosphatidylinositol (DMPI), dipalmitoylphosphatidylinositol (DPPI) and dioleoylphosphatidylinositol (DOPI) were synthesized from yeast PI through the processes of deacylation and reacylation, identified by infrared (IR) and Fourier transform nuclear magnetic resonance (FT-NMR) spectra, and subjected to the action of PI-PLC. All the synthetic PIs were hydrolyzed by this enzyme, with DLPI and DMPI being the best substrates. PI-PLC did not catalyze the hydrolysis of the phosphatidylnucleosides 5'-phosphatidylcytidine, 5'-phosphatidyluridine, 5'-phosphatidylthymidine, 5'-phosphatidyladenosine and 5'-phosphatidyl-2'-deoxyadenosine.     

用桑蚕丝素蛋白固定脲酶的研究及脲酶电极的制备

提出了一种新的酶固定化方法, 即通过甲醇处理, 使蚕丝素蛋白膜的构象由random coil向β-sheet发生根本性的变化, 从而将酶固定在β-sheet所特有的分子间氢键中。利用此方法所制成的脲酶电极, 在合适的操作条件下, 各项响应指标均令人满意, 并且脲酶的耐温性能被大大提高, 电极的有效使用寿命长达三个月以上。此种酶固定化方法原则上能够应用于其他不破坏蚕丝素蛋白分子结构的可溶性酶。     

A new strategy for the synthesis of the pheromones of Lobesia botrana and Bombyx mori

Opening of epoxysilanes by alkenyl cuprates reagents in the presence of BF₃.Et₂O affords β-hydroxysilanes which after basic or acidic treatment give the title pheromones.     

Cloning, characterization, and expression of a new cry2Ab gene from Bacillus thuringiensis strain 14-1

Bacillus thuringiensis is the major source for transfer of genes to impart insect resistance in transgenic plants. Cry2A proteins of B. thuringiensis are promising candidates for management of resistance development in insects owing to their difference from the currently used Cry1A proteins, in structure and insecticidal mechanism. The cry2Ab gene was found to lack a functional promoter and, hence, is cryptic in nature. The cry2Ab7 gene was cloned from a new indigenous B. thuringiensis strain, 14-1. Nucleotide sequencing of the cry2Ab gene cloned from B. thuringiensis strain 14-1 revealed an open reading frame of 1902 bp. The deduced amino acid sequence of Cry2Ab of B. thuringiensis strain 14-1 showed a variation in three amino acid residues in comparison to the holotype sequence, Cry2Ab1. Expression of the newly cloned cry2Ab gene was studied in an acrystalliferous strain of B. thuringiensis (4Q7) by fusing the cry2Ab gene downstream of cry2Aa promoter and orf1+orf2 sequences. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of a spore-crystal mixture obtained from transformants of B. thuringiensis strain 4Q7 showed production of Cry2Ab protein of about 65 kDa. Alkali solubilized Cry2Ab7 protein showed toxicity against Helicoverpa armigera neonates.     

Model calculations for the base-pairing specificity of mutagenic exocyclic DNA adduct 1,N 6-ethenoadenine

1,N ⁶-ethenoadenine (εA) has been studied more extensively due to highly fluorescent and mutagenic nature. We report the model calculations for the base-pairing specificity of mutagenic 1,N ⁶-ethenoadenine adduct. To shed insight into mutagenic process of DNA damage based on geometrical characteristics, electronic properties, B3LYP, M06, B97D, and wB97XD methods have been employed for these model calculations. Single point energy calculation at MP2/6-311++G** levels on corresponding B3LYP, M06, B97D, wB97XD-optimized geometries have also been carried out to better estimate the hydrogen-bonding strengths. The polarisable conductor calculation model (CPCM-UAKS) that accounts for the overall polarizability of the solvent has also been employed. The computed reaction enthalpy values concluded that both guanine and thymine are most favorable candidates to be misincorporated to 1,N ⁶-ethenoadenine adduct, which also in good agreement to experimental report (Leonard, Biochemistry 33: 4755–4761, 79).     

Analysis of the Interaction Between Bacillus coagulans and Bacillus thuringiensis S-layers and Calcium Ions by XRD,Light Microscopy,and FTIR

S-layer is a self-assemble regularly crystalline surface that covers major cell wall component of many bacteria and archaea and exhibits a high metal-binding capacity. We have studied the effect of the calcium ions and type of solid support (glass or mica) on the structure of the S-layers from Bacillus coagulans HN-68 and Bacillus thuringiensis MH14 upon simple methods based on light microscopy and AFM. Furthermore, the Fourier transform infrared spectroscopy (FTIR) study is indicated that the calcium–S-layer interaction occurred mainly through the carboxylate groups of the side chains of aspartic acid (Asp) and glutamic acid (Glu) and nitrogen atoms of Lys, Asn, and histidine (His) amino acids and N–H groups of the peptide backbone. Studied FTIR revealed that inner faces of S-layer are mainly negative, and outer faces of S-layer are mainly positive. Probably, calcium ions with positive charges bound to the carboxyl groups of Glu and Asp. Accordingly, calcium ions are anchored in the space between the inner faces of S-layer with negative charge and the surface of mica with negative charge. This leads to regular arrangement of the S-layer subunits.     

Purification and characterization of a new Bacillus thuringiensis bacteriocin active against Listeria monocytogenes, Bacillus cereus and Agrobacterium tumefaciens

This study reports on the identification, characterization and purification of a new bacteriocin, named Bacthuricin F103, from a Bacillus thuringiensis strain BUPM103. Bacthuricin F103 production began in the early exponential phase and reached a maximum in the middle of the same phase. Two chromatographic methods based on high performance liquid chromatography and fast protein liquid chromatography systems were used to purify Bacthuricin F103. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed that this bacteriocin had a molecular weight of approximately 11 kDa. It also showed a wide range of thermostability of up to 80 °C for 60 min and a broad spectrum of antimicrobial activity over a pH range of 3.0–10.0. This bacteriocin was noted, and for the first time, to exhibit potent antimicrobial activity against Agrobacterium subsp. strains, the major causal agents of crown gall disease in tomato and vineyard crops, and against several challenging organisms in food, such as Listeria monocytogenes and Bacillus cereus. Complete killing with immediate impact on cells was observed within a short period of time. The sequence obtained for Bacthuricin F103 by direct N-terminal sequencing shared considerable homology with hemolysin. Bacthuricin F103 was noted to act through the depletion of intracellular ions, which suggest that the cell membrane was a possible target to Bacthuricin F103.     

Copper Coordination Polymers of N1,N4-Bis(pyridin-2-ylmethyl)terephthalamide: Synthesis,Structures, DNA Binding and Cleavage,and Catalytic Activity for Oxidation of 1-Phenylethanol

Two Cu coordination polymers [Cu(bpmt)Cl₂ · 2DMF]_n ( 1 ), [Cu(bpmt)(μ-Cl) · DMSO]_n ( 2 ) [bpmt = N¹,N⁴-bis(pyridin-2-ylmethyl)terephthalamide)], were synthesized and characterized. X-ray single-crystal diffractions revealed complex 1 exhibits a one-dimensional (1D) chain in which central Cu^II ions are located in a six-coordinate slightly distorted octahedral structure. Complex 2 features 1D chain composed of Cu^I–Cl bridges and all the central Cu^I ions are situated a twisted tetrahedral configurations. Multi-spectroscopic DNA interaction studies indicate that the two complexes may bind to DNA via groove binding modes, and the calculated binding constants are 1.42 × 10⁴ ( 1 -CT-DNA) and 1.41 × 10⁴ ( 2 -CT-DNA). Both complexes are also capable of efficiently cleaving plasmid pBR322 DNA in the presence of H₂O₂ via an oxidative mechanism. Furthermore, 1 and 2 are employed as catalysts for the oxidation of 1-phenylethanol to acetophenone. Complex 1 displays good catalytic potential with the conversion rate reaches 99 % using tert-butyl hydroperoxide (TBHP) as oxidant and CH₃CN as solvent at 60 °C for 2.0 h.     

Homology Modeling of Cyt2Ca1 of Bacillus thuringiensis and Its Molecular Docking with Inositol Monophosphate

Cyt2Ca1 is an insecticidal crystal protein produced by Bacillus thuringiensis ET29 during its stationary phase, and this δ‐endotoxin demonstrates remarkable insecticidal activity against not only insects of the order Coleoptera, but also against fleas, and in particular the larvae of the cat flea, Ctenocephalides felis. The first theoretical model of the three‐dimensional structure of Cyt2Ca1 was predicted and compared with Cyt2Aa, which is lethal to insect larvae. The three‐dimensional structure of the Cyt2Ca1 was obtained by homology modeling on the structures of the Cyt2Aa protein. The deduced model resembles previously reported Cyt2Aa toxin. A binding mode of inositol monophosphate as a polar head group of the putative membrane phospholipid ligand to Cyt2Ca1 was presented using molecular docking. The residues of Leu9, Glu21, Tyr23 and Gln110 of the Cyt2Ca1 toxin are responsible for the interactions with inositol monophosphate via eight hydrogen bonds. Those residues could be important for receptor recognition. This binding simulation will be helpful for the design of mutagenesis experiments aimed at the improvement of toxicity, and lead to a deep understanding of the mechanism of action of Cyt toxins.     

Binding properties of a monoclonal antibody against the Cry1Ab from <Emphasis Type="Italic">Bacillus Thuringensis</Emphasis> for the development of a capillary electrophoresis competitive immunoassay

Experimental work performed was aimed at the assessment of a competitive capillary electrophoresis immunoassay with laser-induced fluorescence (CEIA-LIF) detection for the determination of the Cry1Ab endotoxin from Bacillus thuringensis. The binding constant of a monoclonal antibody, raised against the insecticide protein Cry1Ab, was determined on a microplate by indirect enzyme-linked immunosorbent assay (ELISA) and compared with that obtained in-capillary under nonequilibrium separation conditions. The two binding constants appear comparable—(5.0 ± 1.2) × 10⁶ M⁻¹ and (9.06 ± 5.7) × 10⁶ M⁻¹—reflecting good preservation of the antibody binding behavior in the capillary electrophoresis format. These results allow use of a calibration curve possible between 0.2 and 150 nM of endotoxin protein, with a limit of detection of 0.5 nM (33 μg L⁻¹). Preliminary recovery experiments on maize extracts spiked with known amounts of Cry1Ab endotoxin also showed promising results in detecting the toxin in complex real matrices.     

Mimicking the Vancomycin Carboxylate Binding Site: Synthetic receptors for sulfonates,carboxylates, and N-protected α-amino acids in water

The novel H₂O-soluble cyclophanes 1 and 2 incorporating different anion-recognition sites were prepared in short synthetic routes (Schemes 1 and 2) as first-generation mimics of the natural, D -Ala-D -Ala binding antibiotic vancomycin. The X-ray crystal structure of 1 , a tris(hydrochloride)salt, revealed an open, preorganized cavity of sufficient size for the incorporation of small aliphatic residues (Fig. 3). In the crystal, molecules of 1 are arranged in parallel stacks, generating two types of channels, an ‘intra-stack’ channel passing through the cyclophane cavities and an ‘inter-stack’ channel located between cyclophane stacks (Fig. 4). The strongest intermolecular interactions between macrocycles in the crystal are C?O…?H? N H-bonds between the carboxamide residues of adjacent cyclophanes in neighboring stacks (Fig. 5). The ‘intra-’ and ‘inter-stack’ channels incorporate the three ordered Cl^? counterions and several, partially ordered solvent molecules (4 MeOH, 1 H₂O) (Fig. 6). Counterion Cl(2) is located within the ‘intra-stack’ channel and interacts with a protonated piperazinium N-atom and both ‘intra-stack’ MeOH molecules. The two other counterions, Cl(1) and Cl(3), are located within the ‘inter-stack’ channel. They are connected to two MeOH and one H₂O molecules and also interact both with the NH group of the protonated spiropiperidinium ring in 1 , forming an infinite, chain-like H-bonding network …?Cl(1)…?HOH…?MeOH…?Cl(3)…?HNH…?Cl(1′)…?. Both ‘intra-’ and ‘inter-stack’ MeOH molecules undergo weak CH…?π interactions with neighboring aromatic rings. Cyclophane 1 complexed aromatic sulfonates in 0.5M KCl/DCl buffer in D₂O, whereas the tetrakis(quaternary ammonium) receptor 2 bound the sodium salts of aliphatic and aromatic carboxylates and sulfonates, of N-acylated α-amino acids as well as of N-acetyl-D -alanyl-D -alanine (Ac-D -Ala-D -Ala), a substrate of vancomycin, in pure H₂O. In all of these complexes, ion pairing between the cationic recognition site in the periphery of the cyclophane receptor and the anionic substrates represents the major driving force for host-guest association. The ¹H-NMR analysis of complexation-induced changes in chemical shift clearly demonstrated that, in solution, this ion pairing exclusively takes place outside the cavity. Nevertheless, the macrocyclic bridges are essential for the efficiency of the anion-recognition sites in the two cyclophane receptors 1 and 2 . Control compounds 3 and 4 possess nearly the same anion-recognition sites than 1 and 2 , but lack their macrocyclic preorganization; as a consequence, they do not form stable ion-pairing complexes with mono-anionic substrates in the considered concentration ranges ( < 50 mM ) in D₂O.