Characterization of highly stable extracellular lipase from the extremely halophilic archaeon Halolamina sp

Enzymes of the archaea living in extreme environments are resistant to the challenging conditions. Lipase is among the important enzymes used in the industry and agriculture. In this study, the extracellular lipase from extremely halophilic archaeon Halolamina sp. was characterized for the first time. Optimum temperature for the enzyme activity was determined as 70oC, optimum pH was 7.0, and the optimum salt concentration was 3.6 M. Additionally, more than 70% of the enzyme activity was remained between pH 3.0-10.0 for 48 h as well as incubation of the enzyme at 70oC for 30 min increased its activity for 44%, and no activity loss was observed after incubation at 80oC. Also, presence of the metals increased the enzyme activity up to 88%. The enzyme was highly resistant to the organic solvents acetone, methanol, and DMSO while strong inhibition was caused by n-butanol. Among the detergents, the enzyme kept its activity substantially in the presence of SDS; however, other detergents caused inhibition of the enzyme activity. This characterization study showed that the lipase from the haloarchaeon Halolamina sp. is highly stable at the wide ranges of temperature and pH values as well as in the presence of diverse inhibitors. This enzyme is promising to be used in biotechnological applications.

Comparative molecular studies of halophilic bacteria from saline water and soil in the Saudi environment / Estudos moleculares comparativos de bactérias halofílicas de água salina e solo no meio ambiente saudita

Halophilic bacteria are microorganisms that grow optimally in the presence of the very high concentration of sodium chloride. Halophiles are vital sources of various enzymes including hydrolases, which are very stable and catalytically highly efficient at high salt concentration and other extreme conditions such as high temperature, pH and presence of organic solvents. Several hydrolases such as amylases, proteases, and lipases have been obtained from halophilic bacteria and are commonly used for various industrial applications. We initiated a screening to isolate and characterize the halophilic bacteria from the Red Sea, which is one of the saltiest bodies of water in the world. Water and soil samples, collected from the Red Sea coast, Jeddah, Saudi Arabia, were screened for isolation of halophilic bacteria. Ten bacterial isolates were obtained, which were characterized by biochemical tests and 16S rRNA gene sequencing. Hydrolase producing bacteria among the isolates were screened by plate assay on starch and gelatin agar plates for amylase and protease, respectively. Two bacterial isolates i.e. Bacillus glycinifermentans S3 and Enterobacter cloacae W1were found to possess significant amylase and protease activity.

Bactérias halofílicas são microrganismos que crescem de maneira ideal na presença de uma concentração muito alta de cloreto de sódio. Halófilos são fontes vitais de várias enzimas, incluindo hidrolases, que são muito estáveis e cataliticamente altamente eficientes em alta concentração de sal e outras condições extremas, como alta temperatura, pH e presença de solventes orgânicos. Várias hidrolases como amilases, proteases e lipases foram obtidas a partir de bactérias halofílicas e são comumente usadas para várias aplicações industriais. Iniciamos uma triagem para isolar e caracterizar as bactérias halofílicas do Mar Vermelho, que é um dos corpos de água mais salgados do mundo. Amostras de água e solo, coletadas na costa do Mar Vermelho, Jeddah, na Arábia Saudita, foram examinadas quanto ao isolamento de bactérias halofílicas. Foram obtidos dez isolados bacterianos, caracterizados por testes bioquímicos e seqüenciamento do gene 16S rRNA. As bactérias produtoras de hidrolase entre os isolados foram triadas por ensaio em placa em placas de amido e ágar de gelatina para amilase e protease, respectivamente. Verificou-se que dois isolados bacterianos, isto é, Bacillus glycinifermentans S3 e Enterobacter cloacae W1, possuíam significativa atividade de amilase e protease.

Expression analysis of genes responsible for amino acid biosynthesis in halophilic bacterium Salinibacter ruber.

The degeneracy of the genetic code allows for multiple codons to encode the same amino acid. However, alternative codons and amino acids are used unevenly among genes, a phenomenon termed codon-usage bias. Genes regulating amino acid biosynthesis of Salinibacter ruber, an extremely halophilic bacterium were studied in order to determine the synonymous codon usage patterns. Factors responsible for codon usage variation among the genes were investigated using codon usage indices and multi-variate statistical approach. Overall codon usage data analysis indicated that codons ending in G and/or C were predominant among the genes. Multi-variate statistical analysis showed that there was a single major trend in the codon usage variation among the genes, which had a strong positive correlation (r = 0.93, P<0.01) with (G + C) content of the genes. Further, correlation analysis indicated that genes with higher expression level and showing a greater degree of codon usage bias were GC-rich and preferred codons with C or G nucleotides at the third position. A set of thirteen codons were identified through Chi-square test as optimal codons, which were preferred in highly expressed genes. It could be concluded that mutational bias had a profound effect on codon usage pattern. In addition, translational selections also operated with a proper balance, making the genes translationally more efficient. The frequency of these codons appeared to be correlated with the level of gene expression and might be a useful indicator in the case of genes (or open-reading-frames) whose expression levels are unknown.

Screening and isolation of halophilic bacteria producing industrially important enzymes

Halophiles are excellent sources of enzymes that are not only salt stable but also can withstand and carry out reactions efficiently under extreme conditions. The aim of the study was to isolate and study the diversity among halophilic bacteria producing enzymes of industrial value. Screening of halophiles from various saline habitats of India led to isolation of 108 halophilic bacteria producing industrially important hydrolases (amylases, lipases and proteases). Characterization of 21 potential isolates by morphological, biochemical and 16S rRNA gene analysis found them related to Marinobacter, Virgibacillus, Halobacillus, Geomicrobium, Chromohalobacter, Oceanobacillus, Bacillus, Halomonas and Staphylococcus genera. They belonged to moderately halophilic group of bacteria exhibiting salt requirement in the range of 3-20%. There is significant diversity among halophiles from saline habitats of India. Preliminary characterization of crude hydrolases established them to be active and stable under more than one extreme condition of high salt, pH, temperature and presence of organic solvents. It is concluded that these halophilic isolates are not only diverse in phylogeny but also in their enzyme characteristics. Their enzymes may be potentially useful for catalysis under harsh operational conditions encountered in industrial processes. The solvent stability among halophilic enzymes seems a generic novel feature making them potentially useful in non-aqueous enzymology.

Biodegradation of petroleum hydrocarbons in hypersaline environments

Literature on hydrocarbon degradation in extreme hypersaline media presents studies that point to a negative effect of salinity increase on hydrocarbonoclastic activity, while several others report an opposite tendency. Based on information available in the literature, we present a discussion on the reasons that justify these contrary results. Despite the fact that microbial ability to metabolize hydrocarbons is found in extreme hypersaline media, indeed some factors are critical for the occurrence of hydrocarbon degradation in such environments. How these factors affect hydrocarbon degradation and their implications for the assessment of hydrocarbon biodegradation in hypersaline environments are presented in this review.

Production of 2, 3-butanediol and succinic acid by Salinivibrio YS / 生物工程学报

The production of 2, 3-butanediol and succinic acid by a moderate halophile under anaerobic condition was investigated. This halophile, termed Salinivibrio YS, was isolated from the solid samples collected from Aydingkol Lake. Based on the single factor experiment, the parameters and their values for the production were obtained. Then, the optimum values of these parameters by the orthogonal experiments were obtained: temperature, 33 degrees C; initial pH of fermentation, 8.0; the pH during fermentation, 7.0; the concentration of acetic acid was 3 g/L and NaC1 was 10 g/L. Finally, a 3-L fermentation based on these conditions was carried out. After 108 h of fermentation under anaerobic condition, 35.05 g/L of 2, 3-butanediol and 22.46 g/L of succinic acid were obtained. About 50% of total glucose conversion was achieved. The study on 2, 3-butanediol and succinic acid by a halophile under anaerobic condition will expand the applications of halophiles and open a new area of production of 2, 3-butanediol and succinic acid.

Characterization and stability of extracellular alkaline proteases from halophilic and alkaliphilic bacteria isolated from saline habitat of coastal Gujarat, India

The present study deals with the isolation and characterization of the moderately halophilic-alkaliphilic bacteria from a saline habitat in western India. Eight different bacterial strains were isolated using enrichment techniques at 20 percent (w/v) NaCl and pH 10. The isolates exhibited diversity towards gram's reaction, colony and cell morphology. They were able to grow and produce alkaline protease over a broad range of NaCl, 5-20 percent (w/v) and pH, 8-10. None of the isolates could grow at pH 7, and one could not grow even at pH 8. Crude and partially purified proteases from strain S5 were subjected to characterization with reference to pH, salt stability and protein folding. Optimum protease activity and stability was recorded at 10 percent salt and pH 9-9.5. Denaturation kinetics of S5 alkaline protease along with a reference protease was studied at 8M urea followed by renaturation. The S5 alkaline protease could be partially renatured up to 32 percent of the original activity. Despite of the fact that all the 8 isolates were from the same site, they displayed significant diversity with respect to their salt requirement for growth and enzyme secretion. While the effect of pH was less demarcated on growth, the protease production was significantly affected. Isolate S5 produced substantial amount of halotolerant and alkaline protease. The activity and stability of the alkaline protease in a broader range of pH and salt would definitely make this enzyme an important candidate for various industrial applications.

O presente estudo relata o isolamento e caracterização de bactérias moderadamente halofilicas e alcalífilicas de um habitat salino no oeste da índia. Oito cepas diferentes de bactérias foram isoladas empregando técnicas de enriquecimento em NaCl a 20 por cento (p/v) e pH 10. As cepas apresentaram diversidade em relação à coloração de Gram e à morfologia das colônias e células. As cepas foram capazes de multiplicar e produzir protease alcalina em uma ampla faixa de concentração de NaCl (5 a 20 por cento) e pH (8 a 10). Nenhuma das cepas foi capaz de se multiplicar em pH 7, e uma não se multiplicou nem em pH 8.0. Proteases naturais e parcialmente purificadas da cepa S5 foram submetidas à caracterização com relação ao pH, estabilidade salina, e estrutura protéica. Atividade e estabilidade ótimas da protease foram obtidas com 10 por cento de sal e pH 9-9,5. A cinética de denaturação da protease de S5, juntamente com uma protease de referencia, foi avaliada com uréia 8M seguida de renaturação. A protease alcalina de S5 foi renaturada a 32 por cento da atividade original. Apesar de provenientes do mesmo local, as oito cepas mostraram grande diversidade em relação à exigência de sal para multiplicação e secreção enzimática. Enquanto o efeito do pH na multiplicação foi menos marcante, o efeito na produção de protease foi significativamente afetada. A cepa S5 produziu uma quantidade substancial de protease alcalina e halotolerante. A atividade e estabilidade da protease alcalina em uma faixa mais ampla de pH e sal tornam essa enzima uma importante candidata para diversas aplicações industriais.