Ionic liquid‐based aqueous biphasic systems as a versatile tool for the recovery of antioxidant compounds

The comparative evaluation of distinct types of ionic liquid‐based aqueous biphasic systems (IL‐ABS) and more conventional polymer/salt‐based ABS to the extraction of two antioxidants, eugenol and propyl gallate, is focused. In a first approach, IL‐ABS composed of ILs and potassium citrate (C₆H₅K₃O₇/C₆H₈O₇) buffer at pH 7 were applied to the extraction of two antioxidants, enabling the assessment of the impact of IL cation core on the extraction. The second approach uses ABS composed of polyethylene glycol (PEG) and potassium phosphate (K₂HPO₄/KH₂PO₄) buffer at pH 7 with imidazolium‐based ILs as adjuvants. Their application to the extraction of the compounds allowed the investigation of the impact of the presence/absence of IL, the PEG molecular weight, and the alkyl side chain length of the imidazolium cation on the partition. It is possible to maximize the extractive performance of both antioxidants up to 100% using both types of IL‐ABS. The IL enhances the performance of ABS technology. The data puts in evidence the pivotal role of the appropriate selection of the ABS components and design to develop a successful extractive process, from both environmental and performance points of view. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:70–77, 2015     

Microbial corrosion monitoring by an amperometric microbial biosensor developed using whole cell of Pseudomonas sp.

A microbial biosensor was developed for monitoring microbiologically influenced corrosion (MIC) of metallic materials in industrial systems. The Pseudomonas sp. isolated from corroded metal surface was immobilized on acetylcellulose membrane and its respiratory activity was estimated by measuring oxygen consumption. The microbial biosensor was used for the measurement of sulfuric acid in a batch culture medium contaminated by microorganisms. A linear relationship between the microbial sensor response and the concentration of sulfuric acid was observed. The response time of biosensor was 5 min and was dependent on the immobilized cell loading of Pseudomonas sp., pH, temperature and corrosive environments. The microbial biosensor response was stable, reproducible and specific for sensing of sulfur oxidizing bacterial activity.     

Purification and characterization of 9-hexadecenoic acid cis-trans isomerase from Pseudomonas sp. strain E-3

A 9-hexadecenoic acid cis-trans isomerase (9-isomerase) that catalyzed the cis-to-trans isomerization of the double bond of free 9-cis-hexadecenoic acid [16:1(9c)] was purified to homogeneity from an extract of Pseudomonas sp. strain E-3 and characterized. Electrophoresis of the purified enzyme on both incompletely denaturing and denaturing polyacrylamide gels yielded a single band of a protein with a molecular mass of 80 kDa, suggesting that the isomerase is a monomeric protein of 80 kDa. The 9-isomerase, assayed with 16:1(9c) as a substrate, had a specific activity of 22.8 μmol h^–1 (mg protein)^–1 and a K _m of 117.6 mM. The optimal pH and temperature for catalysis were approximately pH 7–8 and 30° C, respectively. The 9-isomerase catalyzed the cis-to-trans conversion of a double bond at positions 9, 10, or 11, but not that of a double bond at position 6 or 7 of cis-mono-unsaturated fatty acids with carbon chain lengths of 14, 15, 16, and 17. Octadecenoic acids with a double bond at position 9 or 11 were not susceptible to isomerization. These results suggest that 9-isomerase has a strict specificity for both the position of the double bond and the chain length of the fatty acid. The enzyme catalyzed the cis-to-trans isomerization of fatty acids in a free form, and in the presence of a membrane fraction it was also able to isomerize 16:1(9c) esterified to phosphatidylethanolamine. The 9-isomerase was strongly inhibited by catecholic antioxidants such as α-tocopherol and nordihydroguaiaretic acid, but was not inhibited by 1,10-phenanthroline or EDTA or under anoxic conditions. Based on these results, the possible mechanism of catalysis by this enzyme is discussed. Received: 21 May 1997 / Accepted: 5 September 1997     

RpoS as an intermediate in RsmA-dependent regulation of secondary antifungal metabolites biosynthesis in Pseudomonas sp. M18

To investigate the regulatory mechanism governing antifungal metabolite biosynthesis, two kinds of global regulator genes in Pseudomonas sp. M18, an rpoS and an rsmA gene, were cloned and mutated by inserting with an aacC1 cassette, respectively. Two mutants showed the same regulatory mode with repression of phenazine-1-carboxylic acid and remarkable enhancement of pyoluteorin. In the rpoS-mutant, a translational rsmA'-'lacZ fusion was expressed at the same level corresponding to that in the wild-type strain. In the rsmA-mutant, however, expression of the translational rpoS'-'lacZ fusion was only about 30% of that in the wild-type strain. The results indicated that the absence of RsmA leads to repression of the rpoS gene expression, which has further been confirmed with construction of chromosomal rpoS-lacZ fusion in the rsmA-mutant and the wild-type strain, respectively. The findings showed a new regulatory cascade controlling antifungal metabolite production in Pseudomonas sp. M18, suggesting that RpoS may serve as a mediator in RsmA-dependent regulation of secondary metabolite biosynthesis.     

Toluene-induced accumulation of trehalose by Pseudomonas sp. BCNU 106 through the expression of otsA and otsB homologues

AIM: The objective of this study was to investigate toluene-induced accumulation mechanism of trehalose in a toluene-tolerant bacterium Pseudomonas sp. BCNU 106. METHODS AND RESULTS: The accumulation of trehalose by a toluene-tolerant bacterium Pseudomonas sp. BCNU 106 was examined at various cultivation time by measuring the total intracellular trehalose content, trehalase activity and mRNA levels of the trehalose-biosynthetic genes. The pattern of trehalose accumulation corresponded to the mRNA expression pattern of the trehalose-biosynthetic genes with the maximum level at 12 h or 4 h of cultivation with 10% (v/v) toluene, respectively. The trehalose-biosynthetic genes were also cloned and sequenced. Furthermore, the effects of toluene addition on the intracellular osmotic pressure and pH were investigated. It was shown that homeostasis was maintained in the bacterial cells. CONCLUSIONS: In a toluene-tolerant bacterium Pseudomonas sp. BCNU 106, a significant amount of trehalose was accumulated through the toluene-induced expression of the trehalose-biosynthetic genes after the exposure to toluene. SIGNIFICANCE AND IMPACT OF THE STUDY: The accumulation of the high level of intracellular trehalose was preceded by the expression of otsA/B genes in toluene-tolerant bacteria, contributing to the elucidation of the tolerance mechanism.     

GacA对假单胞菌株M18两个phz基因簇和菌群传感的调控

摘要:【目的】假单胞菌株M18（Pseudomonas sp. M18）是从甜瓜根际土壤中分离获得的一株对多种植物病原菌具有显著拮抗作用的菌株,在菌群传感（quorum sensing）系统的调控下,能分泌吩嗪-1-羧酸（PCA）以及多种吩嗪（phz）类衍生物的抗真菌物质。全局性因子GacA是M18菌株吩嗪类物质的合成与菌群传感系统的重要调控因子,本文将就GacA对上述两者的调控做进一步研究。【方法】PCR基因扩增和测序研究M18菌株中PCA合成基因簇,运用RT-PCR及构建phzA-lacZ转录融合手段     

Purification and characterization of the recombinant alginate lyase from Pseudomonas sp. leaked by Escherichia coli upon addition of glycine

Abstract The plasmid pAL205 encodes an alginate lyase gene of Pseudomonas sp. OS-ALG-9, fused in frame to the β-galactosidase α-peptide gene. The alginate lyase (Aly) expressed in Escherichia coli (pAL205) was significantly secreted into the medium by the addition of glycine. The extracellular enzyme isolated from the culture of E. coli JM109 (pAL205) was purified over 15 000-fold by successive chromatography and subjected to amino acid sequence analysis. The sequence determined was identical to that of the intracellular protein. Since the activity and molecular size of the extracellular Aly is identical to the intracellular protein and to the Aly isolated from Pseudomonas , the glycine does not affect or modify the Aly during its leakage into the medium.     

N-Carbamyl-L-Amino Acid Amidohydrolase of Pseudomonas sp. Strain NS671: Purification and Some Properties of the Enzyme Expressed in Escherichia coli

An N-carbamyl-L-amino acid amidohydrolase was purified from cells of Escherichia coli in which the gene for N-carbamyl-L-amino acid amidohydrolase of Pseudomonas sp. strain NS671 was expressed. The purified enzyme was homogeneous by the criterion of SDS–polyacrvlamide gel electrophoresis. The results of gel filtration chromatography and SDS–polyacrylamide gel electrophoresis suggested that the enzyme was a dimeric protein with 45-kDa identical subunits. The enzyme required Mn²⁺ ion (above 1 mM) for the activity. The optimal pH and temperature were 7.5 and around 40°C, respectively, with N-carbamyl-L-methionine as the substrate. The enzyme activity was inhibited by ATP and was iost completely with p-chloromercuribenzoate (1 mM). The enzyme was strictly L-specific and showed a broad substrate specificity for N-carbamyl-L-α-amino acids.     

阿特拉津降解菌Acinetobacter sp. DNS32对无机氮源的响应

【目的】研究Acinetobacter sp.DNS32的生长、阿特拉津降解能力和降解基因转录水平的表达对无机氮素的响应关系,为菌株的工程应用提供指导与理论基础。【方法】以Acinetobactersp.DNS32为对象,采用摇瓶法研究菌株在阿特拉津培养基中菌株生长情况及降解能力对外加硝态氮与铵态氮的响应关系,利用荧光定量PCR技术检测DNS32降解基因表达量对外加无机氮源的响应关系。【结果】外加无机氮源可以促进DNS32菌株的生长,提高阿特拉津降解能力,无机氮源对DNS32菌株的trzN、atzB和atzC 3种降解基因表达均有促进作用,加入无机氮源的试验处理中DNS32菌株trzN基因的表达量最高可达对照的11.252±2.408倍,推断DNS32菌株的这3种降解基因所编码的酶是稳定表达的组成酶。【结论】DNS32降解阿特拉津不受"氮饥饿"诱导机制调控,且无机氮源的存在对菌株的生长与降解有促进作用,因此菌株在土壤修复实践中具有广阔的应用前景。     

Purification and characterization of a Synechococcus sp. strain PCC 7942 polypeptide structurally similar to the stress-induced Dps/PexB protein of Escherichia coli

A stable DNA/protein complex having an apparent molecular mass of approximately 150kDa was purified from nitrate-limited cultures of the cyanobacterium Synechococcus sp. strain PCC 7942. Amino-terminal peptide sequencing indicated that the polypeptide was structurally similar to the Dps protein of Escherichia coli; Dps is also known as the product of the starvation- and stationary-phase-inducible gene, pexB. The 150-kDa complex dissociated into a 22-kDa protein monomer after boiling in 2% SDS. The 150-kDa complex preparation had approximately a 10% nucleic acid content and upon dissociation released DNA fragments that were sensitive to S1 nuclease digestion. Immunoblot data indicated that the complex accumulates during stationary phase and during nitrogen, sulfur, and phosphorus limitation. DNA-binding assays indicated that the protein nonspecifically binds both linear and supercoiled DNA. Circular dichroism spectroscopy revealed that the Synechococcus sp. Dps-like protein contains extensive regions of alpha-helical secondary structure. We propose that the 150-kDa complex represents a hexameric aggregate of the Dps-like protein complexed with single-stranded DNA and serves to bind a portion of the chromosomal DNA under nutrient-limited conditions.     

Treatability of cheese whey for single-cell protein production in nonsterile systems: Part II. The application of aerobic sequencing batch reactor (aerobic SBR) to produce high biomass of Dioszegia sp. TISTR 5792

This study aimed to investigate the efficiency of an aerobic sequencing batch reactor (aerobic SBR) in a nonsterile system using the application of an experimental design via central composite design (CCD). The acidic whey obtained from lactic acid fermentation by immobilized Lactobacillus plantarum sp. TISTR 2265 was fed into the bioreactor of the aerobic SBR in an appropriate ratio between acidic whey and cheese whey to produce an acidic environment below 4.5 and then was used to support the growth of Dioszegia sp. TISTR 5792 by inhibiting bacterial contamination. At the optimal condition for a high yield of biomass production, the system was run with a hydraulic retention time (HRT) of 4 days, a solid retention time (SRT) of 8.22 days, and an acidic whey concentration of 80% feeding. The chemical oxygen demand (COD) decreased from 25,230 mg/L to 6,928 mg/L, which represented a COD removal of 72.15%. The yield of biomass production and lactose utilization by Dioszegia sp. TISTR 5792 were 13.14 g/L and 33.36%, respectively, with a long run of up to 180 cycles and the pH values of effluent were rose up to 8.32 without any pH adjustment.     

Jasminum polyanthum Franch. as a natural source of (−)‐methyl jasmonate: an alternative to the use of the synthetic standard

Introduction – Methyl jasmonate (MJ) contains two chiral centres at C‐3 and C‐7 in its chemical structure, which implies that it can exist in four possible stereoisomeric forms, namely (+)‐MJ, (?)‐MJ, (+)‐epiMJ and (?)‐epiMJ. The absolute configuration of the two side chains of MJ affects the biological activity associated with this compound. Objective – To isolate pure (?)‐MJ from a natural source, Jasminum polyanthum Franch., with the intention of increasing the knowledge about its biological properties, including its effect on the biosynthesis of plant metabolites. Methodology – The method used was based on steam distillation extraction (SDE) as an extraction technique followed by high‐performance liquid chromatography (HPLC) as a purification procedure. The HPLC flow‐rate as well as the number of fractions accumulated were optimised to achieve the concentration and purity required. Results – The employment of 0.3 mL/min as HPLC flow‐rate and the accumulation of three HPLC fractions allowed the required enantiomeric purity (95%) and concentration (0.36 mg/L in each HPLC fraction) to efficiently obtain (?)‐methyl jasmonate from Jasminum polyanthum Franch. to be achieved. Conclusion – The approach proposed may enable the properties and effect of pure (?)‐MJ on plant responses to be studied. The use of a natural source to obtain (?)‐MJ is presented as an alternative to the enantioselective synthesis and enantiomeric resolution from the standard racaemic mixture. Copyright © 2009 John Wiley & Sons, Ltd.