Production of alkaline xylanase by an alkaliphilic Bacillus sp. isolated from an alkalinesoda lake

An alkaline xylanase-producing alkaliphilic Bacillus sp. AR-009 was isolated from analkaline soda lake in Ethiopia. The enzyme was optimally active at pH 9 and was stable over abroad pH range. The optimum temperature for xylanase activity, assayed at pH 9, was60°–65°C. Measured at pH 8 and 9, the enzyme had good stability at 55° and60°C. At both pH values, over 80% of its original activity was retained after heating for2·5 h at 55°C. At 60°C, the enzyme maintained 63% of its original activity after2·5 h incubation while at pH 9 it retained 54% of its original activity after 1 h heating. Theseproperties qualify the enzyme to be novel and potentially important for application in someindustrial processes.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

Thermophilic denitrifying bacteria: A survey of hot springs in Southwestern Iceland

Abstract Samples of water, sediment and bacterial mat from hot springs in Grændalur and Hveragerdi areas in southwestern Iceland were screened at 70°C and 80°C for thermophilic denitrifying bacteria by culturing in anaerobic media containing nitrate or N₂O as the terminal oxidant. The s springs ranged in temperature from 65–100°C and included both neutral (pH 7–8.5) and acidic (pH 2.5–4) types. Nitrate reducing bacteria (nitrate → nitrite) and denitrifiers (nitrate → N₂) were found that grew at 70°C but not at 80°C in nutrient media at pH 8. Samples from neutral springs that were cultured at pH 8 failed to yield a chemolithotrophic, sulfur-oxidizing and nitrate-reducing bacterium, and samples from acidic springs that were cultured at pH 3.5 seemed entirely to lack dissimilatory, nitrate-utilizing bacteria. No sample yielded an organism capable of growth solely by N₂O respiration. The denitrifiers appeared to be Bacillus . Two such Bacillus strains were examined in pure culture and found to exhibit the unusual denitrification phenotype described previously for the mesophile, Pseudomonas aeruginosa , and one other strain of thermophilic Bacillus . The phenotype is characterized by the ability to grow by reduction of nitrate to N₂ with N₂O as an intermediate but a virtual inability to reduce N₂O when N₂O was the sole oxidant.     

New Strains of Bacillus licheniformis and Bacillus coagulans producing Thermostable α-Amylase Active at Alkaline pH

Two species of Bacillus producing thermostable α-amylase with activity optima at alkaline pH are reported here. These organisms were isolated from soil and have been designated as Bacillus licheniformis CUMC 305 and B. coagulans CUMC 512. The enzymes released by these two species were partially purified up to about 81- and 72-fold respectively of the initial activity. The enzyme from B. licheniformis showed a wide temperature-range of activity, with optimum at 91°C. At this temperature it remained stable for 1 h. It retained 40–50% activity at 110°C and showed only 60% of its activity at 30°C. The enzyme showed a broad pH range of activity (4–10) retaining substantial activity on the alkaline side. The optimum pH was 9·5. The enzyme of B. coagulans showed activity up to 90°C, with optimum at 85°C and had a wide pH range with optimum at 7·5–8·5. The hydrolysis pattern of the substrate starch by these enzymes indicated that glucose, maltose, maltotriose and maltotetraose are the principal products rather than higher oligosaccharides.     

Amylase production by a Gram-positive bacterium isolated from fermenting tef (Eragrostis tef)

Bacillus sp. A-001, which produced large amounts of amylase, was isolated from fermenting tef ( Eragrostis tef ) on tryptone soya agar supplemented with 1% starch. The organism grew between pH 4.5 and 10.5 with an optimum at 7–7.5. Growth occurred between 20 and 55°C but the optimum was about 35–40°C. At optimum medium pH (7.5) and a temperature of 35°C the organism entered the stationary phase after about 72 h and amylase production was at its highest (9.6 units ml^-1) at this time. Enzyme activity was optimal at pH 5.5 and 40°C and showed good thermal stability; it required 110 min to lose 50% of its activity at 70°C. The enzyme hydrolysed native starch (flour from tef, corn and kocho) to various oligosaccharides, including maltotriose, maltose and glucose.     

Bacillus subtilis and B. mojavensis strains connected to food poisoning produce the heat stable toxin amylosin

Aim:  To screen and characterize toxic, heat-stable substances produced by food borne strains from Bacillus subtilis group.
Methods and Results:  Using the boar sperm motility inhibition assay, six isolates from two outbreaks, out of the 94 isolates from 26 foods, were found to produce ethanol-soluble heat-stable substances that were toxic to sperm cells by depleting the mitochondrial membrane potentials. The toxic isolates were identified as Bacillus subtilis and B mojavensis. Colon carcinoma cells (Caco-2) were used to model the contact with the human digestive tract. The extract of B. subtilis F 2564/96 depolarized the mitochondria in intact Caco-2 cells similarly as in sperm cells. The substance responsible for these effects was purified using HPLC and identified by electron spray ionization ion trap mass spectrometry analysis as amylosin. The temperature requirement for amylosin production was 21–37°C for B. subtilis and 11–21°C for B. mojavensis . Both species produced amylosin in air as well as in 7–8% CO₂ with 8–9% O_2.
Conclusions:  Food borne illness related strains of B. subtilis and B. mojavensis, produced the heat-stable toxin amylosin.
Significance and Impact of the Study:  This is the first report that suggests a role for the heat-stable, ion-channel forming toxin amylosin, as a virulence factor in food borne Bacillus .     

Effect of temperature on the vegetative growth of type and field strains of Bacillus cereus

Growth of eight selected potentially pathogenic strains of Bacillus cereus was evaluated in a rich medium at different temperatures. No strain grew at 50°C; maximal growth-permissive temperatures were in the range 46–50°C for six strains and under 46°C for one strain. Faster growth occurred at 42°C. Growth may be delayed at 20°C, where the lag phase can reach 7 h. Furthermore at 20°C, cells generally show an aggregation immediately in the early exponential stage, except for two strains. Owing to this aggregation, growth is more difficult to estimate by turbidimetry at lower temperatures. These data describe the behaviour of type and field strains between 50° and 20°C and can help the prediction of shelf-life of potentially contaminated products.     

A highly thermostable amylase from a newly isolated thermophilic Bacillus sp. WN11

A thermostable amylase-producing Bacillus sp. WN11 was isolated from Wondo Genet hot spring in Ethiopia. The enzyme had a temperature optimum of 75–80 °C. Over 80% of its peak activity was in the pH range of 5–8, with an optimum at 5·5. Thermal stability of the enzyme at 105 °C was higher with the addition of starch. The stabilizing effect of starch was concentration-dependent, showing better stability with increasing concentration of starch. At liquefying temperature (105 °C), addition of Ca²⁺ did not result in further improvement of the stabilizing effect of starch. This indicates that in the presence of starch, WN11 amylase does not require Ca²⁺ as a stabilizer at liquefying temperatures as high as 105 °C.     

Growth of Listeria monocytogenes at refrigeration temperatures

The growth of three strains of Listeria monocytogenes at refrigeration temperatures (-0.5 to 9.3°C) in chicken broth and/or UHT milk was determined using a rocking temperature gradient incubator. Minimum growth temperatures ranged from -0.1 to -0.4°C for the three strains. Lag times of 1–3 d and 3 to >34 d were observed with incubation at 5 and 0°C respectively. Corresponding generation times ranged from 13–24 h at 5°C and 62–131 h at 0°C. The type of culture medium had an influence on both the rate and extent of growth. Incubation of cultures at 4°C before inoculation caused a marked reduction in the lag time when compared with cultures which had been previously incubated at 30°C.     

Hemicellulases of Bacillus species: preliminary comparative studies on production and properties of mannanases and galactanases

A range of Bacillus subtilis strains and other Bacillus species were screened for mannanase, β-mannosidase and galactanase activities. Maximum mannanase activity, 106.2 units/ml, was produced by B. subtilis NRRL 356. β-Mannosidase and galactanase activities from all strains were relatively low. The effect of carbon and nitrogen source on mannanase and galactanase production by B. brevis ATCC 8186, B. licheniformis ATCC 27811, B. polymyxa NRRL 842 and B. subtilis NRRL 356 was investigated. Highest mannanase production was observed in the four strains tested when the mannan substrate, locust bean gum, was used as carbon source. Induction was most dramatic in the case of B. subtilis NRRL 356 where only basal enzyme levels were produced in the presence of other carbon sources. β-Mannosidase was induced in the four Bacillus cultures by locust bean gum. Results indicated that galactose acted as an inducer for production of galactanase. Organic and inorganic nitrogen sources resulted in induction of high mannanase titres in B. subtilis. Highest galactanase activity was produced by each organism in media containing sodium nitrate as nitrogen source. Mannanases from B. brevis, B. licheniformis, B. polymyxa and B. subtilis retained 100% residual activity after a 3 h incubation at 65°C, 65°C, 60°C and 55°C respectively. Galactanases retained more than 95% activity at 55°C after 3 h. The pH optima of mannanases ranged from 6.5–6.8 whereas galactanases ranged from 5.1 in the case of B. brevis to 7.0 for B. polymyxa.     

Partial purification and characterization of phospholipase C from Yersinia enterocolitica

About 34% of the strains of Yersinia enterocolitica isolated from raw milk were found to produce lecithinase. A selected strain produced phospholipase C at 22°C and 37°C; production was optimum at 37°C in the stationary phase (14–16 h). A decrease in phospholipase C activity at various storage temperatures (—5°C, 4°C, 37°C) was also observed, although the enzyme was active over a wide range of temperature (5–65°C) and pH (3mD5–7mD5). The phospholipase C was partially purified by ammonium sulphate precipitation and Sephadex column chromatography, and characterized.     

Conservation of antigenic determinants in leucine dehydrogenase from thermophilic and mesophilic Bacillus strains

Abstract Antibodies against the purified octameric l -leucine dehydrogenase (LeuDH) from the mesophilic Bacillus cereus have been used to screen 16 thermophilic Bacillus strains for LeuDH. 4 of these strains, Bacillus sphaericus 461 and Bacillus sp. 405, 406, and 411, showed a particularly strong cross reaction of the partial identity type when examined by Ouchterlony double diffusion assay, thus indicating that they were immunologically related to the B. cereus enzyme. The LeuDH from the thermophilic strains were very stable and highly active at elevated temperatures, and gave a downward bend at about 55°C in the Arrhenius plot. The pH optimum for l -leucine deamination was around pH 11 for all strains examined.     

Direct induction of homozygous diploidization in the fission yeast Schizosaccharomyces pombe by pressure stress

Abstract Hydrostatic pressure stress and a dye plate method were first used to investigate the direct induction of homozygous diploids from the haploid yeast Schizosaccharomyces pombe . Above 100 MPa at 25 °C for 10 min, pressure stress greatly inactivated the haploid strains of JY1 (L972 h ⁻) JY3 (L975 h ⁹⁰) and JY334 ( ade 6-M216 leul h ⁺). At the same time, when pressure stressed cells of these strains at more than 100–200 MPa were spread on a dye plate, some pressure-effected visible colonies were stained violet (variant colonies); the rest were stained pink, similar to colonies originating from haploid cells that were not pressure-stressed. Based on the cell size, DNA content, crosses, and random spore analyses for the segregation of mating types or auxotrophic markers, variant cells originating from color changed colonies of JY1 after pressure stress were very stable and found to be homozygous diploid with an h− / h− genotype at the mating-type locus. From these results we conclude that pressure stress in combination with a dye plate is a simple and useful method for direct induction of homozygous diploid cells with very high stability.     

Simple Detection of Xylosidase Activity in Single Colonies,Using 1-Naphthyl-β-d-xylopyranoside

Since the xylosidase of Bacillus pumilus hydrolyzed 1-naphthyl-β-d-xylopyranoside (naphthyl-X) to produce xylose and 1-naphthol and a chromogenic azo compound is produced by coupling 1-naphthol and Fast Blue Salt B, a simple method for detection of xylosidase activity in single colonies was studied. Escherichia coli JM109 carrying the xylosidase gene of B. pumilus was cultivated at 37°C for 18 h on an LB plate containing 0.5 mg/ml naphthyl-X, and then the plate was overlaid with 3 ml of a top layer containing 24 mg of agar and 6 mg of Fast Blue Salt B. After incubation of the plate at 37°C for 1 h, each colony became reddish-brown. Even a small colony with the xylosidase on the plate was easily distinguished from colonies without the enzyme.     

Cultural conditions for production of glucoamylase from Lactobacillus amylovorus ATCC 33621

Lactobacillus amylovorus ATCC 33621 is an actively amylolytic bacterial strain which produces a cell-bound glucoamylase (EC 3.2.1.3). Conditions of growth and glucoamylase production were investigated using dextrose-free de Man-Rogosa-Sharpe (MRS) medium in a 1.5 I fermenter, with varying dextrin concentration (0.1–1.5% (w/v)), pH (4.5–6.5) and temperature (25–55°C). Cell extracts were prepared by subjecting cells to treatment with a French Pressure cell in order to release intracellular proteins. Glucoamylase activity was then assayed. The effects of pH (4.0–9.0), temperature (15–85°C) and substrate (dextrin and starch, 0–2% w/v) concentration on crude enzyme activity were investigated. Optimal growth was obtained in MRS medium containing 1% (w/v) dextrin, at pH 5.5 and 37°C. Glucoamylase production was maximal at the late logarithmic phase of growth, during 16–18 h. Crude enzyme had a pH optimum of 6.0 and temperature optimum of 60°C. With starch as the substrate, maximal activity was obtained at a concentration of 1.5% (w/v). The effects of ions and inhibitors on glucoamylase activity were also investigated. Enzyme activity was not significantly influenced by Ca²⁺ and EDTA at 1 mmol 1⁻¹ concentration; however Pb²⁺ and Co²⁺ were found to inhibit the activity at concentrations of 1 mmol 1⁻¹. The crude enzyme was found to be thermolabile when glucoamylase activity decreased after about 10 min exposure at 60°C. This property can be exploited in the brewing of low calorie beers where only mild pasteurization treatments are used to inactivate enzymes. The elimination of residual enzyme effect would prevent further maltodextrin degradation and sweetening during long-term storage, thus helping to stabilize the flavour of beer.     

Proteolytic activity from a thermophilic Streptomyces megasporus strain SDP4

Multiple proteases secreted by a thermophilic actinomycete Streptomyces megasporus SDP4 after 18 h of growth at 55 °C are reported. The enzyme preparation exhibited activity over a broad pH and temperature range of pH 6–12 and 25–85 °C, respectively. Optimum activity was observed at pH 8·0, pH 10·0 and 55 °C and was calcium independent. Thermostability was enhanced in the presence of 0·01 mol l⁻¹ calcium ions and half-life was 30 min at 85 °C. The enzyme was active in the presence of SDS. Both, EDTA and PMSF were partially inhibitory, indicating the presence of serine and metal requiring proteases. Three active zones in the range of 90–30 kDa were detected post-electrophoretically.     

Cell-Bound Thiaminase I of Bacillus thiaminolyticus

The distribution of the extracellular enzyme, thiaminase I, was determined for logarithmically growing cultures of Bacillus thiaminolyticus. About 60% of the enzyme is associated with the cells throughout the growth cycle. The remainder of the enzyme is in the culture medium. The release of the cell-bound thiaminase I is examined under a variety of conditions. The rate and extent of release is dependent on the pH and the nature of the incubation solution. The release process appears to be relatively independent of de novo protein synthesis, energy derived from oxidative phosphorylation, or divalent metal ions. The absence of carbon or nitrogen sources has little effect on the release of the enzyme. Cell-bound thiaminase I probably is the immediate precursor for extracellular thiaminase I found in the culture medium. Washed cells continue to release thiaminase I at the expense of cell-bound enzyme. In addition, purified cell-bound thiaminase I is indistinguishable from purified extracellular thiaminase I by a number of physical and kinetic criteria.     

The sporicidal activity and inactivation of chlorhexidine gluconate in aqueous and alcoholic solution

The sporicidal activity of chlorhexidine gluconate in aqueous and alcoholic solution against spores of Bacillus subtilis was examined over a broad temperature range. Activity was not observed at 20°C even with concentrations as high as 10% chlorhexidine. Temperatures of 37°–70°C in combination with such high concentrations were required for reductions in spore viability. No viable spores were recoverable after 4 h contact at 55°C with 10% aqueous chlorhexidine and none after 3 h contact with the alcoholic solution. Because of the high concentrations necessary for activity and the possibility of sporostasis occurring from inefficient chlorhexidine inactivation, existing inactivation systems were examined and modified to obtain satisfactory results. The spores of other Bacillus species examined ( B. cereus, B. megaterium and B. stearothermophilus ) proved to be considerably less resistant than those of B. subtilis. Presence of organic matter had little effect on the activity.     

Production of thiaminase I for analytical purposes

A simple medium enhancing the production of thiaminase I (EC 2.5.1.2) by Bacillus thiaminolyticus was developed. Ca²⁺ stimulated the enzyme production. The activity of extracellular thiaminase I ranged between 1.29 and 1.33 U/ml medium.