Alkaliphilic Bacillus Research Articles

Overexpression and characterization of alkaliphilic Bacillus firmus strain K-1 xylanase

The alkaliphilic Bacillus firmus strain K-1 produces an alkaliphilic xylanase (Xyn11A) which can be stable across a wide pH range and active at high temperatures. However, the enzyme suffers from low activity when isolated directly from the culture broth using corn husk. A method was developed which employed recombinant DNA technology to produce recombinant Xyn11A (rXyn11A) in an Escherichia coli (E. coli) expression system. The thioredoxin-fused Xyn11A (Thio-rXyn11A) could be expressed as a soluble form in BL21 (DE3). The expressed protein was tobacco etch virus (TEV) cleavaged to remove the thioredoxin tag and subjected to further purification using Ni2+-NTA affinity chromatography followed by gel-filtration chromatography. Activity of rXyn11A was shown to be under the same conditions as the native enzyme isolated directly from the K-1, having a broad range of active pH (5.0–12.0), with the maximum activity obtained from pH 5.0 at 60 °C. Interestingly, the obtained rXyn11A exhibited a very large increase in specific activity (3034 U/mg), which was 84-times higher than that reported in the native enzyme when observed under the same conditions. Also seen in the xylan hydrozation assay, the rXyn11A hydrolyzed insoluble xylans around 100-times more effectively than the native enzyme. The results from this study demonstrated a successful method for generating the enzyme rXyn11A with much improved activity, making it feasible for industrial applications.

Ultrasound-assisted improvements in biocatalytic activity and production of organic-solvent stable protease from Bacillus circulans MTCC 7942

This study reports the ultrasound-assisted improvements in biocatalytic activity and production of protease from organic solvent-tolerant, alkaliphilic Bacillus circulans MTCC 7942. The strain withstands 10min ultrasound treatment (33kHz); this treatment promotes the growth of bacterium. The protease production was improved by 1.5-fold on multiple ultrasound treatment at 0, 6, 12h intervals. Although the substrate casein remains unaffected by ultrasound treatment (33kHz, 10min); the treatment had boosted the enzyme activity of purified protease. Also, the ultrasound treated purified protease showed enhancement in enzyme activity in presence of n-dodecane, n-octane, n-hexadecane, n-decane, xylene, butanol, toluene, pyridine and chloroform.

A new GH13 α-glucosidase from alkaliphilic Bacillus pseudofirmus 703 with both exo-α-l, 4-glucosidase and oligo-l, 6-glucosidase activities toward amylopectin

Debranching of (1, 6)-α-linkages in starch is of great significance as it is widely used in different industries. In recent years, identifying a single potential enzyme that could debranch (1, 6)-α-linkages and cleave (1, 4)-α-linkages brought great interest as amylopectin possessed both (1, 4)-α- and (1, 6)-α-linkages. In the present study, a 64\\,kDa exo-α-l, 4-glucosidase from Bacillus pseudofirmus 703, annotated as Amy112, was cloned and characterized. Biochemical analysis indicated that heterologous Amy112 expressed in Escherichia coli exhibited a high activity against amylopectin, with the optimal temperature and pH of 40°C and pH 7.0, respectively. Addition of K+ ions improved the amy112 activity by 12%, but Li+, Ca2+ and Mg2+ ions showed no significant effect. Amy112 sequence homology revealed that it belonged to glycoside hydrolase family 13, showing 65% identity with α-glucosidase GSJ from Geobacillus sp. HTA-462. This is the first report indicating that Amy112 from B. pseudofirmus 703 belongs to GH13 enzyme family, having both exo-α-1, 4-glucosidase and oligo-l, 6-glucosidase activities. However, no transglycosylation activity was detected in LC–MS analysis. Amy112 would be of great significance of being utilized to debranch starch in different industries in a cost effective manner.

Alkaliphilic Bacillus species show potential application in concrete crack repair by virtue of rapid spore production and germination then extracellular calcite formation.

Characterization of alkaliphilic Bacillus species for spore production and germination and calcite formation as a prelude to investigate their potential in microcrack remediation in concrete. Conditions, extent and timing of endospore production was determined by dark-field light microscopy; germination induction and kinetics were assessed by combining reduction in optical density with formation of refractile bodies by phase-contrast microscopy. Bacillus pseudofirmus was selected from several species as the most suitable isolate. Levels and timing of calcium carbonate precipitated invitro by B. pseudofirmus were evaluated by atomic absorption spectroscopy and structural identity confirmed as calcite and aragonite by Raman spectroscopy and FTIR. The isolate produced copious spores that germinated rapidly in the presence of germinants l-alanine, inosine and NaCl. Bacterial cells produced CaCO3 crystals in microcracks and the resulting occlusion markedly restricted water ingress. By virtue of rapid spore production and germination, calcium carbonate formation invitro and insitu, leading to sealing of microcracks, B. pseudofirmus shows clear potential for remediation of concrete on a commercial scale. Microbial sealing of microcracks should become a practicable and sustainable means of increasing concrete durability.

Cloning, evaluation, and high-level expression of a thermo-alkaline pectate lyase from alkaliphilic Bacillus clausii with potential in ramie degumming.

Alkaline pectate lyases (Pels) have potential application in bioscouring of the textile industry. In this study, a thermo-alkaline Pel (BacPelA) gene from an alkaliphilic Bacillus clausii strain was cloned and overexpressed in Escherichia coli. The mature BacPelA exhibited maximum activity at pH10.5 and 70°C and showed high cleavage capability on methylated pectins. BacPelA showed the highest specific activity of 936.2Umg-1 on ≥85% methylated pectin and 675.5Umg-1 on standard substrate polygalacturonic acid (PGA) upon evaluation of the absorbance at 235nm (A235). The K m and k cat values for PGA were 0.54gl-1 and 346.5s-1, respectively. Moreover, the 3,5-dinitrosalicylic acid (DNS) assay, which detects the released reducing oligogalacturonic acids, was confirmed to be inaccurate and unsuitable for endo-acting pectinase activity assay because of the difference in the reducibility by DNS reagent between the standard galacturonic acid and the catalytic oligomer products. Significant ramie fiber weight loss was observed following treatment with BacPelA (24.8%) and combined enzyme-chemical method (30.9%), which indicated that the degumming efficiency of BacPelA was the highest of all alkaline and thermostable Pels reported to date. The total activity of the recombinant mature BacPelA reached 8378.2Uml-1 (A235) by high-cell-density cultivation in fed-batch fermentation with productivity of 239.4Uml-1h-1 using E. coli as host, which represents the highest Pel yield reported to date. Therefore, BacPelA, with promising properties for bioscouring, shows potential applications for ramie degumming in the textile industry.

Characterization and overproduction of a thermo-alkaline pectate lyase from alkaliphilic Bacillus licheniformis with potential in ramie degumming

Abstract A thermo-alkaline pectate lyase (BliPelA) gene from an alkaliphilic Bacillus licheniformis strain was cloned and overexpressed in Escherichia coli . Mature BliPelA exhibited maximum activity at pH 11 and 70 °C, and demonstrated cleavage capability on a broad range of substrates such as polygalacturonic acid, pectins, and methylated pectins. The highest specific activity, of 320 U mg −1 , was towards polygalacturonic acid. Significant ramie ( Boehmeria nivea ) fiber weight loss (21.5%) was obtained following enzyme treatment and combined enzyme-chemical treatment (29.3%), indicating a high ramie degumming efficiency of BliPelA. The total activity of recombinant BliPelA reached 1450.1 U ml −1 with a productivity of 48.3 U ml −1 h −1 under high-cell-density cultivation with a glycerol exponential feeding strategy for 30 h in 1-l fed-batch fermenter, and 1380.1 U ml −1 with a productivity of 57.5 U ml −1 h −1 after 24 h under constant glucose feeding in a 20-l fermenter using E. coli as the host. The enzyme yields reached 4.5 and 4.3 g l −1 in 1-l and 20-l fed-batch fermenters, respectively, which are higher than those of most reported alkaline Pels. Based on these promising properties and high-level production, BliPelA shows great potential for application in ramie degumming in textile industry.

Ion Selectivity of the Flagellar Motors Derived from the Alkaliphilic Bacillus and Paenibacillus Species.

Many bacteria can swim using their flagella, which are filamentous organelles that extend from the cell surface. The flagellar motor is energized by either a proton (H+) or sodium ion (Na+) as the motive force. MotAB-type stators use protons, whereas MotPS- and PomAB-type stators use Na+ as the coupling ions. Recently, alkaliphilic Bacillus alcalophilus was shown to use potassium ions (K+) and rubidium ions (Rb+) for flagellar rotation, and the flagellar motor from Paenibacillus sp. TCA-20 uses divalent cations such as magnesium ions (Mg2+), calcium ions (Ca2+), and strontium ions (Sr2+) for coupling. In this chapter, we focus on how to identify the coupling ions for flagellar rotation of alkaliphilic Bacillus and Paenibacillus species.

Danger lurking in the "unknowns": structure-to-function studies of hypothetical protein Bleg1_2437 from Bacillus lehensis G1 alkaliphile revealed an evolutionary divergent B3 metallo-beta-lactamase.

The effectiveness of β-lactam antibiotics as chemotherapeutic agents to treat bacterial infections is gradually threatened with the emergence of antibiotic resistance mechanism among pathogenic bacteria through the production metallo-β-lactamase (MBL). In this study, we discovered a novel hypothetical protein (HP) termed Bleg1_2437 from the genome of alkaliphilic Bacillus lehensis G1 which exhibited MBL-like properties of B3 subclass; but evolutionary divergent from other circulating B3 MBLs. Domain and sequence analysis of HP Bleg1_2437 revealed that it contains highly conserved Zn2+-binding residues such as H54, H56, D58, H59, H131 and H191, important for catalysis, similar with the subclass B3 of MBL. Built 3-D Bleg1_2437 structure exhibited an αββα sandwich layer similar to the well-conserved global topology of MBL superfamily. Other features include a ceiling and floor in the model which are important for accommodation and orientation of β-lactam antibiotics docked to the protein model showed interactions at varying degrees with residues in the binding pocket of Bleg1_2437. Hydrolysis activity towards several β-lactam antibiotics was proven through an in vitro assay using purified recombinant Bleg1_2437 protein. These findings highlight the presence of a clinically important and evolutionary divergent antibiotics-degrading enzyme within the pools of uncharacterized HPs.

Nonconventional cation-coupled flagellar motors derived from the alkaliphilic Bacillus and Paenibacillus species.

Prior to 2008, all previously studied conventional bacterial flagellar motors appeared to utilize either H+ or Na+ as coupling ions. Membrane-embedded stator complexes support conversion of energy using transmembrane electrochemical ion gradients. The main H+-coupled stators, known as MotAB, differ from Na+-coupled stators, PomAB of marine bacteria, and MotPS of alkaliphilic Bacillus. However, in 2008, a MotAB-type flagellar motor of alkaliphilic Bacillus clausii KSM-K16 was revealed as an exception with the first dual-function motor. This bacterium was identified as the first bacterium with a single stator-rotor that can utilize both H+ and Na+ for ion-coupling at different pH ranges. Subsequently, another exception, a MotPS-type flagellar motor of alkaliphilic Bacillus alcalophilus AV1934, was reported to utilize Na+ plus K+ and Rb+ as coupling ions for flagellar rotation. In addition, the alkaline-tolerant bacterium Paenibacillus sp. TCA20, which can utilize divalent cations such as Ca2+, Mg2+, and Sr2+, was recently isolated from a hot spring in Japan, which contains a high Ca2+ concentration. These findings show that bacterial flagellar motors isolated from unique environments utilize unexpected coupling ions. This suggests that bacteria that grow in different extreme environments adapt to local conditions and evolve their motility machinery.

High level extracellular production of a truncated alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli by the optimization of induction condition and fed-batch fermentation.

To improve the extracellular production of alkaline β-mannanase from alkaliphilic Bacillus sp. N16-5 in Escherichia coli, two truncated recombinant mannanases (32a-ManAR2 and 22b-ManAR2) were obtained. Compared with the full-length mannanases (32a-ManAR1 and 22b-ManAR1), the truncated mannanases not only showed higher secretion rate, but also exhibited higher thermostability and alkalistability. The K m value (11mg/mL) of 32a-ManAR2 was higher than that (1.46mg/mL) of 32a-ManAR1. The specific activity of 22b-ManAR2 was 2.7 times higher than that of 22b-ManAR1. 22b-ManAR2 showed the highest k cat/K m value of 602.7ml/mgs. The parameters of induction for recombinant mannanase production of E. coli BL21 (pET32a-manAR2) and E. coli BL21 (pET22b-manAR2) were subsequently optimized. The yield of soluble mannanase was found to be enhanced with lower induction temperature (25°C), lower IPTG concentration (0.01-0.05mM), and Triton X-100 supplement (0.1%) in a shake flask. Moreover, a one-time feeding strategy and Triton X-100 supplement were applied in production of 22b-ManAR2 in a 10L fermentor. The productivity of the total soluble mannanase reached 9284.64U/mL with the extracellular rate of 74% at 46h of fermentation, which was the highest productive level of alkaline β-mannanase in recombinant E. coli to date.

Effects of genetic modifications and fermentation conditions on 2,3-butanediol production by alkaliphilic Bacillus subtilis

Effects of genetic modifications and fermentation conditions on 2,3-butanediol production by alkaliphilic Bacillus subtilis

Two unexpected promiscuous activities of the iron-sulfur protein IspH in production of isoprene and isoamylene.

BackgroundBacillus species, possessing the methylerythritol phosphate (MEP) pathway for the synthesis of isoprenoid feedstock, are the highest producers of isoprene among bacteria; however, the enzyme responsible for isoprene synthesis has not been identified. The iron–sulfur protein IspH is the final enzyme of the MEP pathway and catalyses the reductive dehydration of (E)-4-hydroxy-3-methyl-2-butenyl diphosphate (HMBPP) to form isopentenyl diphosphate and dimethylallyl diphosphate (DMAPP). In this study, we demonstrated two unexpected promiscuous activities of IspH from alkaliphilic Bacillus sp. N16-5, which can produce high levels of isoprene.ResultsBacillus sp. N16-5 IspH could catalyse the formation of isoprene from HMBPP and the conversion of DMAPP into a mixture of 2-methyl-2-butene and 3-methyl-1-butene. Both reactions require an electron transfer system, such as that used for HMBPP dehydration. Isoprene and isoamylene synthesis in Bacillus sp. N16-5 was investigated and the reaction system was reconstituted in vitro, including IspH, ferredoxin and ferredoxin-NADP+-reductase proteins and NADPH. The roles of specific IspH protein residues were also investigated by site-directed mutagenesis experiments; two variants (H131N and E133Q) were found to have lost the HMBPP reductase activity but could still catalyse the formation of isoprene. Overexpression of IspH H131N in Bacillus sp. N16-5 resulted in a twofold enhancement of isoprene production, and the yield of isoprene from the strain expressing E133Q was increased 300 % compared with the wild-type strain.ConclusionsIspH from Bacillus sp. N16-5 is a promiscuous enzyme that can catalyse formation of isoprene and isoamylene. This enzyme, especially the H131N and E133Q variants, could be used for the production of isoprene from HMBPP.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0476-9) contains supplementary material, which is available to authorized users.

Keratinolytic activities of alkaliphilic Bacillus sp. MBRL 575 from a novel habitat, limestone deposit site in Manipur, India.

Microbial degradation of keratinous wastes is preferred over physicochemical methods as the latter is costlier and not eco-friendly. Novel habitats are promising for discovery of new microbial strains. Towards discovery of novel keratinolytic bacteria, screening of bacterial strains from a novel limestone habitat in Hundung, Manipur, India was done and a promising isolate, MBRL 575, was found to degrade native chicken feather efficiently. It could grow over a broad pH range (Langeveld et al. in J Infect Dis 188:1782–1789, 2003; Park and Son in Microbiol Res 164:478–485, 2009; Zaghloul et al. in Biodegradation 22:111–128, 2011; Takami et al. in Biosci Biotechnol Biochem 56:1667–1669, 1992; Riffel et al. in J Biotechnol 128:693–703, 2007; Wang et al. in Bioresour Technol 99:5679–5686, 2008) and in presence of 0–15 % NaCl. Based on phenotypic characterization and 16S rRNA gene sequence analysis, the new keratinolytic limestone isolate was identified as Bacillus sp. MBRL 575. It produced 305 ± 12 U/ml keratinase and liberated 120 ± 5.5 mg of soluble peptides and 158 ± 4 mg of amino acids per gram of feather after 48 h of incubation at 30 °C in chicken feather medium. The strain could also degrade feathers of other species besides chicken. The cell-free enzyme was also able to degrade feather. Citrate and soybean meal were found to be the best carbon and nitrogen supplements for enhanced enzyme, soluble peptide and amino acid production. In addition to keratinolytic activity, MBRL 575 also exhibited antagonistic activity against two major rice fungal pathogens, Rhizoctonia oryzae-sativae (65 %) and Rhizoctonia solani (58 %).

A Novel Manno-Oligosaccharide Binding Protein Identified in Alkaliphilic Bacillus sp. N16-5 Is Involved in Mannan Utilization.

ManH, a novel substrate-binding protein of an ABC transporter, was identified from the mannan utilization gene cluster of Bacillus sp. N16-5. We cloned, overexpressed, and purified ManH and measured its binding affinity to different substrates by isothermal titration calorimetry. ManH binds to mannotriose, mannotetraose, mannopentose, and galactosyl-mannotriose with dissociation constants in the micromolar range. Deletion of manH led to decreased growth ability of the strain when cultivated in medium with manno-oligosaccharides or mannan as the carbon source. ManH belongs to a manno-oligosaccharide transporter and plays an important role in mannan utilization by Bacillus sp. N16-5.

Editorial: Enzymes from Extreme Environments.

Editorial: Enzymes from Extreme Environments.

Development of novel robust nanobiocatalyst for detergents formulations and the other applications of alkaline protease.

Alkaline protease from alkaliphilic Bacillus sp. NPST-AK15 was immobilized onto functionalized and non-functionalized rattle-type magnetic core@mesoporous shell silica (RT-MCMSS) nanoparticles by physical adsorption and covalent attachment. However, the covalent attachment approach was superior for NPST-AK15 protease immobilization onto the activated RT-MCMSS-NH₂nanoparticles and was used for further studies. In comparison to free protease, the immobilized enzyme exhibited a shift in the optimal temperature and pH from 60 to 65 °C and pH 10.5-11.0, respectively. While free protease was completely inactivated after treatment for 1 h at 60 °C, the immobilized enzyme maintained 66.5% of its initial activity at similar conditions. The immobilized protease showed higher k cat and K m , than the soluble enzyme by about 1.3-, and 1.2-fold, respectively. In addition, the results revealed significant improvement of NPST-AK15 protease stability in variety of organic solvents, surfactants, and commercial laundry detergents, upon immobilization onto activated RT-MCMSS-NH₂nanoparticles. Importantly, the immobilized protease maintained significant catalytic efficiency for ten consecutive reaction cycles, and was separated easily from the reaction mixture using an external magnetic field. To the best of our knowledge this is the first report about protease immobilization onto rattle-type magnetic core@mesoporous shell silica nanoparticles that also defied activity-stability tradeoff. The results clearly suggest that the developed immobilized enzyme system is a promising nanobiocatalyst for various bioprocess applications requiring a protease.

Sustainable biorefining in wastewater by engineered extreme alkaliphile Bacillus marmarensis.

Contamination susceptibility, water usage, and inability to utilize 5-carbon sugars and disaccharides are among the major obstacles in industrialization of sustainable biorefining. Extremophilic thermophiles and acidophiles are being researched to combat these problems, but organisms which answer all the above problems have yet to emerge. Here, we present engineering of the unexplored, extreme alkaliphile Bacillus marmarensis as a platform for new bioprocesses which meet all these challenges. With a newly developed transformation protocol and genetic tools, along with optimized RBSs and antisense RNA, we engineered B. marmarensis to produce ethanol at titers of 38 g/l and 65% yields from glucose in unsterilized media. Furthermore, ethanol titers and yields of 12 g/l and 50%, respectively, were produced from cellobiose and xylose in unsterilized seawater and algal-contaminated wastewater. As such, B. marmarensis presents a promising approach for the contamination-resistant biorefining of a wide range of carbohydrates in unsterilized, non-potable seawater.

Efficient fermentative production of polymer-grade D-lactate by an engineered alkaliphilic Bacillus sp. strain under non-sterile conditions.

BackgroundPolylactic acid (PLA) is one important chemical building block that is well known as a biodegradable and a biocompatible plastic. The traditional lactate fermentation processes need CaCO3 as neutralizer to maintain the desired pH, which results in an amount of insoluble CaSO4 waste during the purification process. To overcome such environmental issue, alkaliphilic organisms have the great potential to be used as an organic acid producer under NaOH-neutralizing agent based fermentation. Additionally, high optical purity property in d-lactic acid is now attracting more attention from both scientific and industrial communities because it can improve mechanical properties of PLA by blending l- or d-polymer together. However, the use of low-price nitrogen source for d-lactate fermentation by alkaliphilic organisms combined with NaOH-neutralizing agent based process has not been studied. Therefore, our goal was the demonstrations of newly simplify high-optical-purity d-lactate production by using low-priced peanut meal combined with non-sterile NaOH-neutralizing agent based fermentation.ResultsIn this study, we developed a process for high-optical-purity d-lactate production using an engineered alkaliphilic Bacillus strain. First, the native l-lactate dehydrogenase gene (ldh) was knocked out, and the d-lactate dehydrogenase gene from Lactobacillus delbrueckii was introduced to construct a d-lactate producer. The key gene responsible for exopolysaccharide biosynthesis (epsD) was subsequently disrupted to increase the yield and simplify the downstream process. Finally, a fed-batch fermentation under non-sterile conditions was conducted using low-priced peanut meal as a nitrogen source and NaOH as a green neutralizer. The d-lactate titer reached 143.99 g/l, with a yield of 96.09 %, an overall productivity of 1.674 g/l/h including with the highest productivity at 16 h of 3.04 g/l/h, which was even higher than that of a sterile fermentation. Moreover, high optical purities (approximately 99.85 %) of d-lactate were obtained under both conditions.ConclusionsGiven the use of a cheap nitrogen source and a non-sterile green fermentation process, this study provides a more valuable and favorable fermentation process for future polymer-grade d-lactate production.

Contribution of intracellular negative ion capacity to Donnan effect across the membrane in alkaliphilic Bacillus spp.

To elucidate the energy production mechanism of alkaliphiles, the relationship between the H(+) extrusion rate by the respiratory chain and the corresponding ATP synthesis rate was determined in the facultative alkaliphile Bacillus cohnii YN-2000 and compared with those in the obligate alkaliphile Bacillus clarkii DSM 8720(T) and the neutralophile Bacillus subtilis IAM 1026. Under high aeration condition, much higher ATP synthesis rates and larger Δψ in the alkaliphilic Bacillus spp. grown at pH10 than those in the neutralophilic B. subtilis grown at pH7 were observed. This high ATP productivity could be attributed to the larger Δψ in alkaliphiles than in B. subtilis because the H(+) extrusion rate in alkaliphiles cannot account for the high ATP productivity. However, the large Δψ in the alkaliphiles could not be explained only by the H(+) translocation rate in the respiratory chain in alkaliphiles. There is a possibility that the Donnan effect across the membrane has the potential to contribute to the large Δψ. To estimate the contribution of the Donnan effect to the large Δψ in alkaliphilic Bacillus spp. grown at pH10, intracellular negative ion capacity was examined. The intracellular negative ion capacities in alkaliphiles grown at pH10 under high aeration condition corresponding to their intracellular pH (pH8.1) were much higher than those in alkaliphiles grown under low aeration condition. A proportional relationship is revealed between the negative ion capacity and Δψ in alkaliphiles grown under different aeration conditions. This relationship strongly suggests that the intracellular negative ion capacity contributes to the formation of Δψ through the Donnan effect in alkaliphilic Bacillus spp. grown at pH10.

Alkaline protease from a new halotolerant alkaliphilic Bacillus agaradhaerens strain ak-r isolated from egyptian soda lakes

Alkaline proteases are hydrolytic enzymes that cleave peptide bonds in proteins and peptides in alkaline conditions, which occupy a pivotal importance with respect to their industrial applications. This study aimed to isolate new alkaline protease producing alkaliphilic bacteria from Egyptian soda lakes and optimize the fermentation process to enhance the enzyme production. The extensive screening process of the samples collected from Egyptian soda lakes resulted in isolation of a potent alkaline protease producing alkaliphilic strain AK-R. The isolate was identified as Bacillus agaradhaerens strain AK-R based on 16S rRNA gene analysis (99%). Wheat bran and gelatin supported maximum alkaline protease production as carbon and nitrogen sources, respectively. Strain AK-R is halo-tolerant thermo-tolerant alkaliphilic bacterium in nature, as it can grow over a wide range of NaCl concentrations (up to 25%) and up to 55 °C, with maximal growth and enzyme production at 2.5-5%, and pH 11 at 35 °C. Among the tested cations, only Mg 2+ and Ca 2+ ions significantly enhanced the enzyme production by about 1.2, and 1.3 fold compared to control, respectively. Alkaline protease secretion was coherent with the growth pattern, reaching maximal yield after about 32 h (mid stationary phase). In conclusion a new halo-tolerant thermo-tolerant alkaliphilic alkaline protease producing Bacillus agaradhaerens strain AK-R was isolated from Egyptian soda lakes. Optimization of the nutritional and cultivation conditions resulted in increase of enzyme yield by 20 fold. Strain AK-R and its extracellular alkaline protease with salt, pH and temperature, tolerance signify their potential application in laundry and pharmaceuticals industries.