Induced protein profile changes in arsenate tolerant and sensitive pseudomonas fluorescens strains

Pseudomonas fluorescens are gram‐negative, motile, rod‐shaped bacteria known for their metabolic versatility. Many strains of this species possess excellent capability to colonize plant roots, promoting plant growth in soils contaminated with toxic metals. P. fluorescens biotype F is a strain tolerant to arsenic which had been isolated previously from soil contaminated with arsenic and other toxic metals and characterized as a promoter of plant growth and accumulation of arsenic. In the present work we studied the protein profile of this strain together with the profile of a sensitive P. fluorescens CECT 378 in the presence and absence of sodium arsenate using two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE). The gels were analyzed by PDQuest, while the statistical significance was proved by ANOVA. We found 9 differentially expressed proteins in the tolerant strain ‐ 4 new proteins, 4 upregulated proteins and 1 downregulated proteins in the presence of 1000 ppm As. In the non‐tolerant strain there were 7 differentially expressed proteins ‐ 1 new protein, 3 upregulated proteins and 3 downregulated proteins. The identification of the proteins with MALDI‐TOF is in progress. Proteino profilio pakeitimai, sukelti arseną toleruojančioje ir jautriose pseudomonas fluorescens atmainose Santrauka Pseudomonas fluorescens yra gramneigiama, judanti lazdelės formos bakterija, žinomas jos metabolinis universalumas. Daugelis šios rūšies atmainų gerai sugeba kolonizuotis augalų šaknyse ir padeda augti augalams toksiniais metalais užterštame dirvožemyje. P. fluorescens biotipas F yra tolerantiškas arsenui, jis buvo atskirtas nuo dirvožemio, užteršto arsenu ir kitais toksiniais metalais ir charakterizuotas kaip augalų augimo aktyviklis bei arseno akumuliatorius. Tirtas šios atmainos proteino profilis kartu su jautriojo P. fluorescens CECT 378 profiliu, kai natrio arsenatas yra ir kai jo nėra, naudojant dviejų dimensijų poliakrilamido gelių elektroforezę (2D-PAGE). Geliai buvo analizuoti PDQuest, statistinė reikšmė patikrinta su ANOVA. Atrasti 9 skirtingai išreikšti proteinai toleruojančioje atmainoje. Netoleruojančioje atmainoje buvo skirtingai išreikšti 7 baltymai. Daug nuveikta identifikuojant proteinus su MALDI-TOF. Reikšminiai žodžiai: arsenas, Pseudomonus fluorescens, toleravimas, proteinai, 2D-elektroforezė, rizobakterijos. Изменения профиля протеина, вызванные толерантными к мышьяку и чувствительными разновидностями pseudomonas fluorescens Резюме Pseudomonas fluorescens – это грамм-отрицательная, движущаяся бактерия в виде палочки. Известна ее метаболическая универсальность. Большинство разновидностей этого вида способны создавать колонии в корнях растений, способствуя росту растений в загрязненной тяжелыми металлами почве. Биотип P. fluorescens толерантен к мышьяку, ранее выделенному из почвы, зараженной мышьяком и другими токсичными металлами, и характеризуется как активатор роста растений и аккумуляции мышьяка. Нами исследовался профиль протеина этой разновидности совместно с профилем чувствительного CECT 378 P. fluorescens в случае с мышьяком и с применением для электрофореза (2D-PAGE) двухдименсионного полиакриламидного геля. Гель был проанализирован в PDQuest, статистическое значение было подтверждено с помощью ANOVA. Были обнаружены 9 дифференцированно выраженных протеинов в толерантной разновидности при 1000 ppm As. В нетолерантной разновидности были дифференцированно выделены 7 протеинов. Идентификация протеинов с помощью MALDI-TOF продолжается. Ключевые слова: мышьяк, Pseudomonas fluorescens, толеранция, протеин, 2-дименсионный электрофорез, ризобактерия, обработка протеинов. First Published Online: 14 Oct 201

A Biofuel Similar to Biodiesel Obtained by Using a Lipase from Rhizopus oryzae, Optimized by Response Surface Methodology

A new biodiesel-like biofuel is obtained by the enzymatic ethanolysis reaction of sunflower oil with ethanol, in free solvent media, by using BIOLIPASE-R, a multipurpose alimentary additive from Biocon®-Spain that is a low cost lipase from a strain of Rhizopus oryzae. This biofuel is composed by two parts of fatty acid ethyl esters (FAEE) and one of monoglyceride (MG), which in this form integrates glycerol, through the application of the 1,3-selective lipases. Thus, this process minimizes waste generation and maximizes the efficiency of the process because no residual glycerol is produced. Response surface methodology (RSM) is employed to evaluate the main reaction parameters (reaction temperature, oil/ethanol ratio and pH) on the sunflower oil conversion. Water content and amount of lipase were also previously investigated. Regarding the results, we found that it operates optimally with a water content of the reaction medium of 0.15%, 0.05%–0.1% lipase by weight relative to the weight of oil used, 20 °C, volume ratio (mL/mL) oil/ethanol 12/3.5 and pH 12 (by addition of 50 μL of 10 N NaOH solution). These results have proven a very good efficiency of the biocatalyst in the studied selective process

Biocatalytic Behaviour of Immobilized Rhizopus oryzae Lipase in the 1,3-Selective Ethanolysis of Sunflower Oil to Obtain a Biofuel Similar to Biodiesel

A new biofuel similar to biodiesel was obtained in the 1,3-selective transesterification reaction of sunflower oil with ethanol using as biocatalyst a Rhizopus oryzae lipase (ROL) immobilized on Sepiolite, an inorganic support. The studied lipase was a low cost powdered enzyme preparation, Biolipase-R, from Biocon-Spain, a multipurpose additive used in food industry. In this respect, it is developed a study to optimize the immobilization procedure of these lipases on Sepiolite. Covalent immobilization was achieved by the development of an inorganic-organic hybrid linker formed by a functionalized hydrocarbon chain with a pendant benzaldehyde, bonded to the AlPO4 support surface. Thus, the covalent immobilization of lipases on amorphous AlPO4/sepiolite (20/80 wt %) support was evaluated by using two different linkers (p-hydroxybenzaldehyde and benzylamine-terephthalic aldehyde, respectively). Besides, the catalytic behavior of lipases after physical adsorption on the demineralized sepiolite was also evaluated. Obtained results indicated that covalent immobilization with the p-hydroxybenzaldehyde linker gave the best biocatalytic behavior. Thus, this covalently immobilized lipase showed a remarkable stability as well as an excellent capacity of reutilization (more than five successive reuses) without a significant loss of its initial catalytic activity. This could allow a more efficient fabrication of biodiesel minimizing the glycerol waste productio

New Biofuel Integrating Glycerol into Its Composition Through the Use of Covalent Immobilized Pig Pancreatic Lipase

By using 1,3-specific Pig Pancreatic lipase (EC 3.1.1.3 or PPL), covalently immobilized on AlPO4/Sepiolite support as biocatalyst, a new second-generation biodiesel was obtained in the transesterification reaction of sunflower oil with ethanol and other alcohols of low molecular weight. The resulting biofuel is composed of fatty acid ethyl esters and monoglycerides (FAEE/MG) blended in a molar relation 2/1. This novel product, which integrates glycerol as monoacylglycerols (MG) into the biofuel composition, has similar physicochemical properties compared to those of conventional biodiesel and also avoids the removal step of this by-product. The biocatalyst was found to be strongly fixed to the inorganic support (75%). Nevertheless, the efficiency of the immobilized enzyme was reduced to half (49.1%) compared to that of the free PPL. The immobilized enzyme showed a remarkable stability as well as a great reusability (more than 40 successive reuses) without a significant loss of its initial catalytic activity. Immobilized and free enzymes exhibited different reaction mechanisms, according to the different results in the Arrhenius parameters (Ln A and Ea). However, the use of supported PPL was found to be very suitable for the repetitive production of biofuel due to its facile recyclability from the reaction mixture