Pseudomonas in environmental bioremediation of hydrocarbons and phenolic compounds- key catabolic degradation enzymes and new analytical platforms for comprehensive investigation.

Pollution of the environment with petroleum hydrocarbons and phenolic compounds is one of the biggest problems in the age of industrialization and high technology. Species of the genus Pseudomonas, present in almost all hydrocarbon-contaminated areas, play a particular role in biodegradation of these xenobiotics, as the genus has the potential to decompose various hydrocarbons and phenolic compounds, using them as its only source of carbon. Plasticity of carbon metabolism is one of the adaptive strategies used by Pseudomonas to survive exposure to toxic organic compounds, so a good knowledge of its mechanisms of degradation enables the development of new strategies for the treatment of pollutants in the environment. The capacity of microorganisms to metabolize aromatic compounds has contributed to the evolutionally conserved oxygenases. Regardless of the differences in structure and complexity between mono- and polycyclic aromatic hydrocarbons, all these compounds are thermodynamically stable and chemically inert, so for their decomposition, ring activation by oxygenases is crucial. Genus Pseudomonas uses several upper and lower metabolic pathways to transform and degrade hydrocarbons, phenolic compounds, and petroleum hydrocarbons. Data obtained from newly developed omics analytical platforms have enormous potential not only to facilitate our understanding of processes at the molecular level but also enable us to instigate and monitor complex biodegradations by Pseudomonas. Biotechnological application of aromatic metabolic pathways in Pseudomonas to bioremediation of environments polluted with crude oil, biovalorization of lignin for production of bioplastics, biofuel, and bio-based chemicals, as well as Pseudomonas-assisted phytoremediation are also considered.

High-quality draft genome sequence of Pseudomonas aeruginosa san ai, an environmental isolate resistant to heavy metals.

The strain Pseudomonas aeruginosa san ai, isolated from an extreme environment (industrial mineral cutting oil, pH 10), is able to survive and persist in the presence of a variety of pollutants such as heavy metals and organic chemicals. The genome of P. aeruginosa san ai is 6.98 Mbp long with a GC content of 66.08% and 6485 protein encoding genes. A large number of genes associated with proteins, responsible for microbial resistance to heavy metal ions and involved in catabolism of toxic aromatic organic compounds were identified. P. aeruginosa san ai is a highly cadmium-resistant strain. Proteome analysis of biomass after cadmium exposal confirmed a high tolerance to sublethal concentrations of cadmium (100 mg/L), based on: extracellular biosorption, bioaccumulation, biofilm formation, controlled siderophore production and a pronounced metalloprotein synthesis. Proteins responsible for survival in osmostress conditions during exposure to elevated concentrations of cadmium (200 mg/L) demonstrate a strong genetic potential of P. aeruginosa san ai for survival and adaptation. Sequencing of P. aeruginosa san ai genome provides valuable insights into the evolution and adaptation of this microbe to environmental extremes at the whole-genome level, as well as how to optimally use the strain in bioremediation of chemically polluted sites.

Phytochemical Properties of Satureja kitaibelii, Potential Natural Antioxidants: a New Insight.

Satureja kitaibelii Wierzb. ex Heuff. has a great importance in Serbian ethnopharmacology/herbal traditional medicine, as well as a flavoring food additive. Ethanol extract of aerial parts of Satureja kitaibelii analyzed by liquid chromatography-mass spectrometry revealed the presence of 18 compounds among which the most abundant were phenolic acids, flavonoids, jasmonic acid derivatives and rosmanol. The extracts were rich in total phenolics and flavonoid contents, while rosmarinic acid was the dominant compound (18.30-29.52 mg/g). As assessments of antioxidant properties of natural extracts are important because of their growing use in medicine and food industry, antioxidant activity of ethanol extracts of Satureja kitaibelii was analyzed by several assays. The half maximal scavenging capacity (SC50) of 2,2'-diphenyl-1-picrylhydrazyl ranging from 71.20 to 125.65 µg/mL, the total antioxidant capacity from 272.37 to 714.12 mg ascorbic acid/g, and ferric ion reducing antioxidant power ranging from 0.74 to 1.94 µmol Fe/mg, clearly imply a significant antioxidant potential of Satureja kitaibelii. The extracts inhibit growth of Micrococcus luteus and Pseudomonas aeruginosa with inhibition zones 20-30 and 16-26 mm, respectively. Antioxidant and antibacterial activity of compounds identified in extracts suggest a great potential for Satureja kitaibelii application as valuable food ingredient.

Total synthesis and biological evaluation of (-)-atrop-abyssomicin C.

Enantioselective synthesis of a marine antibiotic (-)-atrop-abyssomicin C was accomplished in 21 steps, in 1.8% overall yield (4%, based on the recovered starting material). The key steps of the synthesis are the formation of the functionalized cyclohexane core by an organocatalyzed Tsuji-Trost reaction, the formation of a tricyclic spirotetronate unit by a gold-catalyzed reaction sequence and the highly efficient eleven-membered ring closure by a Nozaki-Hiyama-Kishi reaction. Biological tests showed all abyssomicin derivatives to possess strong antibacterial activity against methicillin resistant S. aureus strains; however, they also proved to be cytotoxic, both to malignant and to normal somatic cells.

Cucurbita pepo contains characteristic proteins without a signal peptide in the xylem sap.

Xylem sap is a fluid that transfers water and nutrients from the rhizosphere. This sap contains relatively low concentrations of proteins that originate from the extracellular space among the root cells. One of the characteristic proteins in the xylem sap of the Cucurbitaceae family, which includes cucumber and zucchini, is a major latex-like protein (MLP). MLPs are responsible for crop contamination through the transport of hydrophobic pollutants from the roots. However, detailed information on the content of MLPs in the xylem sap is not available. Proteomic analysis of root and xylem sap proteins from the Cucurbita pepo cultivars Patty Green (PG) and Raven (RA) showed that the xylem sap of cv. RA, a high accumulator of hydrophobic pollutants, contained four MLPs that accounted for over 85% of the total xylem sap proteins in this cultivar. The xylem sap of PG, a low accumulator, mainly contained an uncharacterized protein. The amount of each root protein between the PG and RA cultivars was significantly and positively correlated in spite of being with and without a signal peptide (SP). However, the amount of xylem sap proteins without an SP was not correlated. These results suggest that cv. RA is characterized by MLPs in the xylem sap.

A comprehensive proteomics analysis of the response of Pseudomonas aeruginosa to nanoceria cytotoxicity.

The increased commercial use and spread of nanoceria raises concerns about the risks associated with its effects on living organisms. Although Pseudomonas aeruginosa may be ubiquitous in nature, it is largely found in locations closely linked with human activity. P. aeruginosa san ai was used as a model organism for a deeper understanding of the interaction between biomolecules of the bacteria with this intriguing nanomaterial. A comprehensive proteomics approach along with analysis of altered respiration and production of targeted/specific secondary metabolites was conducted to study the response of P. aeruginosa san ai to nanoceria. Quantitative proteomics found that proteins associated with redox homeostasis, biosynthesis of amino acids, and lipid catabolism were upregulated. Proteins from outer cellular structures were downregulated, including transporters responsible for peptides, sugars, amino acids and polyamines, and the crucial TolB protein of the Tol-Pal system, required for the structural formation of the outer membrane layer. In accordance with the altered redox homeostasis proteins, an increased amount of pyocyanin, a key redox shuttle, and the upregulation of the siderophore, pyoverdine, responsible for iron homeostasis, were found. Production of extracellular molecules, e.g. pyocyanin, pyoverdine, exopolysaccharides, lipase, and alkaline protease, was significantly increased in P. aeruginosa san ai exposed to nanoceria. Overall, nanoceria at sublethal concentrations induces profound metabolic changes in P. aeruginosa san ai and provokes increased secretion of extracellular virulence factors, revealing the powerful influence this nanomaterial has on the vital functions of the microorganism.

Chemical cross-linking, mass spectrometry, and in silico modeling of proteasomal 20S core particles of the haloarchaeon Haloferax volcanii.

A fast and accurate method is reported to generate distance constraints between juxtaposited amino acids and to validate molecular models of halophilic protein complexes. Proteasomal 20S core particles (CPs) from the haloarchaeon Haloferax volcanii were used to investigate the quaternary structure of halophilic proteins based on their symmetrical, yet distinct subunit composition. Proteasomal CPs are cylindrical barrel-like structures of four-stacked homoheptameric rings of α- and ß-type subunits organized in α(7)ß(7) ß(7)α(7) stoichiometry. The CPs of H. volcanii are formed from a single type of ß subunit associated with α1 and/or α2 subunits. Tandem affinity chromatography and new genetic constructs were used to separately isolate α1(7)ß(7)ß(7)α1(7) and α2(7)ß(7)ß(7)α2(7) CPs from H. volcanii. Chemically cross-linked peptides of the H. volcanii CPs were analyzed by high-performance mass spectrometry and an open modification search strategy to first generate and then to interpret the resulting tandem mass spectrometric data. Distance constraints obtained by chemical cross-linking mass spectrometry, together with the available structural data of nonhalophilic CPs, facilitated the selection of accurate models of H. volcanii proteasomal CPs composed of α1-, α2-, and ß-homoheptameric rings from several different possible structures from Protein Data Bank.

A study of the flexibility of the carbon catabolic pathways of extremophilic P. aeruginosa san ai exposed to benzoate versus glucose as sole carbon sources by multi omics analytical platform.

Polyextremophilic, hydrocarbonoclastic Pseudomonas aeruginosa san ai can survive under extreme environmental challenges in the presence of a variety of pollutants such as organic solvents and hydrocarbons, particularly aromatics, heavy metals, and high pH. To date, the metabolic plasticity of the extremophilic P. aeruginosa, has not been sufficiently studied in regard to the effect of changing carbon sources. Therefore, the present study explores the carbon metabolic pathways of polyextremophilic P. aeruginosa san ai grown on sodium benzoate versus glucose and its potential for aromatic degradation. P. aeruginosa san ai removed/metabolised nearly 430 mg/L of benzoate for 48 h, demonstrating a high capacity for aromatic degradation. Comparative functional proteomics, targeted metabolomics and genomics analytical approaches were employed to study the carbon metabolism of the P. aeruginosa san ai. Functional proteomic study of selected enzymes participating in the ß-ketoadipate and the Entner-Doudoroff pathways revealed a metabolic reconfiguration induced by benzoate compared to glucose. Metabolome analysis implied the existence of both catechol and protocatechuate branches of the ß-ketoadipate pathway. Enzymatic study of benzoate grown cultures confirmed the activity of the ortho- catechol branch of the ß-ketoadipate pathway. Even high concentrations of benzoate did not show increased stress protein synthesis, testifying to its extremophilic nature capable of surviving in harsh conditions. This ability of Pseudomonas aeruginosa san ai to efficiently degrade benzoate can provide a wide range of use of this strain in environmental and agricultural application.

Study of the venom proteome of Vipera ammodytes ammodytes (Linnaeus, 1758): A qualitative overview, biochemical and biological profiling.

Vipera ammodytes (Va), is the European venomous snake of the greatest medical importance. We analyzed whole venom proteome of the subspecies V. ammodytes ammodytes (Vaa) from Serbia for the first time using the shotgun proteomics approach and identified 99 proteins belonging to four enzymatic families: serine protease (SVSPs), L-amino acid oxidase (LAAOs), metalloproteinases (SVMPs), group II phospholipase (PLA2s), and five nonenzymatic families: cysteine-rich secretory proteins (CRISPs), C-type lectins (snaclecs), growth factors -nerve (NGFs) and vascular endothelium (VEGFs), and Kunitz-type protease inhibitors (SPIs). Considerable enzymatic activity of LAAO, SVSPs, and SVMPs and a high acidic PLA2 activity was measured implying potential of Vaa to produce haemotoxic, myotoxic, neuro and cardiotoxic effects. Moreover, significant antimicrobial activity of Vaa venom against Gram-negative (Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus) was found. The crude venom shows considerable potential cytotoxic activity on the C6 and HL60 and a moderate level of potency on B16 cell lines. HeLa cells showed the same sensitivity, while DU 145 and PC-3 are less sensitive than as normal cell line. Our data demonstrated a high complexity of Vaa and considerable enzymatic, antibacterial and cytotoxic activity, implying a great medical potential of Vaa venom as a promising source for new antibacterial and cytostatic agents.

Efficient biodegradation of petroleum n-alkanes and polycyclic aromatic hydrocarbons by polyextremophilic Pseudomonas aeruginosa san ai with multidegradative capacity.

Pseudomonas aeruginosa san ai, an alkaliphilic, metallotolerant bacterium, degraded individual selected petroleum compounds, i.e., n-alkanes (n-hexadecane, n-nonadecane) and polycyclic aromatic hydrocarbons (fluorene, phenanthrene, pyrene) with efficiency of 80%, 98%, 96%, 50% and 41%, respectively, at initial concentrations of 20 mg L-1 and in seven days. P. aeruginosa san ai showed a high biodegradative capacity on complex hydrocarbon mixtures, the aliphatic and aromatic fractions from crude oil. The efficiency of P. aeruginosa san ai degradation of crude oil fractions in seven days reached stage 3-4 of the oil biodegradation scale, which ranges from 0 (no biodegradation) to 10 (maximum biodegradation). Identified metabolites concomitant with genomic and enzymatic data indicated the terminal oxidation pathway for the n-alkane degradation, and the salicylate and phthalate pathways for fluorene biodegradation. Polyextremophilic P. aeruginosa san ai, as a biosurfactant producer with multidegradative capacity for hydrocarbons, can be used in an improved strategy for environmental bioremediation of hydrocarbon-contaminated sites, including extreme habitats characterized by low or elevated temperatures, acidic or alkaline pH or high concentrations of heavy metals.

Enzymatic characterization of 30 kDa lipase from Pseudomonas aeruginosa ATCC 27853.

An extracellular lipase from Pseudomonas aeruginosa ATCC 27853 has been purified and its enzymatic characteristics were determined. According to SDS-PAGE and gel filtration molecular mass estimated to be 30 kDa, what classified the lipase in group I.1. Although 14 lipases from P. aeruginosa with similar molecular mass are referred to date, their basic enzymatic properties have not been reported yet. To address the gap we found: the optimal temperature and pH in water solution being 50 degrees C and 9.3, respectively; the lipase was inhibited with Hg2+ ions and sodium dodecylsulphate (SDS), while non-ionic detergent Triton X-100 activated the enzyme; the lipase hydrolyzed more rapidly middle chain triglycerides and it was not regiospecific; the lipase demonstrated naturally occurring stability in different organic solvents with concentrations ranging from 30 to 70%, including good thermal stability in 30% organic solvent solution. Even though strain P. aeruginosa ATCC 27853 was not isolated from extreme environment it showed activity in organic solvent suggesting that this lipase is suitable for variety of applications, including reactions in water restricted medium and bioremediation of contaminations by organic solvents.

A comprehensive study of conditions of the biodegradation of a plastic additive 2,6-di-tert-butylphenol and proteomic changes in the degrader Pseudomonas aeruginosa san ai.

The Pseudomonas aeruginosa san ai strain was investigated for its capability to degrade the 2,6-di-tert-butylphenol (2,6-DTBP) plastic additive, a hazardous and toxic substance for aquatic life. This investigation was performed under different parameter values: 2,6-DTBP concentration, inoculum size, pH, and temperature. The GC-MS study showed that P. aeruginosa efficiently degraded 2,6-DTBP in the pH range of 5-8 at higher temperatures. Under exposure to 2,6-DTBP concentrations of 2, 10, and 100 mg L-1, the strain degraded by 100, 100, and 85%, respectively, for 7 days. Crude enzyme preparation from the biomass of P. aeruginosa san ai showed higher efficiency in 2,6-DTBP removal than that shown by whole microbial cells. Gene encoding for the enzymes involved in the degradation of aromatic compounds in P. aeruginosa san ai was identified. To complement the genomic data, a comparative proteomic study of P. aeruginosa san ai grown on 2,6-DTBP or sunflower oil was conducted by means of nanoLC-MS/MS. The presence of aromatic substances resulted in the upregulation of aromatic ring cleavage enzymes, whose activity was confirmed by enzymatic tests; therefore, it could be concluded that 2,6-DTBP might be degraded by ortho-ring cleavage. A comparative proteomics study of P. aeruginosa san ai indicated that the core molecular responses to aromatic substances can be summarized as the upregulation of proteins responsible for amino acid metabolism with emphasized glutamate metabolism and energy production with upregulated enzymes of glyoxylate bypass. P. aeruginosa san ai has a high capacity to efficiently degrade aromatic compounds, and therefore its whole cells or enzymes could be used in the treatment of contaminated areas.

Genetic and proteomic analyses of a proteasome-activating nucleotidase A mutant of the haloarchaeon Haloferax volcanii.

The halophilic archaeon Haloferax volcanii encodes two related proteasome-activating nucleotidase proteins, PanA and PanB, with PanA levels predominant during all phases of growth. In this study, an isogenic panA mutant strain of H. volcanii was generated. The growth rate and cell yield of this mutant strain were lower than those of its parent and plasmid-complemented derivatives. In addition, a consistent and discernible 2.1-fold increase in the number of phosphorylated proteins was detected when the panA gene was disrupted, based on phosphospecific fluorescent staining of proteins separated by 2-dimensional gel electrophoresis. Subsequent enrichment of phosphoproteins by immobilized metal ion and metal oxide affinity chromatography (in parallel and sequentially) followed by tandem mass spectrometry was employed to identify key differences in the proteomes of these strains as well as to add to the restricted numbers of known phosphoproteins within the Archaea. In total, 625 proteins (approximately 15% of the deduced proteome) and 9 phosphosites were identified by these approaches, and 31% (195) of the proteins were identified by multiple phosphoanalytical methods. In agreement with the phosphostaining results, the number of identified proteins that were reproducibly exclusive or notably more abundant in one strain was nearly twofold greater for the panA mutant than for the parental strain. Enriched proteins exclusive to or more abundant in the panA mutant (versus the wild type) included cell division (FtsZ, Cdc48), dihydroxyacetone kinase-linked phosphoenolpyruvate phosphotransferase system (EI, DhaK), and oxidoreductase homologs. Differences in transcriptional regulation and signal transduction proteins were also observed, including those differences (e.g., OsmC and BolA) which suggest that proteasome deficiency caused an up-regulation of stress responses (e.g., OsmC versus BolA). Consistent with this, components of the Fe-S cluster assembly, protein-folding, DNA binding and repair, oxidative and osmotic stress, phosphorus assimilation, and polyphosphate synthesis systems were enriched and identified as unique to the panA mutant. The cumulative proteomic data not only furthered our understanding of the archaeal proteasome system but also facilitated the assembly of the first subproteome map of H. volcanii.

Cadmium specific proteomic responses of a highly resistant Pseudomonas aeruginosa san ai.

Pseudomonas aeruginosa san ai is a promising candidate for bioremediation of cadmium pollution, as it resists a high concentration of up to 7.2 mM of cadmium. Leaving biomass of P. aeruginosa san ai exposed to cadmium has a large biosorption potential, implying its capacity to extract heavy metal from contaminated medium. In the present study, we investigated tolerance and accumulation of cadmium on protein level by shotgun proteomics approach based on liquid chromatography and tandem mass spectrometry coupled with bioinformatics to identify proteins. Size exclusion chromatography was used for protein prefractionation to preserve native forms of metalloproteins and protein complexes. Using this approach a total of 60 proteins were observed as up-regulated in cadmium-amended culture. Almost a third of the total numbers of up-regulated were metalloproteins. Particularly interesting are denitrification proteins which are over expressed but not active, suggesting their protective role in conditions of heavy metal exposure. P. aeruginosa san ai developed a complex mechanism to adapt to cadmium, based on: extracellular biosorption, bioaccumulation, the formation of biofilm, controlled siderophore production, enhanced respiration and modified protein profile. An increased abundance of proteins involved in: cell energy metabolism, including denitrification proteins; amino acid metabolism; cell motility and posttranslational modifications, primarily based on thiol-disulfide exchange, were observed. Enhanced oxygen consumption of biomass in cadmium-amended culture versus control was found. Our results signify that P. aeruginosa san ai is naturally well equipped to overcome and survive high doses of cadmium and, as such, has a great potential for application in bioremediation of cadmium polluted sites.

Purification and characterization of an alkaline lipase from Pseudomonas aeruginosa isolated from putrid mineral cutting oil as component of metalworking fluid.

Extracellular lipase was isolated and purified from the culture broth of Pseudomonas aeruginosa, an extremophile which naturally grows in water-soluble mineral cutting oil (pH 10) used as metalworking fluid (MWF) for cooling and lubrication in industrial metalworking processes. The molecular mass of the purified lipase was estimated by SDS-PAGE to be 54 kDa. The optimum pH and temperature were 11 and 70 degrees C, respectively. The enzyme is stabile over a broad pH range (pH 4-11.5). The lipase preferably acted on triacylglycerols with medium-chain fatty acids. The lipase was inhibited strongly by Zn(2+), Hg(2+), Cu(2+) and slightly by Ca(2+) and Mg(2+). Non-ionic detergents and sodiumdeoxycholate enhanced lipase activity. Alkaline lipase from P. aeruginosa, capable of growing in a water-restricted medium has excellent properties and good potential for biotechnological applications in the metal industry. Its marked stability and activity in organic solvents suggest that this lipase is highly suitable as a biotechnological tool in a water-restricted medium with a variety of applications including organosynthetic reactions and the control and prevention of MWF putrification in the metal industry.

Proteasomes: perspectives from the Archaea.

The development of whole systems approaches to microbiology (e.g. genomics and proteomics) has facilitated a global view of archaeal physiology. Surprisingly, as archaea respond to environmental signals, the majority of protein concentration changes that occur are not reflected at the mRNA level. This incongruity highlights the importance of post-transcription control mechanisms in these organisms. One of the central players in proteolysis is the proteasome, a multicatalytic energy-dependent protease. Proteasomes serve both proteolytic and non-proteolytic roles in protein quality control and in the regulation of cell function. The proteolytic active sites of these enzymes are housed within a central chamber of an elaborate nanocompartment termed the 20S proteasome or core particle. Axial gates, positioned at each end of this particle, restrict the type of substrate that can access the proteolytic active sites. Assortments of regulatory AAA complexes are predicted to recognize/bind and unfold substrate proteins, open the axial gates, and translocate substrate into the 20S core particle.

Purification and characterization of a protease from Pseudomonas aeruginosa grown in cutting oil.

The Pseudomonas aeruginosa san-ai strain was isolated from water-soluble cutting oil used for cooling and lubrication during industrial metal-working processes. This strain, which is grown in a high alkaline (pH 10) mixture of surfactants and mineral oil, produces an extracellular proteolytic enzyme. We have purified and characterized this 18 kDa protease. The P. aeruginosa san-ai protease functions optimally at pH 9.0 and 60 degrees C. Additionally, it is a Zn-containing metalloenzyme, and its monomeric structure contains at least one disulfide bond. Because the enzyme is stable in the presence of organic solvents, it is suitable for peptide synthesis. Furthermore, the P. aeruginosa san-ai protease could be used in an intelligent drug delivery system (DDS) designed for applications in the metal industry for prevention of putrefaction of cutting oil.

Leucine aminopeptidase from Streptomyces hygroscopicus is controlled by a low molecular weight inhibitor.

In culture filtrate of Streptomyces hygroscopicus a producer of polyketide antibiotics, a leucine aminopeptidase and its autogenous inhibitor were detected. The leucine aminopeptidase was purified 4573-fold with yield of 82% by combination of ion exchange and hydrophobic chromatography. The enzyme is monomeric with a molecular mass of 51 kDa determined by gel chromatography and 67 kDa determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Optimal activity was at pH 8.0 and 40 degrees C. The pI of leucine aminopeptidase is 8.2. The enzyme is strongly inhibited by 1,10-phenantroline, amastatin and dithiothreitol. Atomic absorption spectrometry indicated 2 mols of ion zinc per mol of enzyme. The enzyme is stable at up to 70 degrees C. Leucine aminopeptidase prefers leucine and methionine as N-terminal amino acids. Activity of leucine aminopeptidase is strongly modulated by an autogenous low-molecular weight inhibitor during fermentation, especially during periods of intensive antibiotic production.

Production of lipase and protease from an indigenous Pseudomonas aeruginosa strain and their evaluation as detergent additives: compatibility study with detergent ingredients and washing performance.

An indigenous Pseudomonas aeruginosa strain has been studied for lipase and protease activities for their potential application in detergents. Produced enzymes were investigated in order to assess their compatibility with several surfactants, oxidizing agents and commercial detergents. The crude lipase appeared to retain high activity and stability in the presence of several surfactants and oxidizing agents and it was insusceptible to proteolysis. Lutensol® XP80 and Triton® X-100 strongly activated the lipase for a long period (up to 40 and 30% against the control after 1h) while the protease activity was enhanced by the addition of Triton® WR1339 and Tween® 80. The washing performance of the investigated surfactants was significantly improved with the addition of the crude enzyme preparation. Studies were further undertaken to improve enzymes production. The optimization of fermentation conditions led to an 8-fold increase of lipase production, while the production of protease was enhanced by 60%.

Improvement of two-dimensional gel electrophoresis proteome maps of the haloarchaeon Haloferax volcanii.

Proteins of haloarchaea are remarkably unstable in low-ionic-strength solvents and tend to aggregate under standard two-dimensional (2-D) gel electrophoresis conditions, causing strong horizontal streaking. We have developed a new approach to generate 2-D maps of halophilic proteins which included washing cells with 1.5 M Tris-HCl buffer. In addition, proteins were precipitated with acetone, solubilized with urea and thiourea in the presence of the sulfobetaine detergent 3-[(3-cholamidopropyl)dimethylamino]-1-propanesulfonate (CHAPS), reduced with tributylphosphine (TBP), and separated with microrange strips of immobilized pH gradients (pH 3.9-5.1). This combination enabled the construction of highly reproducible 2-D maps of Haloferax volcanii proteins.