Thermostable chitinase from Cohnella sp. A01: isolation and product optimization

Abstract Twelve bacterial strains isolated from shrimp farming ponds were screened for their growth activity on chitin as the sole carbon source. The highly chitinolytic bacterial strain was detected by qualitative cup plate assay and tentatively identified to be Cohnella sp. A01 based on 16S rDNA sequencing and by matching the key morphological, physiological, and biochemical characteristics. The cultivation of Cohnella sp. A01 in the suitable liquid medium resulted in the production of high levels of enzyme. The colloidal chitin, peptone, and K2HPO4 represented the best carbon, nitrogen, and phosphorus sources, respectively. Enzyme production by Cohnella sp. A01 was optimized by the Taguchi method. Our results demonstrated that inoculation amount and temperature of incubation were the most significant factors influencing chitinase production. From the tested values, the best pH/temperature was obtained at pH 5 and 70 °C, with Km and V max values of chitinase to be 5.6 mg/mL and 0.87 µmol/min, respectively. Ag+, Co2+, iodoacetamide, and iodoacetic acid inhibited the enzyme activity, whereas Mn2+, Cu2+, Tweens (20 and 80), Triton X-100, and EDTA increased the same. In addition, the study of the morphological alteration of chitin treated by enzyme by SEM revealed cracks and pores on the chitin surface, indicating a potential application of this enzyme in several industries.

Evaluación de la persistencia de una formulación comercial de Bacillus sphaericus en criaderos naturales de anofelinos vectores de malaria en estado Bolívar, Venezuela / Evaluation of the persistence of a comercial formulation of Bacillus sphaericus in malaria vectors anopheline larval hábitats in Bolívar state, Venezuela

El municipio Sifontes del estado Bolívar es la principal área endémica a malaria de Venezuela. Durante los últimos cinco años se ha reportado en este municipio un promedio anual cercano a 15 mil casos, alcanzándose durante 2009 la cifra de 20.282, lo que constituye una situación de epidemia. En este municipio se evaluó la persistencia de una formulación comercial de Bacillus sphaericus Neide (Vectolex CG 7,5%) en criaderos naturales de Anopheles marajoara, An. triannulatus y An. braziliensis. Para ello se realizaron dos experimentos en los cuales se evaluaron las dosis de 2 y 3 gr/m2 durante 60 y 90 días. Los resultados indican que hay diferencias en la persistencia por tipo de criadero y especie de anofelino. La tendencia general para todas las especies indica que el porcentaje de reducción de la población larvaria fue de 100% durante la primera semana, luego disminuyó paulatinamente hasta más o menos 40% después de los 28 días, para luego recuperarse a alrededor de 60% a los 45 días, manteniéndose así hasta el día 90 con ambas dosis. Este comportamiento es más evidente para An. marajoara, mientras que con An. triannulatus la eficacia se mantiene alrededor de 80% durante 90 días con 3 gr/m2. Sobre An. braziliensis, la eficacia se mantiene en 100% hasta el día 14 con ambas concentraciones, para luego caer abruptamente sin recuperación. Es la primera vez que se evalúa B. sphaericus sobre estas especies de anofelinos en Venezuela, considerándose estos resultados muy prometedores para el control de vectores

Sifontes municipality in Bolivar state is the main malaria endemic area in Venezuela. During the last five years an annual average of 14,000 cases has been reported, reaching 13,450 during 2008, resulting in an epidemic situation. A commercial formulation of Bacillus sphaericus Neide (Vectolex CG 7.5%), was evaluated in larval habitats of Anopheles marajoara, An. triannulatus and An. braziliensis. Two experiments were carried out in a lagoon and a pasture inundated area, 2 and 3 gr/m2 doses were evaluated during 60 and 90 days. The results point out that there are differences in the persistence depending on the type of larval habitat and anopheline species. The general trend indicated that for all the species the persistence of the Vectolex, therefore the reduction of the larval population was 100% during the first week. However, it felt under 40% after day 28, then recovered by day 45, and remaining around 40% for 90 days with both doses. A similar pattern was observed for An. marajoara, whereas for An. triannulatus the efficacy remained around 80% during 90 days only at 3 gr/m2. With An. braziliensis there was 100% persistence until day 14 with both doses, for then felt abruptly without recovering. This is the first time that B. sphaericus was evaluated for these anopheline species in Venezuela, considering these results promissory for the malaria control program

Production of a bacteriophage, a phage tail-like bacteriocin and an antibiotic by Bacillus azotofixans

Twenty-two nitrogen-fixing Bacillus azotofixans strains shown to produce an inhibition zone against themselves in plate assays. The B. azotofixans type strain P3L-5, chosen for further studies, produced inhibition zones against various Bacillus strains and other bacterial genera. This antibacterial substance was also produced in liquid medium and its production was enhanced in semisolid medium (0.4% agar) after 3 to 5 days of incubation. The substance was suggested to be an antibiotic and its preliminary characterization showed resistance to heat (100§ C, 15 minutes), to trypsin, pronase, deoxyribonuclease I, ribonuclease A, phospoholipase C, ethanol, acetone, and ether, and sensitivity to strong alkali treatment. Its molecular weight was estimated to be between 3500 to 6000. After induction of B. azotofixans P3L-5 with mitomycin C or ultraviolet light, two types of particles were detected in the lysate: one similar to a phage tail and the other, less frequent, similar to a complete bacteriophage. Lysates containing these particles showed a killing effect in some but not all B. azotofixans strains, but neither the other Bacillus species nor Micrococcus were inhibited by these lysates

Taxonomía numérica del género Bacilus / Numerical taxonomy of the genus Bacillus

Se estudiaron por taxonomía numérica 70 cepas del género Bacillus de diferentes orígenes. Se emplearon 81 caracteres morfológicos y fisiológicos. se aplicaron un método de codificación y cuatro de agrupamiento (método de los pares no pesados usando promedios aritméticos, método de los apres pesados usando promedios aritméticos, método de ligamiento simple y método de ligamiento completo) obteniéndose cuatro dendrogramas. Utilziando los datos del primer método de agrupamiento, que presenta el coeficiente de correlación cofenética más alto (0,718), se clasifican las cepas en 11 grupos 14 subgrupos, estableciéndose como grupo al conjunto de cepas que se unen con niveles de similtud mayores de 0,700 y como subgrupo a las que se unen con niveles mayores de 0,800. Se observó un comportamiento muy similar al analizar el dendrograma obtenido utilizando como método de agrupamiento el de los pares pesados usando promedios aritméticos. La variabilidad morfológica y fisiológica de los miembros de este género también se manifiesta en la clasificación por taxonomia numérica