Key criteria for engineering mycotoxin binding aptamers via computational simulations: Aflatoxin B1 as a case study.

Due to the difficulties in monoclonal antibody production specific to mycotoxins, aptameric probes have been considered as suitable alternatives. The low efficiency of the SELEX procedure in screening high affinity aptamers for binding mycotoxins as small molecules can be significantly improved through computational techniques. Previously, we designed five new aptamers to aflatoxin B1 (AFB1) based on a known aptamer sequence (Patent: PCT/CA2010/001 292, Apt1) through a genetic algorithm-based in silico maturation strategy and experimentally measured their affinity to the target toxin. Here, integrated molecular dynamic simulation (MDs) studies with molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) analysis to clarify the binding modes, critical interacting nucleic bases and energy component contributions in the six AFB1-binding aptamers. The aptamer F20, which was selected in the first work, showed the best free binding energy and complex stability compared to other aptamers. The trajectory analysis revealed that AFB1 recognized F20 through the groove binding mode along with precise shape complementarity. The MD simulation results revealed that dynamic water intermediate interactions also play a key role in promoting complex stability. According to the MM-PBSA calculations, van der Waals contacts were identified as dominant energy components in all complexes. Interestingly, a high consistency is observed between the experimentally obtained binding affinities of the six aptamers with their free energy solvation. The computational findings, confirmed via previous experiments, highlighted the binding modes, the dynamic hydration of complex components and the total free interacting energy as the crucial criteria in discovering high functional aptameric probes.

In silico maturation of affinity and selectivity of DNA aptamers against aflatoxin B1 for biosensor development.

A high affinity and selectivity DNA aptamer for aflatoxin B1 (AFB1) was designed through Genetic Algorithm (GA) based in silico maturation (ISM) strategy. The sequence of a known AFB1 aptamer (Patent: PCT/CA2010/001292, Apt1) applied as a probe in many aptasensors was modified using seven GA rounds to generate an initial library and three different generations of ss DNA oligonucleotides as new candidate aptamers. Molecular docking methodology was used to screen and analyze the best aptamer-AFB1 complexes. Also, a new pipeline was proposed to faithfully predict the tertiary structure of all single stranded DNA sequences. By the second generation, aptamer Apt1 sequence was optimized in the local search space and five aptamers including F20, g12, C52, C32 and H1 were identified as the best aptamers for AFB1. The selected aptamers were applied as probes in an unmodified gold nanoparticles-based aptasensor to evaluate their binding affinity to AFB1 and their selectivity against other mycotoxins (aflatoxins B2, G1, G2, M1, ochratoxin A and zearalenone). In addition, a novel direct fluorescent anisotropy aptamer assay was developed to confirm the binding interaction of the selected aptamers over AFB1. The ISM allowed the identification of an aptamer, F20, with up to 9.4 and 2 fold improvement in affinity and selectivity compared to the parent aptamer, respectively.

A computational method for prediction of xylanase enzymes activity in strains of Bacillus subtilis based on pseudo amino acid composition features.

Xylanases are hydrolytic enzymes which based on physicochemical properties, structure, mode of action and substrate specificities are classified into various glycoside hydrolase (GH) families. The purpose of this study is to show that the activity of the members of the xylanase family in the specified pH and temperature conditions can be computationally predicted. The proposed computational regression model was trained and tested with the Pseudo Amino Acid Composition (PseAAC) features extracted solely from the amino acid sequences of enzymes. The xylanases with experimentally determined activities were used as the training dataset to adjust the model parameters. To develop the model, 41 strains of Bacillus subtilis isolated from field soil were screened. From them, 28 strains with the highest halo diameter were selected for further studies. The performance of the model for prediction of xylanase activity was evaluated in three different temperature and pH conditions using stratified cross-validation and jackknife methods. The trained model can be used for determining the activity of newly found xylanases in the specified condition. Such computational models help to scale down the experimental costs and save time by identifying enzymes with appropriate activity for scientific and industrial usage. Our methodology for activity prediction of xylanase enzymes can be potentially applied to the members of the other enzyme families. The availability of sufficient experimental data in specified pH and temperature conditions is a prerequisite for training the learning model and to achieve high accuracy.

Valorisation of untreated cane molasses for enhanced phytase production by Bacillus subtilis K46b and its potential role in dephytinisation.

BACKGROUND: The high cost of phytase production is the most limiting factor in its application in animal feeds. The present study aimed to develop a low-cost medium for production of a novel phytase in submerged fermentation using inexpensive agro-industrial by-products. The applicability of phytase in dephytinisation of commonly used food/feed ingredients, i.e. soybean meal and wheat bran, was also investigated. RESULTS: Using a one-factor-at-a-time approach, soybean meal and cane molasses were identified as significant agro-industrial by-products and these factors were subsequently optimised using response surface methodology (RSM). A central composite design was employed to further enhance phytase yield. Under optimum conditions of soybean meal 22.3 g L-1 , cane molasses 100 g L-1 and 39 h fermentation, phytase production increased to 56.562 U mL-1 , indicating more than 28-fold enhancement. The enzyme efficiently dephytinised wheat bran and soybean meal after 24 h incubation at 56.5 °C and increased inorganic phosphate content by 240% and 155%, respectively. CONCLUSION: Soybean meal and cane molasses were successfully used for enhancement of phytase production as economical carbon, nitrogen and phytic acid sources using RSM. The phytase showed a good capability to dephytinise wheat bran and soybean meal, demonstrating that the enzyme can be considered as a potential candidate for industrial food and feed applications. © 2016 Society of Chemical Industry.

A novel phytase characterized by thermostability and high pH tolerance from rice phyllosphere isolated Bacillus subtilis B.S.46.

In this study, an extracellular alkali-thermostable phytase producing bacteria, Bacillus subtilis B.S.46, were isolated and molecularly identified using 16S rRNA sequencing. Response surface methodology was applied to study the interaction effects of assay conditions to obtain optimum value for maximizing phytase activity. The optimization resulted in 137% (4.627 U/mL) increase in phytase activity under optimum condition (56.5 °C, pH 7.30 and 2.05 mM sodium phytate). The enzyme also showed 60-73% of maximum activity at wide ranges of temperature (47-68 °C), pH (6.3-8.0) and phytate concentration (1.40-2.50 mM). The partially purified phytase demonstrated high stability over a wide range of pH (6.0-10.0) after 24 h, retaining 85% of its initial activity at pH 6 and even interestingly, the phytase activity enhanced at pH 8.0-10.0. It also exhibited thermostability, retaining about 60% of its original activity after 2 h at 60 °C. Cations such as Ca(2+) and Li(+) enhanced the phytase activity by 10-46% at 1 mM concentration. The phytase activity was completely inhibited by Cu(2+), Mg(2+), Fe(2+), Zn(2+), Hg(2+) and Mn(2+) and the inhibition was in a dose dependent manner. B. subtilis B.S.46 phytase had interesting characteristics to be considered as animal feed additive, dephytinization of food ingredients, and bioremediation of phosphorous pollution in the environment.

Evaluation of anti-phytoplasma properties of surfactin and tetracycline towards lime witches' broom disease using real-time PCR.

The anti-phytoplasma activities of surfactin (derived from Iranian native Bacillus subtilis isolates) and tetracycline towards Candidatus "Phytoplasma aurantifolia", the agent of lime Witches' broom disease, were investigated. HPLC was used to quantify the surfactin production in four previously characterized native surfactin-producing strains, and the one producing the highest amount of surfactin (about 1,500 mg/l) was selected and cultivated following optimized production and extraction protocols. Different combinations of purified surfactin and commercial tetracycline were injected into artificially phytoplasmainfected Mexican lime seedlings using a syringe injection system. An absolute quantitative real-time PCR system was developed to monitor the phytoplasma population shifts in the lime phloem during 3 months following the injections. The results revealed that the injections of surfactin or tetracycline had a significant inhibitory effect on Candidatus "P. aurantifolia". However, the combined treatment with both surfactin and tetracycline (1:1) resulted in the highest inhibition due to a synergic effect, which suppressed the phytoplasma population from about 2×10(5) to less than 10 phytoplasma units/g plant tissue.

Molecular and biochemical characterization of Iranian surfactin-producing Bacillus subtilis isolates and evaluation of their biocontrol potential against Aspergillus flavus and Colletotrichum gloeosporioides.

The characterization of surfactin-producing Bacillus subtilis isolates collected from different ecological zones of Iran is presented. Characterization was performed using blood agar, PCR, drop-collapse, and reverse-phase high-performance liquid chromatography (HPLC) analyses, and the isolates' biocontrol effects against the aflatoxin-producing agent Aspergillus flavus and the citrus antracnosis agent Colletotrichum gloeosporioides were studied. In total, 290 B. subtilis isolates were isolated from phylosphere and rhizosphere samples collected from fields and gardens of 5 provinces of Iran. Blood agar assays showed that 185 isolates produced different biosurfactants. Isolates containing the sfp gene, coding for surfactin, were detected using the PCR method. It was found that 14 different isolates contained the sfp gene. Drop-collapse assays, which detect isolates with high production of surfactin, showed that 7 isolates produced high levels of surfactin. It was found from HPLC analysis that the isolates containin the sfp gene produced between 55 and 1610 mg of surfactin per litre of broth medium. Four isolates, named BS119m, BS116l, N3dn, and BS113c, produced more than 1000 mg of surfactin per litre of broth. The highest surfactin production level was observed for isolate BS119m (1610 mg/L). The antagonistic potential of the sfp gene-containing isolates was determined using dual culture and chloroform vapour methods. Our bioassay results indicated that isolate BS119m showed high inhibitory effects against A. flavus (100%) and C. gloeosporioides (88%). Furthermore, the effect of purified surfactin on the growth of A. flavus was evaluated. Mycelia growth was considerably reduced with increasing concentration of surfactin, and 36%, 54%, 84%, and 100% inhibitions of mycelia growth were, respectively, observed at 20, 40, 80, and 160 mg/L after 7 days of incubation.

Molecular detection of nematicidal crystalliferous Bacillus thuringiensis strains of Iran and evaluation of their toxicity on free-living and plant-parasitic nematodes.

The characterization of nematode-effective strains and cry genes in the Iranian Bacillus thuringiensis (Bt) collection (70 isolates) is presented. Characterization was based on PCR analysis using 12 specific primers for cry5, cry6, cry12, cry13, cry14, and cry21 genes encoding proteins active against nematodes, crystal morphology, and protein band patterns as well as their nematicidal activity on root-knot nematode (Meloidogyne incognita) and two free-living nematodes (Chiloplacus tenuis and Acrobeloides enoplus). PCR results with primers for these genes showed that 22 isolates (31.5%) contain a minimum of one nematode-active cry gene. Strains containing the cry6 gene were the most abundant and represent 22.8% of the isolates. Bt strains harboring cry14 genes were also abundant (14.2%). cry21 and cry5 genes were less abundant, found in 4.2% and 2.8% of the strains, respectively. In total, six different nematode-active cry gene profiles were detected in this collection. Four isolates did not show the expected PCR product size for cry5, cry6, and cry21 genes; they might contain potentially novel insecticidal crystal protein genes. Twenty-two Bt isolates containing nematode-active cry genes were selected for preliminary bioassays on M. incognita. Based on these bioassays, four isolates were selected for detailed bioassays. Isolates YD5 and KON4 at 2 x 10(8) CFU/mL concentrations showed 77% and 81% toxicity on M. incognita, respectively. The free-living nematodes C. tenuis and A. enoplus were more susceptible and the highest mortality was observed within 48 h of incubation at all of the concentrations tested. Maximum mortality was recorded for isolates SN1 and KON4 at 2 x 10(8) CFU/mL concentrations and resulted in 68% and 77% adults deaths of C. tenuis and 68% and 72% for A. enoplus, respectively. Our results showed that PCR is a useful technique for toxicity prediction of nematicidal Bt isolates.