Absolute dormancy of pollen induced by soaking in organic solvents

Summary The pollen grains ofChrysanthemum pacificum, andLilium longiflorum retained their viability in organic solvents, and by soaking in the solvents the curve of the pollen longevity was separated into two. Pollen soaked in organic solvents may become absolute dormant temporarily, but recover the initial activity when taken out of the solvents.     

1H nuclear magnetic resonance studies of N-acetyl-L-proline N-methylamide. Molecular conformations,hydrogen bondings,and thermodynamic quantities in various solvents

Concentration and temperature dependences of the ¹H nmr spectra of N-acetyl-L -proline N-methylamide were observed in various solvents [CCl₄, CDCl₃, (CD₃)₂CO, (CD₃)₂SO, H₂O, and D₂O]. The fraction of the cis isomer (with respect to the bond between the acetyl carbonyl carbon and prolyl nitrogen atoms) depends greatly on the solvent used; the fraction of the cis isomer is higher in polar solvents than in nonpolar solvents. It depends also on concentration and temperature in nonpolar solvents but not in polar solvents. In nonpolar solvents the trans isomer mostly exists in the γ-turn structure with an intramolecular hydrogen bond and the cis isomer tends to form molecular aggregates by intermolecular hydrogen bonds. In polar solvents both the cis and trans isomers exist in monomeric forms which interact with solvent molecules. The pH dependences of the N-methyl proton resonances indicate that the γ-turn structure of the trans isomer is present also in aqueous solution, though its population is difficult to determine. Apparent enthalpy and entropy changes for the conversion of the trans isomer to cis isomer are evaluated for various solvents. The results are discussed in terms of the intra- and intermolecular hydrogen bondings.     

Screening of biocompatible organic solvents for enhancement of lipid milking from Nannochloropsis sp.

To extract the microalgal lipid in situ, biocompatible solvents were screened for lipid milking of Nannochloropsis sp. in an aqueous–organic system. The effects of organic solvents on the microalgal growth, the lipid extractability, the dehydrogenases activity and the cell membrane integrity were investigated by UV–visible spectrophotometer, FT-IR spectroscopy, 2,3,5-triphenyltetrazolium chloride (TTC) and Evans Blue stain method, respectively. The results showed that alkane solvents with log P > 5.5 were biocompatible while the hydrophilic solvents with log P < 5.5 were toxic to Nannochloropsis sp. due to the deactivated dehydrogenase and increased cell membrane permeability. As 10% (v/v) hexadecane was used to establish biphasic system, the total lipid production of Nannochloropsis sp. was increased by 28.9% compared to the control. The screened biocompatible solvent hexadecane enhanced not only the algal growth but also the lipid accumulation, showing an effective way to facilitate the process for in situ lipid milking from Nannochloropsis sp.     

Organic solvents for bioorganic synthesis

Summary The influence of solvents on enzymatic activity and stability was investigated. As a model reaction the -chymotrypsin-catalyzed esterification of N-acetyl-l-phenylalanine with ethanol was used. The enzyme was adsorbed on porous glass beads and used in various solvents. Small amounts of water were added to increase the enzymatic activity. These enzyme preparations obeyed. Michaelis-Menten kinetics. K _m,app decreased slightly with the log P value of the solvent while V _app increased markedly with the log P value. Log P values were also useful for generalizing the influence of solvents on enzyme stability. The enzyme preparations showed a markedly higher thermostability in dry solvents having log P values >0.7 than in less hydrophobic solvents.Also the operational stability was better in the more hydrophobic solvents. The amount of water added to the enzyme preparations greatly influenced the initial reaction rates. For some solvents optimal water contents were determined. The thermostability decreased with increasing water content.The observations are summarized in the conclusion that more hydrophobic solvents are preferable to less hydrophobic ones. The log P value gives a good guidance when selecting an organic solvent for enzymatic conversions.     

Modification Effects of Organic Solvents on Microenvironment of the Enzyme in Thermolysin-catalyzed Peptide Synthesis of N-(Benzyloxycarbonyl)-l-phenylalanyl-l-phenylalanine Methyl Ester

Thermolysin-catalyzed peptide synthesis using N-benzyloxycarbonyl)-l-phenylalanine (Z-Phe) and l-phenylalanine methyl ester (Phe-OMe) as substrates was done mainly in a water-organic one phase solvent system. The organic solvent content used was less than the saturation concentration in buffer. With organic solvents with high log P values, the enzymatic activity increased as the organic solvent content increased; but further increases in the organic solvent content decreased the enzymatic activity, showing an “organic activity” profile. On the other hand, with organic solvents of low log P values, the enzymatic reaction was inhibited even by the initial addition of organic solvents. When a correlation between maximum activities and logP values or Hildebrand solubility parameters was investigated, a linear correlation was obtained among the same category of organic solvents, but not between categories. This suggests that the direct effect of organic solvents on the microenvironment of the enzyme largely depends on the molecular structure of the solvents.     

Oxidation of aromatic compounds in organic solvents with laccase from Trametes versicolor

Summary Laccase purified from Trametes versicolor oxidizes 2,6-dimethoxyphenol (2,6-DMP) and syringaldazine in hydrophobic solvents presaturated with water, and in hydrophilic organic solvents provided that a sufficient amount of water is added. Ease of performance of the laccase test in organic solvents is improved after immobilization of the enzyme by entrapping in Sepharose CL-6B during enzyme filtration through the gel beads. The gel-enzyme association has been shown to be stable in water-presaturated solvents. Efficiency of the immobilized laccase in organic solvents containing 7% water was 10%–20% of that in potassium-citrate buffer. Immobilized laccase in organic solvents showed good stability and high tolerance to elevated temperatures.     

Bacteria tolerant to organic solvents

The toxic effects that organic solvents have on whole cells is an important drawback in the application of these solvents in environmental biotechnology and in the production of fine chemicals by whole-cell biotransformations. Hydrophobic organic solvents, such as toluene, are toxic for living organisms because they accumulate in and disrupt cell membranes. The toxicity of a compound correlates with the logarithm of its partition coefficient with octanol and water (log P _ow). Substances with a log P _ow value between 1 and 5 are, in general, toxic for whole cells. However, in recent years different bacterial strains have been isolated and characterized that can adapt to the presence of organic solvents. These strains grow in the presence of a second phase of solvents previously believed to be lethal. Different mechanisms contributing to the solvent tolerance of these strains have been found. Alterations in the composition of the cytoplasmic and outer membrane have been described. These adaptations suppress the effects of the solvents on the membrane stability or limit the rate of diffusion into the membrane. Furthermore, changes in the rate of the biosynthesis of the phospholipids were reported to accelerate repair processes. In addition to these adaptation mechanisms compensating the toxic effect of the organic solvents, mechanisms do exist that actively decrease the amount of the toxic solvent in the cells. An efflux system actively decreasing the amount of solvents in the cell has been described recently. We review here the current knowledge about exceptional strains that can grow in the presence of toxic solvents and the mechanisms responsible for their survival. Received: January 22, 1998 / Accepted: February 16, 1998     

Application of a new amidophosphite ligand to Rh‐catalyzed asymmetric hydrogenation of β‐dehydroamino acid derivatives in supercritical carbon dioxide: Activation effect of protic co‐solvents

New chiral amidophosphite ligand was synthesized and tested in the Rh‐catalyzed asymmetric hydrogenation of (Z)‐β‐(acylamino)acrylates in protic solvents and supercritical carbon dioxide (scCO₂) The catalytic performance is affected greatly by the acidity of the solvents. Better enantioselectivity (up to 88% ee) was achieved in scCO₂ containing 1,1,1,3,3,3‐hexafluoro‐2‐propanol, compared to neat protic solvents. Chirality, 2011. © 2011 Wiley‐Liss, Inc.     

Lipase in aqueous‐polar organic solvents: Activity,structure, and stability

Studying alterations in biophysical and biochemical behavior of enzymes in the presence of organic solvents and the underlying cause(s) has important implications in biotechnology. We investigated the effects of aqueous solutions of polar organic solvents on ester hydrolytic activity, structure and stability of a lipase. Relative activity of the lipase monotonically decreased with increasing concentration of acetone, acetonitrile, and DMF but increased at lower concentrations (upto ～20% v/v) of dimethylsulfoxide, isopropanol, and methanol. None of the organic solvents caused any appreciable structural change as evident from circular dichorism and NMR studies, thus do not support any significant role of enzyme denaturation in activity change. Change in 2D [15N, 1H]‐HSQC chemical shifts suggested that all the organic solvents preferentially localize to a hydrophobic patch in the active‐site vicinity and no chemical shift perturbation was observed for residues present in protein's core. This suggests that activity alteration might be directly linked to change in active site environment only. All organic solvents decreased the apparent binding of substrate to the enzyme (increased K_m); however significantly enhanced the k_cat. Melting temperature (T_m) of lipase, measured by circular dichroism and differential scanning calorimetry, altered in all solvents, albeit to a variable extent. Interestingly, although the effect of all organic solvents on various properties on lipase is qualitatively similar, our study suggest that magnitudes of effects do not appear to follow bulk solvent properties like polarity and the solvent effects are apparently dictated by specific and local interactions of solvent molecule(s) with the protein.     

Effects of polar additives on the enzyme enantioselectivity of an esterification reaction in organic solvents

Summary The enantioselectivity ofcandida cylindracea lipase in esterification between ibuprofen and 1-butanol in organic solvents is influenced by adding a small amount of polar solvents. The results show that both the solvents and the amount added affected the reaction. Addition of 0.05% (v/v) N,N-dimethylformamide increase the ee value of the ester from 57.5% of no addition to that of 91%.     

Multiple allelopathic activity of the crustose coralline algaLithophyllum yessoenseagainst settlement and germination of seaweed spores

A study was made to investigate possible formation by the crustose coralline algaLithophyllum yessoenseof multiple allelopathic-related substances against the settlement and germination of spores of various seaweeds. Seven different solvents (n-hexane, diethyl ether, acetone, ethyl acetate, acetonitrile, methanol, distilled water) and seawater were used to obtain crude extracts and secretory exudates from the coralline alga. The extracts and the algal conditioned seawater were tested for inhibitory activity against the settlement and germination of spores from 17 species representing 15 genera. Spore settlement of 14 species was inhibited over 90% by one or more extracts of the six organic solvents and conditioned seawater. The germination of spores from 13 species was inhibited by one or more extracts of all seven solvents and conditioned seawater. The species where spore settlement was not significantly affected showed strong inhibition of germination, andvice versa.     

Effect of salt on the stability of the pH 4.2 rA8 double helix in a series of organic/aqueous mixed solvents: A test of oligoelectrolyte theory

Temperature-dependent uv absorption spectroscopy has been used to investigate the salt dependence of the order–disorder transition for the pH 4.2 rA₈ double helix in 100% aqueous buffer and in a series of organic/aqueous mixed solvents. Melting temperature, T_m, data were obtained for the transitions in the different solvents by analysis of the uv melting curves. For the pure aqueous buffer solvent, the melting temperature was found to exhibit a reduced salt dependence (?t_m/? log Na⁺) when compared to the corresponding polymer. This reduction is explained in terms of end effects and is shown to be consistent with the theoretical treatments of oligoelectrolyte transitions developed by Record and Lohman [Biopolymers, 17 , 159–166 (1978)]. In the mixed solvents, the salt dependence of the melting temperature (?t_m/? log Na⁺) is shown to exhibit a linear dependence on the bulk dielectric constant of the medium for all of the hydroxyl-containing solvents studied. Significantly, N,N-dimethylformamide demonstrated different behavior.     

Relation between bacterial strain resistance to solvents and biodesulfurization activity in organic medium

Microorganisms used in biodesulfurization of petroleum products have to withstand high concentrations of hydrocarbons. The capacities of seven desulfurizing strains of Rhodococcus to be active in the presence of solvents were evaluated. Octanol and toluene (log P=2.9) were selected as toxic solvents. The effect of the solvents was determined by measuring either inhibition of growth or the decrease in respiratory activity of the cells. Differences among strains in their resistance to solvent responses were observed, but these variations were dependent on the test used. Resistance to solvents was then compared to the capacity of the different strains to retain biodesulfurization activity in the presence of hexadecane. Inhibition of desulfurization by high concentrations of hexadecane was found to be well correlated to the sensitivity of the strains to respiration inhibition by toluene, but not to growth inhibition. This result also showed that the respirometric test was a rapid and reliable test to select solvent-resistant strains for use as resting cells in biocatalysis processes, such as biodesulfurization, in organic media.     

INHIBITION OF FERN SPORE GERMINATION BY LIPOPHILIC SOLVENTS

Several alcohols and other solvents inhibit germination of spores of the fern, Onoclea sensibilis L. The inhibition is reversible when spores are transferred to solvent-free media. The effectiveness of different solvents, measured by the concentration needed to inhibit germination by 50%, increases with their lipid solubility. The activity of straight-chain alcohols from methanol through n-heptanol is highly correlated with lipid solubility, whereas the correlation is weaker for several other solvents. The results indicate that some lipophilic site in the spore is important in germination.     

Stability and structure of Penicillium chrysogenum lipase in the presence of organic solvents

Abstract

The present work describes the enzymatic properties of Penicillium chrysogenum lipase and its behavior in the presence of organic solvents. The temperature and pH optima of the purified lipase was found to be 55?°C and pH 8.0 respectively. The lipase displayed remarkable stability in both polar and non-polar solvents upto 50% (v/v) concentrations for 72?h. A structural perspective of the purified lipase in different organic solvents was gained by using circular dichroism and intrinsic fluorescence spectroscopy. The native lipase consisted of a predominant α-helix structure which was maintained in both polar and non-polar solvents with the exception of ethyl butyrate where the activity was decreased and the structure was disrupted. The quenching of fluorescence intensity in the presence of organic solvents indicated the transformation of the lipase microenviroment P. chrysogenum lipase offers an interesting system for understanding the solvent stability mechanisms which could be used for rationale designing of engineered lipase biocatalysts for application in organic synthesis in non-aqueous media.     

The influence of various organic solvents on the respiration of free and immobilized cells of Tagetes minuta

The influence of several organic solvents (esters, phthalates, alkanes, alcohols and perfluorchemicals) on the oxygen metabolism of Tagetes minuta (marigolds) was tested by incubating the cells in medium mixed with 1, 5 or 10% (v/v) of the organic solvents. The results were in good agreement with the general rules (log P) for the influence of organic solvents on biocatalytic activity. Immobilization of the cells in calcium alginate provided a slight protection of the cells against the toxic solvents.     

Selection of organic solvents for in situ extraction of fermentation products fromClostridium thermohydrosulfuricum cultures

Summary Fifteen organic solvents were examined to determine their biocompatibility for in situ extraction of fermentation products from cultures of the thermophilic anaerobeClostridium thermohydrosul furicum. Five solvents (hexadecane, isooctane, kerosene, oleyl alcohol, Shellsol TD) were found to be non-toxic toClostridium thermohydrosul furicum. Interfacial tensions, phase separation and partition coefficients for ethanol of the biocompatible solvents were compared. With the exception of kerosene, these solvents showed good separation from the aqueous phase. Oleyl alcohol had the highest partition coefficient for ethanol (K_D=0.34 at 65°C) and appears to be suitable for extractive ethanol fermentation.     

Effects of Calcium Ion on the Catalytic Activity of α-Chymotrypsin in Organic Solvents

Addition of small amounts of calcium ion markedly accelerated the transesterification of N-acetyl-l-tyrosine methyl ester to its ethyl ester by the catalysis of α-chymotrypsin in organic solvents. Maximum increase of the reaction rate was about 12-fold in the presence of 25 μm of calcium ion in ethanol. The rate increase was strongly dependent on calcium ion concentration and nature of organic solvents. Esterification of N-acetyl-l-tyrosine and hydrolysis of N-acetyl-l-tyrosine ethyl ester by α-chymotrypsin in organic solvents were also accelerated by calcium ion. The reactions obeyed Michaelis–Menten kinetics, and the acceleration of the reactions was due to the increase in k_cat.     

A caveat for the use of log P values for the assessment of the biocompatibility of organic solvents

The degree of enzyme deactivation for lipases from Candida rugosa and Pseudomonas sp., hydroxynitrile lyase and mandelate racemase upon exposure to organic solvents can be correlated to their respective partition coefficients (log P values). However, three unexpected results were obtained: (1) the deactivation exerted by protic solvents, e.g., methanol, is severely underestimated; (2) little deactivation by an organic solvent cannot neccessarily be correlated to catalytic activity in this medium, and (3) in contrast to other enzymes, hydroxynitrile lyase is exceptionally stable towards deactivation by DMF.     

Effect of solvents on hydrolysis of olive oil by immobilized lipase in reverse phase system

Summary Lipase fromCandida rugosa was immobilized by adsorption on three supports which could contain water available for the hydrolysis of olive oil in a reverse phase system. To select the most suitable solvent for this system, the effect of organic solvents on the stability and catalytic activity of immobilized lipase for the hydrolysis reaction has been examined. The results revealed that isooctane was superior to any other solvents tested in this study for enzymatic fat splitting in a reverse phase system. Also the effect of the solvent polarity on the hydrolysis of olive oil has been examined in detail using various organic solvents mixed with an equivolume of isooctane. It was found that the hydrolysis of olive oil by immobilized lipase was markedly affected by the polarity of reaction solvents.