Thermophilic Bacillus Sp Research Articles

Introduction of a Sodium-Binding Motif into Subunits <i>a</i> and <i>c</i> of <i>Bacillus</i> sp. PS3 Proton F-ATPase Does Not Result in Sodium Specificity of the Enzyme

In bacteria F-type ATPase (F-ATPase) plays a key role in bioenergetics and couples ATP synthesis/hydrolysis with the transport of ions (H+ or Na+) across the membrane. The ion specificity of the enzyme is determined by the amino acid sequence of subunits c and а. Here, we introduced several mutations (7 in subunit c and 6 in subunit a) into F-ATPase of thermophilic bacterium Bacillus sp. PS3 in order to change the ion specificity of the enzyme from proton to sodium. The mutations did not affect the ATPase activity of the enzyme, but led to loss of proton conductivity and impaired the binding of subunit a to the c-subunit oligomer, rather than changed the ion specificity.

Spray drying of cellulases produced by Bacillus sp. SMIA-2

Microbial cellulases present biotechnological potential to be used in several industries, including food, brewery and wine, industrial waste for chemical feedstock, animal feed, pulp and paper, agriculture, textile and detergent production. In this work, cellulases produced by the thermophilic Bacillus sp. SMIA-2 in submerged cultures containing sugarcane bagasse, corn steep liquor and passion fruit rind flour were spray-dried, in an attempt to improve their stability for industrial purposes. The cellulases were spray dried and analyzed before and after the drying process and subsequent storage. A Central Composite Design (CCD) 23 was used to investigate the effect of different concentrations of arabic gum and microcrystalline cellulose, as well as the spray dryer inlet temperature on the cellulase spray drying process. The results evidenced that the combination of 1.0 % (w/v) arabic gum and 1.0% (w/v) microcrystalline cellulose, at inlet temperature of 70 °C, was effective in maintaining the activities of both avicelases (avicel-hydrolyzing enzymes) and carboxymethylcellulases (carboxymethylcellulose-hydrolyzing enzymes - CMCase). The dried avicelase was completely preserved when stored at 5oC, while the CMCase retained 89% of its activity, which indicates promising potential for industrial uses, especially in detergent formulations.

The evaluation of Isolated KH1-29 properties for producing α-amylase from a novel native highly thermostable, thermophilic and alkaline Bacillus sp

The evaluation of Isolated KH1-29 properties for producing α-amylase from a novel native highly thermostable, thermophilic and alkaline Bacillus sp

How Does F1-ATPase Generate Torque?: Analysis From Cryo-Electron Microscopy and Rotational Catalysis of Thermophilic F1.

The F1-ATPase is a rotary motor fueled by ATP hydrolysis. Its rotational dynamics have been well characterized using single-molecule rotation assays. While F1-ATPases from various species have been studied using rotation assays, the standard model for single-molecule studies has been the F1-ATPase from thermophilic Bacillus sp. PS3, named TF1. Single-molecule studies of TF1 have revealed fundamental features of the F1-ATPase, such as the principal stoichiometry of chemo-mechanical coupling (hydrolysis of 3 ATP per turn), torque (approximately 40 pN·nm), and work per hydrolysis reaction (80 pN·nm = 48 kJ/mol), which is nearly equivalent to the free energy of ATP hydrolysis. Rotation assays have also revealed that TF1 exhibits two stable conformational states during turn: a binding dwell state and a catalytic dwell state. Although many structures of F1 have been reported, most of them represent the catalytic dwell state or its related states, and the structure of the binding dwell state remained unknown. A recent cryo-EM study on TF1 revealed the structure of the binding dwell state, providing insights into how F1 generates torque coupled to ATP hydrolysis. In this review, we discuss the torque generation mechanism of F1 based on the structure of the binding dwell state and single-molecule studies.

Optimization of spray-drying conditions for obtaining Bacillus sp. SMIA-2 protease powder

Microbial proteases, especially from Bacillus spp., have enormously been exploited for a broad variety of applications such as for physiological processes, food and feedstuff, detergents, as well as in the pharmaceutical and leather. In this work, proteases produced by the thermophilic Bacillus sp. SMIA-2 submerged cultures were spray-dried in an attempt to improve its stability for applications in industry. The enzymatic extract was dried using drying adjuvants, and optimal conditions for preserving enzymatic activity were studied following a statistical experimental design. The spray process factors studied were the drier air inlet temperature and the adjuvants concentrations. The responses analyzed were the enzymatic activity and mass recovered of the powder after spray drying. Additionally, the stability of the powder was assessed during 180 days at room temperature. The results revealed that satisfactory levels of enzymatic activity were obtained when 0.5% (w/v) carboxymethylcellulose and 1.0% (w/v) maltodextrin were incorporated to enzymes solutions and the spray drier inlet temperature was 110 ºC. Furthermore, this dried protease extracts showed potential for future commercial applications because of their stability at room temperature for 180 days.

Pre-evaluation of urease production by Bacillus sp. SK II-5 thermophilic bacteria using agricultural waste as a substrate

Agricultural waste contain nutritive minerals including nitrogen, phosphorus, and potassium which help plants grow faster and increase yields, but when released into the open environment can leads the aquatic environment to eutrophication. Agricultural waste, which is mostly contain urea, can be utilized for the growth media of microorganisms to produce urease. Urease is an enzyme that functions to hydrolyze urea to ammonia and carbonate. Naturally, some organisms can produce urease, one of which is the thermophilic bacteria of the genus Bacillus. The purpose of this research is determine the results of the viability test of thermophilic bacteria Bacillus sp. SK II-5 using small island agricultural waste as media. The viability test of Bacillussp. SK II-5 is carried out using the streak plate and turbidimetry methods. The results of this research were thermophilic bacteria Bacillus sp. SK II-5 can grow at 50°C and 60°C which have been tested for cell viability in agricultural waste extract agar media at 37°C.

Purification of Amylase Produced by Thermophilic Bacillus sp. TS9

Amylases are the starch degrading enzymes obtained from diverse sources such as plants, animals and microorganisms including bacteria and fungi. But the microbial amylases are preferred over other sources. The thermophilic alpha amylase obtained from bacterial species has applications in many industries. Six thermophilic bacteria Microbacterium sp. TS1, Pseudomonas sp. TS2, Alishewanella sp.TS3, Rheinheimera sp. TS5, Bacillus sp. TS9 and halophilic Bacillus sp. HS7 were isolated from Shahdrah and Kheiwera regions of Pakistan and their 16s RNA identified through molecular techniques. All the isolates were screened for amylase quantitatively and qualitatively. The Thermophilic Bacillus sp. TS9, based on its higher production of amylase, was selected for purification and characterization. The bacteria produced clearance zone of 2.7cm in diameter on 1% starch supplemented agar plate and the maximum specific activity of 2.45U/mg was obtained after 72 hours incubation at 45° C and pH 8. The extracellular amylase was then purified by ammonium sulphate precipitation and gel permeation chromatography. The specific activity of amylase obtained from Bacillus sp. TS9 increased to 4.47U/mg (1.82 fold) through partial purification, and 15.92U/mg (6.48 fold) through column chromatography. The current Study points out a valuable approach to purifiy the amylase and consequently improves the activity of amylase which have numerous applications in many industries

Characterization of Amylase Produced by Thermophilic Bacillus sp. TS9

Amylases constitute the second largest group of enzymes in the world enzyme market and are produced by many bacterial and fungal strains. The potent producer of amylase is Bacillus due to the production of thermostable enzymes that can withstand harsh conditions in industrial bioprocess. The α-amylase produced by Bacillus sp. TS9 was purified through gel filtration chromatography and then crude, partially purified and purified α-amylase was characterized. The crude, partially purified and purified amylase showed stability to a wide range of temperature (35-80°C) and pH (6-9) with optimum temperature and pH is 55°C and 9 respectively. The purified amylase also retained 70% of its activity at 100°C after incubation of 3 hours. The crude, partially purified and purified amylase showed stability to Na +1 and Mg +2 , methanol, and commercial detergents, less affected by Zn+2 , (NH4)2SO4, Triton-X-100, Tween-80 and SDS and some solvents but its activity was reduced by Ca +2 and Hg +2 . The amylase obtained from Bacillus sp. TS9 is Ca+2 independent as don’t require Ca +2 ions for its activity, but still the purified amylase was sensitive to EDTA to some extent. Its activity was completely inhibited by mercapto-ethanol revealing that histidine residues are present at the active site of an enzyme. As the amylase obtained from thermophilic Bacillus sp. TS9 showed stability to high temperature and pH, solvents, metal ions, detergents, and surfactants, so can be utilized in starch processing, detergent, textile, and food industries.

ATP-binding affinity of the ε subunit of thermophilic F1-ATPase under label-free conditions

The ε subunits of several bacterial F1-ATPases bind ATP. ATP binding to the ε subunit has been shown to be involved in the regulation of F1-ATPase from thermophilic Bacillus sp. PS3 (TF1). We previously reported that the dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C is 4.3 μM by measuring changes in the fluorescence of the dye attached to the ε subunit (Kato, S. et al. (2007) J. Biol. Chem.282, 37618). However, we have recently noticed that this varies with the dye used. In this report, to determine the affinity for ATP under label-free conditions, we have measured the competitive displacement of 2′(3′)-O-N′-methylaniloyl-aminoadenosine-5′-triphosphate (Mant-ATP), a fluorescent analog of ATP, by ATP. The dissociation constant for ATP of wild-type ε subunit of TF1 at 25 °C was determined to be 0.29 μM, which is one order of magnitude higher affinity than previously reported values.

Endoglucanase gene of M42 aminopeptidase/endoglucanase family from thermophilic Bacillus sp. PW1 and PW2 isolated from Tattapani hot spring, Himachal Pradesh, India

BackgroundThermostable cellulases are in constant demand for several biotechnological applications. Two thermophilic bacterial strains PW1 and PW2 isolated from Tattapani hot spring were found to have cellulolytic activity. Subsequently, PW1 and PW2 were identified and mined for genes encoding cellulase activity. ResultsSequencing of the 16S rDNA of PW1 and PW2 identified them as Bacillus sp. PW1 (Acc no. KU711837) and Bacillus sp. PW2 (Acc no. KU711838), respectively, which clustered in the clades containing thermophilic members of Bacillus sp. and Geobacillus species. Phylogenetic analysis revealed that despite the morphological and sequence identities, Bacillus sp. PW1 and Bacillus sp. PW2 are different at the genetic level. The cellulase genes (~ 1.1 kb) of the two bacterial strains were amplified using primers designed against related thermophilic cellulases. Sequencing of the cellulase gene amplicons of PW1 and PW2 revealed that they encode proteins of 280 and 206 amino acid residues, respectively. Sequence and domain analysis of the protein products of PW1 and PW2 revealed that they belong to M42 family of aminopeptidase/endoglucanase. The PW2 endoglucanase coding sequence was submitted to Genbank under accession no. MH049504. The structures of putative endoglucanases of PW1 and PW2 were generated using 1VHE.A as template, which showed the presence of vast proportion of random coils. Molecular docking of the modeled endoglucanase proteins with various substrates and products of cellulases showed that carboxymethyl cellulose and maltose exhibit the highest binding affinity, while xylan and glucose the least. ConclusionsThe two thermophilic bacteria PW1 and PW2 and their endoglucanase gene can be further utilized for recombinant production of thermostable cellulases for their application in industries.

Production of highly thermo-tolerant laccase from novel thermophilic bacterium Bacillus sp. PC-3 and its application in functionalization of chitosan film

In this study, a novel thermophilic bacterial strain was isolated from Tattapani hot spring located in the Chhattisgarh state of India. The laccase was produced via submerged fermentation and purified by ammonium sulfate precipitation and anion exchange chromatography up to 13.7 fold. The 16S rRNA gene sequence and biochemical analysis revealed that the bacterial isolate is Bacillus sp. strain PC-3. The activity of extracellular crude laccase was determined to be 11.2 U/mL using 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a substrate. The SDS-PAGE revealed that the enzyme consists of single subunit with molecular size of 36kDa. The laccase exhibited the maximum enzyme activity at temperature of 60°C and pH 7. Moreover, the laccase retained 99.1% of its original activity for 180min and exhibited half-life of 3.75hat 60°C. Similarly, the laccase retained 95% activity at pH 7 for 240min and displayed significant activity at wider pH range. In addition, the laccase was used for functionalization of chitosan film and characterized for antioxidant and antimicrobial activity. Interestingly, the functionalized chitosan film showed the improved antioxidant and antimicrobial activity.

Plastic degradation by thermophilic Bacillus sp. BCBT21 isolated from composting agricultural residual in Vietnam

Three different kinds of plastic bags HL, VHL, and VN1 with different chemical nature were degraded by a novel thermophilic bacterial strain isolated from composting agricultural residual in Vietnam in shaking liquid medium at 55 °C after 30 d. The new strain was classified in the Bacillus genus by morphological property and sequence of partial 16Sr RNA coding gene and named as Bacillus sp. BCBT21. This strain could produce extracellular hydrolase enzymes including lipase, CMCase, xylanase, chitinase, and protease with different level of activity in the same media. After a 30-d treatment at 55 °C with Bacillus sp. BCBT21, all characteristics including properties and morphology of treated plastic bags had been significantly changed. The weight loss, structure and surface morphology of these bags as well as the change in the average molecular weight of VHL bag were detected. Especially, the average molecular weight of VHL bag was significantly reduced from 205 000 to 116 760. New metabolites from the treated bags indicated biodegradation occurring with the different pathways. This finding suggests that there is high potential to develop an effective integrated method for plastic bags degradation by a combination of extracellular enzymes from bacteria and fungi existing in the composting process.

A thermostable and alkaline GDSL-motif esterase from Bacillus sp. K91: crystallization and X-ray crystallographic analysis.

The esterase Est8 from the thermophilic bacterium Bacillus sp. K91 belongs to the GDSL family and is active on a variety of acetylated compounds, including 7-aminocephalosporanic acid. In contrast to other esterases of the GDSL family, the catalytic residues Asp182 and His185 were more pivotal for the catalytic activity of Est8 than the Ser11 residue. To better understand the biochemical and enzymatic properties of Est8, recombinant Est8 protein was purified and crystallized. Crystals of Est8 were obtained by the hanging-drop vapour-diffusion method using 2.0 M ammonium sulfate, 5%(v/v) 2-propanol as the crystallization solution. X-ray diffraction data were collected to a resolution of 2.30 Å with an Rmerge of 16.4% from a crystal belonging to space group P41212 or P43212, with unit-cell parameters a = b = 68.50, c = 79.57 Å.

Amylase Production from Thermophilic Bacillus sp. BCC 021-50 Isolated from a Marine Environment

The high cost of fermentation media is one of the technical barriers in amylase production from microbial sources. Amylase is used in several industrial processes or industries, for example, in the food industry, the saccharification of starchy materials, and in the detergent and textile industry. In this study, marine microorganisms were isolated to identify unique amylase-producing microbes in starch agar medium. More than 50 bacterial strains with positive amylase activity, isolated from marine water and soil, were screened for amylase production in starch agar medium. Bacillus sp. BCC 021-50 was found to be the best amylase-producing strain in starch agar medium and under submerged fermentation conditions. Next, fermentation conditions were optimized for bacterial growth and enzyme production. The highest amylase concentration of 5211 U/mL was obtained after 36 h of incubation at 50 °C, pH 8.0, using 20 g/L molasses as an energy source and 10 g/L peptone as a nitrogen source. From an application perspective, crude amylase was characterized in terms of temperature and pH. Maximum amylase activity was noted at 70 °C and pH 7.50. However, our results show clear advantages for enzyme stability in alkaline pH, high-temperature, and stability in the presence of surfactant, oxidizing, and bleaching agents. This research contributes towards the development of an economical amylase production process using agro-industrial residues.

Production and Partial Characterization of α-Amylase Enzyme from Bacillus sp. BCC 01-50 and Potential Applications.

Amylase is an industrially important enzyme and applied in many industrial processes such as saccharification of starchy materials, food, pharmaceutical, detergent, and textile industries. This research work deals with the optimization of fermentation conditions for α-amylase production from thermophilic bacterial strain Bacillus sp. BCC 01-50 and characterization of crude amylase. The time profile of bacterial growth and amylase production was investigated in synthetic medium and maximum enzyme titer was observed after 60 h. In addition, effects of different carbon sources were tested as a substrate for amylase production and molasses was found to be the best. Various organic and inorganic compounds, potassium nitrate, ammonium chloride, sodium nitrate, urea, yeast extract, tryptone, beef extract, and peptone, were used and beef extract was found to be the best among the nitrogen sources used. Temperature, pH, agitation speed, and size of inoculum were also optimized. Highest enzyme activity was obtained when the strain was cultured in molasses medium for 60 h in shaking incubator (150 rpm) at 50°C and pH 8. Crude amylase showed maximal activity at pH 9 and 65°C. Enzyme remained stable in alkaline pH range 9-10 and 60–70°C. Crude amylase showed great potential for its application in detergent industry and saccharification of starchy materials.

Coproduction of protease and amylase by thermophilic Bacillus sp. BBXS-2 using open solid-state fermentation of lignocellulosic biomass

•Untreated lignocellulosic biomass is fermented to produce bacterial enzymes.•An open (nonaseptic) solid-state fermentation is used to minimize costs.•Thermophilic/alkalophilic fermentation conditions could control contamination.•Much higher titers of enzymes were produced compared to the literature.•Enzymes added to laundry detergents improved stain removal by ∼2.5-fold.

Green Extracellular Synthesis of the Silver Nanoparticles Using Thermophilic Bacillus Sp. AZ1 and its Antimicrobial Activity Against Several Human Pathogenetic Bacteria.

Silver nanoparticles (AgNPs) are among the most effective antimicrobial agents that are used in the medicine and pharmaceutics. During the past decades, metal nanoparticles synthesis through application of the biological methods has increasingly been used, as the biologically synthesized particles are mostly non-toxic as well as effective. The main goal for undertaking the present investigation was to evaluate the extracellular synthesis of the AgNPs by a native thermophilic Bacillus Sp. AZ1 that was isolated from a hot spring in Ardebil province. Subsequently the antimicrobial potentials of the nanoparticle was evaluated against several human pathogenic organisms. The biosynthesized AgNPs were confirmed visually by appearance of a dark brown color formation in the mixture as well as silver surface plasmon resonance band by using UV-Visible spectroscopy. The AgNPs were further characterized by SEM, EDX and TEM. The antimicrobial activity of the AgNPs was investigated using Salmonella typhi, Escherichia coli, Staphylococcus epidermis, and Staphylococcus aureus, by applying disk diffusion method. Identification of the strain AZ1 by the 16S rRNA sequence analysis showed 99% sequence homology between this strain and B. licheniformis. The obtained UV-Visible spectrum of the aqueous medium containing silver ion, showed a peak at 425 nm which indicates a correspondence to the plasmon absorbance of the silver nanoparticles. The biosynthesized AgNPs were found to be in the size range of ~7-31 nm with spherical the shape. Studies regarding the antibacterial effect of the particles showed the highest inhibitory effect against the two strains; E. coli, and S. typhi, respectively. Our study presents a simple green synthesis process for the production of an extracellular nanoparticles which is environmental friendly. Biosynthesis of the AgNPs by a thermophilic bacillus from the hot spring (Qeynarjeh, Ardebil) in Iran with the highest similarity to Bacillus licheniformis is reported for the first time.

Properties of a newly identified esterase from Bacillus sp. K91 and its novel function in diisobutyl phthalate degradation.

The widely used plasticizer phthalate esters (PAEs) have become a public concern because of their effects on environmental contamination and toxicity on mammals. However, the biodegradation of PAEs, especially diisobutyl phthalate (DiBP), remains poorly understood. In particular, genes involved in the hydrolysis of these compounds were not conclusively identified. In this study, the CarEW gene, which encodes an enzyme that is capable of hydrolyzing ρ-nitrophenyl esters of fatty acids, was cloned from a thermophilic bacterium Bacillus sp. K91 and heterologously expressed in Escherichia coli BL21 using the pEASY-E2 expression system. The enzyme showed a monomeric structure with a molecular mass of approximately 53.76 kDa and pI of 4.88. The enzyme exhibited maximal activity at pH 7.5 and 45°C, with ρ-NP butyrate as the best substrate. The enzyme was fairly stable within the pH range from 7.0 to 8.5. High-pressure liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS) were employed to detect the catabolic pathway of DiBP. Two intermediate products were identified, and a potential biodegradation pathway was proposed. Altogether, our findings present a novel DiBP degradation enzyme and indicate that the purified enzyme may be a promising candidate for DiBP detoxification and for environmental protection.

PRODUCTION AND CHARACTERIZATION OF THERMOPHILIC CARBOXYMETHYL CELLULASE SYNTHESIZED BY Bacillus sp. GROWING ON SUGARCANE BAGASSE IN SUBMERGED FERMENTATION

The production and characterization of cellulase from thermophilic strain Bacillus sp. C1AC5507 was studied. For enzyme production, sugarcane bagasse was used as carbon source. The produced carboxymethyl cellulase (CMCase) had a molecular weight around 55 kDa and its activity varied between 0.14 and 0.37 IU mL-1 in conditions predicted by Response Surface Methodology. The optimum temperature and pH for the CMCase production were 70 °C and 7.0, respectively. The enzyme activity was inhibited mostly by Cu+2 and activated mostly by Co+2, Mn2+, Ca+2 and Fe+3. Our findings provide a contribution to the use of natural wastes such as sugarcane bagasse as substrate for growth and production of thermophilic CMCase. Further optimization to increase the production of cellulase enables the use in industrial applications.

Purification and characterization of thiol dependent, oxidation-stable serine alkaline protease from thermophilic Bacillus sp.

Alkaline serine protease was purified to homogeneity from culture supernatant of a thermophilic, alkaliphilic Bacillus sp. by 80% ammonium sulphate precipitation followed by CM-cellulose and DEAE-cellulose ion exchange column chromatography. The enzyme was purified up to 16.5-fold with 6900 U/mg activity. The protease exhibited maximum activity towards casein at pH 8.0 and at 80 °C. The enzyme was stable at pH 8.0 and 80 °C temperature up to 2 h. The Ca2+ and Mn2+ enhanced the proteolytic activity up to 44% and 36% as compared to control, respectively. However, Zn2+, K+, Ba2+, Co2+, Hg2+ and Cu2+ significantly reduced the enzyme activity. PMSF (phenyl methyl sulphonyl fluoride) completely inhibited the protease activity, whereas the activity of protease was stimulated up to two folds in the presence of 5 mM 2-mercaptoethanol. The enzyme was also stable in surfactant (Tween-80) and other commercial detergents (SDS, Triton X-100).