Showing papers on "Bacillus licheniformis published in 2000"

Phosphate-solubilizing microorganisms associated with the rhizosphere of mangroves in a semiarid coastal lagoon

Abstract: The phosphate-solubilizing potential of the rhizosphere microbial community in mangroves was de- monstrated when culture media supplemented with in- soluble, tribasic calcium phosphate, and incubated with roots of black (Avicennia germinans L.) and white (La- guncularia racemosa (L.) Gaertn.) mangrove became transparent after a few days of incubation. Thirteen phosphate-solubilizing bacterial strains were isolated from the rhizosphere of both species of mangroves: Ba- cillus amyloliquefaciens, Bacillus licheniformis, Bacillus atrophaeus, Paenibacillus macerans, Vibrio proteolyti- cus, Xanthobacter agilis, Enterobacter aerogenes, Ente- robacter taylorae, Enterobacter asburiae, Kluyvera cryo- crescens, Pseudomonas stutzeri, and Chryseomonas lu- teola. One bacterial isolate could not be identified. The rhizosphere of black mangroves also yielded the fungus Aspergillus niger. The phosphate-solubilizing activity of the isolates was first qualitatively evaluated by the for- mation of halos (clear zones) around the colonies grow- ing on solid medium containing tribasic calcium phos- phate as a sole phosphorus source. Spectrophotometric quantification of phosphate solubilization showed that all bacterial species and A. niger solubilized insoluble phosphate well in a liquid medium, and that V. proteo- lyticus was the most active solubilizing species among the bacteria. Gas chromatographic analyses of cell-free spent culture medium from the various bacteria de- monstrated the presence of 11 identified, and several unidentified, volatile and nonvolatile organic acids. Those most commonly produced by different species were lactic, succinic, isovaleric, isobutyric, and acetic acids. Most of the bacterial species produced more than one organic acid whereas A. niger produced only suc- cinic acid. We propose the production of organic acids by these mangrove rhizosphere microorganisms as a possible mechanism involved in the solubilization of in- soluble calcium phosphate.

Detection of the dipicolinic acid biomarker in Bacillus spores using Curie-point pyrolysis mass spectrometry and Fourier transform infrared spectroscopy.

Abstract: Thirty-six strains of aerobic endospore-forming bacteria confirmed by polyphasic taxonomic methods to belong to Bacillus amyloliquefaciens, Bacillus cereus, Bacillus licheniformis, Bacillus megaterium, Bacillus subtilis (including Bacillus niger and Bacillus globigii), Bacillus sphaericus, and Brevi laterosporus were grown axenically on nutrient agar, and vegetative and sporulated biomasses were analyzed by Curie-point pyrolysis mass spectrometry (PyMS) and diffuse reflectance−absorbance Fourier-transform infrared spectroscopy (FT-IR). Chemometric methods based on rule induction and genetic programming were used to determine the physiological state (vegetative cells or spores) correctly, and these methods produced mathematical rules which could be simply interpreted in biochemical terms. For PyMS it was found that m/z 105 was characteristic and is a pyridine ketonium ion (C6H3ON+) obtained from the pyrolysis of dipicolinic acid (pyridine-2,6-dicarboxylic acid; DPA), a substance found in spores but not in ...

Structural analysis of a chimeric bacterial alpha-amylase. High-resolution analysis of native and ligand complexes.

Abstract: Several chimeric alpha-amylases genes were constructed by an in vivo recombination technique from the Bacillus amyloliquefaciens and Bacillus licheniformis genes. One of the fusion amylases (hereafter BA2), consisting of residues 1-300 from B. amyloliquefaciens and 301-483 from B. licheniformis, has been extensively studied by X-ray crystallography at resolutions between 2.2 and 1.7 A. The 3-dimensional structure of the native enzyme was solved by multiple isomorphous replacement, and refined at a resolution of 1.7 A. It consists of 483 amino acids, organized similarly to the known B. lichiniformis alpha-amylase structure [Machius et al. (1995) J. Mol. Biol. 246, 545-559], but features 4 bound calcium ions. Two of these form part of a linear cluster of three ions, the central ion being attributed to sodium. This cluster lies at the junction of the A and B domains with one calcium of the cluster structurally equivalent to the major Ca(2+) binding site of fungal alpha-amylases. The third calcium ion is found at the interface of the A and C domains. BA2 contains a fourth calcium site, not observed in the B. licheniformis alpha-amylase structure. It is found on the C domain where it bridges the two beta-sheets. Three acid residues (Glu261, Asp328, and Asp231) form an active site similar to that seen in other amylases. In the presence of TRIS buffer, a single molecule of TRIS occupies the -1 subsite of the enzyme where it is coordinated by the three active-center carboxylates. Kinetic data reveal that BA2 displays properties intermediate to those of its parents. Data for crystals soaked in maltooligosaccharides reveal the presence of a maltotriose binding site on the N-terminal face of the (beta/alpha)(8) barrel of the molecule, not previously described for any alpha-amylase structure, the biological function of which is unclear. Data for a complex soaked with the tetrasaccharide inhibitor acarbose, at 1.9 A, reveal a decasaccharide moiety, spanning the -7 to +3 subsites of the enzyme. The unambiguous presence of three unsaturated rings in the (2)H(3) half-chair/(2)E envelope conformation, adjacent to three 6-deoxypyranose units, clearly demonstrates synthesis of this acarbose-derived decasaccharide by a two-step transglycosylation mechanism.

Production of poly-γ-glutamic acid by fed-batch culture of Bacillus licheniformis

Abstract: Fed-batch cultures of Bacillus licheniformis produced poly-γ-glutamic acid (PGA), a water-soluble biodegradable polymer. PGA reached 35 g l−1 with a productivity of 1 g l−1 h−1 by pulsed-feeding of citric acid (1.44 g h−1) and l-glutamic acid (2.4 g h−1) when citric acid was depleted from the culture medium.

Oxygen-transfer strategy and its regulation effects in serine alkaline protease production by Bacillus licheniformis.

Abstract: The effects of oxygen transfer on the production and product distribution in serine alkaline protease (SAP) fermentation by Bacillus licheniformis and oxygen-transfer strategy in relation to the physiology of the bacilli were investigated on a defined medium with citric acid as sole carbon source in 3.5-dm3 batch bioreactor systems. By forming a 3 × 3 matrix with the parameters air-inlet rates of QO/VR = 0.2, 0.5, 1.0 vvm, and agitation rates of N = 150, 500, 750 min−1, the effects of oxygen transfer were investigated at nine different conditions. The concentrations of the product SAP and by-products, i.e., neutral protease, α-amylase, amino acids, and organic acids, and SAP activities were determined throughout the bioprocess. Among the constant air-flow and agitation-rate fermentations, QO/VR = 0.5 vvm, N = 750 min−1 oxygen-transfer conditions produced maximum SAP activity that was 500 U cm−3, at t = 37 h. With the increase in QO/VR and/or N, Damkohler number that is the oxygen-transfer limitation decreases; and the process passes from oxygen-transfer limited conditions to biochemical-reaction limited conditions. Further increase in SAP activity, A = 680 U cm−3 was achieved by applying an oxygen-transfer strategy based on the analysis of the data obtained with the constant oxygen-transfer condition experiments, with a step increase in air-inlet rate, from QO/VR = 0.2 to QO/VR = 0.5 vvm at N = 750 min−1 constant agitation rate at t = 24 h. Organic acids and amino acids that were excreted to the fermentation medium varied depending on the oxygen-transfer conditions. With the increase in oxygen-transfer rate acetic acid concentration increased; contrarily, with the decrease in the oxygen-transfer rate the TCA-cycle organic acids α-ketoglutaric and succinic acids, and gluconic acid were excreted to the fermentation broth; nevertheless, the application of the oxygen-transfer strategy prevented the increase in acetic acid concentration between t = 35–38 h. Under all the oxygen-transfer conditions, the amino acid having the highest concentration and the amino acid that was not excreted to the fermentation broth were lysine and asparagine, respectively; both of which belong to the aspartic acid-group amino acids. Further, this result indicates the requirement of the genetic regulation directed to the aspartic acid-group enzymes for the progress in SAP production in B. licheniformis. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 69: 301–311, 2000.

Studies on the extracellular tannase from newly isolated Bacillus licheniformis KBR 6.

Abstract: A tannase producing bacterial strain KBR 6 has been isolated from lateritic soil and identified as Bacillus licheniformis. It is capable of producing tannase in the medium containing only tannic acid. The rapid degradation of tannic acid and production of extracellular tannase was observed in three different media containing tannic acid (M1), tannic acid + basal salt (M2) and tannic acid + basal salt + glucose (M3). Maximum enzyme production and growth of the organism was obtained at 18-21 h and 30-36 h, respectively. The increased order of enzyme production in relation to different media is as per the following sequence, M3 > M2 > M1. The maximum growth and enzyme production was observed at pH 5.0. The pH and temperature optima of the enzyme activity were found to be at 5.75 and 60 degrees C respectively. Paper chromatographic analysis indicates that gallic acid is the enzymatic degradative product of tannic acid.

Subtilisins of Bacillus spp. hydrolyze keratin and allow growth on feathers.

Abstract: Keratinase is a serine protease produced by Bacillus licheniformis PWD-1 that effectively degrades keratin and confers the ability to grow on feathers to a protease-deficient B. subtilis strain. St...

Toxic lactonic lipopeptide from food poisoning isolates of Bacillus licheniformis

Abstract: Toxins from three Bacillus licheniformis strains connected to a fatal food poisoning were isolated and their structures elucidated Toxins were purified from methanol extracts of the B licheniformis biomass using boar sperm cells as the toxicity indicator The HPLC purified toxins showed protonated masses m/z 1007, 1021 and 1035 in MALDI-TOF-MS The toxins isolated from the strains of different origins contained the same three components of which and each had a same amino-acid residues l-Gln, l-Leu, d-Leu, l-Val, l-Asp, d-Leu and l-Ile in that order Toxins were identified as lichenysin A, a cyclic lactonic heptalipopeptide in which the main 3-hydroxy fatty acids are 13–15 carbons in length We showed that the toxins from food and food poisoning isolates of B licheniformis were identical to lichenysin A both in the structure and in the toxic symptoms induced to boar spermatozoa Confocal laser scanning microscopy showed that the acrosome and the plasma membrane of boar spermatozoa were the targets of lichenysin A toxicity

Tannase production by Bacillus licheniformis

Abstract: Bacillus licheniformis KBR 6 produced maximum extracellular tannase activity at 0.21 U ml−1 with 1.5% (w/v) tannic acid either in the absence or presence of glucose (1 g l−1) after 18–21 h growth though the organism did not attain maximum growth until 36 h.

Purification and characterization of β-mannanase from Bacillus licheniformis for industrial use

Abstract: An easily scaled-up technique has been designed to purify β-mannanase from Bacillus licheniformis Using flocculation, ultrafiltration and ion-exchange chromatography, the enzyme was purified 33-fold with a final recovery of 47% and a specific activity of 4341 U mg−1protein The enzyme had maximum activity at 60 °C and pH 70 It was stable at 50 °C and pH 60 for 6 h, but lost all of its activity when held at 70 °C and pH 60 for 1 h

Cell-associated degradation affects the yield of secreted engineered and heterologous proteins in the Bacillus subtilis expression system.

Abstract: A series of chimeric α-amylase genes derived from amyL, which encodes the liquefying α-amylase from Bacillus licheniformis, were constructed in vitro using gene splicing techniques. The gene constructs were cloned in Bacillus subtilis, where their ability to direct the synthesis and secretion of active α-amylase was determined. Detectable α-amylase activity was observed for some, but not all, of the chimeric proteins. Studies on the secretion of wild-type AmyL and its chimeric derivatives revealed that, whilst these proteins were stable in the extracellular milieu, all were subject to some degree of degradation during secretion. The chimeric enzymes were degraded to a greater extent than the native enzyme. These findings suggest that cell-associated proteolysis is a significant problem affecting the use of B. subtilis as host bacterium for the production of heterologous proteins.

Short chain flavour ester synthesis by a new esterase from Bacillus licheniformis

Abstract: The newly characterized esterase from Bacillus licheniformis was used for ethyl ester synthesis in n-heptane by direct esterification with fatty acids of different chain lengths. The highest reaction rates and yields were obtained with caproic (C6) and caprylic (C8) acids reflecting the enzyme specificity for mid-chain length fatty acids. The rate of ethyl caproate formation increased linearly with the enzyme concentration in the range 0–40 mg/ml. The enzyme displayed maximum activity with 0.1% (v/v) addition of water. The initial reaction rate was maximal at 67°C but the highest yield (95%) was obtained at 45°C. Kinetic parameters were determined for caproic acid: KM of 38.4 mM and VM of 8.3 μmol/min/ml (at 0.25 M ethanol), and for ethanol KM of 12.3 mM and VM of 1.3 μmol/min/ml (at 0.25 M caproic acid). The highest activities and yields were observed for solvents having log P≥3.2. The enzyme was used three times without activity loss.

The role of the bacitracin ABC transporter in bacitracin resistance and collateral detergent sensitivity

Abstract: The bacitracin resistance of Bacillus licheniformis, a producer of bacitracin, is mediated by the ABC transporter Bcr. Bacillus subtilis cells carrying bcr genes on high-copy number plasmids developed collateral detergent sensitivity, as did human cells with overexpressed multidrug resistance P-glycoprotein. Resistance against bacitracin and sensitivity of resistant cells to detergents were shown to be inseparable phenomena associated with the membrane part of Bcr transporter, namely protein BcrC. A fused protein, consisting of ATP-binding protein BcrA and membrane component BcrC was constructed. It resembled a half molecule of P-glycoprotein and was capable of providing a significant degree of antibiotic resistance and detergent sensitivity.

Screening of Microorganisms for Microbial Enhanced Oil Recovery Processes

Abstract: The objective of this study is to screen effective microorganisms for the Microbial Enhanced Oil Recovery process (or simply as MEOR). Samples of drilling cuttings, formation water, and soil were collected from domestic drilling sites and oil fields. Moreover, samples of activated-sludge and compost were collected from domestic sewage treatment facility and food treatment facility. At first, microorganisms in samples were investigated by incubation with different media; then they were isolated. By two stage-screening based on metabolizing ability, 4 strains (Bacillus licheniformis TRC-18-2-a, Enterobacter cloacae TRC-322, Bacillus subtilis TRC-4118, and Bacillus subtilis TRC-4126) were isolated as effective microorganisms for oil recovery. B. licheniformis TRC-18-2-a is a multifunctional microorganism possessing excellent surfactant productivity, and in addition it has gas, acid and polymer productivities. E. cloacae TRC-322 has gas and acid producing abilities. B. subtilis TRC-4118 and TRC-4126 are effective biosurfactant producers, and they reduce the interfacial tension to 0.04 and 0.12dyne/cm, respectively.

The dppA gene of Bacillus subtilis encodes a new d‐aminopeptidase

Abstract: Summary Different strains of Bacillus were screened for their ability to hydrolyse D-alanyl-p-nitroanilide. Activity was detected in Bacillus pumilus, Bacillus brevis, Bacillus licheniformis 749I and Bacillus subtilis 168. The last strain was the best producer and was selected for the production and purification of the enzyme. The determination of the N-terminal sequence identified the enzyme as the product of the dppA gene (previously named dciAA) belonging to the dipeptide ABC transport (dpp) operon expressed early during sporulation. Open reading frames (ORFs) encoding putative related proteins were found in the genomes of a variety of Archaea and both sporulating and non-sporulating bacteria. The enzyme behaves as a D-aminopeptidase and represents the prototype of a new peptidase family. Among the tested substrates, the highest activities were found with D-Ala-D-Ala and D-Ala-Gly-Gly. The active enzyme behaves as an octamer of identical 30 kDa subunits. It exhibits a broad pH optimum, extending between pH 9 and 11. It is reversibly inhibited in the presence of Zn 21 chelators, and the sequence comparisons highlight the conservation of potential Zn-binding residues. As it has been shown by others that null mutations in the dpp operon do not inhibit spore formation, the physiological role of DppA is probably an adaptation to nutrient deficiency.

Potential of spore-forming bacteria as biocontrol agents of wheat foliar diseases under laboratory and greenhouse conditions

Abstract: The potential of Bacillus subtilis, Bacillus cereus, Bacillus licheniformis, Bacillus pumilus, Brevibacillus laterosporus and Paenibacillus polymyxa as biocontrol agents of four foliar necrotrophic pathogens of wheat in Argentina has been evaluated. The following assays were used: (a) effect of the bacterial antagonists on fungal growth in the central disk test with paired cultures, (b) effect of the antagonists on the germination of fungal spores in the paired suspension assay, and (c) reduction of disease severity in greenhouse experiments. The observed effects were specific for the antagonist-pathogen combination investigated. In the paired culture test, most of the bacterial antagonists inhibited the mycelial growth of the four pathogens tested at 14 days of the evaluation. B. licheniformis showed the best results for controlling Septoria tritici and Drechslera tritici-repentis whereas it was the worst against Bipolaris sorokiniana. In the case of Alternaria triticimaculans, the best were B. laterosporus, P. polymyxa and B. licheniformis. Microscopic examination of excised pieces of the fungal colony perimeters showed clear differences among treatments and controls in hyphal morphology (plasmolysis and vacuolation of hyphae, irregular texture in the cytoplasm, formation of chlamydospores). In the central disk test, mycelial necrosis of A. triticimaculans was induced by B. laterosporus, P. polymyxa and B. pumilus. Similar results were obtained with B. sorokiniana in the presence of B. licheniformis and B. subtilis and D. tritici-repentis with B. pumilus. In some combinations, the presence of inhibition haloes or bacterial chemotaxis were observed. In the paired suspension assay, all the bacteria except B. cereus, inhibited the conidial germination of S. tritici. Only P. polymyxa, B. licheniformis and B. laterosporus inhibited the conidial germination of A. triticimaculans, B. laterosporus being the most effective. Although all the bacteria inhibited the conidial germination of B. sorokiniana, the most efficient were B. licheniformis, B. cereus and B. laterosporus. In greenhouse experiments, P. polymyxa and B. cereus were the most efficient to reduce disease severities produced by S. tritici and A. triticimaculans; on the contrary, these bacteria were not effective against B. sorokiniana and D. tritici-repentis. B. laterosporus BLA 170 showed a good behaviour against all the fungal pathogens, particularly for D. tritici-repentis and B. sorokiniana. This is the first report on the efficacy of B. laterosporus against fungal wheat pathogens.

The influence of secretory-protein charge on late stages of secretion from the Gram-positive bacterium Bacillus subtilis.

Abstract: Following their secretion across the cytoplasmic membrane, processed secretory proteins of Bacillus subtilis must fold into their native conformation prior to translocation through the cell wall and release into the culture medium. The rate and efficiency of folding are critical in determining the yields of intact secretory proteins. The B. subtilis membrane is surrounded by a thick cell wall comprising a heteropolymeric matrix of peptidoglycan and anionic polymers. The latter confer a high density of negative charge on the wall, endowing it with ion-exchange properties, and secretory proteins destined for the culture medium must traverse the wall as the last stage in the export process. To determine the influence of charge on late stages in the secretion of proteins from this bacterium, we have used sequence data from two related alpha-amylases, to engineer the net charge of AmyL, an alpha-amylase from Bacillus licheniformis that is normally secreted efficiently from B. subtilis. While AmyL has a pI of 7.0, chimaeric enzymes with pI values of 5.0 and 10.0 were produced and characterized. Despite the engineered changes to their physico-chemical properties, the chimaeric enzymes retained many of the enzymic characteristics of AmyL. We show that the positively charged protein interacts with the cell wall in a manner that influences its secretion.

Separation of the protease enzymes of Bacillus licheniformis from the fermentation medium by crossflow ultrafiltration

Abstract: The separation from fermentation medium of extracellular serine alkaline protease (SAP) enzyme produced by Bacillus licheniformis was investigated using a crossflow ultrafiltration system. SAP was separated from the high molecular weight neutral protease (NP) and amylase (AMY) enzymes and from the low molecular weight organic acids and amino acids in a crossflow ultrafiltration system with 30 000 Da and 10 000 Da MWCO polysulfone membranes, respectively. The effects of transmembrane pressure (TMP), recirculation velocity (v), and initial enzyme concentration (CE) on the permeate flux, on the activities of SAP, NP and AMY enzymes, and on the recovery of SAP were investigated. High permeate flux was obtained at high recirculation velocity and TMP, but at low initial enzyme concentration. SAP enzyme recovery and activity measured in the system also showed alterations with hydrodynamic conditions. The best operation conditions for the separation of SAP from NP and AMY were TMP = 20 kPa, v = 0.50 ms−1 and CE = 0.28 gdm−3. The separation of SAP from the organic and amino acids was best performed at TMP = 100 kPa, v = 0.33 ms−1 and CE = 0.40 gdm−3. © 2000 Society of Chemical Industry

Novel endo-beta-1,4-glucanases

Abstract: An enzyme exhibiting endo-s-1,4-glucanase activity which belongs to family 9 of glycosyl hydrolases is obtainable from or endogeneous to a strain belonging to the genus Bacillus such as Bacillus licheniformis, ATCC 14580; an isolated polynucleotide (DNA) molecule encoding an enzyme or enzyme core (the catalytically active domain of the enzyme) exhibiting endo-s-1,4-glucanase activity selected from (a) polynucleotide molecules comprising a nucleotide sequence as shown in SEQ ID NO:1 from nucleotide 76 to nucleotide 1455 or from nucleotide 76 to nucleotide 1941, (b) polynucleotide molecules that encode a polypeptide being at least 75% identical to the amino acid sequence of SEQ ID NO:2 from amino acid residue 26 to amino acid 485 or from amino acid residue 26 to amino acid residue 646, and (c) degenerate nucleotide sequences of (a) or (b), the expressed endoglucanase enzyme being useful in various industrial applications.

Genetic Characterization of a New Thermotolerant Bacillus licheniformis Strain

Abstract: A potentially new thermotolerant B. licheniformis strain (code name I89), producer of an antibiotic active against Gram-positive bacteria, was genetically characterized and compared with the type strain B. licheniformis ATCC 10716, producer of bacitracin. Studies on DNA base composition (G + C content) and DNA reassociation revealed that the two strains show around 76% homology. Nevertheless, results obtained by rRNA hybridization, with a heterologous probe coding for most of the 16S region of the rRNA operon of Bacillus subtilis, revealed differences in the number of copies for that gene and in the hybridization pattern. Additionally, a different restriction digestion pattern was obtained when DNA was digested with the enzymes NotI, SmaI and analyzed by PFGE. The I89 strain holds a 7.6-kb plasmid not present in the reference strain. The existence of various unique restriction sites and also the stability of this plasmid make it ideal for the future development of a cloning and expression vector.

Cytotoxicity of alkaline-tolerant dairy-associated Bacillus spp.

Abstract: The cytotoxicity of five Bacillus spp. isolated from alkaline cleaning solutions in South African dairies was evaluated against McCoy mouse cells using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)-based assay, confocal scanning laser microscopy and scanning electron microscopy. According to the MTT-based assay, two of the Bacillus isolates (Bacillus licheniformis 5 and B. pumilus 122) were cytotoxic to McCoy cells and the cytotoxic components were heat labile. Confocal scanning laser microscopy combined with fluorescent staining using propidium iodide and fluorescein diacetate indicated that cytotoxic effects occurred within 3 h, appeared to be membrane active and resulted in cell necrosis. Scanning electron microscopy showed that McCoy cells exposed to the cytotoxic components exhibited morphological damage.

Oligosaccharide synthesis by reversed catalysis using α-amylase from Bacillus licheniformis

Abstract: Evidence is provided for the synthesis of hetero-oligosaccharides by Termamyl, the thermotolerant α-amylase from Bacillus licheniformis. In the presence of soluble starch and added non-starch sugars (isomaltose, cellobiose, mannose, melibiose, stachyose, xylose and 1- o -methyl-β- d -glucopyranoside), oligosaccharides were produced by the reversed catalytic reaction. New peaks of homo- and hetero-oligosaccharides (DP1-10) were detected by high performance anion exchange chromatography (HPAEC) in equilibrium reaction mixtures. Attempts to increase the extent of synthesis of oligosaccharides using organic solvents (ethanol, methanol, N - propanol, propanediol, N - butanol, dioxane and dimethyl sulfoxide (DMSO)) to lower the water activity of the reaction mixture were unsuccessful. Raising the temperature of the reaction mixture from 37°C to 55°C was ineffective.

Lactobacillus plantarum amylase acting on crude starch granules. Native isoforms and activity changes after limited proteolysis.

Abstract: The microheterogeneous native amylolytic complex secreted by the isolate A6 of Lactobacillus plantarum revealed a selective enzyme specificity loss when submitted to a limited proteolysis under a suboptimum pH condition. A clear electrophoretic profile change toward just one shorter, more acidic, and equally active polypeptide fragment resulted from the pronase E pretreatment. Although the whole enzyme activity remained apparently unaffected for soluble starch, the native parallel activity on intact and non-gelatinized starch granules either from cereals or tubers was dramatically reduced. This phenomenon was more clearly documented by scanning electron microscopy using the easiest accessible native substrate: wheat starch granules. The anion-exchange-purified native enzymes from L. plantarum displayed a different optimum pH curve when compared with the thermotolerant α-amylase from Bacillus licheniformis. The α-amylases from the lactic-acid-producing A6 isolate presented an electrophoretic profile easily distinguishable from those from B. liqueniformis and B. subtilis species.

Microbiological investigation on black crusts from open-air stone monuments of Bologna (Italy).

Abstract: Samples of black crusts taken from open-air stone monuments in Bologna dating back to between the twelfth and the nineteenth century were considered. The microbiological test procedures isolated from a high number of chemoorganotrophic bacteria, each of the samples although it was expected to find chemo-lithotrophic (specially sulfur-oxidizing and nitrifying) bacteria on account of the great amounts of sulphur, carbon, nitrogen dioxides and ammonia in the air. Sporogenous and asporogenous species were found among the isolated bacteria: the first were recognized as Bacillus subtilis, Bacillus brevis, Bacillus licheniformis, Bacillus mycoides and Bacillus megaterium; the latter were Corynebacterium glutamicum, Actinomyces sp., Alcaligenes denitrificans subsp. denitrificans, Flavobacterium breve, Acinetobacter calcoaceticus, Enterobacter cloacae, Pseudomonas stutzeri. These species were differently associated in the samples studied with a frequency of more than sixty-four per cent. This finding leads us to believe that their presence in the black crusts is not occasional, but the result of an ecologically significant process of colonization. Although behavioural tests (solubilization of calcium carbonate, of calcium sulphate and marble dust) did not reveal lithic activity among the bacteria studied, one can presume that their behaviour in nature - as a more or less complex microbial consortium - can be totally different and exert a decomposing action with regard to several minerals, most of them acidify the media in which they develop.

The Effect of Oxidizing Biocides on Desulfurication and Denitrification Processes

Abstract: The effect of oxidizing microbiocides (DCDMH and BCDMH) on the activity of bacteria in the processes of denitrification (Bacillus licheniformis) and desulfurication (Desulfotomaculum ruminis) is studied. Desulfotomaculum ruminis bacteria have been found to be more resistant than Bacillus licheniformis bacteria to the inhibiting effect of active halogen released from the studied halogenodimethylhydantoines. The study has shown that the compounds tested can be used as a safe source of slowly released halogen for disinfection.

Enzymatic formation of Glu-Xaa and Asp-Xaa bonds using Glu/Asp-specific endopeptidase from Bacillus licheniformis in frozen aqueous systems.

Abstract: The capability of Glu/Asp-specific endopeptidase from Bacillus licheniformis to form Glu/Asp-Xaa bonds in frozen aqueous systems was investigated. Under frozen state conditions, the enzyme was able to catalyse peptide bond formation more effectively than in liquid reaction mixtures. The acceptance of amino components which were completely inefficient nucleophiles at room temperature indicates a changed specificity of Glu/Asp-specific endopeptidase under frozen state conditions. Protease-catalysed coupling of two acidic amino acids was demonstrated for the first time. The utilization of Glu/Asp-specific endopeptidase from Bacillus licheniformis in frozen aqueous systems offers new possibilities in enzyme-catalysed peptide synthesis.