Thermotolerant Bacillus Sp Research Articles

Highly efficient production and characterization of the exopolysaccharides from a thermotolerant Bacillus sp.

One thermotolerant Bacillus strain SFLV-ZM107 which could produce large amounts of extracellular polysaccharides (EPS) at 50°C was identified as Bacillus licheniformis by 16S rDNA sequencing and physiological and biochemical experiments. The EPS was identified as levan fructan by monosaccharides determination, NMR, and FTIR. Strain SFLV-ZM107 can be grown in the range of 35-55°C and it is efficient to produce levan from 400 to 500 g l-1 of sucrose. The production of levan can reach 158 g l-1 in a 5 L tank fermentation with an initial sucrose concentration of 450 g l-1 at 50°C for 12 hours with a maximum productivity of 13.17 g l-1 h-1. To the best of our knowledge, the strains obtained in this study are the most productive and efficient, which has great prospects for industrial application.

Screening and characterization of bioflocculant isolated from thermotolerant Bacillus sp. ISTVK1 and its application in wastewater treatment

Bioflocculants are extracellular polymeric substances (EPS) that are produced by several microorganisms including bacteria. They find application in wastewater treatment wherein they are employed as flocculating agents for removal of suspended particles during the secondary clarification process. In this study, EPS produced by a previously studied thermotolerant strain Bacillus sp. ISTVK1 was analyzed for its flocculation property. The bacteria was cultured in basal and mineral media and the EPS extracted from the two types of cultures were analyzed for their structure, composition, and flocculation efficiency (%), which was tested on kaolin in terms of parameters like duration, pH, temperature and salts. Both types of EPS showed highest flocculating activity on 6th day, at pH 6, and temperature 32.7 °C and with salt AlCl3. When tested on real wastewater, physico-chemical analysis showed reduction in organics in samples treated with EPS and EPS+AlCl3. Aromatic contaminants like Diisobutyl benzene-1,2-dicarboxylate (Retention Time (R.T.) 22.55) and 2-Phenyl-n,n-bis(1-phenylethyl)-1,1-cyclobutanedicarboxamid (R.T. 44.21) were not detected in the bioflocculant treated samples. The study highlighted the applicability of bacterial EPS as a cost-effective bioflocculant for real wastewater.

Alkaline protease production by thermotolerant Bacillus sp. KU-K2, from non-rubber skim latex through the non-sterile system and its enzymatic characterization

The non-rubber skim latex was used as a substrate for alkaline protease production under a non-sterile system. Among 108 isolates of aerobic spore-forming bacteria, the isolate of KU-K2 was selected as a potent bacterial strain for alkaline protease production. The 16s rRNA gene sequence analysis showed that the KU-K2 strain was closely related to the B. licheniformis with 99.39% similarity. The alkaline protease production from non-rubber skim latex serum was optimized using the Plackett-Burman design (PB) and response surface methodology based on central composite design (CCD). The results showed that the optimized medium consisted of 9.82 g/L skim milk, 0.97 g/L glucose, 0.82 g/L K2HPO4, and 0.02 g/L CoCl2·6H2O gave the highest enzyme production at 6.14 ± 0.13 U/mL. The upscale production in the 5.0 L stirrer fermenter was achieved at 6.78 ± 0.32 U/mL when incubated at 37 °C, 250 rpm with 2.0 vvm of aeration rate for 48 h, which increased by 2.90 folds compared to the un-optimized medium. The optimal pH and temperature for alkaline protease activity were 10.0 and 60 °C, respectively. Zn2+, Fe3+, and Hg2+ metal ions strongly inhibited the crude alkaline protease. This study showed the potential of thermotolerant Bacillus sp. For alkaline protease production using non-rubber skim latex as a substrate which could be applied to reduce the industrial waste from latex industries.

Screening and Optimization of Thermo-Tolerant Bacillus Sp. For Amylase Production and Antifungal Activity

Amylases are starch degrading enzymes which are produced by plants, animals and microorganisms. Amylases produced by microorganisms have a wide range of industrial applications such as in pharmaceutical, food, textile and paper industries. However, there are still limitations in the isolation of amylase producing microorganisms. The objective of this study was to isolate the potent amylase producing Bacillus sp. from soil samples and evaluate their abilities for inhibiting the aflatoxin producing Aspergillus flavus. In this study, 30 soil samples were used. For the screening and identification of Bacillus strain, morphological and biochemical tests were performed. Iodine assay was done to screen the potent amylase producers. Two parameters (pH and temperature) were used to optimize the cultural conditions for the production of amylase. To determine the total reducing sugar, dinitrosalicylic acid (DNS) assay was used. Altogether 29 colonies were selected and identified as Bacillus spp out of which 16 were selected to determine enzyme activity by cup plate method. Four isolates (DK9, DK10, IM4 and KD7) showing highest amylolytic activities (16 mm, 12 mm, 14 mm and 14 mm zone of hydrolysis) were subjected for further study. Isolate KD7 showed the highest amylolytic activity (0.19 U/mL) compared to other isolates. Maximum amylase production was found at pH 6 and temperature 50° C (0.19 U/mL). Among these 4 isolates, DK9 and KD9 showed strong antagonistic activity against Aspergillus flavus while DK10 and IM4 showed moderate antifungal activities. Thus, the bacterial isolate KD7 was identified as the most potent strain for maximum amylase production.

Purification, characterization, and overexpression of an endo-1,4-β-mannanase from thermotolerant Bacillus sp. SWU60.

Endo-β-1,4-mannanases are important catalytic agents in several industries. The enzymes randomly cleave the β-1,4-linkage in the mannan backbone and release short β-1,4-mannooligosaccharides and mannose. In the present study, mannanase (ManS2) from thermotolerant Bacillus sp. SWU60 was purified, characterized, and its gene was cloned and overexpressed in Escherichia coli. ManS2 was purified from culture filtrate (300ml) by using hydrophobic, ion-exchange, and size-exclusive liquid chromatography. The apparent molecular mass was 38kDa. Optimal pH and temperature for enzyme activity were 6.0 and 60 °C, respectively. The enzyme was stable up to 60 °C for 1h and at pH 5-9 at 4 °C for 16h. Its enzyme activity was inhibited by Hg2+. The full-length mans2 gene was 1,008bp, encoding a protein of 336 amino acids. Amino acid sequence analysis revealed that it belonged to glycoside hydrolase family 26. Konjac glucomannan was a favorable substrate for recombinant ManS2 (rManS2). rManS2 also degraded galactomannan from locust bean gum, indicating its potential for production of glucomanno- and galactomanno-oligosaccharides. Both native and recombinant ManS2 from Bacillus sp. SWU60 can be applied in several industries especially food and feed.

Optimization of digested food waste for lactic acid production by thermotolerant Bacillus sp. NF11

Methane production capacity in mesophilic conditions of waste from two food industry plants was assessed in a semi-pilot (6 L, fed-batch) and pilot (300 L, semi-continuous) scale. This was carried out in order to evaluate the convenience of producing heat and electricity in a full scale anaerobic digester. The pilot test was performed in order to obtain more reliable results for the design of the digester. Methane yield, returned from the pilot scale test, was approximately 80% of that from the smaller scale test. This outcome was in line with those from other studies performed in different scales and modes and indicates the success of the pilot scale test. The net electricity produced from the digester accounted for 30–50% of the food industry plants’ consumption. The available thermal energy could cover from 10% to 100% of the plant requirements, depending on the energy demand of the processes performed.

Study of optimization of wastewater contaminant removal along with extracellular polymeric substances (EPS) production by a thermotolerant Bacillus sp. ISTVK1 isolated from heat shocked sewage sludge

The present work involved study of wastewater contaminant removal along with EPS production by a thermotolerant bacterium Bacillus sp. ISTVK1, isolated from heat shocked sewage sludge. EPS production in basal and mineral medium containing 50% filter sterilized wastewater and 0.5% sucrose was found to be 0.83±0.12gL−1 and 0.31±0.10gL−1 culture, respectively. GC–MS analysis of EPS revealed the presence of β-d-glucose, α-d-galactose and β-d-arabinose. FT-IR spectrum confirmed the presence carbohydrates. Box–Behnken design was used to optimize process parameters for enhanced EPS production along with % COD reduction of wastewater. The optimised conditions when used in a 1.5L bioreactor showed EPS production of 1.67±0.06gL−1 culture and 93.0±0.21 % COD removal.

Efficient open fermentative production of polymer-grade L-lactate from sugarcane bagasse hydrolysate by thermotolerant Bacillus sp. strain P38.

Lactic acid is one of the top 30 potential building-block chemicals from biomass, of which the most extensive use is in the polymerization of lactic acid to poly-lactic-acid (PLA). To reduce the cost of PLA, the search for cheap raw materials and low-cost process for lactic acid production is highly desired. In this study, the final titer of produced L-lactic acid reached a concentration of 185 g·L−1 with a volumetric productivity of 1.93 g·L−1·h−1 by using sugarcane bagasse hydrolysate as the sole carbon source simultaneously with cottonseed meal as cheap nitrogen sources under the open fed-batch fermentation process. Furthermore, a lactic acid yield of 0.99 g per g of total reducing sugars was obtained, which is very close to the theoretical value (1.0 g g−1). No D-isomer of lactic acid was detected in the broth, and thereafter resulted in an optical purity of 100%, which exceeds the requirement of lactate polymerization process. To our knowledge, this is the best performance of fermentation on polymer-grade L-lactic acid production totally using lignocellulosic sources. The high levels of optically pure l-lactic acid produced, combined with the ease of handling and low costs associated with the open fermentation strategy, indicated the thermotolerant Bacillus sp. P38 could be an excellent candidate strain with great industrial potential for polymer-grade L-lactic acid production from various cellulosic biomasses.

Cloning, Expression, and Characterization of Thermotolerant Manganese Superoxide Dismutase from Bacillus sp. MHS47

A superoxide dismutase gene from thermotolerant Bacillus sp. MHS47 (MnSOD47) was cloned, sequenced, and expressed. The gene has an open reading frame of 612 bp, corresponding to 203 deduced amino acids, with high homology to the amino acid sequences of B. thuringiensis (accession no. EEN01322), B. anthracis (accession no. NP_846724), B. cereus (accession no. ZP_04187911), B. weihenstephanensis (accession no. YP_001646918), and B. pseudomycoides. The conserved manganese-binding sites (H28, H83, D165, and H169) show that MnSOD47 has the specific characteristics of the manganese superoxide dismutase (MnSOD) enzymes. MnSOD47 expressed an enzyme with a molecular weight of approximately 22.65 kDa and a specific activity of 3537.75 U/mg. The enzyme is active in the pH range 7–8.5, with an optimum pH of 7.5, and at temperatures in the range 30–45 °C, with an optimum temperature of 37 °C. Tests of inhibitors and metal ions indicated that the enzyme activity is inhibited by sodium azide, but not by hydrogen peroxide or potassium cyanide. These data should benefit future studies of MnSODs in other microorganisms and the biotechnological production of MnSOD47, and could also be used to develop a biosensor for the detection of antioxidants and free radical activity. In the future, this basic knowledge could be applicable to the detection of cancer risks in humans and therapeutic treatments.

Purification and Characterization of Organic Solvent and Detergent Tolerant Lipase from Thermotolerant Bacillus sp. RN2

The aim of this study was to characterize the organic solvent and detergent tolerant properties of recombinant lipase isolated from thermotolerant Bacillus sp. RN2 (Lip-SBRN2). The isolation of the lipase-coding gene was achieved by the use of inverse and direct PCR. The complete DNA sequencing of the gene revealed that the lip-SBRN2 gene contains 576 nucleotides which corresponded to 192 deduced amino acids. The purified enzyme was homogeneous with the estimated molecular mass of 19 kDa as determined by SDS-PAGE and gel filtration. The Lip-SBRN2 was stable in a pH range of 9–11 and temperature range of 45–60 °C. The enzyme was a non metallo-monomeric protein and was active against pNP-caprylate (C8) and pNP-laurate (C12) and coconut oil. The Lip-SBRN2 exhibited a high level of activity in the presence of 108% benzene, 102.4% diethylether and 112% SDS. It is anticipated that the organic solvent and detergent tolerant enzyme secreted by Bacillus sp. RN2 will be applicable as catalysts for reaction in the presence of organic solvents and detergents.

Active site characterization of the exo- N-acetyl-β- d-glucosaminidase from thermotolerant Bacillus sp. NCIM 5120: involvement of tryptophan, histidine and carboxylate residues in catalytic activity

The exo- N-acetyl-β- d-glucosaminidase (EC 3.2.1.30) from thermotolerant Bacillus sp. NCIM 5120 is a homotetramer with a molecular mass of 240 000 kDa. Chemical modification studies on the purified exo- N-acetyl-β- d-glucosaminidase revealed the involvement of a single tryptophan, histidine and carboxylate, per monomer, in the catalytic activity of the enzyme. Spectral analysis and maintenance of total enzyme activities indicated that N-acetylglucosamine (competitive inhibitor) and p-nitrophenyl- N-acetyl-β- d-glucosaminide (substrate) prevented the modification of a single essential tryptophan, histidine and carboxylate residue. Kinetic parameters of partially inactivated enzyme (by NBS/HNBB) showed the involvement of tryptophan in substrate binding while that of histidine (by photooxidation/DEPC) and carboxylate (by EDAC/WRK) in catalysis. The Bacillus sp. NCIM 5120 exo- N-acetyl-β- d-glucosaminidase deviates from the reported N-acetyl-β- d-glucosaminidases and β-hexosaminidases that utilize anchimeric assistance in their hydrolytic mechanism.

Characterization of a novel exo- N-acetyl-β- d-glucosaminidase from the thermotolerant Bacillus sp. NCIM 5120

An exo- N-acetyl-β- d-glucosaminidase from the thermotolerant Bacillus sp. NCIM 5120 was purified to homogeneity by chromatography on CM-cellulose, Sephacryl S-300 and phenyl–Sepharose. The enzyme has a M r of 230 000 as determined by size exclusion chromatography on Sephacryl S-300/Sephadex G-200 and exhibited a relative subunit M r of 60 000 on denaturing gel electrophoresis. It is a neutral protein with a p I of 6.79. The optimum pH and temperature for the enzyme activity are 6.0 and 70°C, respectively. Determination of the reaction stereochemistry indicates that the enzyme is a retaining glycosidase with the β anomer of GlcNAc formed as the initial product. Determination of the energy of activation with different leaving groups ( p-nitrophenol and 4-methyl-umbelliferone) reveals that the enzyme exhibits a biphasic Arrhenius plot with two characteristic energy of activation with an inflection temperature of 50°C. The activation energy at temperatures below the inflection point was found to be higher than that above the inflection point. The energy of activation for 4-Me-Umb-β- d-GlcNAc was higher at temperatures below the inflection point than for pNP-β- d-GlcNAc (60.3 and 43.2 kJ mol −1, respectively). It hydrolyzes specifically, terminally linked β(1–4) GlcNAc residues from the non-reducing end of oligosaccharides. Comparative studies on the hydrolysis of chito-oligosaccharides by the exo- N-acetyl-β- d-glucosaminidase indicates that chitobiose is the best substrate with a K m and k cat of 0.34 mM and 24 μmol min −1 mg −1, respectively. It also exhibits strict substrate specificity with respect to the glycone substitution as well as anomeric linkage.

Cloning and nucleotide sequence of the gene coding for citrate synthase from a thermotolerant Bacillus sp.

The structural gene coding for citrate synthase from the gram-positive soil isolate Bacillus sp. strain C4 (ATCC 55182) capable of secreting acetic acid at pH 5.0 to 7.0 in the presence of dolime has been cloned from a genomic library by complementation of an Escherichia coli auxotrophic mutant lacking citrate synthase. The nucleotide sequence of the entire 3.1-kb HindIII fragment has been determined, and one major open reading frame was found coding for citrate synthase (ctsA). Citrate synthase from Bacillus sp. strain C4 was found to be a dimer (Mr, 84,500) with a subunit with an Mr of 42,000. The N-terminal sequence was found to be identical with that predicted from the gene sequence. The kinetics were best fit to a bisubstrate enzyme with an ordered mechanism. Bacillus sp. strain C4 citrate synthase was not activated by potassium chloride and was not inhibited by NADH, ATP, ADP, or AMP at levels up to 1 mM. The predicted amino acid sequence was compared with that of the E. coli, Acinetobacter anitratum, Pseudomonas aeruginosa, Rickettsia prowazekii, porcine heart, and Saccharomyces cerevisiae cytoplasmic and mitochondrial enzymes.

Electron microscopic analysis and biochemical characterization of a novel methanol dehydrogenase from the thermotolerant Bacillus sp. C1

Methanol dehydrogenase from the thermotolerant Bacillus sp. C1 was studied by electron microscopy and image processing. Two main projections can be distinguished: one exhibits 5-fold symmetry and has a diameter of 15 nm, the other is rectangular with sides of 15 and 9 nm. Subsequent image processing showed that the 5-fold view possesses mirror symmetry. The rectangular views can be divided into two separate classes, one of which has 2-fold rotational symmetry. It is concluded that methanol dehydrogenase is a decameric molecule, and a tentative model is presented. The estimated molecular weight is 430,000, based on a subunit molecular weight of 43,000. The enzyme contains one zinc and one to two magnesium ions per subunit. N-terminal amino acid sequence analysis revealed substantial similarity with alcohol dehydrogenases from Saccharomyces cerevisiae, Zymomonas mobilis, Clostridium acetobutylicum, and Escherichia coli, which contain iron or zinc but no magnesium. In view of the aberrant structural and kinetic properties, it is proposed to distinguish the enzyme from common alcohol dehydrogenases (EC 1.1.1.1) by using the name NAD-dependent methanol dehydrogenase.