Storage of Pollen Grains in Organic Solvents: Effect of Organic Solvents on Leaching of Phospholipids and its Relationship to Pollen Viability

In vitro germinability and membrane integrity (as revealed bythe fluorochromatic reaction (FCR) test) of pollen grains ofCrotalaria retusa L. stored in various organic solvents forsix months at –20±2 °C were studied and correlatedwith leaching of lipids, phospholipids, sugars and free aminoacids from pollen grains into organic solvents during storage.Pollen grains stored in organic solvents with low dielectricconstants (a measure of their non-polar nature), such as hexane,cyclohexane and diethyl ether, showed high scores for germinationand FCR and very little leaching of phospholipids, sugars andamino acids. Pollen grains stored in solvents with high dielectricconstants (a measure of their polar nature) such as isopropanoland methanol did not show germination or positive FCR scores,but showed extensive leaching of phospholipids, sugars and freeamino acids. The viability of pollen grains stored in organicsolvents seems to be determined largely by the effect of theorganic solvents on pollen phospholipid composition, which inturn affects membrane integrity and consequently pollen viability. Crotalaria retusa, organic solvents, pollen storage, viability, phospholipids     

Acceleration of the Growth of Camellia sasanqua Pollen by Soaking in Organic Solvent

Camellia sasanqua pollen that had been soaked in acetone or diethyl ether for only 5 minutes grew three to four times longer pollen tubes than unsoaked pollen. Although the acceleration of pollen tube growth was observed when the pollen had been soaked in cold solvents (5 C, −15 C) for 100 days, soaking in warm solvents (30 C, 24 C) caused it to decrease gradually, and the pollen eventually lost the ability to germinate. The acceleration may be caused by removing inhibitor from the pollen grain by the organic solvents.     

SOLUBILITY OF POLLEN EXINES

The sporopollenin of pollen exines of Ambrosia trifida is soluble in fused potassium hydroxide, in strong oxidizing solutions, and in certain organic bases. It is insoluble in other organic and inorganic acids and bases, in lipid solvents, and in detergents. The outer exine layer of gymnosperm and angiosperm pollen dissolves in 2-aminoethanol. The inner exine layer, as well as the exine of pteridophyte spores, is insoluble. The exine dissolution process in 2-aminoethanol involves swelling and disintegration of exine structures, leaving some residual globules. Sporopollenin shares some solubility properties with lignin and cutin but appears to be chemically distinct from these substances.     

Retaining the viability of Camellia japonica pollen in various organic solvents

Pollen viability in 31 organic solvents was studied. Pollengrains of Camellia japonica which had been soaked in 28 organicsolvents for 3 days retained their viability and grains in 19solvents such as ethyl acetate, n-amyl alcohol and petroleumether grew longer pollen tubes than fresh pollen. (Received July 20, 1972; )     

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.     

On the size of Corylus avellana L. pollen mounted in silicone oil

A considerable variation in the size of modern Corylus avellana pollen mounted in silicone oil was noticed. It turned out that a residue of the silicone oil solvent (benzene) prevents shrinkage of the pollen grains, and that size-variations may be due to more or less incomplete evaporation of the solvent. Evaporation at 50[ddot]C is more effective than evaporation at room temperature. Diffusion of solvents from a plastic spatula and from the slide-sealing material may cause a swelling of the pollen. The size of pollen grains compressed by the cover slip may increase slightly due to deformation. Size changes with storage up to 17 years are random, compressed grains do not swell, and the average changes are insignificant. The size of Corylus pollen from various modern collections is compared.     

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.     

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.     

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.     

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     

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.     

Circular dichroism study of modified nucleosides

CD study of four modified nucleosides, constituents of tRNA molecules, revealed that 2-thio-5-methyluridine and 5-methyluridine in aqueous solution, 0.1N HCl, and organic solvents essentially occur in an anti-conformation. 5-Methylcytidine also occurs in an anti-conformation similar to cytidine in aqueous solution and organic solvents, while 2-thiocytidine dihydrate appears to occur in an anti-conformation. It is stressed that the CD data of thionucleosides might be applied to the successfully conformational analysis of tRNA molecules.     

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.     

Lipid Composition and Lipid Molecular Species of Lipomyces starkeyi Cultivated in Medium Containing 1,2-Propanediol

Spore suspensions of 15 strains in 15 species of Micromonospora prepared with ultrasonication-technique were tested for resistance to moist heat, acid, alkali, and organic solvents (5 alcohols, 4 ketones and ether). More than 50% spore-survival was found in most organisms heated at 60°C for 20min, but less than 0.5% survived at 80°C. The spore-viability did not change at pH 6 to 8, but decreased beyond this range, and remarkably at acidic pH. A maximum reduction in viability was found with most organic solvents at a concentration of around 80%, and the spores were more resistant to ketone than alcohols and dioxane. Several Streptomyces species were also studied, and their spores were less resistant to heat and organic solvents than those of Micromonospora.     

Enzymatic dehalogenation of 4-chlorobenzoate by 4-chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 in organic solvents

Summary 4-Chlorobenzoate dehalogenase from Pseudomonas sp. CBS3 showed dehalogenating activity in various organic solvents. In alcohols like methanol (150%) or ethanol (120%) higher activities than in water (100%) were obtained. In apolar solvents like petroleum ether (5%) and nhexane (5%) only trace activities were observed. The solvents did not increase the stability of the enzyme. 4-Chlorobenzoic acid methylester, a substance not soluble in water, was not dehalogenated in organic solvents.     

Permeabilization of<Emphasis Type="Italic">Ochrobactrum anthropi</Emphasis> SY509 cells with organic solvents for whole cell biocatalyst

Permeabilization is known to overcome cell membrane barriers of whole cell biocatalysts. The use of organic solvents is advantageous in terms of cost, simplicity, and efficiency. In this study,Ochrobactrum anthropi SY509 was permeabilized with various organic solvents. Treatment with organic solvents resulted in lower permeability barriers due to falling out lipids of the cell membrane. Therefore, permeabilized cells showed higher enzyme activity with no cell viability. Among various organic solvents, 0.5% (v/v) chloroform was selected as the most efficient permeabilizing reagent. Changes in the cell membrane structure were observed and the residual amounts of phospholipids of the cell membrane were measured to investigate the mechanism of the improved permeability.     

Isolation of new toluene-tolerant marine strains of bacteria and characterization of their solvent-tolerance properties

Aims:  To isolate and characterize new marine bacteria capable of tolerating high concentrations of organic solvents, and to understand the toxic effects of these chemicals on marine bacteria. Methods and Results:  Five marine bacteria able to tolerate 0·1% (v/v) toluene were isolated and characterized on the basis of their growth and survival rates in the presence of different organic solvents. The toluene-tolerant marine bacteria identified in this study could not grow in the presence of 0·1% (v/v) of several organic solvents with a log P_ow higher than that of the toluene (which in theory should be less toxic than toluene). The mechanisms underlying solvent tolerance were explored. Conclusions:  Isolates of four different genera were identified as toluene-tolerant. Toxicity of a second phase of an organic solvent toward these isolates could not be predicted on the basis of the solvents’ log P_ow. Significance and Impact of the Study:  To improve the biodegradation rate of some water-insoluble compounds, double-phase bioreactors can be used. This type of bioreactor will require strains able to grow in a salt-containing environment and able to tolerate a second phase of an organic solvent.     

Effects of oxygen and nitrate on growth of Escherichia coli and Pseudomonas aeruginosa in the presence of organic solvents

Escherichia coli and Pseudomonas aeruginosa grown in the presence of certain harmful organic solvents become susceptible to these solvents during the cultivation. This susceptibility is conspicuous in the stationary phase of growth. The organic solvent tolerance levels of these microorganisms were maintained when the oxygen concentration was kept high. The tolerance levels were maintained also when these organisms were grown with nitrate present under anaerobic respiratory conditions. Received: 21 March, 1997 / Accepted: July 20, 1997     

Improvement in organic solvent tolerance by double disruptions of proV and marR genes in Escherichia coli

Aims: To investigate the involvement of osmoprotectant transporters in organic solvent tolerance in Escherichia coli and to construct an E. coli strain with high organic solvent tolerance. Methods and Results: The organic solvent tolerance of ΔbetT, ΔproV, ΔproP or ΔputP single‐gene knockout mutants of E. coli K‐12 strain was examined. Among these mutants, the organic solvent tolerance of the ΔproV mutant remarkably increased compared with that of the parent strain. It has been known that a marR mutation confers tolerance on E. coli to organic solvents. A ΔproV and ΔmarR double‐gene mutant was more tolerant to organic solvents than the ΔproV or ΔmarR single‐gene mutant. The n‐hexane amount accumulated in E. coli cells was examined after incubation in an n‐hexane‐aqueous medium two‐phase system. The intracellular n‐hexane level in the ΔproV and ΔmarR double‐gene mutant was kept lower than those of the parent strain, ΔproV mutant and ΔmarR mutant. Conclusions: The organic solvent tolerance level in E. coli highly increased by dual disruption of proV and marR. Significance and Impact of the Study: This study suggests a new strategy for increasing the organic solvent tolerance level in E. coli to improve the usability of the whole‐cell biocatalysts in two‐phase systems employing organic solvents.