Involvement of etfA gene during CaCO3 precipitation in Bacillus subtilis biofilm

The eftA gene in Bacillus subtilis has been suggested to be involved in the oxidation/reduction reactions during fatty acid metabolism. Interestingly etfA deletion in B. subtilis results in impairment in CaCO₃ precipitation on the biofilm. Comparisons between the wild type B. subtilis 168 and its etfA mutant during in vitro CaCO₃ crystal precipitation (calcite) revealed changes in phospholipids membrane composition with accumulation of up to 10% of anteiso-C_17:0 and 11% iso-C_17:0 long fatty acids. Ca²⁺ nucleation sites such as dipicolinic acid and teichoic acids seem to contribute to the CaCO₃ precipitation. etfA mutant strain showed up to 40% less dipicolinic acid accumulation compared with B. subtilis 168, while a B. subtilis mutant impaired in teichoic acids synthesis was unable to precipitate CaCO₃. In addition, B. subtilis etfA mutant exhibited acidity production leading to atypical flagella formation and inducing extensive lateral growth on the biofilm when grown on 1.4% agar. From the ecological point of view, this study shows a number of physiological aspects that are involved in CaCO₃ organomineralization on biofilms.     

Anatomical and Biochemical Changes Associated with In Vitro Rhizogenesis in Dendrocalamus giganteus

Successful micropropagation protocol of a difficult-to-root bamboo species, Dendrocalamus giganteus (10–15 years old) along with the analysis of anatomical and biochemical changes during in vitro rhizogenesis was accomplished. Proliferated axillary shoots from nodal segments of 10–15 years old field culms exhibited shoot necrosis during multiple shoot formation phase and was controlled by subculturing in modified MS liquid medium having 825 mg ^l?1 NH₄NO₃, 3800 mg ^l?1 KNO₃, 740 mg ^l?1 MgSO₄ and 9% coconut water, 26.64 μM 6-benzylaminopurine (BA) and 0.46 μM kinetin. These multiple shoots proliferated from field grown culms, failed to root and hence callus was induced on MS solid medium containing 4.44 μM BA, 4.52 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.37 μM naphthalene acetic acid (NAA). Organogenesis from the callus was achieved upon transfer to MS medium with 11.10 μM BA and 2.32 pM kinetin. The callus-derived shoots multiplied on modified MS medium were rooted the best (91%) by culturing 3 days on MS medium having glucose (0.5%), sucrose (2.5%) and 98.41 μM indolebutyric acid (IBA) and subsequently to IBA-free MS medium containing 3% sucrose. Studies on peroxidase and IAA oxidase activity and endogenous free- and bound-IAA content showed that IAA oxidase and peroxidase oxidize endogenous IAA resulting in root initials formation. Anatomical studies confirmed the root primordia formation from 3^rd day of IBA treatment and primordia were visible over the surface on 8^th to 10^th day. However, the shoot necrosis symptoms which started on 6^th day of treatment intensified by 10^th day leading to the death of the whole shoot system by 12^th–15^th day. Nevertheless, on the root formation medium with 9.84 μM IBA, new shoot buds were emerged and showed shoot growth in 60% of the rooted cultures, which were successfully acclimatized in shade-house with 100% survival. The present study establishes rooting of callus-derived shoots as the best way for the successful propagation of the difficult-to-root bamboo, D. giganteus when compared to axillary bud proliferated shoots.     

Selection methods in bacilli for recombinants and transformants of intra- and interspecific fused protoplasts

The intraspecific fusion frequencies obtained with the direct selection method on a semi-synthetic regeneration medium between strains of B. subtilis and B. licheniformis were distribution from 9.9×10^-2 to 4.5×10^-3, which was one or two orders higher than those of interspecific recombinations between B. subtilis and B. licheniformis.The regeneration media were also useful for selecting interspecific transformants from plasmid carrier to non-carrier. This selection could be used as a primary selection method for inter-and intraspecific recombinants obtained by protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - Cm chloramphenicol - SMM 0.5 M sucrose-0.02 M MgCl₂-0.02 M maleate buffer, pH 6.5     

Characterisation,screening and selection of Bacillus subtilis isolates for its biocontrol efficiency against major rice diseases

A Soil bacterium, Bacillus subtilis, isolated from the rhizosphere of rice, showed high biocontrol activity against blast, sheath blight and bacterial blight. In the present study, four B. subtilis strains isolated from paddy soil were studied under laboratory, greenhouse and field conditions. Among the four strains assayed, UASP17 gave maximum inhibition of paddy pathogens and was validated under field trials. B. subtilis (UASP17) under different doses and methods of applications was evaluated for two seasons at Agriculture Research Station (UAS, Raichur). UASP17 was effective in reducing the severity of blast (9.00% and 15.57%), bacterial leaf blight (BLB) (5.00% and 6.11%) and sheath blight (11.93% and 4.17%) diseases for two seasons. The application of bioagent also increased the paddy grain yield (61.00 and 64.30?Q/ha) in the two seasons, respectively. Taken together, these results indicate that B. subtilis UASP17 as seedling dip for 30?min (10?mL/L of water) prior to transplanting and 2.50?L/ha foliar application was effective in managing the diseases of paddy.     

LHRH-receptor-regulated Ca2+-ATPase activity in murine pituitary gland

Addition of luteinizing hormone releasing hormone (LHRH) in vitro (10^–5–5×10^–9 M) to murine pituitary membranes resulted in a dose-related decrease in Ca²⁺-ATPase activity within 15 min. Inhibitory effects of LHRH (10^–7 M) occurred after 90 sec, and appeared maximal by 120 sec. Eadie-Hofstee analysis at 10^–7 M LHRH, at varying [Ca²⁺]_free, resulted in aK _m=0.89±0.06 M and aV _max=18.8±0.71 nmol/mg per 2 min, compared to aK _m=0.69±0.06 M and aV _max=32.8±1.21 nmol/mg per 2 min for controls. Pre-incubation for 5 min with LHRH antagonist (10^–8 M) significantly attenuated (50%) the inhibitory effects of 10^–7 M LHRH on pituitary Ca²⁺ ATPase activity with aK _m=0.97±0.24 M and aV _max=28.1±2.8 nmol/mg per 2 min. The addition of LHRH (10^–7 M) to pituitary homogenates significantly increased luteinizing hormone (LH) release already at 10 and up to 40 sec compared to basal LH release. Systemic administration of 50 ng LHRH (i.p.), significantly (P<0.05) reduced pituitary Ca²⁺-ATPase after 30, 60 and 90 min, with a return to control levels by 120 min. Pituitary LH content was reduced slightly at 15 min, but was increased significantly at 90 and 120 min post-treatment. Plasma LH levels were elevated by 5 min, reached a peak by 15 min and returned to control within 60 min. The present findings indicate that LHRH receptor activation may influence cytosolic Ca²⁺ transport through effects on membrane Ca²⁺-ATPase activity. These actions may regulate LHRH-induced synthesis, storage and release of LH from pituitary gonadotropes.     

Biological control of root rot fungus <Emphasis Type="Italic">Macrophomina phaseolina</Emphasis> and growth enhancement of <Emphasis Type="Italic">Pinus roxburghii</Emphasis> (Sarg.) by rhizosphere competent <Emphasis Type="Italic">Bacillus subtilis</Emphasis> BN1

Bacterial isolates having antifungal and good plant growth-promoting attributes were isolated from chir-pine (Pinus roxburghii) rhizosphere. An isolate, Bacillus subtilis BN1 exhibited strong antagonistic activity against Macrophomina phaseolina, and other phytopathogens including Fusarium oxysporum and Rhizoctonia solani. It was characterized and selected for the present studies. BN1 resulted in vacuolation, hyphal squeezing, swelling, abnormal branching and lysis of mycelia. The cell-free culture filtrate of BN1 inhibited the growth of M. phaseolina. Pot trial study resulted in statistically significant increase in seedling biomass besides reduction in root rot symptoms in chir-pine seedlings. BN1 treatment resulted in 43.6% and 93.54% increases in root and shoot dry weights respectively, as compared to control. Also, 80–85% seed viability was recorded in treatments receiving BN1 either alone or in the presence of M. phaseolina, compared to 54.5% with M. phaseolina. Bioinoculant formulation study suggested that maximum viability of bacteria was in a sawdust-based carrier. B. subtilis BN1 produced lytic enzymes, chitinase and β-1,3-glucanase, which are known to cause hyphal degradation and digestion of the cell wall component of M. phaseolina. In the presence of M. phaseolina, population of B1 was 1.5 × 10⁴ c.f.u. g⁻¹ root after one month, which increased to 4.5 × 10⁴ c.f.u. g⁻¹ root in three months. Positive root colonization capability of B. subtilis BN1 proved it as a potent biocontrol agent.     

Enzymic Dissociation of Zea Shoot Cell-Wall Polysaccharides V. Dissociation of Xyloglucan by Urea

A procedure has been devised to extract and identify structuralcomponents of the xyloglucan of Zea mays L. (hybrid B73 ? Mo17) shoot cell-walls. A water-insoluble fraction of Zea shootcell-walls, after pretreatment with purified Bacillus subtilis(1 3),(1 4)-ß-D-glucan 4-glucanohydrolase, purifiedB. subtilis endo-(l 4)-ß-xylanase and an enzyme preparationfrom B. subtilis enriched in glucuronoxylanase (Kato and Nevins1984a, Nishitani and Nevins 1991), was subsequently treatedwith 7 M urea. The carbohydrates (0.8% of the water-insolublefraction of Zea shoot cell-walls) liberated by the urea treatment,were comprised of xyloglucan polymers with molecular weightswhich varied from 1.0 ? 10⁴ to 4.0 7times; 10⁴ Da. Other wallfragments associated with the isolated polymer suggest covalentbonding of xyloglucan to other polysaccharides. Structural analysesof the xyloglucan polymers reveal a cellulose-like backbonewith about 35% of the C-6 positions substituted with xyloseand other sugars. About 80% of xyloglucan present in the enzyme-pretreatedwater-insoluble fraction of Zea shoot cell-walls was liberatedby the urea treatment. The procedure avoids the use of alkaliin the solubilization of xyloglucan. ¹Supported in part by National Science Foundation research grantsPCM 7818588 and DMB 8505901. (Received September 10, 1990; Accepted May 15, 1991)     

Microbacterium suwonense sp. nov., isolated from cow dung

An actinomycete strain, designated M1T8B9^T, was isolated from cow dung in Suwon, Republic of Korea. The isolate was a Gram-positive, nonmotile, and non-spore-forming bacterium. Phylogenetic evaluation based on 16S rRNA gene sequence similarity showed that this isolate belongs to the genus Microbacterium, with its closest neighbors being Microbacterium soli DCY17^T (98.2%) and Microbacterium esteraromaticum DSM 8609^T (98.0%). The polar lipid pattern consisted of diphosphatidylglycerol, phosphatidylglycerol, and one unknown glycolipid. Strain M1T8B9^T contained the major fatty acids C_15:0 anteiso, C_16:0 iso, C _17:0 anteiso, and C_15:0 iso, and the cell-wall peptidoglycan was of type B2β. According to DNA-DNA hybridization studies, strain M1T8B9^T showed 42% and 26% relatedness with M. soli DCY17^T and M. esteraromaticum DSM 8609^T, respectively. On the basis of the data presented, strain M1T8B9^T is considered to represent a novel species of the genus Microbacterium, for which the name Microbacterium suwonense sp. nov. is proposed. The type strain is M1T8B9^T (=KACC 14058^T =NBRC 106310^T).     

Direct induction of protocorm-like bodies from shoot tips, plantlet formation, and clonal fidelity analysis in Anthurium andreanum cv. CanCan

Protocorm-like bodies (PLBs) were induced directly at high frequency from wounded surface of Anthurium andreanum cv. CanCan shoot tip-ends, used as explants. In order to obtain PLB directly, the influence of different types and concentrations of cytokinins were evaluated. Amid the cytokinins, N⁶-(?²-isopentenyl)-adenine (2-iP) at a concentration of 15?μM was most effective in inducing PLB whereby ~98 (97.8)?% of explants induced PLB with an average of 120 PLBs per shoot tip within 50?days of culture. Stereomicroscopic observation meticulously revealed the sequential changes from initiation to maturation of PLB gradually forming shoot apical meristem, shoot primordia and leaf primordia. Mature PLBs showed significant shoot proliferation (98.4?%) in media containing 10?μM 6-furfurylaminopurine forming 17 shoots per PLB within 30?days. The inclusion of activated charcoal (AC) in media containing auxin had promotive effect on rooting whereby 5?μM indole-3-butyric acid plus 500?μM AC resulted in highest number and length of roots. Successfully acclimatized plants, subjected to random amplified polymorphic DNA assessment for genetic fidelity, did not show any variation. Thus, this complete study has successfully outlined a rapid, high frequency direct induction of PLB of Anthurium from shoot tips inclusive of shoot proliferation, rooting and acclimatization.     

Further survey of chromosomal polymorphisms in the freshwater planarian Polycelis auriculata

Although the chromosomal number of Polycelis (Seidlia) auriculata Ijima et Kaburaki is basically 2n = 6, this species shows remarkable chromosomal polymorphisms in different populations. Among worms collected at 30 stations in the central part of Japan's Main Island and in Hokkaido, we found five new karyotypes, considered to be variations of tetraploidy and diploidy: 4n?fis¹. + 1 = 3SM + 2A + 3T₁ + 2T₂ + 4M = 14, 4n?fis².(B) = 2SM + 2A + 4T₁ + 2T₂ + 4M + (0?2B) = 14 –16, 4n?fis². + 1 = 2SM + 2A + 4T₁ + 2T₂+5M= 15, 4n?fis².?2 = 2SM + 2A + 4T₁ + 4M = 12, and 2nM- = 2SM + 2A = 4 (where SM = submetacentric; A = acrocentric; T₁ and T₂ = telocentric; M = metacentric; B = B-chromosome; fis¹. and fis². = occurrence of one or two centric fissions, respectively; and M- = deletion of a metacentric chromosome). These karyotypes exist singly in one individual or as various mixoploids within the same individual. No individual having only karyotype 2nM- has yet been found. This brings the total number of known karyotypes in P. auriculata to 17; these can be arranged in an order that we believe reflects their evolution.     

Genetic and substrate-level modulation of Bacillus subtilis physiology for enhanced extracellular human interferon gamma production

Human interferon-gamma (hIFNG) production is limited by various gene-level bottlenecks including translation, protein folding, and secretion which depends upon the physiological state of the organism. In this study gene-level and substrate-level modulations have been used to control Bacillus subtilis physiology for >15 fold extracellular soluble hIFNG production. Two variants of the native human interferon-gamma gene (hifng) were designed and synthesized, namely, cohifng_his and cohifng having codon adaptation index 25.33 and 26.89% higher than the native gene, respectively. BScoIFNG and BScoIFNGhis with ΔG of ?100.0 and ?113.7?kcal?mol^?1 resulted in 30 and 6.5% higher hIFNG compared to the native gene in complex medium. BScoIFNG produced 1.53 fold higher hIFNG using glucose-based defined medium as compared to the complex medium by modulating the physiological parameter growth rate from 0.35 to 0.26?hr^?1. Further modulatory effect of various phosphotransferase transport system (PTS) and no-PTS sugars, sugar alcohols, and organic acids was quantified on the physiology of B. subtilis WB800N for extracellular hIFNG production. Sorbitol and glycerol emerged as the best hIFNG producers with lowest growth and substrate consumption rates. BScoIFNG produced maximum 3.15?mg?L^?1 hIFNG at 50?g?L^?1 glycerol with highest hIFNG yield (Y_p/x?=?0.136) and lowest substrate uptake rate (q_s?=?0.26).     

Multifarious potential applications of keratinase of Bacillus subtilis K-5

Abstract

Bacillus subtilis K-5, an isolate from compost, utilized a wide range of keratinous wastes viz. diverse feather types, nails, hair, scales, etc. for growth and produced a thermostable alkaline protease (keratinase) with broad proteolytic activity. Optimization of cultural and environmental variables using a Plackett–Burman design and response surface methodology resulted in enhanced keratinase production (89%). Keratinase was partially purified (15-fold) by ammonium sulfate precipitation and carboxymethyl cellulose chromatography. The optimum pH and temperature for keratinase activity were 9.0 and 60°C, however, considerable activity and stability was observed over broad pH (5–10) and temperature range (50–90°C). B. subtilis K-5 keratinase exhibited excellent stability toward detergents (cetyl trimethylammonium bromide, Tween 80, and sodium dodecyl sulfate) and organic solvents (benzene, acetonitrile, phenylmethylsulfonyl fluoride); however, metal ions like Mn²⁺, Cu²⁺, Na⁺, Hg²⁺, K⁺, Ca²⁺, and Zn²⁺ inhibited the activity. B. subtilis K-5 protease showed remarkable potential for diverse applications like blood stain removal, gelatin hydrolysis from waste X-ray films and dehairing of animal hide.     

Effects of levobupivacaine and bupivacaine on intracellular calcium signaling in cultured rat dorsal root ganglion neurons

Bupivacaine and levobupivacaine have been shown to be effective in the treatment of pain as local anesthetics, although the mechanisms mediating their antinociceptive actions are still not well understood. The aim of this study was to investigate the effects of bupivacaine and levobupivacaine on intracellular calcium ([Ca²⁺]_i) signaling in cultured rat dorsal root ganglion (DRG) neurons. DRG neuronal cultures loaded with 5?μM Fura-2/AM and [Ca²⁺]_i transients for stimulation with 30?mM KCl (Hi K⁺) were assessed by using fluorescent ratiometry. DRGs were excited at 340 and 380?nm, emission was recorded at 510?nm, and responses were determined from the change in the 340/380 ratio (basal-peak) for individual DRG neurons. Data were analyzed by using Student’s t-test. Levobupivacaine and bupivacaine attenuated the KCl-evoked [Ca²⁺]_i transients in a reversible manner. [Ca²⁺]_i increase evoked by Hi K⁺ was significantly reduced to 99.9?±?5.1% (n?=?18) and 62.5?±?4.2% (n?=?15, P?<?0.05) after the application of 5 and 50?µM levobupivacaine, respectively. Bupivacaine also inhibited Hi K⁺-induced [Ca²⁺]_i responses, reduced to 98.7?±?4.8% (n?=?10) and 69.5?±?4.5% (n?=?9, P?<?0.05) inhibition of fluorescence ratio values of Hi K⁺-induced responses at 5 and 50?μM, respectively. Our results indicate that bupivacaine and levobupivacaine, with no significant differences between both agents, attenuated KCl-evoked calcium transients in a reversible manner. The inhibition of calcium signals in DRG neurons by levobupivacaine and bupivacaine might contribute to the antinociceptive effects of these local anesthetics.     

The contribution of the cell wall to a transmembrane calcium gradient could play a key role in Bacillus subtilis protein secretion

A weak Ca²⁺-binding site (K_a= 0.8× 10³ M^?1, at pH7) was identified in the mature part of levansucrase. An amino acid substitution (Thr-236 →lle) in this site alters simultaneously the affinity for calcium, the folding transition and the efficiency of the secretion process of levansucrase. Moreover, the ability of the Bacillus subtilis cell wall to concentrate calcium ions present in the culture medium was studied. We confirm the results of Beveridge and Murray who showed that the concentration factor is about 100 to 120 times. This property preserves a high concentration of Ca²⁺ (>2 mM) on the external side of the cytoplasmic membrane, even in the absence of further Ca2+ supplementation in the growth medium. Such local conditions allow the spontaneous unfolding folding transition of levansucrase en route for secretion. Since several exocellular proteins of B. subtilis are calcium-binding proteins, we propose that the high concentration of calcium ion in the microenvironment of the cell wall may play a key role in the ultimate step of their secretion process.     

Inhibition of Legionella pneumophila by Bacillus sp.

This study presents the potential of a biological control of Legionella pneumophila. It was verified to what extent the enrichment of various Bacillus species in water may decrease or prevent L. pneumophila from growing in water. During in vitro tests, B. subtilis BS104 was able to induce an average decrease in L. pneumophila numbers of 1.9 + 0.2 log units after 120 h. Furthermore, the spore and cell free filtrate of B. subtilis BS104 also decreased L. pneumophila by 2.6 + 0.4 log units after 120 h. An addition of Bacillus BS104 to a cooling tower test system with a water volume of 8 m³ resulted in a L. pneumophila level below 1000 CFU/L after 3 weeks, whereas the starting point was 5.3 × 10⁴ CFU/L. Further experiments also indicated that B. subtilis BS104 might inhibit the internal replication of L. pneumophila in Acanthamoeba castellanii. This paper demonstrates that certain strains of Bacillus have the potential of reducing the number of viable L. pneumophila in water, or at least prevent its increase. These results are the first indication that a biological abatement of L. pneumophila could be possible.     

Sorption of Cadmium and Lead by Bacteria–Ferrihydrite Composites

The sorptive behavior of bacteria—iron oxide composites was investigated in batch metal sorption assays using ferrihydrite in isolation (0.13 and 0.14 g/L ferrihydrite in cadmium and lead systems, respectively) as well as in combination with Bacillus subtilis (0.25 g/L adsorbent mixture) and Escherichia coli (0.27 g/L adsorbent mixture). A pH range from 3.0 to 6.5 was studied using total metal concentrations of 1.0 × 10 ^{? 4.0} and 3.2 × 10 ^{? 5} M with adsorbent mixtures proportioned on a 1:1 mass/volume basis. The log of the apparent surface complex formation constants (log K ^S _M ) and sorption capacity (S _max ) values were determined by fitting the experimental data to one-site Langmuir sorption isotherms. The one-site model effectively described the sorption data (r ² > 0.9), where Cd ^{2 +} exhibited somewhat lower sorption affinities (log K ^S _M = ?3 for ferrihydrite, ?1.7 for B. subtilis–ferrihydrite, and ?1.1 for E. coli–ferrihydrite) than Pb ^{2 +} (log K ^S _M = ?0.9 for ferrihydrite, ? 0.2 forB. subtilis–ferrihydrite, and –0.1 for E. coli–ferrihydrite). The corresponding S _max values for Cd ^{2 +} and Pb ^{2 +} on ferrihydrite were 0.78 mmole/g and 1.34 mmole/g, respectively. For the B. subtilis–ferrihydrite composites, Cd ^{2 +} and Pb ^{2 +} S _max values were lower at 0.29 mmole/g and 0.5 mmole/g, respectively. Similar values were determined for the E. coli–ferrihydrite composites (0.15 mmole/g and 0.68 mmole/g for Cd ^{2 +} and Pb ^{2 +} , respectively). The sorption of Cd ^{2 +} and Pb ^{2 +} by each of the sorbent systems exhibited a strong dependence on pH with sorption edges in the range of pH 4.0 to 7.3. The observed S _max of the composites were lower than values predicted upon available site additivity (Cd ^{2 +} _{B. subtilis ?ferrihydrite} : 0.29 mmole/g (observed) < 0.57 mmole/g (calculated); Cd ^{2 +} _{E. coli ?ferrihydrite} : 0.15 mmole/g (observed) < 0.44 mmole/ g (calculated); Pb ^{2 +} _{B. subtilis ?ferrihydrite} : 0.5 mmole/g (observed) < 0.805 mmole/g (calculated); Pb ^{2 +} _{E. coli –ferrihydrite} : 0.68 mmole/g (observed) < 0.775 mmole/g (calculated)), implying that a masking of reactive surface sites by attachment had occurred between the bacteria and ferrihydrite. Electrophoretic mobility analysis indicated that the ferrihydrite surface properties dominate the net surface charge for each composite system with lesser contributions from the bacteria.     

Ovicidal activity of Couroupita guianensis (Aubl.) against cotton bollworm Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae)

There is a great demand for food with increasing population of the world. Many chemical fertilisers and pesticides are used to increase crop productivity. Over use of pesticides and fertilisers has affected the environment and non-target organisms severely; minor pests have become major destructive pests. Hence, eco-friendly approaches are needed. The present study reports the effect of hexane, chloroform and ethyl acetate extract of Couroupita guianensis on the eggs of Helicoverpa armigera. All the extracts showed ovicidal activity, and among them hexane extract showed 64.28% ovicidal activity with LC₅₀ value of 2.62% and regression of (R ²) 83.5%. All the original data showed normality and homogeneity. The active hexane extract was fractionated; finally, eight fractions were obtained and studied at different concentrations. Among the fractions, fraction 8 showed 75% ovicidal activity with R ² of 90%. It showed LC₅₀ value of 322.94?ppm with χ ² value at 0.5%. C. guianensis could be used in pest management programme.     

Isolation and characterization of a cold-active,alkaline, detergent stable α-amylase from a novel bacterium Bacillus subtilis N8

A cold-active alkaline amylase producer Bacillus subtilis N8 was isolated from soil samples. Amylase synthesis optimally occurred at 15°C and pH 10.0 on agar plates containing starch. The molecular weight of the enzyme was found to be 205?kDa by performing SDS-PAGE. While the enzyme exhibited the highest activity at 25°C and pH 8.0, it was highly stable in alkaline media (pH 8.0–12.0) and retained 96% of its original activity at low temperatures (10–40°C) for 24?hr. While the amylase activity increased in the presence of β-mercaptoethanol (103%); Ba²⁺, Ca²⁺, Na⁺, Zn²⁺, Mn²⁺, H₂O₂, and Triton X-100 slightly inhibited the activity. The enzyme showed resistance to some denaturants: such as SDS, EDTA, and urea (52, 65, and 42%, respectively). N8 α-amylase displayed the maximum remaining activity of 56% with 3% NaCl. The major final products of starch were glucose, maltose, and maltose-derived oligosaccharides. This novel cold-active α-amylase has the potential to be used in the industries of detergent and food, bioremediation process and production of prebiotics.     

Promotion ofin vitro shoot formation from excised roots of silktree (Albizzia julibrissin) by an oxime ether derivative and other ethylene inhibitors

Summary This report describes the regeneration response of excised seedling roots of silktree (Albizzia julibrissin) to added ethylene precursors/generators (1-amino-cyclopropane-1-carboxylic acid [ACC], 2-chloroethylphosphonic acid [CEPA]), biosynthesis inhibitors (aminoethoxyvinylglycine [AVG], an oxime ether derivative [OED={[(ispropylidene)-amino]oxy}-acetic acid-2-(methoxy)-2-oxoethyl ester], CoCl² [Co⁺⁺]), and an ethylene action inhibitor (AgNO₃ [Ag⁺]). When placed on B5 medium, about 50% of the control explants formed shoot buds within 15 days. Addition of ACC or CEPA (1–10 µM) to the culture medium decreased both the percentage of cultures forming shoots and the number of shoots formed per culture. In contrast, AVG and OED (1–10 µM) increased shoot formation to almost 100% and increased the number of shoots formed per culture. Likewise, both Co⁺⁺ and Ag⁺ (1–10 µM) increased shoot regeneration, but the number of shoots produced after 30 days was less than with AVG or OED. The inhibitors of ethylene biosynthesis were partially effective in counteracting the inhibitory effect of ACC on shoot formation. These results suggest that modulation of ethylene biosynthesis and/or action can strongly influence the formation of adventitious shoots from excised roots of silktree.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - CEPA 2-chloroethylphosphonic acid - OED oxime ether derivative