Medium composition and aeration to high (R,R)-2,3-butanediol and acetoin production by Paenibacillus polymyxa in fed-batch mode.

Concerning the potential application of the optically active isomer (R,R)-2,3-butanediol, and its production by a non-pathogenic bacterium Paenibacillus polymyxa ATCC 842, the present study evaluated the use of a commercial crude yeast extract Nucel®, as an organic nitrogen and vitamin source, at different medium composition and two airflows (0.2 or 0.5 vvm). The medium formulated (M4) with crude yeast extract carried out with the airflow of 0.2 vvm (experiment R6) allowed for a reduction in the cultivation time and kept the dissolved oxygen values at low levels until the total glucose consumption. Thus, the experiment R6 led to a fermentation yield of 41% superior when compared to the standard medium (experiment R1), which was conducted at airflow of 0.5 vvm. The maximum specific growth rate at R6 (0.42 h-1) was lower than R1 (0.60 h-1), however, the final cell concentration was not affected. Moreover, this condition (medium formulated-M4 and low airflow-0.2 vvm) was a great alternative to produce (R,R)-2,3-BD at fed-batch mode, resulting in 30 g.L-1 of the isomer at 24 h of cultivation, representing the main product in the broth (77%) and with a fermentation yield of 80%. These results showed that both medium composition and oxygen supply have an important role to produce 2,3-BD by P. polymyxa.

Purification and characterization of two new antimicrobial molecules produced by an endophytic strain of Paenibacillus polymyxa.

An endophytic bacterium inhibiting pathogenic bacteria was isolated and the strain was genetically identified as Paenibacillus polymyxa. Biochemical characterization of fermentation broth indicated the presence of peptidic antimicrobial molecules. Liquid-liquid partition resulted in an organic fraction (OF) and an aqueous fraction (AF). OF presented a broad spectrum of activity against a panel of pathogenic bacteria and a fungus whereas the AF was active only against Gram-negative bacteria. AF was sequentially submitted to ion-exchange, desalting and reverse phase (RP) chromatography. A molecule with an RT of 2.45 min exhibited activity against all Gram-negative pathogenic strains tested beside P. mirabilis. The primary structure of the molecule, named AMP-Pp, was determined as Gly-Glu-Hyp-Gly-Ala by N-terminal sequencing. The molecular mass and amino acid sequence were confirmed by MS/MS. With a molecular mass of 463 Da, AMP-Pp is one of the smallest active natural peptides reported, yet. RP chromatography of OF resulted in four peaks. The first three peaks corresponded to known antimicrobials. MS analysis of peak 4 revealed the presence of an ion with m/z 3,376.4 Da, whose proposed molecular formula is C182H321N29O29. The compound, named polycerradin, showed a spectrum of activity against Gram-positive bacteria, Gram-negative bacteria (beside P. mirabilis) and a fungus.

Antigenotoxic potential of the fermentation broth produced by Paenibacillus polymyxa RNC-D in vitro.

Aim: Evaluate the chemopreventive potential of the extract from P. polymyxa RNC-D. Methods: Concentrations of P. polymyxa RNC-D extract were tested in HepG2/C3A cells to assess their genotoxic (comet assay), mutagenic (micronucleus test) and antigenotoxic potential (comet assay) in vitro. Results: 400 and 40 µg/ml concentrations induced DNA lesions, whereas the 4 µg/ml induced a desmutagenic effect. Complementary tests indicated that the extract minimized the formation of reactive oxygen species induced by methyl methanesulfonate and normalized the loss of membrane potential. The quantification of cytokines indicated that TNF-α was immunostimulated by the extract. However, when administered in conjunction with the methyl methanesulfonate, the extract blocked the TNF-α release. Conclusion: The fermentation broth from P. polymyxa RNC-D showed an antigenotoxic effect, and thus the potential to be used as chemopreventive compound.

Paenibacillus polymyxa NMA1017 as a potential biocontrol agent of Phytophthora tropicalis, causal agent of cacao black pod rot in Chiapas, Mexico.

Cacao represents an important source of income for farmers in the south of Mexico. However, phytosanitary problems have disrupted the production over the years. The use of antagonistic microorganisms as biocontrol agents might improve the production of cacao. In this study, Paenibacillus polymyxa NMA1017, isolated from the rhizosphere of Opuntia ficus-indica L., was used as a biocontrol agent for black pod rot of Theobroma cacao L. cultivated in Chiapas, Mexico. The experiments were carried in vitro and in vivo using pear fruit (Pyrous communis) as model and cacao pods in the field, respectively. The effect of NMA1017 on the phytopathogen was observed by electron microscopy and the production of enzymes was tested as a potential mechanism of action. The bacterium inhibited the radial growth of Phytophthora tropicalis PtCa-14 by 85.9 ± 0.12%. The strain NMA1017 affected mycelial development, as observed by the damage to the cell wall of the oomycete. In pear fruit, the biocontrol agent controlled the production of mycelium on the pear fruit surface, indicating an inhibitory effect exerted. Cacao pods infected with P. tropicalis in the field resulted in a reduction in disease incidence from 86 to 33% and in infection from 68 to 6%. Moreover, strain NMA1017 produced hydrolytic enzymes such as cellulases, xylanases, chitinases and proteases. The results obtained highlight P. polymyxa NMA1017 as an organism of interest for the biocontrol of P. tropicalis, as a method to rescue this important crop in Mexico.

Glutantßase: a database for improving the rational design of glucose-tolerant ß-glucosidases.

Β-glucosidases are key enzymes used in second-generation biofuel production. They act in the last step of the lignocellulose saccharification, converting cellobiose in glucose. However, most of the ß-glucosidases are inhibited by high glucose concentrations, which turns it a limiting step for industrial production. Thus, ß-glucosidases have been targeted by several studies aiming to understand the mechanism of glucose tolerance, pH and thermal resistance for constructing more efficient enzymes. In this paper, we present a database of ß-glucosidase structures, called Glutantßase. Our database includes 3842 GH1 ß-glucosidase sequences collected from UniProt. We modeled the sequences by comparison and predicted important features in the 3D-structure of each enzyme. Glutantßase provides information about catalytic and conserved amino acids, residues of the coevolution network, protein secondary structure, and residues located in the channel that guides to the active site. We also analyzed the impact of beneficial mutations reported in the literature, predicted in analogous positions, for similar enzymes. We suggested these mutations based on six previously described mutants that showed high catalytic activity, glucose tolerance, or thermostability (A404V, E96K, H184F, H228T, L441F, and V174C). Then, we used molecular docking to verify the impact of the suggested mutations in the affinity of protein and ligands (substrate and product). Our results suggest that only mutations based on the H228T mutant can reduce the affinity for glucose (product) and increase affinity for cellobiose (substrate), which indicates an increment in the resistance to product inhibition and agrees with computational and experimental results previously reported in the literature. More resistant ß-glucosidases are essential to saccharification in industrial applications. However, thermostable and glucose-tolerant ß-glucosidases are rare, and their glucose tolerance mechanisms appear to be related to multiple and complex factors. We gather here, a set of information, and made predictions aiming to provide a tool for supporting the rational design of more efficient ß-glucosidases. We hope that Glutantßase can help improve second-generation biofuel production. Glutantßase is available at http://bioinfo.dcc.ufmg.br/glutantbase .

Production optimization, purification, expression, and characterization of a novel α-L-arabinofuranosidase from Paenibacillus polymyxa

Background: α-L-Arabinofuranosidase (EC 3.2.1.55) catalyzes the hydrolysis of terminal α-L-1,2-, -1,3-, and -1,5- arabinofuranosyl residues in arabinose-containing polymers, and hence, it plays an important role in hemicellulose degradation. Herein, the bacterium Paenibacillus polymyxa, which secretes arabinofuranosidase with high activity, was selected for enzyme production, purification, and characterization. Results: Medium components and cultural conditions were optimized by the response surface method using shake flask cultures. Arabinofuranosidase production reached 25.2 U/mL under optimized conditions, which were pH 7.5, 28°C, and a basic medium supplemented with 1.5 g/L mannitol and 3.5 g/L soymeal. Furthermore, the arabinofuranosidase secreted by P. polymyxa, named as PpAFase-1, was partially purified from the supernatant using a DEAE Sepharose Fast Flow column and a hydroxyapatite column. The approximate molecular mass of the purified PpAFase-1 was determined as 56.8 kDa by SDS-PAGE. Protein identification by mass spectrometry analysis showed that the deduced amino acid sequence had significant similarity to the glycosyl hydrolase family 51. The deduced gene of 1515 bp was cloned and expressed in Escherichia coli BL21 (DE3) cells. Purified recombinant PpAFase-1 was active toward p-nitrophenyl-α-L-arabinofuranoside (pNPAraf). The Km and kcat values toward pNPAraf were 0.81 mM and 53.2 s−1 , respectively. When wheat arabinoxylan and oat spelt xylan were used as substrates, PpAFase-1 showed poor efficiency. However, a synergistic effect was observed when PpAFase-1 was combined with xylanase from Thermomyces lanuginosus. Conclusion: A novel GH51 enzyme PpAFase-1 was cloned from the genome of P. polymyxa and expressed in E. coli. This enzyme may be suitable for hemicellulose degradation on an industrial scale.

Paenibacillus polymyxa Associated with the Stingless Bee Melipona scutellaris Produces Antimicrobial Compounds against Entomopathogens.

Social insects are frequently observed in symbiotic association with bacteria that produce antimicrobial natural products as a defense mechanism. There is a lack of studies on the microbiota associated with stingless bees and their antimicrobial compounds. To the best of our knowledge, this study is the first to report the isolation of Paenibacillus polymyxa ALLI-03-01 from the larval food of the stingless bee Melipona scutellaris. The bacterial strain was cultured under different conditions and produced (L)-(-)-3-phenyllactic acid and fusaricidins, which were active against entomopathogenic fungi and Paenibacillus larvae. Our results indicate that such natural products could be related to colony protection, suggesting a defense symbiosis between P. polymyxa ALLI-03-01 and Melipona scutellaris.

Cytotoxic Effects and Production of Cytokines Induced by the Endophytic Paenibacillus polymyxa RNC-D In Vitro.

BACKGROUND: Prominent among all the organisms that have a potential value for the production of new medicines, are endophytes, fungi and bacteria that live inside plants without harming them. In this study, a total lyophilized extract (TLE) of Paenibacillus polymyxa RNC-D was used. The P. polymyxa lineages are known for their capacity to segregate a large number of extracellular enzymes and bioactive substances. METHODS: The TLE of Paenibacillus polymyxa RNC-D was tested in cell viability assays for cytotoxicity and cytokine production in BALB/3T3 and J774A.1 cell lineages. RESULTS: A 50% mortality rate of fibroblasts (BALB/3T3) was observed in the 1.171±0.161 mg/mL and 0.956±0.112 mg/mL doses after 48 and 72 hours, respectively, as well as a 50% mortality rate of macrophage cells (J774A.1) in the 0.994±0.170 mg/mL and 0.945±0.280 mg/mL doses after 48 and 72 hours, respectively. The ≈1 mg/mL concentration significantly affected the kinetic of growth in all the measured periods. The extract induced apoptosis and necrosis 24 hours after the ≈1 mg/mL concentration in both tested lineages. The treatment with the ≈1 mg/mL concentration led to the production of TNF-α and IFN-γ cytokines in 24 hours. IL-12 and IL-10 began to be detected as a result of the treatment with 0.1 mg/mL. However, with the 0.5 mg/mL dose in 24 hours, a significant reduction in IL-10 was observed. CONCLUSION: Our data suggest that the TLE of P. polymyxa RNC-D modulated the production of cytokines with different patterns of immune response in a dose-dependent way.