Caracterización bioinformática y producción de L-asparaginasa de Bacillus sp. M62 aislado de las salinas de Maras, Cusco, Perú

El objetivo del estudio fue realizar la caracterización bioinformática, así como optimizar la producción de L-asparaginasa extracelular de Bacillus sp. M62 aislada de las salinas de Maras (Cusco). Para ello, se verificó la producción de L-asparaginasa mediante el viraje del medio M9 modificado con azul de bromofenol 0.0075%, pH 7.4 a 37 °C por 72 h. A la vez, se extrajo el ADN genómico para amplificar los genes ribosómicos 16S y el gen ansA3. La secuencia aminoacídica codificada por el gen ansA3 se predijo mediante análisis bioinformático. La producción de L-asparaginasa intracelular y extracelular se evaluó a diferentes niveles de glucosa, L-asparagina, NaCl y pH en el medio M9 modificado. Adicionalmente, las actividades enzimáticas de L-asparaginasa y L-glutaminasa se determinaron mediante cuantificación del amonio liberado por el método de Nessler. Así, Bacillus sp. M62 produjo el viraje del medio M9 modificado, obtuvo alta similitud y cercanía evolutiva con Bacillus licheniformis, se encontró que el gen ansA3 amplificado codificaba para 319 aa, dentro de la cual se predijo una secuencia patrón del sitio activo (GFVITHGTDTM ) y 15 sitios inmunogénicos. La producción de L-asparaginasa extracelular fue superior a la intracelular, la que se optimizó de 0.37 U/mL (0.24 U/mg) a 2.15 ± 0.39 U/mL (0.63 U/mg). Finalmente, se encontró que Bacillus sp. M62 presenta L-asparaginasa extracelular con mínima actividad de L-glutaminasa.

The aim of this study was to perform bioinformatics characterization and optimize the production of extracellular L-asparaginase from Bacillus sp. M62, isolated from the Maras salt ponds (Cusco). To achieve this, the production of L-asparaginase was verified by the change in color of modified M9 medium, containing 0.0075% bromophenol blue, at pH 7.4 and 37°C for 72 hours. Genomic DNA was extracted to amplify the 16S ribosomal genes and the ansA3 gene. The amino acid sequence encoded by the ansA3 gene was predicted using bioinformatic analysis. The production of intracellular and extracellular L-asparaginase was evaluated at different levels of glucose, L-asparagine, NaCl, and pH in modified M9 medium. Additionally, the enzymatic activities of L-asparaginase and L-glutaminase were determined by quantifying the released ammonium using the Nessler method. Bacillus sp. M62 showed the change in color of the modified M9 medium, high similarity, and evolutionary closeness to Bacillus licheniformis. The amplified ansA3 gene was found to encode for 319 amino acids, with a predicted active site pattern (GFVITHGTDTM) and 15 immunogenic sites. The production of extracellular L-asparaginase was found to be higher than intracellular L-asparaginase and was optimized from 0.37 U/mL (0.24 U/mg) to 2.15 ± 0.39 U/mL (0.63 U/mg). Finally, it was found that Bacillus sp. M62 presents extracellular L-asparaginase with minimal L-glutaminase activity.

JMT-1: a novel, spherical lytic halotolerant phage isolated from Yuncheng saline lake

ABSTRACT This work described a novel halotolerant phage, JMT-1, with a spherical morphology. JMT-1, which was isolated from a hypersaline lake, could produce clear plaques on Chromohalobacter sp. LY7-3. The purified virions are spherical, have no visible tail, and are about 3050 nm in diameter. JMT-1 has a wide host range, and this study showed that the phage can infect at least five halophilic bacteria. The proteins of JMT-1 were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and six proteins were detected. Results show that JMT-1 is a bacteriophage with a linear double-stranded DNA. Meanwhile, the genome is approximately 23 kb in length and is sensitive to the restriction endonucleases Bam I, EcoR I, Hind III and Kpa I. JMT-1 has a high titer, approaching 1.5 × 109 pfu/mL after dilution to 106 pfu/mL. The phage is also sensitive to chloroform but not to temperature, pH, and lowered salt concentration. JMT-1 is a spherical lytic halotolerant phage with a wide host range and has the tolerance to specific extreme environments. These data could provide references for studying phage resources in extreme environments and would also provide the useful methods for isolation and identification of other valuable phage in the salt lake environment.

JMT-1: a novel, spherical lytic halotolerant phage isolated from Yuncheng saline lake

ABSTRACT This work described a novel halotolerant phage, JMT-1, with a spherical morphology. JMT-1, which was isolated from a hypersaline lake, could produce clear plaques on Chromohalobacter sp. LY7-3. The purified virions are spherical, have no visible tail, and are about 30-50 nm in diameter. JMT-1 has a wide host range, and this study showed that the phage can infect at least five halophilic bacteria. The proteins of JMT-1 were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and six proteins were detected. Results show that JMT-1 is a bacteriophage with a linear double-stranded DNA. Meanwhile, the genome is approximately 23 kb in length and is sensitive to the restriction endonucleases Bam I, EcoR I, Hind III and Kpa I. JMT-1 has a high titer, approaching 1.5 × 109 pfu/mL after dilution to 10−6 pfu/mL. The phage is also sensitive to chloroform but not to temperature, pH, and lowered salt concentration. JMT-1 is a spherical lytic halotolerant phage with a wide host range and has the tolerance to specific extreme environments. These data could provide references for studying phage resources in extreme environments and would also provide the useful methods for isolation and identification of other valuable phage in the salt lake environment.

Alleviation of salt stress by halotolerant and halophilic plant growth-promoting bacteria in wheat (Triticum aestivum)

ABSTRACT In the current study, 18 halotolerant and halophilic bacteria have been investigated for their plant growth promoting abilities in vitro and in a hydroponic culture. The bacterial strains have been investigated for ammonia, indole-3-acetic acid and 1-aminocyclopropane-1-carboxylate-deaminase production, phosphate solubilisation and nitrogen fixation activities. Of the tested bacteria, eight were inoculated with Triticum aestivum in a hydroponic culture. The investigated bacterial strains were found to have different plant-growth promoting activities in vitro. Under salt stress (200 mM NaCl), the investigated bacterial strains significantly increased the root and shoot length and total fresh weight of the plants. The growth rates of the plants inoculated with bacterial strains ranged from 62.2% to 78.1%.Identifying of novel halophilic and halotolerant bacteria that promote plant growth can be used as alternatives for salt sensitive plants. Extensive research has been conducted on several halophilic and halotolerant bacterial strains to investigate their plant growth promoting activities. However, to the best of my knowledge, this is the first study to inoculate these bacterial strains with wheat.

Structure and Functional Relationship of Halotolerant Proteins- A Review.

With increasing need for various industries for stable enzymes which can outperform under harsh conditions of pH, temperature and saline environments evoke the need for extensive research on extremophilic microorganisms which found to be a suitable source for enzymes with novel properties. Especially, enzymes capable of functioning over a wide range of salt concentrations are in great demand for industries which mainly include proteases, xylanases, mannases, lipases, amylases and cellulases. In case of halophilic, extremely halophilic or halotolerant organisms it has been scientifically proven that there is a correlation between the salt requirement and bacterial growth and its enzymatic activities. When compared to halophilic organism, halotolerant bacteria can survive from low to moderate level of salinity, which gives them unique property over halophilic organisms. The halotolerant organisms synthesize enzymes with greater conformational stability and they can be potentially employed in many industrial processes even under harsher conditions over a range of salinity conditions where enzymes from non-halophilic and halophilic organisms found to be unfit. Less research has been carried out on halotolerant organisms when compared to thermophilic, alkaliphilic and halophilic organisms and so yet to explore the biotechnological applications of these halotolerant enzymes to use them in industrial applications.

Scale up of production in a bioreactor of a halotolerant protease from moderately halophilic Bacillus sp. isolated from soil

Studies were conducted on the production of protease by moderately halophilic Bacillus sp. on agro-industrial waste materials. The bacterium could efficiently use many agro wastes as substrates but wheat bran supported maximum enzyme production. To ascertain the performance of the process in shake flasks and lab scale bioreactor, experiments were conducted to analyse protease activity utilizing wheat bran as cost effective substrate. The studies unveiled that pH 7.0, temperature 30°C and static conditions were optimal for enzyme production in flask level fermentation. In scale-up fermentation, at optimal pH and temperature, agitation rate of 50 rpm was best for protease production. The enzymatic nature was studied in 10% SDS gels with BSA (2.5 mg/mL) as substrate and banding pattern was compared with undigested BSA as control. The endoprotease nature and the kinetics of protease activity were confirmed. The enzyme retained 37% of its activity even at 5 M NaCl concentration. The proteolytic activity was also confirmed by casein zymogram analysis. The fermentation medium containing inexpensive substrates, physical conditions and ability of Bacillus sp. to exhibit protease activity on a large scale could collectively be useful for commercial production.

Biosynthesis and characterization of keratinolytic protease from actinobacterium sp. in solid state culture.

The aim of this study was to use agro-industrial residues for the production of a halotolerant keratinolytic- protease by Actinobacterium sp. in solid-state fermentation. Among various agro-industrial residues that were evaluated, apple pomace supported maximum protease production (8400 U/g material). The optimum conditions required for enzyme production were a fermentation period of 72 h, 10% (w/v) NaCl, pH 7.0, 120% (v/w) moisture and 10% (v/w) inoculum. The enzyme exhibited activity to a range of pH (7.0-9.0) and temperature (30-45°C), with optima at 8.0 and 40 °C, respectively. Most of the divalent ions tested stimulated the protease activity and Ca2+ ion was required for its activity and stability. The enzyme was widely active at the range of NaCl concentration (5%-15%, w/v) and effectively degraded chicken feather. This protease could be useful in fish sauce fermentation and also in feed industry.

Characterization of novel extracellular protease produced by marine bacterial isolate from the Indian Ocean

Out of the vast pool of enzymes, proteolytic enzymes from microorganisms are the most widely used in different industries such as detergent, food, peptide production etc. Several marine microorganisms are known to produce proteases with commercially desirable characteristics. We have isolated nine different cultures from marine samples of the Indian Ocean. All of them were i) motile ii) rod shaped iii) non spore forming iv) catalase and amylase positive v) able to grow in presence of 10 percent NaCl. They produced acid from glucose, fructose and maltose and grew optimally at 30 0C temperature and pH 7.0-8.0. None of them could grow above 45 0C and below 15 0C. Only one of them (MBRI 7) exhibited extracellular protease activity on skim milk agar plates. Based on 16S rDNA sequencing, it belonged to the genus Marinobacter (98 percent sequence similarity, 1201 bp). The cell free extract was used to study effects of temperature and pH on protease activity. The optimum temperature and pH for activity were found to be 40 0C and 7.0 respectively. The crude enzyme was stable at temperature range of 30-80 0C and pH 5.0-9.0. It retained 60 percent activity at 80 0C after 4 h and more than 70 percent activity at 70 0C after 1 h. D value was found to be 342 minutes and 78 minutes for 40 0C and 80 0C respectively. Interestingly the enzyme remained 50 percent active at pH 9.0 after 1 h. Comparison with other proteases from different microbial sources indicated that the neutral protease from the halotolerant marine isolate MBRI 7 is a novel enzyme with high thermostability.

Detergent stable, halotolerant -amylase from bacillus aquimaris vitp4 exhibits reversible unfolding.

Halotolerant Bacillus aquimaris VITP4, isolated from Kumta coast (India), was used to produce extracellular α-amylase. The production was found to be maximal after 24 h of growth at pH 8.0 and 40 oC. Optimal activity of the purified enzyme was in the pH range of 7.5 – 9.5 at 40 oC. The enzyme was found to retain more than 60% of the initial activity even at NaCl concentration of 3.5 M, indicating that it is halotolerant. Calcium ion (0.01 mM) and CTAB (10 mM) enhanced the activity whereas EDTA decreased the enzymatic activity. Thermal inactivation kinetics suggested that the enzyme exhibits reversible unfolding even at high temperature (till 90 oC) and the t1/2 at 90 oC was found to be 43.5 min. These results suggests that the α-amylase from Bacillus aquimaris strain VITP4 is halotolerant, metal ion dependent enzyme which is stable in the presence of cationic detergent and moderate temperature conditions.

Isolation,Identification and Degradation Characteristics of a Slight Halophilic Denitrifying Bacteria / 微生物学通报

A slight halophilic denitrifying bacteria(designated GYL)was screened from the activated sludge which was used to treat high-salinity wastewater.According to the results of morphological observation,physiological and biochemical test,sequence analysis of the 16S rDNA,strain GYL was identified as Halo-monas sp..This strain could survive at 10% salinity and the optimal salinity range for growth was 2%~7%.The suitable pH value for growth was 7.5~8.5 and sucrose was the most effective carbon source.The nitro-gen removal efficiency exceeded 80% when the temperature ranged from 25?C to 30?C.Meanwhile hetero-trophic nitrification characteristics of this strain were measured.Results showed that this strain was able to realize SND and ammonia removal rate was 98.3%.It showed that this strain could perform the whole proc-ess of bacteria denitrification independently.

Salt mediated changes in some enzymes of carbohydrate metabolism in halotolerant Cladosporium sphaerospermum.

Cladosporium sphaerospermum, isolated from salt pans was halotolerant. When grown in the presence of salt, the activities of invertase, isocitrate lyase, fructose-1,6 diphosphate aldolase and malate dehydrogenase were found to be increased and that of amylase decreased. Both, enzyme activation as well as an increase in de novo synthesis of enzymes were found to be some of the mechanisms of salt mediated changes. This may be one of the adaptive mechanisms, in halotolerant Cladosporium sphaerospermum.