Bacillus Alcalophilus Research Articles

Purification and Characterization of an Alkaline Protease from <i>Bacillus alcalophilus</i>

The purification and characterization of an alkaline protease produced by Bacillus alcalophilus were investigated. The enzyme was purified in two steps: concentrating the crude enzyme using ammonium sulfate precipitation, followed by cation-exchange chromatography. The purified protease had a molecular mass of approximately 28 kDa, was highly active over an alkaline pH range of 10.0 to 11.0, and remained stable over a pH range of 7.0 to 12.0. The optimum temperature for the enzyme activity was found to be 40~60°C, while the thermotolerance of the enzyme was poor. Therefore, these characteristics of the protease indicate its potential for a wide range of commercial applications.

Bioresolution of benzyl glycidyl ether using whole cells of Bacillus alcalophilus

The incubation of whole Bacillus alcalophilus cells grown on a mineral supplemented medium (MSM) containing 1% (w/v) sucrose as carbon source, 1.2% (w/v) tryptone as nitrogen source at pH 6.5 and temperature 30 °C in 24 h kinetically resolved benzyl glycidyl ether (1 mg/ml) to provide (S)-benzyl glycidyl ether with 30% ee and (R)-3-benzyloxypropane-1,2-diol with 40% ee.

Purification and biochemical characterization of a serine alkaline protease TC4 from a new isolated Bacillus alcalophilus TCCC11004 in detergent formulations

An extracellular alkaline protease producing strain was isolated from alkaline soil and identified as Bacillus alcalophilus TCCC11004 on the basis of 16S rDNA gene sequencing and biochemical properties. The most appropriate medium for the protease production was composed of (g/l): maltodextrin 110, yeast extract 17.5, cotton seed meal 29.3, K 2 HPO 4 18, trisodium citrate 3.3 and CaCl 2 2.6. The alkaline protease TC4 was purified from the culture supernatant by ammonium sulfate precipitation, Sephadex G-75 gel filtration and SP-Sepharose HP ion exchange chromatography, with a 6.8 fold increase in specific activity and 15.2% recovery. The molecular weight was estimated to be 26 kDa on SDS-PAGE. The protease was highly active from pH 9.0-12.0 with an optimal at pH 11.0. It was active at 30 - 60°C and exhibited maximal activity at 50°C. The thermostability of the protease was increased by the addition of CaCl 2 . It retained 70 and 81% of its initial activity after heating for 2 h at 50°C, in the absence or presence of 2 mM CaCl 2 , respectively. The enzyme was inactivated by diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride, suggesting that it is a serine protease. The protease was stable in 0.5% SDS and retained 70.3% of its initial activity after 1 h of incubation. It was active in the presence of 3% Triton X-100 with 100% activity and stable towards oxidizing agent with 69.2% activity in the presence of 1% H 2 O 2 . The enzyme showed excellent compatibility with commercial detergents such as TaiZi, BiLang, DiaoPai and TianQing, retaining more than 90% of its initial activity in the tested detergents after 1 h of preincubation at 40°C. Keywords : Serine alkaline protease, Bacillus alcalophilus , stability, detergent compatibility.

Purification and characterization of a surfactant-stable high-alkaline protease from Bacillus sp. B001

The newly isolated alkalophilic Bacillus sp. B001 produced a high level of proteolytic activity (34277 U/mL) when grown in production medium, and a 28 kDa protease, designated AprB, was purified from the culture supernatant. Partial amino acid sequences were obtained by tandem mass spectrometry (MS/MS) and a pair of degenerate primers was developed to amplify a 467-bp genomic sequence. The observed and predicted amino acid sequences showed similarity with sequences of high-alkaline proteases from Bacillus clausii, Bacillus alcalophilus, and Bacillus lentus. High stability of AprB towards surfactants and oxidizing agents, an optimal pH of 10.0, and an optimal temperature of 60 °C suggest that this high-alkaline protease has potential applications for various industrial processes.

Cloning and Characterization of ectABC Cluster from Bacillus alcalophilus DTY1

Bacillus alcalophilus DTY1, a moderate halophytic bacterium isolated from saline soil in Loess Plateau of China, was characterized with efficient production of ectoine. In this study, the gene cluster ectABC taking in charge of biosynthesizing ectoine was cloned from the genomic library of strain DTY1. Nucleotide sequencing indicated that ectA, ectB and ectC were predicted to encode peptides of 169, 428 and 132 amino acids, respectively. The deduced amino acid sequences of EctA, EctB and EctC share 59%, 81% and 81% identity to 2,4-diaminobutyric acid acetyltransferase, 2,4-diaminobutyric acid transaminase and ectoine synthase of B. halodurans C-125, respectively. A fragment containing ectABC genes was introduced into B. cereus Z, which made the transgenic Z cells increased tolerance to salt, remarkably. HPLC analysis of ectoine in the transgenic Z cells revealed that 70.1 mg/g ectoine was detected in 1.0% NaCl medium and 118.6 mg/g ectoine in 5.0% NaCl medium. Furthermore, as the concentration of salt increased, transgenic Z cells accumulated more ectoine. These results indicate that ectoine is an important facet in B. alcalophilus DTY1 to high-osmolarity surroundings, and the expression of ectABC is induced by salt strength.

Structural basis of enzyme adaptation to alkaline pH

pH is one of the key parameters that affect stability and function of proteins. The crystal structure of the vitamin B 6-dependent enzyme phosphoserine aminotransferase from the obligatory alkaliphile Bacillus alcalophilus has been determined at 1.08 A resolution [1], the highest resolution for any PLP-dependent enzyme so far. The final model contains 5824 protein atoms and 949 water molecules, and has an R-factor of 11.7% (Rfree = 13.9%) after anisotropic B-factor refinement with SHELX. The enzyme displays a narrow pH optimum of enzymatic activity at pH 9.0. The final structure was compared to the previously reported structure of themesophilic phosphoserine aminotransferase from Escherichia coli [2] and to that of phosphoserine aminotransferase froma facultative alkaliphile, Bacillus circulans subsp. alkalophilus [3]. All three enzymes are homodimers with each monomer comprising a two-domain architecture. Despite the high structural similarity, the alkaliphilic representatives possess a set of distinctive structural features. Two residues directly interacting with pyridoxal-5’-phosphate are replaced, and an additional hydrogen bond to theO3’ atomof the cofactor is present in alkaliphilic phosphoserine aminotransferases. The number of hydrogen bonds and hydrophobic interactions at the dimer interface is increased. Hydrophobic interactions between the two domains in the monomers are enhanced. Moreover, the number of negatively charged amino acid residues increases on the solvent accessible molecular surface and fewer hydrophobic residues are exposed to the solvent. Further, the total amount of ion pairs and ion networks is significantly reduced in theBacillus enzymes, while the total number of hydrogen bonds is increased. The mesophilic enzyme fromEscherichia coli contains two additional s-strands in a surface loop with a third s-strand being shorter in the structure. The identified structural features are proposed to be possible factors implicated in the alkaline adaptation of phosphoserine aminotransferase.

Bacillus alcalophilus AX2000 유래 xylanase 유전자 (XynT)의 Cloning과 염기서열 분석

A gene coding for xylanase from alkali-tolerant Bacillus alcalophilus AX2000 was cloned into Escherichia coli using pUC19. Among 2,000 transformants, one transformant showed clear zone on the detection agar plate containing oat-spells xylan. Its recombinant plasmid, named pXTY99, was found to carry 7.0 kb insert DNA fragment. When the nucleotide sequence of the cloned xylanase gene (xynT) was determined, xynT gene was found to consist of 1,020 base-pair open reading frame coding for a poly-peptide of 340 amino acids with a deduced molecular weight of 40 kDa. The coding sequence was preceded by a putative ribosome binding site, and the transcription initiation signals. The deduced amino acid sequence of xylanase is similar to those of the xylanases from Bacillus sp. Nl37 and B. stearothermophilus 21 with and identical residues, respectively

Bacillus bogoriensis sp. nov., a novel alkaliphilic, halotolerant bacterium isolated from a Kenyan soda lake

Strain LBB3(T) isolated from Bogoria soda lake in Kenya is an alkaliphilic, Gram-positive, strictly aerobic, non-motile, spore-forming bacterium. It was identified as a member of the genus Bacillus on the basis of phenotypic and phylogenetic analyses. The organism grows optimally at 37 degrees C and pH 10. The G+C content of the genomic DNA is 37.5 mol%. 16S rRNA gene sequence analysis showed 95 and 96 % sequence similarity with Bacillus pseudofirmus (DSM 8715(T)) and Bacillus alcalophilus (DSM 485(T)), respectively. Furthermore, DNA-DNA hybridization against these two Bacillus species showed 39.0 and 55.5 % similarity, respectively. Based on our observations, strain LBB3(T) is proposed to represent a novel species of the genus Bacillus, for which the name Bacillus bogoriensis sp. nov. is proposed. The type strain of B. bogoriensis is LBB3(T) (=ATCC BAA-922(T)=LMG 22234(T)).

Enzyme adaptation to alkaline pH: Atomic resolution (1.08 Å) structure of phosphoserine aminotransferase from Bacillus alcalophilus

The crystal structure of the vitamin B(6)-dependent enzyme phosphoserine aminotransferase from the obligatory alkaliphile Bacillus alcalophilus has been determined at 1.08 A resolution. The model was refined to an R-factor of 11.7% (R(free) = 13.9%). The enzyme displays a narrow pH optimum of enzymatic activity at pH 9.0. The final structure was compared to the previously reported structure of the mesophilic phosphoserine aminotransferase from Escherichia coli and to that of phosphoserine aminotransferase from a facultative alkaliphile, Bacillus circulans subsp. alkalophilus. All three enzymes are homodimers with each monomer comprising a two-domain architecture. Despite the high structural similarity, the alkaliphilic representatives possess a set of distinctive structural features. Two residues directly interacting with pyridoxal-5'-phosphate are replaced, and an additional hydrogen bond to the O3' atom of the cofactor is present in alkaliphilic phosphoserine aminotransferases. The number of hydrogen bonds and hydrophobic interactions at the dimer interface is increased. Hydrophobic interactions between the two domains in the monomers are enhanced. Moreover, the number of negatively charged amino acid residues increases on the solvent-accessible molecular surface and fewer hydrophobic residues are exposed to the solvent. Further, the total amount of ion pairs and ion networks is significantly reduced in the Bacillus enzymes, while the total number of hydrogen bonds is increased. The mesophilic enzyme from Escherichia coli contains two additional beta-strands in a surface loop with a third beta-strand being shorter in the structure. The identified structural features are proposed to be possible factors implicated in the alkaline adaptation of phosphoserine aminotransferase.

Bacillus alcalophilus peptidoglycan induces IFN-α-mediated inhibition of vaccinia virus replication

Bacterial products such as cell walls (CW) and peptidoglycan (PGN) are known to activate macrophages and NK cells during microbial infections. In this report, we demonstrated that whole CW and PGN of four Gram-positive bacteria are capable of enhancing the anti-poxviral activity of murine macrophage RAW 264.7 cells. Among the major Bacillus alcalophilus CW components, PGN contributes the most to antiviral activity and induces remarkably higher levels of IFN-α. Anti-IFN-α/β antibody, but not anti-IFN-γ, anti-IFN-γ receptor, or anti-IL-12, reversed the PGN-induced inhibition of vaccinia virus replication and reduced nitric oxide (NO) production. Our data thus suggest that PGN induce antiviral activity through IFN-α and to a lesser extent, through NO production.

Lipase-producing microorganisms from a Kenyan alkaline soda lake.

Lipolytic enzyme production of 150 isolated strains from samples of Lake Bogoria (Kenya) was examined. Among these, fifteen isolates were selected on the basis of their lipolytic activities and subjected to morphological and 16S rRNA gene sequencing analyses for their identification. All the microorganisms have been selected under culture conditions with pH ranges between 7-10 and temperatures of 37-55 degrees C. Most of them showed optimal growth at 37 degrees C and tolerated salinity up to 10% (w/v). Ten of the isolates were Gram-negative, nine of which were closely related to the Pseudomonas cluster and one to the Halomonas cluster sharing high similarity profile with Halomonas desiderata. The remaining Gram-positive isolates were closely related to the Bacillus cluster, and were grouped with Bacillus halodurans, Bacillus alcalophilus and Bacillus licheniformis. Four members of the Bacillus cluster and the Halomonas sp. produced lipolytic activity under alkaline conditions, while others did so at neutral pH values.

Wide-range distribution of insertion sequences identified in B. halodurans among bacilli and a new transposon disseminated in alkaliphilic and thermophilic bacilli.

All of the insertion sequences (ISs) except for IS663 and a group II intron identified in the alkaliphilic Bacillus halodurans C-125 genome were also detected in nine other strains of the same species by PCR and Southern blot analysis. The transposase of IS 653 identified in the genomes of the 10 strains of B. halodurans was found to have become the most diversified of all ISs identified in the genomes of 10 strains. A new IS element designated IS661 belonging to the IS1380 family with inverted repeats (IRs) 17 bp in length was present within IS658 identified in the genome of B. halodurans A59. In addition, a new transposon designated Tn3271bh was identified within the IS642 element in the A59 genome, which is similar to a transposon identified in thermophilic Geobacillus stearothermophilus T-6. The new transposon, Tn3271bh, generated an 8-bp duplication of the target site sequence and carries a 21-bp IR. On the other hand, all kinds of ISs except for IS643 and IS658 were distributed in the genome of obligately alkaliphilic Bacillus alcalophilus. Three ISs (IS652, IS653, and IS660) and a group II intron (Bh.Int) were widely dispersed in other Bacillus species without a correlation with the phylogenetic placement based on 16S rDNA sequences.

Bacillus krulwichiae sp. nov., a halotolerant obligate alkaliphile that utilizes benzoate and m-hydroxybenzoate.

Obligate alkaliphilic strains, AM31D(T) and AM11D, that utilize benzoate and m-hydroxybenzoate were isolated from soil obtained from Tsukuba, Ibaraki, Japan. The isolates grew at pH 8-10, but not at neutral pH. They were Gram-positive, facultatively anaerobic, straight rods with peritrichous flagella and produced ellipsoidal spores. The isolates reduced nitrate to nitrite and grew in 0-14 % NaCl, but not in higher concentrations. The major isoprenoid quinones were menaquinone-5, -6 and -7, and the cellular fatty acid profile consisted of significant amounts of 15-C branched-chain acids, isoC(15 : 0) and anteisoC(15 : 0). Phylogenetic analysis based on 16S rRNA gene sequencing indicated that strain AM31D(T) was a member of group 6 (alkaliphiles) in the genus Bacillus. DNA-DNA hybridization revealed a low relatedness of the isolates with several phylogenetically close neighbours, including Bacillus alcalophilus and Bacillus pseudalcaliphilus (less than 19.3 %). Based on phenotypic characteristics, phylogenetic data and DNA-DNA relatedness data, it was concluded that these isolates merited classification as a new species, for which the name Bacillus krulwichiae is proposed. The type strain of this species is AM31D(T) (=NCIMB 13904(T)=JCM 11691(T)=IAM 15000(T)).

Molecular characterization and identification of Bacillus clausii Strains marketed for use in oral bacteriotherapy.

A substantial number of Bacillus species have been marketed for use in oral bacteriotherapy because of their purported ability to prevent or treat various gastrointestinal disorders. Recently, some of the Bacillus strains in Enterogermina, which is made up of aqueous suspensions of viable Bacillus spores, have been partially characterized and aligned with members of the Bacillus alcalophilus subgroup rather than with Bacillus subtilis, as previously reported. With a view toward verifying the original taxonomic position of the Enterogermina strains, we catalogued both phenotypic and genotypic traits exhibited by the four Bacillus strains isolated from the spore mixtures found in original commercial preparations dated 1975 and 1984 and commercial preparations now being propagated industrially. Analyses of physiological and biochemical traits, complete 16S rRNA gene sequences, DNA-DNA reassociation, tRNA intergenic spacer length polymorphism, single-strand conformation polymorphism of PCR-amplified spacer regions of tRNA genes, and randomly amplified polymorphic DNA led to the finding that all of the Enterogermina strains belong to a unique genospecies, which is unequivocally identified as the alkalitolerant species Bacillus clausii. Moreover, we provide evidence that in contrast to several reference strains of B. clausii, the strains constituting Enterogermina are characterized by a notable low level of intraspecific genome diversity and that each strain has remained the same for the last 25 years.

Identification of facultatively alkaliphilic Bacillus sp. strain YN-2000 and its fatty acid composition and cell-surface aspects depending on culture pH.

Facultatively alkaliphilic Bacillus sp. strain YN-2000 was isolated from an indigo ball. Although the strain has been extensively investigated as a representative strain of alkaliphilic bacillus, its taxonomic position is not yet known. Morphological, biochemical, and physiological characteristics and chemotaxonomic properties indicated that the strain was closely related to Bacillus cohnii; this was confirmed by the high homology of the 16S rRNA sequence and the construction of a phylogenetic tree on the basis of the 16S rRNA sequence and DNA-DNA relatedness data. Strain YN-2000 contained a larger amount of unsaturated fatty acids compared with Bacillus subtilis and the obligate alkaliphile, Bacillus alcalophilus, regardless of its culture pH. When the cells were grown at pH 10, the unsaturated fatty acid content and anteiso-/iso-branched fatty acid ratio became lower than those at pH 7. This result suggests that membrane fluidity decreases when the cells are grown at pH 10 compared to those of pH 7. In the cells of strain YN-2000 grown at pH 10, the cell-surface aspect was rougher, the cell shape was longer, and the cell-surface layer was thicker compared with those of the cells grown at pH 7. The cell-surface structural change might be related to adaptation to an alkaline environment.

Bacillus pseudomycoides sp. nov.

Previous DNA relatedness studies showed that strains identified as Bacillus mycoides segregated into two genetically distinct yet phenotypically similar groups, one being B. mycoides sensu stricto and the other, an unclassified taxon. In the present study, the taxonomic position of this second group was assessed by measuring DNA relatedness and determining phenotypic characteristics of an increased number of B. mycoides strains. Also determined was the second group's 16S RNA gene sequence. The 36 B. mycoides strains studied segregated into two genetically distinct groups showing DNA relatedness of about 30%; 18 strains represented the species proper and 18 the second group with intragroup DNA relatedness for both groups ranging from 70 to 100%. DNA relatedness to the type strains of presently recognized species with G+C contents of approximately 35 mol% (Bacillus alcalophilus, Bacillus cereus, Bacillus circulans, Bacillus lentus, Bacillus megaterium and Bacillus sphaericus) ranged from 22 to 37%. Although shown to be genetically distinct taxa, the two B. mycoides groups exhibited highly similar (98%) 16S RNA sequences. Phylogenetic analyses showed that both B. mycoides and the second group clustered closely with B. cereus. Although not distinguishable by physiological and morphological characteristics, the two B. mycoides groups and B. cereus were clearly separable based on fatty acid composition. The data established that the second B. mycoides group merits recognition as a new species for which the name Bacillus pseudomycoides is proposed. The type stain is NRRL B-617(T).

Comparative 16S rDNA sequence analysis of some alkaliphilic bacilli and the establishment of a sixth rRNA group within the genusBacillus

Comparative sequence analysis of the 16S rDNA of 14 alkaliphilic or alkalitolerant, Gram-positive, aerobic, endo-spore forming bacterial strains was performed. Bacillus alcalophilus DSM 485T and Bacillus cohnii DSM 6307T were included to represent the two validly described alkaliphiles assigned to the genus Bacillus. The majority of isolates (8 strains) clustered with B. alcalophilus DSM 485T forming a distinct phylogenetic group (rRNA group 6) within the radiation of the genus Bacillus and related taxa. Bacillus cohnii DSM 6307T and two of the isolates, DSM 8719 and DSM 8723, grouped with B. fastidiosus and B. megaterium and are allocated to rRNA group 1. The remaining two strains DSM 8720 and DSM 8721 show an equidistant relationship to both groups.

Membrane ultrastructure of alkaliphilic Bacillus species studied by rapid-freeze electron microscopy.

Cells of Bacillus firmus OF4 and Bacillus alcalophilus were examined by rapid-freeze freeze-fracture and freeze-substitution electron microscopy. No special vesicular structures linked to growth at alkaline pH were found, either within or associated with the cytoplasmic membrane. The cytoplasmic membranes of the alkaliphilic bacilli and the neutrophilic Bacillus subtilis BD99 were indistinguishable. Distinctive intramembrane particle rings, presumed to be flagellar structures on the basis of distribution and morphological characteristics, were found in all of these species. These observations indicate that the adaptations required to effect oxidative phosphorylation and flagellar rotation at extreme alkaline pH occur without gross morphological rearrangement.

Crystal structure of the high-alkaline serine protease PB92 from Bacillus alcalophilus.

The crystal structure of a serine protease from the alkalophilic strain Bacillus alcalophilus PB92 has been determined by X-ray diffraction at 1.75 A resolution. The structure has been solved by molecular replacement using the atomic model of subtilisin Carlsberg. The model of the PB92 protease has been refined to an R-factor of 14.0% and contains 1882 protein atoms, two calcium ions and 188 water molecules. The overall folding of the polypeptide chain closely resembles that of the subtilisins. Furthermore, almost all of the secondary structure elements found in subtilisin Carlsberg are also present in the PB92 protease. The major differences between the two structures are located around the deletion regions (residues 37 and 158-161 in subtilisin Carlsberg) and in two loops which are known to be the most variable parts of subtilisin structures. Flexibility of one of these loops (residues 126-130 in the PB92 protease) is believed to account for the induced-fit mechanism of substrate binding.

Molecular cloning and sequencing of a gene from alkaliphilic Bacillus firmus OF4 that functionally complements an Escherichia coli strain carrying a deletion in the nhaA Na+/H+ antiporter gene.

A gene has been cloned from a DNA library from alkaliphilic Bacillus firmus OF4 that functionally complements a mutant strain of Escherichia coli, NM81, that carries a deletion for one of that strain's Na+/H+ antiporter genes (delta nhaA). The cloned alkaliphile gene restored to NM81 the ability to grow at pH 7.5 in the presence of 0.6 M NaCl and on 100 mM Li+ in the presence of melibiose, and concomitantly led to an increase in the membrane associated Na+/H+ antiport activity. The biologically active alkaliphile DNA was identified as an incomplete open reading frame, the sequence of which would encode a hydrophobic protein. The insert was used to isolate clones containing the complete open reading frame, which would be predicted to encode a protein with a molecular weight of 42,960 and multiple membrane spanning regions. When the open reading frame was expressed under the control of the T7 promoter, the gene product was localized in the membrane. Southern analysis indicated no homology between the alkaliphile gene, which we propose to call nhaC, and the nhaA gene of Escherichia coli, nor with other genes in digests of DNA from E. coli, Bacillus subtilis, or Bacillus alcalophilus. Although there was also no significant similarity between the deduced protein products of the alkaliphile gene and the nhaA gene of E. coli, there was a small region of significant similarity between the deduced alkaliphile gene product and the protein encoded by a human Na+/H+ antiporter gene (Sardet, C., Franchi, A., and Pouyssegur, J. (1989) Cell 56, 271-280).