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1.
Sci Rep ; 11(1): 14114, 2021 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-34239009

RESUMO

Plant-parasitic nematodes wreak havoc on crops by root parasitism worldwide. An approach to combat nematode root parasitism is the application of antagonistic microbes like the rhizobacterium Bacillus firmus I-1582 which is promoted as biological control agent. Although B. firmus is a known nematode antagonist in general, the underlying mechanisms about its interaction with nematodes and plants have not yet been elucidated. Therefore, we explored the influence of B. firmus I-1582 as well as its extracellular and secreted molecules on plant-nematode interaction utilizing the plant-pathogen system Arabidopsis thaliana-Heterodera schachtii. We demonstrated that B. firmus I-1582 is attracted by A. thaliana root exudates, particularly by those of young plants. The bacterium colonized the root and showed a strictly pH-dependent development and plant growth promotion effect. Our results revealed that root colonization by B. firmus I-1582 significantly protected A. thaliana from infestation by the beet cyst nematode whereas dead bacterial cells or the culture supernatant were not effective. The bacterium also negatively affected nematode reproduction as well as pathogenicity and development of next generation nematodes. The obtained results highlight B. firmus I-1582 as a promising biocontrol agent that is well suited as an element of integrated control management strategies in sustainable agriculture.


Assuntos
Arabidopsis/crescimento & desenvolvimento , Arabidopsis/parasitologia , Bacillus firmus/fisiologia , Desenvolvimento Vegetal , Doenças das Plantas/parasitologia , Tylenchoidea/fisiologia , Animais , Quimiotaxia , Concentração de Íons de Hidrogênio , Exsudatos de Plantas , Raízes de Plantas/parasitologia , Tylenchoidea/patogenicidade , Virulência
2.
Appl Microbiol Biotechnol ; 105(11): 4589-4598, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34027563

RESUMO

We used agricultural residue, corn cob, with biorefinery and bioeconomy concepts. At short-time cultivation in corn cob (12 h), Bacillus firmus K-1 produced cellulase-free xylanolytic enzyme, with xylooligosaccharides (XOSs), X5 and X6, as the main products, which can be used in a variety of applications. The xylanolytic enzyme produced from B. firmus K-1 effectively degraded xylan in corn cob, which was examined by chemical composition, scanning electron microscope (SEM), and Fourier transform infrared spectroscopy (FTIR). After cultivation, the xylan contained in the corn cob residue was decreased (as biological pretreatment), causing morphological and structural changes, including creating porosity and increasing the surface area and the exposure of cellulose of pretreated corn cob. These results lead to an improvement of cellulose access by cellulases. Commercially available cellulases, Accellerase® 1500 and Cellic® CTec2, yielded significantly higher glucose concentrations from pretreated corn cob compared to untreated corn cob. After saccharification, the lignin-rich corn cob residue can be used as a raw material for other purposes. Moreover, the B. firmus cells, with a low risk to human health, can be used in some applications. This study presents an efficient method for producing high-value-added products from agricultural residue (corn cob) through biological processes which are environmentally friendly and economically viable. KEY POINTS: • High-value-added products were efficiently produced from corn cob by B. firmus K-1. • After biological pretreatment by B. firmus K-1, cellulase can better reach cellulose. • XOSs and cellulose-derived glucose were the main products from corn cob.


Assuntos
Bacillus firmus , Celulase , Celulases , Humanos , Hidrólise , Zea mays
3.
Microb Pathog ; 157: 105007, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34044052

RESUMO

Screening of halophiles with antimicrobial activity in saltpan soil samples from Nagapattinam district, Tamil Nadu, revealed isolate VE-2 as the most potent, identified as Bacillus firmus strain VE-2 through 16s rRNA gene sequencing. It had an optimum growth condition (OD 3.1) and antimicrobial protein (AMP) production (450 µg/mL) at 37 °C, pH 8, 25% NaCl, and 36 h incubation. SDS-PAGE analysis of the purified AMP showed the molecular weight of 36 kDa. HPLC analysis of the purified AMP showed different amino acids, such as asparagines, alanine, lysine, proline, threonine, glycine, cysteine, serine, aspartic acid leucine, and valine. Further characterization and identification using FT-IR, 2D-PAGE, MALDI-TOF, and in-silico analysis showed that the isolated AMP had the highest similarity to Subtilisin-A. It showed antibacterial activity against clinical bacterial pathogens like S. aureus, S. pyogenes, C. diphtheria, E. coli, and P. aeruginosa with the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration of 2.5 µg/mL and 20 µg/mL and also against various fungal pathogens such as A. niger, A. flavus, C. albicans, C. tropicalis and C. parapsilosis with the MIC and minimum fungicidal concentrations of 1.25-80 µg/mL. The purified AMP had excellent antioxidant potential, showed a scavenging effect against DPPH and Nitric oxide radicals, and displayed anticancer activity against HeLa cell lines with the IC50 values 53 µg/mL. Hence, the purified bioactive antimicrobial peptides (AMP) could also be used in anticancer therapies.


Assuntos
Bacillus firmus , Subtilisina/farmacologia , Antibacterianos/farmacologia , Escherichia coli , Células HeLa , Humanos , Índia , Testes de Sensibilidade Microbiana , RNA Ribossômico 16S/genética , Espectroscopia de Infravermelho com Transformada de Fourier , Staphylococcus aureus
4.
Int J Biol Macromol ; 170: 240-247, 2021 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-33359611

RESUMO

The purpose of this study was to gain an insight into the effects of mutation-induced binding pocket tilting of the Xyn11A xylanase from Bacillus firmus K-1 in producing a unique hydrolysis characteristic. In this study, the wildtype Xyn11A and its K40L mutant were compared for their hydrolysis patterns on beechwood xylan and xylooligosaccharides of sizes 2 to 6. According to our thin-layer chromatography experiment, the K40L mutant produced a larger amount of xylotetraose leftover than the wildtype. Kinetic determination of the WT and K40L mutant suggested that the higher X4 leftover on TLC was reflected in the decreasing catalytic efficiency (kcat/Km) between enzyme and X4. The mechanisms underlying this efficiency loss were examined through atomistic molecular dynamics (MD) simulations. The MD trajectory analysis showed that the mutation-induced binding pocket tilting resulted in an additional hydrophobic contact between the reducing end of X4 and Trp128. Meanwhile, the interactions between the non-reducing end and the Arg112 residue near the active site became lost, which could decrease the catalytic efficiency. This work suggested that the protein engineering to fine-tune the hydrolysis pattern for some desired xylooligosaccharide products was possible.


Assuntos
Endo-1,4-beta-Xilanases/genética , Xilanos/química , Xilanos/metabolismo , Bacillus firmus/genética , Bacillus firmus/metabolismo , Domínio Catalítico , Endo-1,4-beta-Xilanases/metabolismo , Escherichia coli/genética , Glucuronatos/química , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Cinética , Simulação de Dinâmica Molecular , Oligossacarídeos/química , Engenharia de Proteínas/métodos , Especificidade por Substrato
5.
J Mol Model ; 26(6): 124, 2020 May 09.
Artigo em Inglês | MEDLINE | ID: mdl-32388588

RESUMO

Lignin and phenolic compounds have been shown as the main recalcitrance for biomass decomposition, as they inhibit a number of lignocellulose-degrading enzymes. Understanding the inhibition mechanisms and energetic competitions with the native substrate is essential for the development of lignin resistive enzymes. In this study, atomistic detail of the size-dependent effects and binding modes of monomeric coniferyl alcohol, dimeric oligolignol, and tetrameric oligolignol made from coniferyl alcohols on the GH11 xylanase from Bacillus firmus strain K-1 was investigated by using molecular docking and atomistic molecular dynamics (MD) simulations. From the MD simulation results on the docked conformation of oligolignol binding within the "Cleft" and the "N-terminal," changes were observed both for protein conformations and positional binding of ligands, as binding with "Thumb" regions was found for all oligolignin models. Moreover, the uniquely stable "N-terminal" binding of the coniferyl alcohol monomer had no effect on the highly fluctuated Thumb region, showing no sign of inhibitory effect, and was in good agreement with recent studies. However, the inhibitory effect of oligolignols was size dependent, as the estimated binding energy of the tetrameric oligolignol became stronger than that of the xylohexaose substrate, and the important binding residues were identified for future protein engineering attempts to enhance the lignin resistivity of GH11. Graphical Abstract Size-dependent binding modes of coniferyl alcohol monomers (upper panels) and the dimers (lower panels). Uniquely stable "N-terminal" binding of the monomer is shown to have no effect on the binding pocket, and hence no sign of inhibition, which was in good agreement with some recent studies.


Assuntos
Bacillus firmus/enzimologia , Modelos Moleculares , Fenóis/farmacologia , Xilosidases/antagonistas & inibidores , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Lignina/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Fenóis/química , Polímeros/química , Polímeros/farmacologia , Ligação Proteica , Conformação Proteica , Xilosidases/metabolismo
6.
Environ Pollut ; 261: 114124, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32078878

RESUMO

Groundwater arsenic pollution causes millions of deaths worldwide. Long term natural and anthropogenic activities have increased arsenic levels in groundwater causing higher threats of arsenic exposure. Arsenic hyper-tolerant Firmicute Bacillus firmus L-148 was isolated from arsenic limiting Lonar lake soil, which tolerated more than 3 M arsenic and could oxidize 75 mM arsenite [As(III)] in 14 days. It oxidized As(III) in presence of heavy metals and had unusual pH optima at 9.2. B. firmus L-148 was studied at the biochemical, protein, genomic and transcript level for understanding its arsenic oxidizing machinery. The proteomic and transcript analysis exhibited the presence of ars and aio operon and supported the inducible nature of ars operon. Robust, hyper-tolerant, fast As(III) oxidizing, least nutrient requiring and multi-metal resistance qualities of the strain were used in microcosm studies for bioremediation. Artificial groundwater mimicking microcosm with 75 mM As(III) was developed. Modulation of carbon source, iron and multi metals affected growth and As(III) oxidation rate. The As(III) oxidation was recorded to be 77% in 15 days in presence of sodium acetate and Fe ions. This microcosm study can be explored for bioremediation of arsenic contaminated water and followed by precipitation using other methods.


Assuntos
Arsênio/análise , Bacillus firmus , Água Subterrânea , Poluentes Químicos da Água/análise , Oxirredução , Proteômica
7.
Arch Biochem Biophys ; 672: 108068, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31401092

RESUMO

Synergistic effect of distal site-directed mutations and molecular mechanisms on the enhanced thermostability of GH11 xylanase from B. firmus Strain K-1 (xyn11A) was investigated through enzyme activity assays and atomistic molecular dynamics (MD) simulation. From the experiment, single N-terminal leucine substitution at K40L caused a significant drop in enzymatic activity. However, the addition of a disulphide bond at S100C/N147C, along with the K40L mutation enhanced the enzymatic activity at room temperature. Molecular mechanisms on the improvement of enzymatic activity were addressed through atomistic molecular dynamics (MD) simulations of enzyme-substrate complexes. Conformational analysis of the right-hand-shaped GH11 protein structures showed that K40L mutation 'tilted' the Palm region away from the Pinky finger at N-terminus and S100C/N147C tilted the Palm region towards the Pinky finger at N-terminus, which destabilized the binding complexes. The extended hydrophobic cluster formed within the K40L/S100C/N147C mutant stabilized the loops associated with the N-terminus and the Thumb region, which facilitated substrate binding and corresponded to the enhanced activity. This proposed mechanism could serve as a scheme for protein engineering to enhance enzymatic activity of GH11 enzymes at low temperatures.


Assuntos
Proteínas de Bactérias/química , Dissulfetos/química , Endo-1,4-beta-Xilanases/química , Bacillus firmus/enzimologia , Proteínas de Bactérias/genética , Sítios de Ligação , Biocatálise , Cisteína/química , Endo-1,4-beta-Xilanases/genética , Ensaios Enzimáticos , Escherichia coli/genética , Interações Hidrofóbicas e Hidrofílicas , Simulação de Dinâmica Molecular , Mutagênese Sítio-Dirigida , Mutação , Conformação Proteica
8.
Bioprocess Biosyst Eng ; 42(4): 621-629, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30604010

RESUMO

Bacillusfirmus strain 37 produces the cyclomaltodextrin glucanotransferase (CGTase) enzyme and CGTase produces cyclodextrins (CDs) through a starch cyclization reaction. The strategy for the cloning and expression of recombinant CGTase is a potentially viable alternative for the economically viable production of CGTase for use in industrial processes. The present study used Bacillus subtilis WB800 as a bacterial expression host for the production of recombinant CGTase cloned from the CGTase gene of B. firmus strain 37. The CGTase gene was cloned in TOPO-TA® plasmid, which was transformed in Escherichia coli DH5α. The subcloning was carried out with pWB980 plasmid and transformation in B. subtilis WB800. The 2xYT medium was the most suitable for the production of recombinant CGTase. The enzymatic activity of the crude extract of the recombinant CGTase of B. subtilis WB800 was 1.33 µmol ß-CD/min/mL, or 7.4 times greater than the enzymatic activity of the crude extract of CGTase obtained from the wild strain. Following purification, the recombinant CGTase exhibited an enzymatic activity of 157.78 µmol ß-CD/min/mL, while the activity of the CGTase from the wild strain was 9.54 µmol ß-CD/min/mL. When optimal CDs production conditions for the CGTase from B. firmus strain 37 were used, it was observed that the catalytic properties of the CGTase enzymes were equivalent. The strategy for the cloning and expression of CGTase in B. subtilis WB800 was efficient, with the production of greater quantities of CGTase than with the wild strain, offering essential data for the large-scale production of the recombinant enzyme.


Assuntos
Bacillus firmus , Bacillus subtilis , Proteínas de Bactérias , Clonagem Molecular , Glucosiltransferases , Análise de Sequência de DNA , Bacillus firmus/enzimologia , Bacillus firmus/genética , Bacillus subtilis/enzimologia , Bacillus subtilis/genética , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Expressão Gênica , Glucosiltransferases/biossíntese , Glucosiltransferases/química , Glucosiltransferases/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
9.
Plant Physiol Biochem ; 132: 375-384, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30268029

RESUMO

Soil salinity is an adverse abiotic factor which reduces plant growth, yield and quality. Plant growth-promoting rhizobacteria (PGPR) have a great potential to enhance growth and alleviate saline stress effects without harming the environment via regulating physiological and molecular processes in plants. This study aimed at investigating Bacillus firmus SW5 effects on the performance of soybean (Glycine max L.) subjected to salt stress (0, 40 and 80 mM NaCl). Salinity stress mitigated the growth and biomass yield, root architecture traits, nutrient acquisition, chlorophyll level, transpiration rate (E), photosynthesis rate (Pn), stomatal conductance (gs), soluble proteins content, soluble sugars content and total phenolics and flavonoid contents of soybean plants. High salinity augmented the levels of osmolytes (glycine betaine and proline), hydrogen peroxide (H2O2), malondialdehyde (MDA) and the activities of antioxidant enzymes (APX, CAT, SOD and POD) in soybean plants. High salinity also induced the expression of antioxidant enzyme-encoding genes (APX, CAT, POD, Fe-SOD) and genes conferring tolerance to salinity (GmVSP, GmPHD2, GmbZIP62, GmWRKY54, GmOLPb, CHS) in soybean plants. On the other hand, inoculation of NaCl-stressed soybean plants with Bacillus firmus SW5 promoted the growth and biomass yield, chlorophyll synthesis, nutrient uptake, gas exchange parameters, osmolytes levels, total phenolic and flavonoid contents, and antioxidant enzymes activities, in comparison with the plants treated with NaCl alone. Bacillus firmus SW5 inoculation also significantly reduced the IC50 values for both DPPH and ß-carotene-linoleic acid assays and indicated higher antioxidant activities in salt-stressed plants. Furthermore, contents of H2O2 and MDA were alleviated in salinity-stressed soybean plants inoculated with Bacillus firmus SW5, in comparison with those in plants exposed to NaCl alone. The antioxidant enzyme-encoding genes and stress-related genes exhibited the highest expression levels in soybean plants inoculated with Bacillus firmus SW5 and treated with 80 mM NaCl. Taken together, our results demonstrate the crucial role of Bacillus firmus SW5 in ameliorating the adverse effects of high salinity on soybean growth and performance via altering the root system architecture and inducing the antioxidant defense systems and stress-responsive genes expression.


Assuntos
Antioxidantes/metabolismo , Bacillus firmus/metabolismo , Regulação da Expressão Gênica de Plantas , Raízes de Plantas/anatomia & histologia , Tolerância ao Sal/genética , Soja/genética , Soja/fisiologia , Estresse Fisiológico/genética , Betaína/metabolismo , Biomassa , Clorofila/metabolismo , Flavonoides/metabolismo , Gases/metabolismo , Genes de Plantas , Peróxido de Hidrogênio/metabolismo , Malondialdeído/metabolismo , Nitrogênio/análise , Fenóis/metabolismo , Fósforo/análise , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Prolina/metabolismo , Salinidade , Solubilidade , Soja/crescimento & desenvolvimento , Açúcares/análise
10.
PLoS One ; 13(2): e0193119, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29447287

RESUMO

Bacillus amyloliquefaciens QST713 and B. firmus I-1582 are bacterial strains which are used as active ingredients of commercially-available soil application and seed treatment products Serenade® and VOTiVO®, respectively. These bacteria colonize plant roots promoting plant growth and offering protection against pathogens/pests. The objective of this study was to develop a qPCR protocol to quantitate the dynamics of root colonization by these two strains under field conditions. Primers and TaqMan® probes were designed based on genome comparisons of the two strains with publicly-available and unpublished bacterial genomes of the same species. An optimized qPCR protocol was developed to quantify bacterial colonization of corn roots after seed treatment. Treated corn seeds were planted in non-sterile soil in the greenhouse and grown for 28 days. Specific detection of bacteria was quantified weekly, and showed stable colonization between ~104-105 CFU/g during the experimental period for both bacteria, and the protocol detected as low as 103 CFU/g bacteria on roots. In a separate experiment, streptomycin-resistant QST713 and rifampicin-resistant I-1582 strains were used to compare dilution-plating on TSA with the newly developed qPCR method. Results also indicated that the presence of natural microflora and another inoculated strain does not affect root colonization of either one of these strains. The same qPCR protocol was used to quantitate root colonization by QST713 and I-1582 in two corn and two soybean varieties grown in the field. Both bacteria were quantitated up to two weeks after seeds were planted in the field and there were no significant differences in root colonization in either bacteria strain among varieties. Results presented here confirm that the developed qPCR protocol can be successfully used to understand dynamics of root colonization by these bacteria in plants growing in growth chamber, greenhouse and the field.


Assuntos
Bacillus amyloliquefaciens , Bacillus firmus , Desenvolvimento Vegetal/fisiologia , Raízes de Plantas/microbiologia , Solo , Zea mays/microbiologia , Raízes de Plantas/crescimento & desenvolvimento , Sementes/crescimento & desenvolvimento , Sementes/microbiologia , Zea mays/crescimento & desenvolvimento
11.
Plant Dis ; 102(1): 107-113, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30673448

RESUMO

Successful management of the soybean cyst nematode Heterodera glycines is limited by increased virulence of nematode populations on resistant soybean cultivars and persistence of the nematode in the soil in the absence of hosts. Seed treatments are now available for H. glycines management. However, it is unclear how these treatments affect specific life stages of the nematode. The objectives of this study were to assess the effects of ILeVO (with active ingredient fluopyram) and VOTiVO (with active ingredient Bacillus firmus I-1582) seed treatments on H. glycines reproduction and important processes in the nematode life cycle, such as second-stage juvenile (J2) hatching, motility, and root penetration. The effects of seed treated with formulated (ILeVO and VOTiVO) and nonformulated active ingredient (fluopyram and B. firmus I-1582) on H. glycines reproduction were conducted in a greenhouse. Nematode reproduction on plants grown from seed treated with ILeVO or technical fluopyram (active ingredient only) was reduced by 35 to 97% relative to the nontreated control, suggesting that the fluopyram active ingredient was affecting H. glycines directly and was not an inert ingredient in the seed treatment formulation. Hatching, motility, and root penetration experiments also were conducted using only the formulated seed treatments. Exudates collected from ILeVO-treated seed reduced J2 hatching and motility by more than 95% in laboratory assays. Exudates from radicles grown from ILeVO-treated seed reduced hatching in vitro by 48% in one run but had no significant effect in the second run compared with the nontreated control exudates. There also were no consistent effects of radicle exudates, regardless of treatment, on hatching and motility of the J2. ILeVO reduced root penetration of H. glycines J2 at different inoculation densities in a growth chamber experiment. VOTiVO did not affect any of the processes or life stages of the nematode studied. The results of this study indicate that the use of nematode-protectant seed treatments may be useful in controlling H. glycines; however, additional investigations into the precise effects of ILeVO and VOTiVO on H. glycines life processes and in different parts of the soil profile are necessary.


Assuntos
Antinematódeos/farmacologia , Bacillus firmus/química , Benzamidas/farmacologia , Piridinas/farmacologia , Soja/parasitologia , Tylenchoidea/efeitos dos fármacos , Animais , Exsudatos e Transudatos/fisiologia , Comportamento Alimentar/efeitos dos fármacos , Reprodução/efeitos dos fármacos , Sementes
12.
J Biomol Struct Dyn ; 36(15): 3978-3992, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29129140

RESUMO

Local conformational changes and global unfolding pathways of wildtype xyn11A recombinant and its mutated structures were studied through a series of atomistic molecular dynamics (MD) simulations, along with enzyme activity assays at three incubation temperatures to investigate the effects of mutations at three different sites to the thermostability. The first mutation was to replace an unstable negatively charged residue at a surface beta turn near the active site (D32G) by a hydrophobic residue. The second mutation was to create a disulphide bond (S100C/N147C) establishing a strong connection between an alpha helix and a distal beta hairpin associated with the thermally sensitive Thumb loop, and the third mutation add an extra hydrogen bond (A155S) to the same alpha helix. From the MD simulations performed, MM/PBSA energy calculations of the unfolding energy were in a good agreement with the enzyme activities measured from the experiment, as all mutated structures demonstrated the improved thermostability, especially the S100C/N147C proved to be the most stable mutant both by the simulations and the experiment. Local conformational analysis at the catalytic sites and the xylan access region also suggested that mutated xyn11A structures could accommodate xylan binding. However, the analysis of global unfolding pathways showed that structural disruptions at the beta sheet regions near the N-terminal were still imminent. These findings could provide the insight on the molecular mechanisms underlying the enhanced thermostability due to mutagenesis and changes in the protein unfolding pathways for further protein engineering of the GH11 family xylanase enzymes.


Assuntos
Bacillus firmus/química , Proteínas de Bactérias/química , Endo-1,4-beta-Xilanases/química , Simulação de Dinâmica Molecular , Mutação , Engenharia de Proteínas/métodos , Bacillus firmus/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico , Clonagem Molecular , Endo-1,4-beta-Xilanases/genética , Endo-1,4-beta-Xilanases/metabolismo , Ensaios Enzimáticos , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Cinética , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia Estrutural de Proteína , Termodinâmica
13.
World J Microbiol Biotechnol ; 34(1): 10, 2017 Dec 18.
Artigo em Inglês | MEDLINE | ID: mdl-29255935

RESUMO

Nitrite is generated from the nitrogen cycle and its accumulation is harmful to environment and it can be reduced to nitric oxid by nitrite reductase. A novel gene from Bacillus firmus GY-49 is identified as a nirK gene encoding Cu-containing nitrite reductase by genome sequence. The full-length protein included a putative signal peptide of 26 amino acids and shown 72.73% similarity with other Cu-containing nitrite reductase whose function was verified. The 993-bp fragment encoding the mature peptide of NirK was cloned into pET-28a (+) vector and overexpressed as an active protein of 36.41 kDa in the E.coli system. The purified enzyme was green in the oxidized state and displayed double gentle peaks at 456 and 608 nm. The specific activity of purified enzyme was 98.4 U/mg toward sodium nitrite around pH 6.5 and 35 °C. The K m and K cat of NirK on sodium nitrite were 0.27 mM and 0.36 × 103 s-1, respectively. Finally, homology model analysis of NirK indicated that the enzyme was a homotrimer structure and well conserved in Cu-binding sites for enzymatic functions. This is a first report for nitrite reductase from Bacillus firmus, which augment the acquaintance of nitrite reductase.


Assuntos
Bacillus firmus/enzimologia , Bacillus firmus/genética , Cobre/química , Genes Bacterianos/genética , Nitrito Redutases/química , Nitrito Redutases/genética , Nitrito Redutases/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Ativação Enzimática , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Vetores Genéticos , Concentração de Íons de Hidrogênio , Íons , Cinética , Metais , Modelos Moleculares , Nitritos/metabolismo , Oxirredução , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Alinhamento de Sequência , Análise de Sequência de Proteína , Temperatura
14.
Cell Mol Biol (Noisy-le-grand) ; 63(6): 68-72, 2017 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-28968212

RESUMO

Rhodobacter sphaeroides NMBL-02, photosynthetic purple non sulfur (PNS) bacteria and associated Bacillus firmus NMBL-03 were isolated from water sample collected from 15-20 inches beneath the surface of ponds from Northern region of India in modified Sistrom's media (120 ml) containing 3 g/L malate and 1.2 g/L ammonium sulfate. The isolation was done in air tight serum bottles (120 ml) under tungsten bulb (1.8 kLux light intensity) at 30 oC ± 2 oC. The PNS and heterotrophic bacteria associated with the culture was purified by clonal selection method and characterized by 16S rDNA sequencing. The PNS isolate was identified as Rhodobacter sphaeroides NMBL-02 (ID: 1467407, Accession BANKIT: JN256030) and associated heterotroph as Bacillus firmus NMBL-03 (Gene Bank Accession no.: JN 256029). The effect of initial medium pH on optimization of hydrogen production was investigated in batch process. The maximum hydrogen potential and hydrogen production rate was 2310 ± 55 ml/L and 4.75 ml/L culture/h respectively using glutamate (1.7 mmol/L) as nitrogen source and malate (22.38 mmol/L) as carbon source with 76.39% malate conversion efficiency at initial medium pH 5.0. This co-culture has the ability to produce significant amount of hydrogen in the pH range of 5.0 to 10.0 with 76.39% to 35.71% malate conversion respectively.


Assuntos
Bacillus firmus/metabolismo , Bacillus firmus/fisiologia , Fermentação/fisiologia , Hidrogênio/metabolismo , Rhodobacter sphaeroides/metabolismo , Rhodobacter sphaeroides/fisiologia , Carbono/metabolismo , Técnicas de Cocultura/métodos , Ácido Glutâmico/metabolismo , Concentração de Íons de Hidrogênio , Índia , Luz , Malatos/metabolismo , Nitrogênio/metabolismo
15.
Cell Mol Biol (Noisy-le-grand) ; 63(6): 93-99, 2017 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-28968216

RESUMO

In this study photo-hydrogen production from cheese whey dark fermentation (DF) effluent by the co-culture of Rhodobacter sphaeroides -NMBL-01 and Bacillus firmus - NMBL-03 has been reported. The effect of pH, initial chemical oxygen demand (COD) and the concentration effect of FeSO4.7H2O on photo-hydrogen production have been investigated. The end products of dark fermentation effluent of cheese whey were mainly comprised of soluble organic acids, i.e. butyric acid and lactic acid. The batch process was carried out under light intensity of 2.5 kLux at 32 ± 2oC without any addition of extra carbon and nitrogen source. The single parameter optimization studies revealed optimum pH 6.5, initial COD 4.71 g/L and supplementation of Fe2+ concentration 100 mg/L. The maximum cumulative hydrogen production and yield were found to be 469 ± 45.8 ml H2/L and 146.56 ± 14.31 ml H2/g COD reduced (67.9% reduction in COD) respectively. The mutual interactions among the process parameters were also investigated by three factorial Box-Behnken design of response surface methodology. The optimized experimental values were found concurrent with the calculated values obtained from the theoretical model.


Assuntos
Bacillus firmus/metabolismo , Queijo/microbiologia , Fermentação/fisiologia , Hidrogênio/metabolismo , Rhodobacter sphaeroides/metabolismo , Soro do Leite/microbiologia , Ácido Butírico/metabolismo , Carbono/metabolismo , Técnicas de Cocultura/métodos , Concentração de Íons de Hidrogênio , Ácido Láctico/metabolismo , Nitrogênio/metabolismo , Soro do Leite/metabolismo
16.
J Colloid Interface Sci ; 483: 249-260, 2016 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-27560497

RESUMO

We describe herein a unique approach to synthesize zinc oxide-silica-silver (ZnO-SiO2-Ag) nanocomposite, in a simple, one-pot process. The typical process for ZnO synthesis by alkaline precipitation of zinc salts has been tweaked to replace alkali by alkaline sodium silicate. The free acid from zinc salts helps in the synthesis of silica nanoparticles, whereas the alkalinity of sodium silicate precipitates the zinc salts. Addition of silver ions into the reaction pot prior to addition of sodium silicate, and subsequent reduction by borohydride, gives additional functionality of metallic centres for catalytic applications. The synthesis strategy is based on our recent work typically involving acid-base type of cross-reactions and demonstrates a novel strategy to synthesize nanocomposites in a one-pot approach. Each component in the composite offers a unique feature. ZnO besides displaying mild catalytic and anti-bacterial behaviour is an excellent and a cheap 3-D support for heterogeneous catalysis. Silver nanoparticles enhance the catalytic & anti-bacterial properties of ZnO. Silica is an important part of the composite; which not only "glues" the two nanoparticles thereby stabilizing the nanocomposite, but also significantly enhances the surface area of the composite; which is an attractive feature of any catalyst composite. The nanocomposite is found to show excellent catalytic performance with very high turnover frequencies (TOFs) when studied for catalytic reduction of Rhodamine B (RhB) and 4-Nitrophenol (4-NP). Additionally, the composite has been tested for its anti-bacterial properties on three different bacterial strains i.e. E. coli, B. Cereus and Bacillus firmus. The mechanism for enhancement of catalytic performance has been probed by understanding the role of silica in offering accessibility to the catalyst via its porous high surface area network. The nanocomposite has been characterized by a host of different analytical techniques. The uniqueness of our product and process stems from the novel synthesis strategy, the choice and combination of the three moieties, increased surface area offered by silica, and cost effectiveness, thereby making our product and process commercially viable and sustainable for industrial applications.


Assuntos
Antibacterianos/síntese química , Nanopartículas Metálicas/química , Nanocompostos/química , Dióxido de Silício/química , Prata/química , Óxido de Zinco/química , Álcalis/química , Antibacterianos/farmacologia , Bacillus cereus/efeitos dos fármacos , Bacillus cereus/crescimento & desenvolvimento , Bacillus firmus/efeitos dos fármacos , Bacillus firmus/crescimento & desenvolvimento , Boroidretos/química , Catálise , Precipitação Química , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Concentração de Íons de Hidrogênio , Nanopartículas Metálicas/ultraestrutura , Testes de Sensibilidade Microbiana , Nanocompostos/ultraestrutura , Nitrofenóis/química , Oxirredução , Rodaminas/química , Silicatos/química , Propriedades de Superfície
17.
Sci Rep ; 6: 25012, 2016 04 27.
Artigo em Inglês | MEDLINE | ID: mdl-27118554

RESUMO

Plant-parasitic nematodes (PPNs) cause serious harm to agricultural production. Bacillus firmus shows excellent control of PPNs and has been produced as a commercial nematicide. However, its nematicidal factors and mechanisms are still unknown. In this study, we showed that B. firmus strain DS-1 has high toxicity against Meloidogyne incognita and soybean cyst nematode. We sequenced the whole genome of DS-1 and identified multiple potential virulence factors. We then focused on a peptidase S8 superfamily protein called Sep1 and demonstrated that it had toxicity against the nematodes Caenorhabditis elegans and M. incognita. The Sep1 protein exhibited serine protease activity and degraded the intestinal tissues of nematodes. Thus, the Sep1 protease of B. firmus is a novel biocontrol factor with activity against a root-knot nematode. We then used C. elegans as a model to elucidate the nematicidal mechanism of Sep1, and the results showed that Sep1 could degrade multiple intestinal and cuticle-associated proteins and destroyed host physical barriers. The knowledge gained in our study will lead to a better understanding of the mechanisms of B. firmus against PPNs and will aid in the development of novel bio-agents with increased efficacy for controlling PPNs.


Assuntos
Anti-Helmínticos/isolamento & purificação , Anti-Helmínticos/farmacologia , Bacillus firmus/enzimologia , Nematoides/efeitos dos fármacos , Serina Proteases/isolamento & purificação , Serina Proteases/farmacologia , Animais , Anti-Helmínticos/metabolismo , Bacillus firmus/genética , Genoma Bacteriano , Proteínas de Helminto/metabolismo , Proteólise , Análise de Sequência de DNA , Serina Proteases/genética , Serina Proteases/metabolismo
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