Genes encoding a novel thermostable bacteriocin in the thermophilic bacterium Aeribacillus pallidus PI8.

AIM: Aeribacillus pallidus PI8 is a Gram-positive thermophilic bacterium that produces thermostable antimicrobial substances against several bacterial species, including Geobacillus kaustophilus HTA426. In the present study, we sought to identify genes of PI8 with antibacterial activity. METHODS AND RESULTS: We isolated, cloned, and characterized a thermostable bacteriocin from A. pallidus PI8 and named it pallidocyclin. Mass spectrometric analyses of pallidocyclin revealed that it had a circular peptide structure, and its precursor was encoded by pcynA in the PI8 genome. pcynA is the second gene within the pcynBACDEF operon. Expression of the full-length pcynBACDEF operon in Bacillus subtilis produced intact pallidocyclin, whereas expression of pcynF in G. kaustophilus HTA426 conferred resistance to pallidocyclin. CONCLUSION: Aeribacillus pallidus PI8 possesses the pcynBACDEF operon to produce pallidocyclin. pcynA encodes the pallidocyclin precursor, and pcynF acts as an antagonist of pallidocyclin.

Biodegradation of aliphatic and polycyclic aromatic hydrocarbons by the thermophilic bioemulsifier-producing Aeribacillus pallidus strain SL-1.

The utilization of thermophilic hydrocarbon-degrading microorganisms is a suitable strategy for improving biodegradation of petroleum hydrocarbons and PAHs, as well as enhancing oil recovery from high-temperature reservoirs. In this study, the thermophilic strain Aeribacillus pallidus SL-1 was evaluated for the biodegradation of crude oil and PAHs at 60 °C. Strain SL-1 was found to preferentially degrade short-chain n-alkanes (

Purification and biochemical characterization of a novel thermostable serine alkaline protease from Aeribacillus pallidus C10: a potential additive for detergents.

An extracellular thermostable alkaline serine protease enzyme from Aeribacillus pallidus C10 (GenBank No: KC333049), was purified 4.85 and 17. 32-fold with a yield of 26.9 and 19.56%, respectively, through DE52 anion exchange and Probond affinity chromatography. The molecular mass of the enzyme was determined through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), with approximately 38.35 kDa. The enzyme exhibited optimum activity at pH 9 and at temperature 60 °C. It was determined that the enzyme had remained stable at the range of pH 7.0-10.0, and that it had preserved more than 80% of its activity at a broad temperature range (20-80 °C). The enzyme activity was found to retain more than 70% and 55% in the presence of organic solvents and commercial detergents, respectively. In addition, it was observed that the enzyme activity had increased in the presence of 5% SDS. KM and Vmax values were calculated as 0.197 mg/mL and 7.29 µmol.mL-1.min-1, respectively.

Suitability of the alkalistable carbonic anhydrase from a polyextremophilic bacterium Aeribacillus pallidus TSHB1 in biomimetic carbon sequestration.

Carbonic anhydrase (CA) was produced from the polyextremophilic (halotolerant, moderately thermophilic and alkaliphilic) bacterium Aeribacillus pallidus TSHB1 isolated from water and sediment samples of Choti Anhoni hot spring of Pipariya, Madhya Pradesh (India), is being reported to be suitable for carbon sequestration. Growth and CA production were inhibited at higher CO2 concentration (5-10 %). Under optimized culture variables (tryptone 0.8 %, yeast extract 0.08 %, glucose 1 %, micronutrient solution 1 %, inoculums size 1.10 %, agitation 200 at pH 8, and temperature 55 °C), 3.7-fold higher CA production was attained than that under unoptimized conditions. The zymogram analysis of the partially purified CA revealed an activity band corresponding to 32 kDa. The enzyme is stable in the pH range between 8.0 and 11.0 with T 1/2 of 40, 15, and 8 min at 60, 70, and 80 °C, respectively. The CA of A. pallidus displayed a marked enhancement in the rate of CaCO3 precipitation from aqueous CO2. The CA-aided formation of CaCO3 was 42.5 mg mg(-1) protein. Scanning electron microscopy revealed the formation of rhomboid calcite crystals. This is the first report on the production and applicability of CA from the polyextremophilic A. pallidus in carbon sequestration.

Continuous cultivation of a thermophilic bacterium Aeribacillus pallidus 418 for production of an exopolysaccharide applicable in cosmetic creams.

AIMS: The aim of this study was to evaluate the effectiveness of continuous cultivation approach for exopolysaccharide (EPS) production by a thermophilic micro-organism and the potential of the synthesized EPS for application in cosmetic industry. METHODS AND RESULTS: Study on the ability of Aeribacillus pallidus 418, isolated as a good EPS producer, to synthesize the polymer in continuous cultures showed higher production in comparison with batch cultures. The degree of the EPS in the precipitate after continuous cultivation significantly increased. Non-Newtonian pseudoplastic and thixotropic behaviour of EPS determines the ability of the received cream to become more fluid after increasing time of application on the skin. CONCLUSIONS: This study demonstrates a highly efficient way for production of EPS from a continuous growth culture of A. pallidus 418 that have many advantages and can outperform batch culture by eliminating time for cleaning and sterilization of the vessel and the comparatively long lag phases before the organisms enter a brief period of high productivity. The valuable physico-chemical properties of the synthesized EPS influenced positively the properties of a commercial cream. SIGNIFICANCE AND IMPACT OF THE STUDY: EPSs from thermophilic micro-organisms are of special interest due to the advantages of the thermophilic processes and nonpathogenic nature of the polymer molecules. However, their industrial application is hindered by the comparatively low biomass and correspondingly EPS yield. Suggested continuous approach for EPS could have an enormous economic potential for an industrial scale production of thermophilic EPSs.

Characterization of a Thermostable Endolysin of the Aeribacillus Phage AeriP45 as a Potential Staphylococcus Biofilm-Removing Agent.

Multidrug-resistant Gram-positive bacteria, including bacteria from the genus Staphylococcus, are currently a challenge for medicine. Therefore, the development of new antimicrobials is required. Promising candidates for new antistaphylococcal drugs are phage endolysins, including endolysins from thermophilic phages against other Gram-positive bacteria. In this study, the recombinant endolysin LysAP45 from the thermophilic Aeribacillus phage AP45 was obtained and characterized. The recombinant endolysin LysAP45 was produced in Escherichia coli M15 cells. It was shown that LysAP45 is able to hydrolyze staphylococcal peptidoglycans from five species and eleven strains. Thermostability tests showed that LysAP45 retained its hydrolytic activity after incubation at 80 °C for at least 30 min. The enzymatically active domain of the recombinant endolysin LysAP45 completely disrupted biofilms formed by multidrug-resistant S. aureus, S. haemolyticus, and S. epidermidis. The results suggested that LysAP45 is a novel thermostable antimicrobial agent capable of destroying biofilms formed by various species of multidrug-resistant Staphylococcus. An unusual putative cell-binding domain was found at the C-terminus of LysAP45. No domains with similar sequences were found among the described endolysins.

Draft genome sequences of two thermophilic, spore-forming Aeribacillus pallidus strains isolated from dairy products.

The presence of thermophilic spore-forming bacteria is challenging in industrial food processing. The presented genome sequences of Aeribacillus pallidus, isolated from raw milk and cocoa powder, provide insights into how to prevent damage to minimally processed foods and products with extended shelf life, such as milk products.

Safety evaluation of an extension of use of the food enzyme 4-α-glucanotransferase from the non-genetically modified Aeribacillus pallidus strain AE-SAS.

The food enzyme 4-α-glucanotransferase (1,4-α-d-glucan:1,4-α-d-glucan 4-α-d-glycosyltransferase, EC 2.4.1.25) is produced with the non-genetically modified Aeribacillus pallidus strain AE-SAS by Amano Enzyme Inc. A safety evaluation of this food enzyme was made previously, in which EFSA concluded that this food enzyme did not give rise to safety concerns when used in two food manufacturing processes. Subsequently, the applicant requested to extend its use to two additional processes. In this assessment, EFSA updated the safety evaluation of this food enzyme for use in a total of four food manufacturing processes. As the food enzyme-total organic solids (TOS) is removed from the final foods in one food manufacturing process, the dietary exposure to the food enzyme-TOS was estimated only for the remaining three processes. Dietary exposure was up to 0.040 mg TOS/kg body weight (bw) per day in European populations. When combined with the no observed adverse effect level reported in the previous opinion (900 mg TOS/kg bw per day, the highest dose tested), the Panel derived a margin of exposure of at least 22,500. Based on the data provided for the previous evaluation and the revised margin of exposure, the Panel concluded that this food enzyme does not give rise to safety concerns under the revised intended conditions of use.

Recombinase polymerase amplification using novel thermostable strand-displacing DNA polymerases from Aeribacillus pallidus and Geobacillus zalihae.

Recombinase polymerase amplification (RPA) is an isothermal DNA amplification reaction at around 41 °C using recombinase (Rec), single-stranded DNA-binding protein (SSB), and strand-displacing DNA polymerase (Pol). Component instability and the need to store commercial kits in a deep freezer until use are some limitations of RPA. In a previous study, Bacillus stearothermophilus Pol (Bst-Pol) was used as a thermostable strand-displacing DNA polymerase in RPA. Here, we attempted to optimize the lyophilization conditions for RPA with newly isolated thermostable DNA polymerases for storage at room temperature. We isolated novel two thermostable strand-displacing DNA polymerases, one from a thermophilic bacterium Aeribacillus pallidus (H1) and the other from Geobacillus zalihae (C1), and evaluated their performances in RPA reaction. Urease subunit ß (UreB) DNA from Ureaplasma parvum serovar 3 was used as a model target for evaluation. The RPA reaction with H1-Pol or C1-Pol was performed at 41 °C with the in vitro synthesized standard UreB DNA. The minimal initial copy numbers of standard DNA from which the amplified products were observed were 600, 600, and 6000 copies for RPA with H1-Pol, C1-Pol, and Bst-Pol, respectively. Optimization was carried out using RPA components, showing that the lyophilized RPA reagents containing H1-Pol exhibited the same performance as the corresponding liquid RPA reagents. In addition, lyophilized RPA reagents with H1-Pol showed almost the same activity after two weeks of storage at room temperature as the freshly prepared liquid RPA reagents. These results suggest that lyophilized RPA reagents with H1-Pol are preferable to liquid RPA reagents for onsite use.

Functional analysis of a gene cluster for putative bacteriocin deduced from the genome sequence of Aeribacillus pallidus PI8.

Bacteriocins are a large family of peptides synthesized ribosomally by a variety of bacterial species. The genome of one of the thermophilic Gram-positive bacteria, Aeribacillus pallidus PI8, was found to possess an operon comprising five genes possibly involved in the production of a putative bacteriocin that was named pcnABCDE for the production of "pallidocyclicin." This study investigated the function of the pcn operon experimentally. The heterologous expression of the entire pcn operon from the plasmid was toxic to Escherichia coli but not to Bacillus subtilis. However, when the entire pcn operon was expressed constitutively, even the growth of B. subtilis was impaired, and at least pcnA was implied to serve as the precursor of pallidocyclicin. In addition, a strain of B. subtilis expressing the entire pcn operon from the plasmid showed toxicity to another thermophilic species, Geobacillus kaustophilus, at elevated temperatures, whereas another strain lacking pcnE alone from the pcn operon lost the toxicity, suggesting that pcnE might be involved in the biosynthesis of pallidocyclicin when it is produced in B. subtilis.

Potential application of thermophilic bacterium Aeribacillus pallidus MRP280 for lead removal from aqueous solution.

Bacteria used for application of lead (Pb) removal is usually kept under suboptimal growth conditions. Certain application of Pb removal may be carried out under different condition, such as under aqueous and high temperature conditions. It is, therefore, of interest to examine the Pb removal capacity of the bacteria under adverse environmental conditions. In the present study, Aeribacillus pallidus MRP280, a lead-tolerant thermophilic bacterium was used as an absorbent for the removal of Pb from aqueous solution. The Pb removal and uptake capacity of living and non-living bacterial cells of A. pallidus MRP280 was investigated in 100 mg/L Pb solution. The optimum condition was examined based on several analytical parameters, including temperature, pH, contact time, and cell density. Biosorbent analysis and characterization was carried out using Fourier Transform Infrared (FT-IR) spectroscopy, Scanning Electron Microscope (SEM)-Energy Dispersive X-ray (EDX), and Transmission Electron Microscope (TEM). The results showed that the maximum Pb removal of 96.78 ± 0.19% and 88.64 ± 0.60% were obtained using living and non-living biomass, respectively at 55 °C, pH 6, OD6000.5 for 100 min. Meanwhile, the maximum uptake capacity of 86.47 ± 1.32 mg/g and 85.31 ± 1.37 mg/g by living and non-living cells was reached at 55 °C, pH 6, OD6000.25 for 60 min. Moreover, Pb removing activity was facilitated by the biosorption and bioaccumulation process. Overall, it is shown that A. pallidus MRP280 is effective when applied as biosorbent in removing Pb from contaminated wastewater at high temperatures.

Efficacy of polyextremophilic Aeribacillus pallidus on bioprocessing of beet vinasse derived from ethanol industries.

This work aimed to evaluate the applicability of Aeribacillus pallidus for the aerobic treatment of the concentrated beet vinasse with high chemical oxygen demand (COD 685 g.L-1) that is defined as an environmental pollutant. This bacterium is a polyextremophilic strain and grow aerobically up to 7.5% vinasse at high temperature (50 °C). In the bioreactor and under controlled conditions, A. pallidus reduced the soluble COD content of 5% vinasse up to 27% during 48 h and utilized glucose and glycerol, completely. Furthermore, a reduction of manganese, copper, aluminum, and nickel concentrations was observed in the treated vinasse with A. pallidus. The obtained results make this strain as an appropriate alternative to be used for the aerobic bioprocessing of the vinasse.

Purification, biochemical and molecular study of lipase producing from a newly thermoalkaliphilic Aeribacillus pallidus for oily wastewater treatment.

Treatment of oily wastewater is constantly a challenge; biological wastewater treatment is an effective, cheap and eco-friendly technology. A newly thermostable, haloalkaline, solvent tolerant and non-induced lipase from Aeribacillus pallidus designated as GPL was purified and characterized of biochemical and molecular study for apply in wastewater treatment. The GPL showed a maximum activity at 65°C and pH 10 after 22 h of incubation, with preference to TC4 substrates. Pure enzyme was picked up after one chromatographic step. It displayed an important resistance at high temperature, pH, NaCl, at the presence of detergents and organic solvents. In fact, GPL exhibited a prominent stability in wide range of organic solvents at 50% (v/v) concentration for 2 h of incubation. The efficiency of the GPL in oil wastewater hydrolysis was established at 50°C for 1 h, the oil removal efficiency was established at 96, 11% and the oil biodegradation was confirmed through fourier transform infrared (FT-IR) spectroscopy. The gene that codes for this lipase was cloned and sequenced and its open reading frame encoded 236 amino acid residues. The deduced amino acids sequence of the GPL shows an important level of identity with Geobacillus lipases.

Enhancement of Aeribacillus pallidus strain VP3 lipase catalytic activity through optimization of medium composition using Box-Behnken design and its application in detergent formulations.

Lipases are hydrolytic enzymes owing much importance in industrial applications. These enzyme-based detergents are ecofriendly and produce a wastewater with low level of COD (chemical oxygen demand). In the present work, a novel halophilous, thermoalkaline, and detergent-tolerant lipase produced by a newly isolated Aeribacillus pallidus strain VP3 was studied. Considerable interest has been given to this lipase by the improvement of its catalytic activity through the optimization of the pH, the (C/N) ratio, and the inoculum size, using the response surface methodology based on the Box-Behnken design of experiments. A total of 16 experiments were conducted, and the optimized pH, (C/N) ratio, and inoculum size were 10, 1, and 0.3, respectively. The results of the analysis of variance (ANOVA) test indicated that the established model was significant (p value < 0.05). The optimization of the production conditions leads to 2.83-fold of increase in the catalytic activity calculated as the ratio of the activity obtained after optimization (68 U) and the initial activity before optimization (24 U). All in all, the lipase of Aeribacillus pallidus could be considered as a potential candidate to be incorporated in detergent formulations since it shows a good stability towards detergents and wash performance.

Safety evaluation of the food enzyme 4-α-glucanotransferase from Aeribacillus pallidus (strain AE-SAS).

The food enzyme 4-α-glucanotransferase (1,4-α-d-glucan:1,4-α-d-glucan 4-α-d-glycosyltransferase, EC 2.4.1.25) is produced with a non-genetically modified Aeribacillus pallidus (previously identified as Geobacillus pallidus) strain from Amano Enzyme Inc. The food enzyme is intended to be used in baking processes and in starch processing for the production of modified dextrins. For baking processes, based on the maximum use levels recommended and individual data from the EFSA Comprehensive European Food Database, dietary exposure to the food enzyme-Total Organic Solids (TOS) was estimated to be up to 0.050 mg TOS/kg body weight (bw) per day. Exposure assessment for the modified dextrins was not considered necessary. Genotoxicity tests did not raise a safety concern. Systemic toxicity was assessed by a repeated dose 90-day oral toxicity study in rats. From this study, the Panel identified a no observed adverse effect level (NOAEL) of at least 900 mg TOS/kg bw per day, the highest dose tested. When the NOAEL value is compared to the estimated dietary exposure to the food enzyme used in baking, this results in a Margin of Exposure (MOE) of at least 18,000. The Panel considers that any additional exposure to the food enzyme from the use of modified dextrins will be covered by the above MOE. A search was made for similarity of the amino acid sequence of the food enzyme with those of known allergens. One match was found with a known respiratory allergen, an α-amylase. The Panel considered that an allergic reaction upon oral ingestion of 4-α-glucanotransferase produced by A. pallidus AE-SAS in individuals respiratory sensitised to α-amylase cannot be excluded, but the likelihood is considered to be low. Overall, the Panel concluded that, under the intended conditions of use and based on the data provided, this food enzyme does not give rise to safety concerns.

Characterization of a novel protease from Aeribacillus pallidus strain VP3 with potential biotechnological interest.

The present study investigates the purification and physico-chemical characterization of an extracellular protease from the Aeribacillus pallidus strain VP3 previously isolated from a geothermal oil-field (Sfax, Tunisia). The maximum protease activity recorded after 22h of incubation at 45°C was 3000U/ml. Pure enzyme, designated as SPVP, was obtained after ammonium sulfate fractionation (40-60%)-dialysis followed by heat-treatment (70°C for 30min) and UNO Q-6 FPLC anion-exchange chromatography. The purified enzyme is a monomer of molecular mass about 29kDa. The sequence of the 25 NH2-terminal residues of SPVP showed a high homology with those of Bacillus proteases. The almost complete inhibition by PMSF and DIFP confirmed that SPVP is a member of serine protease family. Its optima of pH and temperature were pH 10 and 60°C, respectively. Its half-life times at 70 and 80°C were 8 and 4h, respectively. Its catalytic efficiency was higher than those of SAPCG, Alcalase Ultra 2.5L, and Thermolysin type X. SPVP exhibited excellent stability to detergents and wash performance analysis revealed that it could remove blood-stains effectively and high resistance against organic solvents. These properties make SPVP a potential candidate for applications in detergent formulations and non-aqueous peptide biocatalysis.

Utility of thermo-alkali-stable γ-CA from polyextremophilic bacterium Aeribacillus pallidus TSHB1 in biomimetic sequestration of CO2 and as a virtual peroxidase.

Aeribacillus pallidus TSHB1 polyextremophilic bacterium produces a γ-carbonic anhydrase (ApCA), which is a homotrimeric biocatalyst with a subunit molecular mass of 32 ± 2 kDa. The enzyme is stable in the pH range between 8.0 and 11.0 and thus alkali-stable and moderately thermostable with T1/2 values of 40 ± 1, 15 ± 1, and 8 ± 0.5 min at 60, 70, and 80 °C, respectively. Activation energy for irreversible inactivation "E d " of carbonic anhydrase is 67.119 kJ mol-1. The enzyme is stable in the presence of various flue gas contaminants such as SO32-,SO42-, and NO3- and cations Mg2+, Mn2+, Ca2+, and Ba2+. Fluorescence studies in the presence of N-bromosuccinimide and fluorescence quenching using KI and acrylamide revealed the importance of tryptophan residues in maintaining the structural integrity of the enzyme. ApCA is more efficient than the commercially available bovine carbonic anhydrase (BCA) in CO2 sequestration. The enzyme was successfully used in biomineralization of CO2 from flue gas. Replacement of active site Zn2+ with Mn2+ enabled ApCA to function as a peroxidase which exhibited alkali-stability and moderate thermostability like ApCA.

Production and characterization of a new antibacterial peptide obtained from Aeribacillus pallidus SAT4.

A novel thermophilic bacterial strain of the genus Aeribacillus was isolated from Thar Dessert Pakistan. This strain showed significant antibacterial activity against Micrococcus luteus, Staphylococcus aureus, and Pseudomonasaeruginosa. The strain coded as 'SAT4' resembled with Aeribacillus pallidus in the morphological, biochemical and molecular tests. The production of antibacterial metabolites by SAT4 was optimized. These active metabolites were precipitated by 50% ammonium sulphate and purified through sephadex G-75 gel permeation chromatography and reverse phase HPLC. The molecular weight of 37 kDa was examined by SDS-PAGE. The structural elucidation of the purified product was studied by FTIR, 1H and 13C NMR. The X-ray diffractions study showed that the crystals belonged to the primitive orthorhombic lattice (a = 12.137, b = 13.421, c = 14.097 Å) and 3D structure (proposed name: Aeritracin) was determined. This new peptide antibacterial molecule can get a position in pharmaceutical and biotechnological industrial research.