Enhancing the biocatalytic potential of carbonyl reductase of Candida viswanathii using aqueous-organic solvent system

In the present work, the toxic effect of various solvents with different Log P values was studied on the whole cells of Candida viswanathii. Experiments showed that the lower concentrations of some solvent increased both the activity retention and enzyme activity as compared to the control while this was not the case with higher concentrations of the same solvents. The model compound taken in the present study was 1-acetophenone. The percentage conversion improved from 76 to 94%. Addition of 2-propanol increased the substrate tolerance, giving the conversion of 90% compared to 9% in control at a substrate concentration of 70 mM in 1h. The operational stability increased at higher temperatures with the addition of 2-propanol in the reaction mixture with good conversion (90%) and enantiomeric excess (>99%) at 45 degrees C and 50 degrees C. The effect was also found to be prominent in other tested substrates. In order to further stabilize the cells for long term use in higher concentration of organic solvents, the cells were further immobilized, and were found to have higher activity retention than that of free cells.     

Growth of Gram-negative bacteria in the presence of organic solvents

The growth behavior of Gram-negative bacteria when exposed to high concentrations (50% v/v) of water-insoluble organic solvents was investigated. The solvents were chosen according to their polarity values as denoted by a logarithmically expressed parameter log P, where P is the partition coefficient of a given solvent in an equimolar mixture of octanol and water. The cell growth was measured by the number of colonies developed on a solid agar medium in direct contact with the solvents. All 31 strains tested showed characteristic growth patterns. The survival and subsequent growth of bacteria increased with the increase in the log P value and was found to be strain specific. For all the strains, 100% cell growth was reached from 0% within 0.1–0.4 log P units. Log P₅₀ values, defined as the log P values at which 50% of the cells form colonies, were determined for each bacterial strain. On the whole, Pseudomonas strains were found to be more resistant to apolar solvents than all other bacteria tested. This resistance was dependent not only on the polarities but also on the toxic nature of different organic solvents, the cell membrane components, and to a limited extent, the growth medium. A tenfold increase in the Mg²⁺ concentration in the growth medium enhanced the solvent resistance of E. coli but had no such effect on Pseudomonads. In general, different growth temperatures had no impact on the solvent resistance of the Gram-negative bacteria tested.     

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.     

Limitations in the measurement of c-myc oncoprotein and other nuclear antigens by flow cytometry

Recent developments in cell fixation, quantitative immunocytochemistry, and flow cytometry allow for the quantification of a variety of oncoproteins and other proliferation-associated antigens in both fresh and archival pathology material. These studies provide evidence that the standard tissue deparaffinization/dissociation technique significantly reduces the amount of c-myc oncoprotein remaining for analysis. To examine the factor(s) responsible for this observation, individual variables of the deparaffinization/dissociation technique including type of fixative, pepsin concentration, pepsinization times, pH, and exposure to organic solvents were examined in HeLa-S3 cells. The cells were stained with monoclonal antibodies either to the c-myc oncoprotein or to p105, a prolifera-tion-associated nuclear antigen. Protein-levels were measured on the basis of anti-c-myc or anti-p105 immunofluorescence by flow cytometry and were found not be affected significantly by type of fixative, exposure to organic solvents, acid pH solution, or mechanical disruption. Levels of c-myc oncoprotein were reduced by over 50%, however, when cells were exposed to 0.5% pepsin, whereas p105 was more resilient with only an approximately 7% reduction following the same treatment. Thus, careful examination of aspects of the deparaffinization/dissociation technique appears to be a necessary prerequisite for quantification of specific nuclear proteins from dissociated tissue specimens.     

Effect of organic solvents on the yield of solvent-tolerant Pseudomonas putida S12.

Solvent-tolerant microorganisms are useful in biotransformations with whole cells in two-phase solvent-water systems. The results presented here describe the effects that organic solvents have on the growth of these organisms. The maximal growth rate of Pseudomonas putida S12, 0.8 h-1, was not affected by toluene in batch cultures, but in chemostat cultures the solvent decreased the maximal growth rate by nearly 50%. Toluene, ethylbenzene, propylbenzene, xylene, hexane, and cyclohexane reduced the biomass yield, and this effect depended on the concentration of the solvent in the bacterial membrane and not on its chemical structure. The dose response to solvents in terms of yield was linear up to an approximately 200 mM concentration of solvent in the bacterial membrane, both in the wild type and in a mutant lacking an active efflux system for toluene. Above this critical concentration the yield of the wild type remained constant at 0.2 g of protein/g of glucose with increasing concentrations of toluene. The reduction of the yield in the presence of solvents is due to a maintenance higher by a factor of three or four as well as to a decrease of the maximum growth yield by 33%. Therefore, energy-consuming adaptation processes as well as the uncoupling effect of the solvents reduce the yield of the tolerant cells.     

烟草废料中绿原酸的提取工艺研究

讨论了从烟草废料中提取绿原酸的优势和甲醇、乙醇、丙酮、水等不同溶剂经超声波辅助提取绿原酸的效果：研究结果表明,用体积分数40％的甲醇得到的浸提液中,绿原酸质量浓度为2．11mg／mL,比以水为溶剂时高出近50％.不同浓度的甲醇溶液中,体积分数50％的甲醇提取绿原酸的浓度最高。对树脂的吸附动力学分析表明,大孔树脂CN-101对烟草浸提液中绿原酸的吸附遵循Freundlich等温方程,吸附和解析分离所得的绿原酸收率为87．6％.在超声辅助条件下,利用甲醇等有机溶剂提取烟草中的绿原酸,进而用大孔树脂进行吸附分离的方法可行。     

东北红豆杉细胞两液相培养中紫杉醇释放行为研究

在东北红豆杉细胞悬浮培养中,分别研究了稀土化合物(硫酸铈铵)、有机溶剂(油酸和邻苯二甲酸二丁脂)和稀土化合物与有机溶剂的协同作用对紫杉醇释放的影响。在此基础上深入研究了在东北红豆杉细胞两液相培养中,紫杉醇释放率随不同的有机溶剂(烷烃、有机酸、醇和脂)、有机溶剂的体积分数、有机溶剂的加入时间和有机溶剂相毒性的变化规律。结果表明分别加入稀土化合物和有机溶剂都明显促进紫杉醇的释放,特别是有机溶剂更显著促进紫杉醇的释放。但在东北红豆杉细胞两液相培养中,稀土化合物加入不能进一步促进紫杉醇的释放。因此两液相培养中有机溶剂本身就是很好的产物释放剂。紫杉醇的释放率由对照组的40%提高到75%以上。     

Stability and activity of a thermostable malic enzyme in denaturants and water-miscible organic solvents

A study was made of the effects of common protein denaturants and water-miscible organic solvents on both the stability and activity of the malic enzyme [(S)-malate:NADP+ oxidoreductase (oxaloacetate-decarboxylating); EC 1.1.1.40] from the extreme thermoacidophilic archaebacterium Sulfolobus solfataricus. At 25 degrees C, the enzyme was not inactivated in 4 M urea or 0.05% SDS over 24 h, while the half-life was 30 min in 6 M guanidine hydrochloride and 5 h in 0.075% SDS. The enzyme stability in water-miscible organic solvents at 25 degrees C is somewhat surprising: after a 24-h incubation, the enzyme was completely active in 50% dimethylformamide; it lost 15% of its initial activity in 50% methanol or 15% ethanol. However, the resistance to organic solvents was greatly reduced at higher temperatures. The enzyme was able to catalyze the malate conversion even in the presence of 1.5% Triton X-100 or sodium deoxycholate. A number of solvents were found to stimulate the malic activity independent of time. Studies with 50% methanol revealed that the activation was reversible and inversely related to the temperature; moreover, the solvent was demonstrated to exclusively affect the maximal velocity of catalysis, the Km values for both substrates being unchanged. Investigation was made to find out whether there was a correlation between enzyme stability, as well as activation, and hydrophobicity of the organic medium. The residual malic activity after incubation in the water/organic medium correlated inversely with the logarithm of the partition coefficient in octanol/H2O of the mixture used as a hydrophobicity index. On the other hand, the extent of activation depended directly on the logarithm of the molar concentration of the organic solvent required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents required for maximal enzymatic activation. Because of its remarkable resistance to organic solvents and protein denaturants in general, the malic enzyme from Sulfolobus solfataricus can be considered suitable for biotechnological applications.     

Screening and isolation of an organic solvent-tolerant bacterium for high-yield production of organic solvent-stable protease

Forty-three strains were screened from crude oil-contaminated samples by toluene and cyclohexane enrichment in medium. Ten of these strains demonstrated high protease activity on skim-milk agar. Among them, the PT121 isolate, identified as Pseudomonas aeruginosa, was selected based on its extracellular protease stability in the presence of hydrophilic organic solvents. The crude protease also retained most of its activity up to at least 14 days in the presence of various organic solvents at 50% concentration, and the protease activity in production medium was 10,876U/ml after 72h incubation. This protease showed high activity as a catalyst for aspartame precursor Cbz-Asp-Phe-NH2 synthesis in the presence of 50% dimethylsulfoxide (DMSO).     

Utilization of tannery solid waste for protease production by Synergistes sp. in solid‐state fermentation and partial protease characterization

Synergistes sp. DQ560074 produced a protease in submerged fermentation (SmF) at 400–420 U/mL and in solid‐state fermentation (SSF) at 745–755 U/g. The protease, which belongs to the aspartic protease class, was active over a wide range of pH (5–7) and at high temperatures (25–45°C). The protease is stable and active in various polar protic solvents (50% v/v) like ethanol, isopropanol, n–butanol, in polar aprotic solvents (50% v/v) like acetonitrile, and in non‐polar solvents (50% v/v) such as ethylacetate and toluene, but not in hydrophilic organic solvents (methyl alcohol and acetone). As far as we know, this is the first contribution to the production of a mesophilic protease with solvent stability in SSF using a proteinaceous solid waste.     

Relationship between the presence of the citrate permease plasmid and high electron-donor surface properties of Lactococcus lactis ssp. lactis biovar. diacetylactis

Some strains of Lactococcus lactis subspecies possess a citrate permease that enables them to utilize citrate and to produce diacetyl. Such strains are classified as diacetylactis biovariants (L. lactis ssp. lactis biovar. diacetylactis). We investigated the electron-donor surface properties of L. lactis strains and observed that the diacetylactis biovariants presented increased adhesion to electron-acceptor solvents (microbial adhesion to solvents electron-donor characteristics of cells of <27% for L. lactis and about 50% for L. lactis ssp. lactis biovar diacetylactis). We investigated the properties of a pCitP- derivative and observed for a diacetylactis biovariant strain a loss of the electron-donor characteristics falling from 47% for a pCitP+ strain to 8% for its pCitP- derivative. This suggests that the presence of high electron-donor characteristics on the surface of L. lactis results to a large extent from the presence of the citrate permease plasmid.     

Application of solvent extraction to ethanol fermentation

Summary The basic problems of applying solvent extraction to ethanol fermentation were investigated. The selection of solvents was based on the selectivity ratio, which was expressed as the ratio of the ethanol distribution coefficient to the water distribution coefficient. Solvents with high selectivity ratios of more than 50 were found mainly among the alcohols and esters. However, most of these solvents were toxic to ethanol-producing microorganisms. We tried to make a barrier to solvent molecules beneath the surface of gel beads immobilizing the cells as a protection against solvent toxicity. Porapack Q was found to be an effective barrier, and the ethanol production rate of immobilized cells protected with Porapack Q did not change event after the production of eight batches in medium saturated with sec-octanol, which was the most toxic solvent used in our experiments.     

Selective release of superoxide dismutase from Saccharomyces cerevisiae with isopropanol

Summary A new and inexpensive approach by which the release and purification of intracellular protein can be carried out simultaneously has been developed. Alcoholic solvents were used to selectively release superoxide dismutase (SOD) from Saccharomyces cerevisiae. 90%(v/v) isopropanol was used to treat the cells for 120 minutes and then 50 mM phosphate buffer(pH7.0) was added after removal of the solvent by filtration. The recovery of SOD was up to 90% , while the specific activity of SOD increased 25 times compared to traditional methods such as ultrasonic method.     

Phytochemical Screening,Antioxidant Assay and Cytotoxic Profile for Different Extracts of Chrysopogon zizanioides Roots

The Chrysopogon zizanioides plant possesses multiple traditional uses, especially in therapeutics, but only a few articles have reported its biological activity. Hence, the present study was planned to explore the phytochemical constituents, cytotoxic potential, radical scavenging activity, and GC/MS (Gas chromatography & Mass spectrometry) analysis of the vetiver root extracts. The roots extracted with different solvents exhibited more significant phytochemical constituents in polar solvents in comparison to non-polar ones, favoring the extraction of a greater number of components in highly polar solvents. All the extracts were tested for their cytotoxicity using SRB (Sulforhodamine B) assay. They confirmed ethanolic extract as a potent extract with GI₅₀ 56±0.5 μg/ml in oral cancer (SCC-29B) along with no cytotoxicity in healthy cells (Vero cells), making it a safer therapeutic option in comparison to standard Adriamycin. This extract was also analyzed for its antioxidant potential by DPPH (1,1-Diphenyl-2-picrylhydrazyl) assay with IC₅₀ value 10.73 μg/ml, which was quite comparable to Ascorbic acid having IC₅₀ value 4.61 μg/ml. The quantitative analysis of ethanolic extract exhibited 107 compounds amongst which Khusenic acid, Ascorbic acid, Junipen, gamma-Himachalene, alpha-Guaiene were the majorly occurring compounds that can be explored further for their cytotoxic activity.     

HydroLink gel electrophoresis (HLGE). I. Matrix characterization

A new gelatinous matrix is reported, having intermediate properties between those of polyacrylamide and agarose gels. The matrix has the unique property of being amphiphilic, i.e. of swelling in both plain water and polar organic solvents, and seems particularly well suited for electrophoresis of DNA. The compatibility with organic solvents includes 50% dimethyl sulphoxide, 50% tetramethyl urea, 50% acetonitrile and 50% tetrahydrofuran, the latter having a dielectric constant of 20. The matrix is hypothesized to consist of brush-like pillars, having a hydrophobic core and a hydrophilic coating. The latter is formed by short chains protruding in the surrounding liquid and able to coordinate large amounts of water.     

Production of S-lactoylglutathione by organic-solvent-extracted glyoxalase I from Hansenula mrakii

Summary Glyoxalase I was extracted from Hansenula mrakii IFO 0895 by incubating the cells with buffer solution containing 50% acetone (enzyme activity 35 units/g cells) or 50% ethyl acetate (enzyme activity 28 units/g cells) at 30°C for 10 h. Glyoxalase II was also extracted from the cells, although the activity of the enzyme was lost during incubation with organic solvents, especially at higher temperature (30°C). By using the organic-solvent-extracted fraction of H. mrakii, enzymatic production of S-lactoylglutathione was studied, and approximately 82 mmol/l (30 g/l) of S-lactoylglutathione was produced from 120 mmol/l glutathione. Offprint requests to: A. Kimura     

Biochemical determination of tubulin-microtubule equilibrium in cultured cells.

The relative amount of free and microtubule-associated tubulin in tissue culture cells was determined by colchicine binding. Both microtubules and tubulin were stabilized in a dilute homogenate containing 50% glycerol and 5% dimethylsulfoxide. Microtubules were separated by sedimentation at 100,000g for 10 min in a benchtop ultracentrifuge and then depolymerized to tubulin. Colchicine binding to free tubulin could be performed only after dilution of the organic solvents present to prevent a 70% reduction in apparent affinity of tubulin for colchicine. Tubulins purified from rat brain, human skin fibroblasts, and rat GH₃ cells were each homogeneous and similar in molecular weight, affinity for DEAE-cellulose, and apparent affinity for colchicine. Microtubules contained 34–41% of tissue culture cell tubulin. Colchicine (10^?6 to 10^?5m) and incubation at 4°C reduced microtubule-derived tubulin to less than 6% of expected.     

Isolation and Characterization of Antioxidation Enzymes from Cells of Zedoary (Curcuma zedoaria Roscoe) Cultured in a 5-l Bioreactor

In this study, a cell suspension culture system for zedoary (Curcuma zedoaria Roscoe) was developed, using 50 g/l of fresh weight inoculum in a batch culture. The highest cell biomass obtained from a 5-l bioreactor equipped with three impellers after 14 days of culture was utilized to extract secondary metabolites (essential oil and curcumin) and determine the activities of antioxidant enzymes (peroxidase, superoxide dismutase, and catalase). For essential oil and curcumin, zedoary extracts were recovered via a variety of methods: steam distillation, volatile solvents, and Soxhlet. After 14 days of culture using volatile solvents, the optimal yield of essential oil (1.78%) was obtained when using petroleum ether at 40°C in 6 h of extraction, and the best curcumin yield (9.69%) was obtained at 60°C in 6 h via extraction with 90% ethanol. The activities of antioxidant enzymes from zedoary cells were also assessed. The specific activities of peroxidase, superoxide-dismutase, and catalase reached maximum values of 0.63 U/mg of protein, 16.60 U/mg of protein, and 19.59 U/mg of protein after 14 days of culture, respectively.     

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.