Organic solvent adaptation of Gram positive bacteria: applications and biotechnological potentials

Organic-solvent-tolerant bacteria are considered extremophiles with different tolerance levels that change among species and strains, but also depend on the inherent toxicity of the solvent. Extensive studies to understand the mechanisms of organic solvent tolerance have been done in Gram-negative bacteria. On the contrary, the information on the solvent tolerance mechanisms in Gram-positive bacteria remains scarce. Possible shared mechanisms among Gram-(−) and Gram-(+) microorganisms include: energy-dependent active efflux pumps that export toxic organic solvents to the external medium; cis-to-trans isomerization of unsaturated membrane fatty acids and modifications in the membrane phospholipid headgroups; formation of vesicles loaded with toxic compounds; and changes in the biosynthesis rate of phospholipids to accelerate repair processes. However, additional physiological responses of Gram-(+) bacteria to organic solvents seem to be specific. The aim of the present work is to review the state of the art of responsible mechanisms for organic solvent tolerance in Gram-positive bacteria, and their industrial and environmental biotechnology potential.     

Biocatalytic synthesis and antitumor activities of novel silybin acylated derivatives with dicarboxylic acids

Novel silybin acylated derivatives with dicarboxylic acids were prepared in various organic solvents using immobilized Candida antarctica lipase B (Novozym 435(?)). The reaction parameters affecting the silybin conversion, such as the nature of the organic solvent and the acyl donor used were investigated. The antiproliferative effects of silybin monoesters, and their ability to modulate the secretion of vascular endothelial growth factor (VEGF) were estimated using K562 human lymphoblastoma cells and compared to the parental compound. The synthesized esters retained the biological function of silybin and in some cases were more effective, indicating that target biotransformation may generate novel compounds with improved antitumor and antiangiogenic activities.     

用于SCAR检测的基因组DNA简易提取法

用一种简单的方法,无需接触有毒的有机试剂,无需离心,即可从小麦叶片中提取完整的基因组DNA,质量满足SCAR检测的要求,该方法可在短时间内制备大量样品,适于进行遗传连锁性分析时对F2分离群体的单株检测和分子标记辅助选择时筛选育种材料,操作简单,实用性强,值得推广。     

Enhancement of stability and activity of phospholipase A(1) in organic solvents by directed evolution.

We attempted to apply the directed evolution approach to enhancing enzyme properties in the presence of organic solvents, in which enzyme stability and activity were often drastically reduced. Stability and catalytic activity of phospholipase A(1) in the presence of an organic solvent were enhanced by error-prone polymerase chain reaction (PCR) and DNA shuffling followed by a filter-based visual screening. Three mutants (SA8, SA17 and SA20) were isolated on indicator plates (i.e., 1% phosphatidylcholine gels containing 30% dimethyl sulfoxide (DMSO)) after a second mutant library was treated in 50% DMSO for 36 h. The half-life values of the three mutants exhibited an approximately 4-fold increase. The three mutants also exhibited increased stability in all organic solvents tested compared with the wild-type enzyme. Thus, an enzyme variant having superior catalytic efficiency in most of the organic solvents could be obtained by using any solvent suitable for designing the efficient screening system, regardless of the properties of the particular solvent.     

Optimized and automated protocols for high-throughput screening of amylosucrase libraries

This article describes the design and validation of a general procedure for the high-throughput isolation of amylosucrase variants displaying higher thermostability or increased resistance to organic solvents. This procedure consists of 2 successive steps: an in vivo selection that eliminates inactive variants followed by automated screening of active variants to isolate mutants displaying enhanced features. The authors chose an Escherichia coli expression vector, allowing a high production rate of the recombinant enzyme in miniaturized culture conditions. The screening assay was validated by minimizing variability for various parameters of the protocol, especially bacterial growth and protein production in cultures in 96-well microplates. Recombinant amylosucrase production was normalized by decreasing the coefficient of variance from 27% to 12.5%. Selective screening conditions were defined to select variants displaying higher thermostability or increased resistance to organic solvents. A first-generation amylosucrase variant library, constructed by random mutagenesis, was subjected to this procedure, yielding a mutant displaying a 25-fold increased stability at 50 degrees C compared to the parental wild-type enzyme.     

Lipase-catalyzed transesterification in several reaction systems: An application of room temperature lonic liquids for bi-phasic production ofn-butyl acetate

Organic solvents are widely used in biotransformation systems. There are many efforts to reduce the consumption of organic solvents because of their toxicity to the environment and human health. In recent years, several groups have started to explore novel organic solvents called room temperature ionic liquids in order to substitute conventional organic solvents. In this work, lipase-catalyzed transesterification in several uni-and bi-phasic systems was studied. Two representative hydrophobic ionic liquids based on 1-butyl-3-methylimidazolum coupled with hexafluorophosphate ([BMIM][PF₆]) and bis[(trifluoromethylsulfonyl) imide] ([BMIM] [Tf₂N]) were employed as reaction media for the transesterification ofn-butanol. The commercial lipase, Novozym 435, was used for the transesterification reaction with vinyl acetate as an acyl donor, The conversion yield was increased around 10% in a water/[BMIM][Tf₂N], bi-phasic system compared with that in a water/hexane system. A higher distribution of substrates into the water phase is believed to enhance the conversion yield in a water/[BMIM][Tf₂N] system. Partion coefficients of the substrates in the water/[BMIM][Tf₂N] bi-phasic system were higher than three times that found in the water/hexane system, while n-butyl acetate showed a similar distribution in both systems. Thus, RTILs appear to be a promising substitute of organic solvents in some biotransformation systems.     

Extremophiles: radiation resistance microbial reserves and therapeutic implications

Micro‐organisms with the ability to survive in extreme environmental conditions are known as ‘extremophiles’. Currently, extremophiles have caused a sensation in the biotechnology/pharmaceutical industries with their novel compounds, known as ‘extremolytes’. The potential applications of extremolytes are being investigated for human therapeutics including anticancer drugs, antioxidants, cell cycle‐blocking agents, anticholesteric drugs, etc. It is hypothesized that the majority of ultraviolet radiation (UVR)‐resistant micro‐organisms can be used to develop anticancer drugs to prevent skin damage from UVR. The metabolites from UVR‐resistant microbes are a great source of potential therapeutic applications in humans. This article aims to discuss the potentials of extremolytes along with their therapeutic implications of UVR extremophiles. The major challenges of therapeutic development using extremophiles are also discussed.     

Detection of aromatic catabolic gene expression in heterogeneous organic matter used for reduction of volatile organic compounds (VOC) by biofiltration

A qualitative procedure of purified DNA/RNA co-extraction from complex organic matter, used as biofilter support for removing volatile organic compounds, was set up and applied to detect xylene monooxygenase gene expression by RT-PCR. A DNA/RNA extraction protocol based on a combination of sample lyophilization pre-treatment and CTAB––phenol/chloroform extraction procedure was optimized for the recovery of purified nucleic acids [100–500 ng DNA (10 kb) and 0.5–2 μg of rRNA 16S from 100 mg matrix]. PCR and RT-PCR protocols were established to detect xylene monooxygenase gene expression starting from differentially induced organic matrices obtained by biofiltration technology. This work allowed the microbial degradation activities in heterogeneous organic solid media to be studied and suggests a rapid method to follow specific biological activities during solid and/or semisolid organic substrates biotransformation.     

Effect of organic solvents on penicillin acylase-catalyzed reactions: interaction of organic solvents with enzymes

The effects of various organic solvents on penicillin acylase-catalyzed synthesis of β-lactam antibiotics (pivampicillin and ampicillin) have been investigated in water-solvent mixtures. The rates of penicillin acylase-catalyzed reactions were found to be significantly reduced by the presence of a small amount of organic solvent. In particular, the rate of enzyme catalysis was extremely low in the presence of ring-structured solvents and acids while enzyme activities were fully restored after removing the solvents. This indicates that interactions between the solvents and the enzyme are specific and reversible. To correlate the inhibitory effects of organic solvents with solvent properties the influence of solvent hydrophobicities and solvent activity on the rate of pivampicillin synthesis was examined. The reaction rate was found to decrease with increasing solvent hydrophobicities, and a better correlation was observed between the reaction rate and solvent activity. The effects of ionic strength on the synthesis of pivampicillin and ampicillin were also examined. The ionic strength dependence indicates that electrostatic interactions are involved in the binding of ionic compounds to the enzyme. On the basis of the active site structure of penicillin acylase, a possible mechanism for molecular interactions between the enzyme and organic solvents is suggested.     

Biotransformation in double-phase systems: physiological responses of Pseudomonas putida DOT-T1E to a double phase made of aliphatic alcohols and biosynthesis of substituted catechols

Pseudomonas putida strain DOT-T1E is highly tolerant to organic solvents, with a logP(ow) (the logarithm of the partition coefficient of a solvent in a two-phase water-octanol system of > or =2.5. Solvent tolerant microorganisms can be exploited to develop double-phase (organic solvent and water) biotransformation systems in which toxic substrates or products are kept in the organic phase. We tested P. putida DOT-T1E tolerance to different aliphatic alcohols with a logP(ow) value between 2 and 4, such as decanol, nonanol, and octanol, which are potentially useful in biotransformations in double-phase systems in which compounds with a logP(ow) around 1.5 are produced. P. putida DOT-T1E responds to aliphatic alcohols as the second phase through cis-to-trans isomerization of unsaturated cis fatty acids and through efflux of these aliphatic alcohols via a series of pumps that also extrude aromatic hydrocarbons. These defense mechanisms allow P. putida DOT-T1E to survive well in the presence of high concentrations of the aliphatic alcohols, and growth with nonanol or decanol occurred at a high rate, whereas in the presence of an octanol double-phase growth was compromised. Our results support that the logP(ow) of aliphatic alcohols correlates with their toxic effects, as octanol (logP(ow) = 2.9) has more negative effects in P. putida cells than 1-nonanol (logP(ow) = 3.4) or 1-decanol (logP(ow) = 4). A P. putida DOT-T1E derivative bearing plasmid pWW0-xylE::Km transforms m-xylene (logP(ow) = 3.2) into 3-methylcatechol (logP(ow) = 1.8). The amount of 3-methylcatechol produced in an aliphatic alcohol/water bioreactor was 10- to 20-fold higher than in an aqueous medium, demonstrating the usefulness of double-phase systems for this particular biotransformation.     

A protease stable in organic solvents from solvent tolerant strain of Pseudomonas aeruginosa

A solvent tolerant strain of Pseudomonas aeruginosa (PseA) was isolated from soil samples by cyclohexane enrichment in medium. The strain was able to sustain and grow in a wide range of organic solvents. The adaptation of P. aeruginosa cell towards solvents was seen at membrane level in transmission electron micrographs. It also secreted a novel protease, which exhibited remarkable solvent stability and retained most of the activity at least up to 10 days in the presence of hydrophobic organic solvents (log P > or = 2.0) at 25% (v/v) concentrations. The protease was able to withstand as high as 75% concentration of solvents at least up to 48 h. P. aeruginosa strain and its protease, both seem promising for solvent bioremediation, wastewater treatment and carrying out biotransformation in non-aqueous medium.     

Recent advances of enzymatic reactions in ionic liquids

The tremendous potential of room temperature ionic liquids as an alternative to environmentally harmful ordinary organic solvents is well recognized. Ionic liquids, having no measurable vapor pressure, are an interesting class of tunable and designer solvents, and they have been used extensively in a wide range of applications including enzymatic biotransformation. In fact, ionic liquids can be designed with different cation and anion combinations, which allow the possibility of tailoring reaction solvents with specific desired properties, and these unconventional solvent properties of ionic liquids provide the opportunity to carry out many important biocatalytic reactions that are impossible in traditional solvents. As compared to those observed in conventional organic solvents, the use of enzymes in ionic liquids has presented many advantages such as high conversion rates, high enantioselectivity, better enzyme stability, as well as better recoverability and recyclability. To date, a wide range of pronounced approaches have been taken to further improve the performance of enzymes in ionic liquids. This review presents the recent technological developments in which the advantages of ionic liquids as a medium for enzymes have been gradually realized.     

Bioengineering of Nitrilases Towards Its Use as Green Catalyst: Applications and Perspectives

Nitrilases are commercial biocatalysts used for the synthesis of plastics, paints, fibers in the chemical industries, pharmaceutical drugs and herbicides for agricultural uses. Nitrilase hydrolyses the nitriles and dinitriles to their corresponding carboxylic acids and ammonia. They have a broad range of substrate specificities as well as enantio-, regio- and chemo-selective properties which make them useful for biotransformation of nitriles to important compounds because of which they are considered as ‘Green Catalysts’. Nitriles are widespread in nature and synthesized as a consequence of anthropogenic and biological activities. These are also present in certain plant species and are known to cause environmental pollution. Biotransformation using native organisms as catalysts tends to be insufficient since the enzyme of interest has very low amount in the total cellular protein, rate of reaction is slow along with the instability of enzymes. Therefore, to overcome these limitations, bioengineering offers an alternative approach to alter the properties of enzymes to enhance the applicability and stability. The present review highlights the aspects of producing the recombinant microorganisms and overexpressing the enzyme of interest for the enhanced stability at high temperatures, immobilization techniques, extremes of pH, organic solvents and hydrolysing dintriles to chiral compounds which may enhance the possibilities for creating specific enzymes for biotransformation.     

Translation of the copper/bipyridine-promoted Petasis reaction to solid phase-coupled DNA for encoded library synthesis

The Petasis three-component reaction gives rise to diverse substituted α-aryl glycines from readily available amines, boronic acids and glyoxalic acid. Thus, this reaction is highly attractive for DNA-encoded small molecule screening library synthesis. The Petasis reaction is for instance promoted by a potentially DNA damaging copper(I)/bipyridine reagent system in dry organic solvents. We found that solid phase-coupled DNA strands tolerated this reagent system at elevated temperature allowing for synthesis of diverse substituted DNA-tagged α-aryl glycines from DNA-conjugated secondary amines.     

Application of organic mono-phase and organic–aqueous two-liquid-phase systems in microalgal conversion of androst-4-en-3,17-dione by Nostoc muscorum

Organic mono-phase and organic–aqueous two-phase systems were applied for 17-carbonyl reduction of androst-4-en-3,17-dione to testosterone by whole cells of the microalga Nostoc muscorum (Nostocaceae). To investigate the correlation between solvent hydrophobicity and biotransformation yield in mono- and biphasic systems, a range of 16 organic solvents with log P_octanol values (logarithm of the solvent partition coefficient in the n-octanol/water system) between ? 1.1 and 8.8 were examined. Organic solvents with log P_octanol values greater than 7, such as hexadecane and tetradecane, provided the best biocompatibility with the bioconversion by algal cells. The data also indicated that the highest yields were obtained using organic–aqueous (1:1, v/v) biphasic systems. The optimum volumetric phase ratio, reaction temperature and substrate concentration were 1:1, 30°C and 0.5 mg mL^?1, respectively. Under the mentioned conditions a fourfold increase in biotransformation yield (from 7.8±2.3 to 33.4±1.8%) was observed.     

Metal-induced inhibition of anaerobic metabolism of volatile fatty acids and hydrogen

The effects of copper (Cu), chromium (Cr), cadmium (Cd), lead (Pb) and zinc (Zn) on the biotransformation of organic acids (acetate, propionate and butyrate) and H₂ were assessed in serum-bottle microcosms. Experiments were performed over a range of metal concentrations (20–200 mg/1) using biomass from an anaerobic bioreactor fed continuously with ethanol distillery waste as inoculum. In general, the added metals inhibited the biotransformation of organic acids with increasing metal concentration. However, the extent of inhibition varied for the different biotransformations and for the different metals tested. For example, the concentration of CuCl₂ effecting a 50% reduction in the rate constant for biotransformation of acetate, propionate and butyrate was 60, 75 and 30 mg/1, respectively. Cu and Cr (VI) were the most inhibitory metals in organic acid transformation, whereas Pb was the least toxic. The rate of biotransformation of acetate was reduced by half at Cu and Cr concentrations of 60 and 40 gm/1 respectively, whereas Cd, Pb, and Zn concentrations of 160 to 200 mg/l had little effect. The activities of hydrogenotrophic methanogens were much less affected by the same metals and metal concentrations.     

Rapid characterization of microbial biodegradation pathways by FT-IR spectroscopy

Fourier transform-infrared (FT-IR) spectroscopy has become an important tool for rapid analysis of complex biological samples. The infrared absorbance spectrum could be regarded as a "fingerprint" which is characteristic of biochemical substances. In this study, Pseudomonas putida NCIMB 9869 was grown with either 3,5-xylenol or m-cresol as the sole carbon source, each inducing different metabolic pathways for m-cresol biotransformation. FT-IR spectroscopy was capable of differentiating both induced cultures of P. putida NCIMB 9869 as well as the resulting biotransformation product mixtures. FT-IR spectral analysis indicated that carboxylic acids were key chemicals responsible for distinguishing the products of the two catabolic pathways. Gas chromatography-mass spectrometry (GC-MS) was performed to validate the FT-IR analysis, indicating that two carboxylic acids, 3-hydroxybenzoic acid and 2,5-dihydroxybenzoic acid, were present as m-cresol biotransformation products from 3,5-xylenol-grown cells, but were absent in m-cresol-grown cells. The ability to use FT-IR to rapidly distinguish between biotransformation product mixtures as well as differentially induced bacterial strains suggests this approach might be a valuable tool for screening large biotransformation assays for novel products and metabolic mutants.     

Characterization of an organic-solvent-tolerant Brevibacillus agri strain 13 able to stabilize solvent/water emulsion

Brevibacillus agri strain 13 was isolated and characterized as a Gram-positive organic-solvent-tolerant bacterium able to grow at 45 °C. It can tolerate high concentrations (5% and 20%, v/v) of various organic solvents with a broad range of log P _ow when the organic solvent was provided as a nonaqueous layer. Although it can tolerate a number of aromatic solvents, it cannot utilize them as a sole carbon source. The surface characteristics of cells exposed to organic solvent were investigated using the bacterial adhesion to hydrocarbon test, a contact angle measurement, ζ potential determination, and fluorescence microscopy analysis and compared with that of nonexposed cells. The results showed that although it has a hydrophilic cell surface, it has a unique indigenous cell surface characteristic in which the cells can stabilize solvent-in-water emulsion by adhering to the solvent–water interface of the solvent droplets. The tolerance and predilection of B. agri strain 13 toward organic solvents may suggest its potential application as a whole-cell biocatalyst for the biotransformation process of water-immiscible substrate(s).     

Whole cell yeast biotransformations in two-phase systems: Effect of solvent on product formation and cell structure

Summary Biotransformation of benzaldehyde and pyruvate to (R)-phenylacetyl carbinol bySaccharomyces cerevisiae was investigated in two-phase aqueous-organic reaction media. With hexane as organic solvent, maximum biotransformation activity was observed with a moisture content of 10%. Of the organic solvents tested, highest biotransformation activities were observed with hexane and hexadecane, and lowest activities occurred with chloroform and toluene. Biocatalyst samples from biphasic media containing hexane, decane and toluene manifested no apparent cell structural damage when examined using scanning electron microscopy. In contrast, cellular biocatalyst recovered from two-phase systems containing chloroform, butylacetate and ethylacetate exhibited damage in the form of cell puncturing after different incubation periods. Phospholipids were detected in reaction media from biocatalytic systems which exhibited cell damage in electron micrographs. Phospholipid release was much lower in the two-phase systems containing toluene or hexane or in 100% aqueous biocatalytic system.