Amplificação isotérmica mediada por loop para detecção de patógenos de plantas

Disease control is crucial to minimize potential losses in agriculture and thereby maintain high crop yield. However, for its effectiveness, the pathogen must be detected early and correctly in the production fields. Different methods of diagnosis can be used, from those based on symptoms to molecular tests. Loop-mediated isothermal amplification (LAMP) is a molecular technique that has been widely used in several biological fields, due to the ease with which it can be applied. The reaction can be carried out in a single thermal condition, due to the use of Bst DNA polymerase, isolated from the bacterium Bacillus stearothermophilus, which has high displacement activity. LAMP is a highly exponential amplification method that produces the target DNA in amounts 109 -1010 times between 45 and 60 minutes at 60-65°C. Its advantages are the visualization of results directly with the naked eye and the fact that it does not need sophisticated equipment for its application. In phytopathology, the technique has been gaining prominence in the detection of fungi, viruses, bacteria, nematodes and phytoplasmas, as well as in the monitoring of fungicide-resistant fungi. LAMP can benefit agriculture so that early, accurate and sensitive diagnostics can be carried out in the fields of cultivation and minimize losses caused by diseases. In this review, we present and discuss LAMP tests, developed for plant pathogens detection, which can be useful for researchers who wish to use the technique in their research area

Porous poly(urethane urea) microparticles for immobilization of maltogenic α amylase from Bacillus stearothermophilus

Maltogenic α amylase from Bacillus stearothermophilus (BsMa) was immobilized by covalent attachment and physical adsorption onto porous poly(urethane urea) (PUU) microparticles obtained from poly(vinyl alcohol) (PVA) and 4,4’-methylenebis(cyclohexyl diisocyanate) (H12DI) by onepot synthesis. The influence of PUU synthesis parameters such as PVA and H12DI molar ratio, synthesis time and temperature on porosity, surface area, structure of microparticles and catalytic activity and stability of immobilized BsMa was investigated. The highest efficiency of the immobilization of BsMa onto the PUU carrier was 97% and the highest residual stability of the immobilized enzyme reached 95% after 28 days of storage at 4°C. The optimal activity temperature of immobilized BsMa was at 80°C and it was higher than that of native enzyme. Effects of ionic strength and repetitive batch processing cycles on the activity of immobilized BsMa were also studied. Immobilization of BsMa onto PUU carriers has a great potential for biotechnology and food industries.

Evaluation of Biocompatibility and Antagonistic Properties of Microorganisms Isolated from Natural Sources for Obtaining Biofertilizers Using Microalgae Hydrolysate

Determination of the biocompatibility of microorganisms isolated from natural sources (Kemerovo Oblast—Kuzbass) resulted in the creation of three microbial consortia based on the isolated strains: consortium I (Bacillus pumilus, Pediococcus damnosus, and Pediococcus pentosaceus), consortium II (Acetobacter aceti, Pseudomonas chlororaphis, and Streptomyces parvus), and consortium III (Amycolatopsis sacchari, Bacillus stearothermophilus; Streptomyces thermocarboxydus; and Streptomyces thermospinisporus). The nutrient media composition for the cultivation of each of the three studied microbial consortia, providing the maximum increase in biomass, was selected: consortium I, nutrient medium 11; consortium II, nutrient medium 13; for consortium III, nutrient medium 16. Consortia I and II microorganisms were cultured at 5–25 °C, and consortium III at 50–70 °C. Six types of psychrophilic microorganisms (P. pentosaceus, P. chlororaphis, P. damnosus, B. pumilus, A. aceti, and S. parvus) and four types of thermophilic microorganisms (B. stearothermophilus, S. thermocarboxydus, S. thermospinisporus, and A. sacchari) were found to have high antagonistic activity against the tested pathogenic strains (A. faecalis, B. cinerea, E. carotovora, P. aeruginosa, P. fluorescens, R. stolonifera, X. vesicatoria. pv. Vesicatoria, and E. aphidicola). The introduction of microalgae hydrolyzate increased the concentration of microorganisms by 5.23 times in consortium I, by 4.66 times in consortium II, by 6.6 times in consortium III. These data confirmed the efficiency (feasibility) of introducing microalgae hydrolyzate into the biofertilizer composition.

Spore Forming Bacteria Responsible for Food Spoilage: A Review

In this article we investigate about the different sort of spore framing microorganisms engaged with deterioration of different edibles either cooked or without cooked. The organisms which are exceptionally heat safe can be disconnected in low - corrosive canned nourishments.The species which are dominating species are by and large Geo bacillus stearothermophilus, Morellathermoacetica and Thermonaro bacterium spp. The items which are in pastry shop or distillery are explicitly ruined by Bacillus species. The primary microorganism revealed for ruining the bread kitchen stuffed food are Bacillus amyloliquefaciens. The different sorts of vacuum pressed meats which are refrigerated are exceptionally get ruined by Clostridium species. Milk is another crude material to be ruined effectively by the microorganisms, presently how it is being ruined by them? So essentially they make ascend in the measure of corrosiveness in the milk itself which makes the milk and its items much preceding turn sour without any problem. It is a direct result of the two genera which is been distinguished from milk when it goes through additional means like sanitization, cleansing, drying out or aging and furthermore likewise can be separated based on heat treatment and capacity temperature. In this review article investigation we came across many research papers. Different types of food and edibles are surveyed giving us various samples about different type of spoilage due to spore in them. We have examined various dairy products meat, fish, canned foods various bakery products and many more things. Our investigation showed that a slimy layer, foul smell sour odour, changes in the colour and structure, changes due to weather conditions proved as a boon for various bacterial spores to hit them with spoilage. This work concludes the study of all the above mentioned topics.

Effect of Leu277 on Disproportionation and Hydrolysis Activity in Bacillus stearothermophilus NO2 Cyclodextrin Glucosyltransferase

The disproportionation activity of cyclodextrin glucosyltransferase (CGTase, EC 2.4.1.19) can be used to convert small molecules into glycosides, thereby enhancing their solubility and stability. However, CGTases also exhibit a competing hydrolysis activity. The +2 subsite of the substrate binding cleft plays an important role in both the disproportionation and hydrolysis activities, but almost all known mutations at this site decrease disproportionation activity. In this study, Leu277 of the CGTase from Bacillus stearothermophilus NO2, located near both the +2 subsite and the catalytic acid/base Glu253, was modified to assess the effect of side chain size at this position on disproportionation and hydrolysis activities. The best mutant, L277M, exhibited a reduced Km for the acceptor substrate maltose (0.48 mM versus 0.945 mM) and an increased kcat/Km (1175 s−1mM−1 versus 686.1 s−1mM−1), compared with those of the wild-type enzyme. The disproportionation to hydrolysis ratio of L277M was 2.4-fold greater than that of the wild-type. Existing structural data were combined with a multiple sequence alignment and Gly282 mutations to examine the mechanism behind the effects of the Leu277mutations. The Gly282 mutations were included to aid a molecular-dynamics (MD) analysis and the comparison of crystal structures. They reveal that changes to a hydrophobic cluster near Glu253 and the hydrophobicity of the +2 subsite combine to produce the observed effects. Importance In this study, mutations that enhance the disproportionation to hydrolysis ratio of a CGTase have been discovered. For example, the disproportionation to hydrolysis ratio of the L277M mutant of Bacillus stearothermophilus NO2 CGTase was 2.4-fold greater than that of the wild-type. The mechanism behind the effects of these mutations is explained. This paper opens up other avenues for future research into the disproportionation and hydrolysis activities of CGTases. Productive mutations are no longer limited to the acceptor subsite, since mutations that indirectly affect the acceptor subsite also enhance enzymatic activity.

Constructing an Efficient Bacillus subtilis Spore Display by Using Cohesin−Dockerin Interactions

Bacillus subtilis spore display has become a field of increasing interest in the past two decades. To improve the efficiency of B. subtilis spore display, its directed modification was performed based on the cellulosome architecture by introducing onto them divergent cohesin (Coh) modules that can specifically bind to the target enzyme bearing the matching dockerins (Doc). In this study, five different pairs of cohesins and dockerins, selected from four cellulolytic microbes, were examined for their capabilities in displaying a tetrameric enzyme β-galactosidase from Bacillus stearothermophilus IAM11001 on the surface of B. subtilis WB600 spores. Immunofluorescence microscopy, western blotting, dot blotting, and enzyme assay was applied to confirm its surface expression. All the resultant five Coh–Doc based spore display can hydrolyze o-nitrophenyl-β-D-galactopyranoside. Further, the optimized Coh–Doc based spore display exhibited the highest display efficiency. Overall, the results of current study may open new perspectives on the use of Coh–Doc interaction, which will find application in improving the efficiency of B. subtilis spore display.

A Novel Thermostable and Alkaline Protease Produced from Bacillus stearothermophilus Isolated from Olive Oil Mill Sols Suitable to Industrial Biotechnology

This study was conducted to identify a new alkaline and thermophilic protease (Ba.St.Pr) produced from Bacillus stearothermophilus isolated from olive oil mill sols and to evaluate its culture conditions, including temperature, pH, carbon and nitrogen sources, and incubation time. The optimum culture conditions for cell growth (10 g/L) and protease production (5050 U/mL) were as follows: temperature 55 °C, pH 10, inoculation density 8 × 108 CFU/mL, and incubation time 24 h. The use of 3% yeast extract as the nitrogen sources and galactose (7.5 g/L) as the carbon sources enhanced both cell growth and protease production. Using reversed-phase analytical HPLC on C-8 column, the new protease was purified with a molecular mass of approximately 28 kDa. The N-terminal sequence of Ba.St.Pr exhibited a high level of identity of approximately 95% with those of Bacillus strains. Characterization under extreme conditions revealed a novel thermostable and alkaline protease with a half-life time of 187 min when incubated with combined Ca2+/mannitol. Ba.St.Pr demonstrated a higher stability in the presence of surfactant, solvent, and Ca2+ ions. Consequently, all the evaluated activity parameters highlighted the promising properties of this bacterium for industrial and biotechnological applications.