Pressurized solvent extraction of pure food grade starch

A commercial pressurized solvent extractor was used to remove lipid and non-lipid material from cornstarch using n-propanol/water and ethanol/water mixtures. Yields and chemical composition of the extract fractions were determined. Cornstarch samples were characterized using pasting properties and shear storage modulus measurements. The n-propanol/water extracted slightly higher amounts of both lipids and non-lipids. The lipid fractions contained mostly linoleic, palmitic and oleic free fatty acids. The non-lipid fraction contained mostly protein in the form of zein. The extracted starch had lower peak and setback viscosities than did the unextracted starch. The starch extracted with n-propanol/water had the lowest shear storage modulus values. Conversely, the samples extracted with ethanol/water had the highest shear storage modulus values. It is hypothesized that low amounts of zein present in conjunction with the starch is responsible for this observed effect.     

Effect of alcohols and their interaction with ethylene on the ripening of epidermal pericarp discs of tomato fruit

Ethanol concentrations that were induced in pericarp discs of mature-green tomato fruit (Lycopersicon esculentum Mill, cv Castlemart) either by anaerobic metabolism or by exposure to ethanol vapor inhibited ripening without increasing the rate of ion leakage. Inhibition of ripening (i.e. lycopene synthesis) of excised tomato pericarp tissue by ethanol vapor was reversed by increasing concentrations of the plant hormone ethylene. A Lineweaver-Burk plot indicated noncompetitive interaction between ethanol and ethylene. Methanol and n-propanol also inhibited lycopene synthesis without significantly increasing ion leakage. The similar inhibitory effects of methanol, ethanol, and n-propanol at concentrations which did not stimulate ion leakage, and the relationship between activity and lipophilia of the alcohols suggest that their mode of action was through disruption of membranes associated with ethylene action.     

Metabolic engineering of Propionibacterium freudenreichii for n-propanol production

Propionibacteria are widely used in industry for manufacturing of Swiss cheese, vitamin B₁₂, and propionic acid. However, little is known about their genetics and only a few reports are available on the metabolic engineering of propionibacteria aiming at enhancing fermentative production of vitamin B₁₂ and propionic acid. n-Propanol is a common solvent, an intermediate in many industrial applications, and a promising biofuel. To date, no wild-type microorganism is known to produce n-propanol in sufficient quantities for industrial application purposes. In this study, a bifunctional aldehyde/alcohol dehydrogenase (adhE) was cloned from Escherichia coli and expressed in Propionibacterium freudenreichii. The mutants expressing the adhE gene converted propionyl- coenzyme A, which is the precursor for propionic acid biosynthesis, to n-propanol. The production of n-propanol was limited by NADH availability, which was improved significantly by using glycerol as the carbon source. Interestingly, the improved propanol production was accompanied by a significant increase in propionic acid productivity, indicating a positive effect of n-propanol biosynthesis on propionic acid fermentative production. To our best knowledge, this is the first report on producing n-propanol by metabolically engineered propionibacteria, which offers a novel route to produce n-propanol from renewable feedstock, and possibly a new way to boost propionic acid fermentation.     

Production of Solvents by Clostridium acetobutylicum Cultures Maintained at Neutral pH

The formation of acetone and n-butanol by Clostridium acetobutylicum NCIB 8052 (ATCC 824) was monitored in batch culture at 35°C in a glucose (2% [wt/vol]) minimal medium maintained throughout at either pH 5.0 or 7.0. At pH 5, good solvent production was obtained in the unsupplemented medium, although addition of acetate plus butyrate (10 mM each) caused solvent production to be initiated at a lower biomass concentration. At pH 7, although a purely acidogenic fermentation was maintained in the unsupplemented medium, low concentrations of acetone and n-butanol were produced when the glucose content of the medium was increased (to 4% [wt/vol]). Substantial solvent concentrations were, however, obtained at pH 7 in the 2% glucose medium supplemented with high concentrations of acetate plus butyrate (100 mM each, supplied as their potassium salts). Thus, C. acetobutylicum NCIB 8052, like C. beijerinckii VPI 13436, is able to produce solvents at neutral pH, although good yields are obtained only when adequately high concentrations of acetate and butyrate are supplied. Supplementation of the glucose minimal medium with propionate (20 mM) at pH 5 led to the production of some n-propanol as well as acetone and n-butanol; the final culture medium was virtually acid free. At pH 7, supplementation with propionate (150 mM) again led to the formation of n-propanol but also provoked production of some acetone and n-butanol, although in considerably smaller amounts than were obtained when the same basal medium had been fortified with acetate and butyrate at pH 7.     

Thermolabile antifreeze protein produced in Escherichia coli for structural analysis

Inclusion bodies of recombinant human growth hormone (r-hGH) were isolated from Escherichia coli, enriched and solubilized in 100 mM Tris buffer containing 6 M n-propanol and 2 M urea. Around 4 mg/ml of r-hGH from inclusion bodies were solubilized in 6 M n-propanol-based buffer containing 2 M urea. Existence of native-like secondary structure of r-hGH in 6 M n-propanol solution was confirmed by CD and fluorescence spectra. Solubilized r-hGH was subsequently refolded by pulsatile dilution, purified to homogeneity and found to be functionally active. Tris buffer containing 6 M n-propanol and 2 M urea also effectively solubilized a number of proteins expressed as inclusion bodies in E. coli. Mild solubilization of inclusion body proteins, chaotropic effect of n-propanol at high concentration and kosmotropic effect at lower concentration helped in improved refolding of the solubilized protein. Around 40% of the r-hGH in the form of inclusion body aggregates was refolded into bioactive form while using n-propanol as solubilization agent. Solubilization with 6 M n-propanol solution thus can be a viable alternative for achieving high throughput recovery of bioactive protein from inclusion bodies of E. coli.     

Microbial Oxidation of Gaseous Hydrocarbons: Production of Secondary Alcohols from Corresponding n-Alkanes by Methane-Utilizing Bacteria

Over 20 new strains of methane-utilizing bacteria were isolated from lake water and soil samples. Cell suspensions of these and of other known strains of methane-utilizing bacteria oxidized n-alkanes (propane, butane, pentane, hexane) to their corresponding secondary alcohols (2-propanol, 2-butanol, 2-pentanol, 2-hexanol). The product secondary alcohols accumulated extracellularly. The rate of production of secondary alcohols varied with the organism used for oxidation. The average rate of 2-propanol, 2-butanol, 2-pentanol, and 2-hexanol production was 1.5, 1.0, 0.15, and 0.08 μmol/h per 5.0 mg of protein in cell suspensions, respectively. Secondary alcohols were slowly oxidized further to the corresponding methylketones. Primary alcohols and aldehydes were also detected in low amounts (rate of production were 0.05 to 0.08 μmol/h per 5.0 mg of protein in cell suspensions) as products of n-alkane (propane and butane) oxidation. However, primary alcohols and aldehydes were rapidly metabolized further by cell suspensions. Methanol-grown cells of methane-utilizing bacteria did not oxidize n-alkanes to their corresponding secondary alcohols, indicating that the enzymatic system required for oxidation of n-alkanes was induced only during growth on methane. The optimal conditions for in vivo secondary alcohol formation from n-alkanes were investigated in Methylosinus sp. (CRL-15). The rate of 2-propanol and 2-butanol production was linear for the 40-min incubation period and increased directly with cell protein concentration up to 12 mg/ml. The optimal temperature and pH for the production of 2-propanol and 2-butanol were 40°C and pH 7.0. Metalchelating agents inhibited the production of secondary alcohols. The activities for the hydroxylation of n-alkanes in various methylotrophic bacteria were localized in the cell-free particulate fractions precipitated by centrifugation between 10,000 and 40,000 × g. Both oxygen and reduced nicotinamide adenine dinucleotide were required for hydroxylation activity. The metal-chelating agents inhibited hydroxylation of n-alkanes by the particulate fraction, indicating the involvement of a metal-containing enzyme system in the oxidation of n-alkanes. The production of 2-propanol from the corresponding n-alkane by the particulate fraction was inhibited in the presence of methane, suggesting that the subterminal hydroxylation of n-alkanes may be catalyzed by methane monooxygenase.     

Membrane permeability changes: A component of phytochrome-mediated anthocyanin synthesis in Sinapis alba

Dark synthesis of anthocyanin in Sinapis alba seedlings is greatly promoted by short treatments with n-propanol. The effect of n-propanol treatment is reversed by subsequent far-red light pulses. Photoreversibility kinetics suggest that n-propanol and red light act in the same way. However, phytochrome measurements and other control experiments suggest that n-propanol treatment does not lead to significant Pfr production, nor does it increase the effectiveness of any ‘dark’ Pfr present in the seedlings. The findings are difficult to explain in terms of Pfr as the sole effector of this phytochrome-mediated response.     

β-d-glucosidase activity in developing leaves of Nicotiana tabacum

Tobacco (Nicotiana tabacum L. cv. Burley 21) leaves were assayed for β-d-glucosidase activity, using esculin as substrate. The enzymatically produced esculetin was silylated and quantitatively measured by GLC, using a tritium foil electron-capture detector. In field-grown plants, the activity in mid-stalk leaves increased with plant maturation; conversely, the activity in the top leaves decreased.     

Production of Methyl Ketones from Secondary Alcohols by Cell Suspensions of C2 to C4n-Alkane-Grown Bacteria

Nineteen new C₂ to C₄n-alkane-grown cultures were isolated from lake water from Warinanco Park, Linden, N.J., and from lake and soil samples from Bayway Refinery, Linden, N.J. Fifteen known liquid alkane-utilizing cultures were also found to be able to grow on C₂ to C₄n-alkanes. Cell suspensions of these C₂ to C₄n-alkane-grown bacteria oxidized 2-alcohols (2-propanol, 2-butanol, 2-pentanol, and 2-hexanol) to their corresponding methyl ketones. The product methyl ketones accumulated extracellularly. Cells grown on 1-propanol or 2-propanol oxidized both primary and secondary alcohols. In addition, the activity for production of methyl ketones from secondary alcohols was found in cells grown on either alkanes, alcohols, or alkylamines, indicating that the enzyme(s) responsible for this reaction is constitutive. The optimum conditions for in vivo methyl ketone formation from secondary alcohols were compared among selected strains: Brevibacterium sp. strain CRL56, Nocardia paraffinica ATCC 21198, and Pseudomonas fluorescens NRRL B-1244. The rates for the oxidation of secondary alcohols were linear for the first 3 h of incubation. Among secondary alcohols, 2-propanol and 2-butanol were oxidized at the highest rate. A pH around 8.0 to 9.0 was found to be the optimum for acetone or 2-butanone formation from 2-alcohols. The temperature optimum for the production of acetone or 2-butanone from 2-propanol or 2-butanol was rather high at 60°C, indicating that the enzyme involved in the reaction is relatively thermally stable. Metal-chelating agents inhibit the production of methyl ketones, suggesting the involvement of a metal(s) in the oxidation of secondary alcohols. Secondary alcohol dehydrogenase activity was found in the cell-free soluble fraction; this activity requires a cofactor, specifically NAD. Propane monooxygenase activity was also found in the cell-free soluble fraction. It is a nonspecific enzyme catalyzing both terminal and subterminal oxidation of n-alkanes.     

Natural products from Tolpis barbata (L.) Gaertn. (Asteraceae,Cichorieae)

One sesquiterpene lactone – 9α-hydroxy-3-deoxyzaluzanin C, three benzopyrans: desmethoxyencecalin (6-acetyl-2,2-dimethylchromene), desacetylripariochromen B and 6-(1-hydroxyethyl)-2,2-dimethylchromene, one coumarin – scopoletin and two eugenol derivatives were isolated from the roots of Tolpis barbata (L.) Gaertn, hitherto unexamined species. In the extract from aerial parts of the plant, five known phenolic compounds, namely: esculin, esculetin, chlorogenic acid (5-CQA), luteolin 7-O-glucoside and 3,5-dicaffeoylquinic acid (3,5-DCQA) were identified as major constituents. Except for the two coumarins – scopoletin and esculetin, which were previously obtained from Tolpis webbii Sch.Bip. and T. proustii Pit., the isolated and identified compounds have not been previously reported as constituents of Tolpis spp. Though benzopyrans were found in numerous species of the Asteraceae, their occurrence in the tribe Cichorieae has not been demonstrated before.     

Esculetin induces apoptosis and inhibits adipogenesis in 3T3-L1 cells

Objective: To determine the effects of esculetin, a plant phenolic compound with apoptotic activity in cancer cells, on 3T3‐L1 adipocyte apoptosis and adipogenesis. Research Methods and Procedures: 3T3‐L1 pre‐confluent preadipocytes and lipid‐filled adipocytes were incubated with esculetin (0 to 800 μM) for up to 48 hours. Viability was determined using the Cell Titer 96 Aqueous One Solution cell proliferation assay; apoptosis was quantified by measurement of single‐stranded DNA. Post‐confluent preadipocytes were incubated with esculetin for up to 6 days during maturation. Adipogenesis was quantified by measuring lipid content using Nile Red dye; cells were also stained with Oil Red O for visual confirmation of effects on lipid accumulation. Results: In mature adipocytes, esculetin caused a time‐ and dose‐related increase in adipocyte apoptosis and a decrease in viability. Apoptosis was increased after only 6 hours by 400 and 800 μM esculetin (p < 0.05), and after 48 hours, as little as 50 μM esculetin increased apoptosis (p < 0.05). In preadipocytes, apoptosis was detectable only after 48 hours (p < 0.05) with 200 μM esculetin and higher concentrations. However, results of the cell viability assay indicated a reduction in preadipocyte number in a time‐ and dose‐related manner, beginning as early as 6 hours with 400 and 800 μM esculetin (p < 0.05). Esculetin also inhibited adipogenesis of 3T3‐L1 preadipocytes. Esculetin‐mediated inhibition of adipocyte differentiation occurred during the early, intermediate, and late stages of the differentiation process. In addition, esculetin induced apoptosis during the late stage of differentiation. Discussion: These findings suggest that esculetin can alter fat cell number by direct effects on cell viability, adipogenesis, and apoptosis in 3T3‐L1 cells.     

Catalytic properties of cross-linked enzyme crystals in organic media

The activity and the enantioselectivity of cross-linked enzyme crystals (CLECs) of subtilisin in the transesterification between N-acetyl-l-phenylalanine ethyl ester and n-propanol have been examined in various organic solvents. The activity of CLECs of subtilisin in decane was 780 times greater than that in triethylamine. CLECs of subtilisin preferred l-enantiomer in the transesterification between N-acetyl-phenylalanine ethyl ester and n-propanol, and the (k_cat/K_M)_L/(k_cat/K_M)_D ratio was 20 000 in cyclohexane.     

Glucosylation of esculetin by plant cell suspension cultures

Cell suspension cultures of Lithospermum erythrorhizon, Gardenia jasminoides and Nicotiana tabacum were capable of glucosylating esculetin to esculin (7-hydroxycoumarin-6-O-β-D-glucoside). Especially, a culture strain of Lithospermum erythrorhizon was superior in the esculetin glucosylating capability; 40 to 50% of esculetin administered to the culture medium at early stationary growth stage was converted into esculin within 24 h. The rate of glucosylation was also dependent on the growth stage and the medium composition especially growth hormones and sugar.     

Effect of n-Alkanols on Acidification Curves of Aureobasidium pullulans Suspensions

Moragues, M. D., Estevez, J. J., Rementerı́a, A., and Sevilla, M. J. 1994. Effect of n-alkanols on acidification curves of Aureobasidium pullulans suspensions. Experimental Mycology 18, 1-6. n -Alkanols, from methanol to 1-hexanol, induce yeast-to-hyphae transition in the dimorphic fungus Aureobasidium pullulans. In order to elucidate whether triggering of the morphogenetic event is membrane related, we have studied the effect of the morphogenetic n-alkanols on the pH of suspensions of A. pullulans, with no external carbon source. n -Alkanols, at their hyphal inducing concentration or higher, caused a decrease in the initial acidification rate (C) of yeast-phase cell suspensions of A. pullulans. From this effect on C, an inhibition coefficient (K) was deduced, specific for each alcohol. These coefficients were directly related to the lipid/buffer partition coefficients of the alkanols. On the other hand, germ tubes of A. pullulans, obtained in the presence of n-propanol, showed a much slower acidification rate in water than yeast cells. Moreover, 1-propanol or 1-butanol did not significantly affect the initial acidification rate of germ tubes. The latter observation was interpreted as an adaptation of cells grown in the presence of alcohol. The results of all these experiments support our hypothesis that the plasma membrane is a target of the morphogenetic effects of n-alkanols.     

Transglucosylases in Cichorium intybus converting cichoriin to esculin

The heads of Cichorium intybus contain two enzymes concerned in the formation of esculin (esculetin 6-glucoside) from cichoriin (esculetin 7-glucoside). Both enzymes can catalyse two reactions, i.e. hydrolysis (HD) of cichoriin to give esculetin, and transglucosylation (TG) from this glucoside to the liberated aglucone forming esculin. One of them, designated enzyme A, is a high molecular weight protein with predominantly TG activity, and dissociates during isolation into the other enzyme having higher HD activity. Enzyme A shows high substrate specificity and different pH optima in HD and TG reactions, as in the case with the transglucosylase from Daphne odora.     

A TLC-GLC procedure for the quantitation of algal photosynthetic pigments and their degradation products

A procedure employing and GLC techniques for the analysis of algal chlorophylls and their degradation products has been developed and evaluated. Algal pigments were separated on MN 300 cellulose plates developed in an ascending solvent system of hexane saturated with acetonitrile and n-propanol (100:0,4, v:v). Quantitation of chlorophyll a, b and pheophytin a, b were accomplished by GLC analysis of their phytol, following alkaline methanolic hydrolysis of the individual pigments. The amount of chlorophyllides/pheophorbides in a sample was estimated by its free phytol content. This technique is especially valuable for the evaluation of the pigment contents of near sediment phytoplankton and periphyton samples, where large quantities of chlorophyll degradation products and/or carotenoid pigments are generally present which may interfere significantly with the routine analytical methods.     

A semi-empirical conformational analysis of the interaction of n-alkanols with dipalmitoylphosphatidylcholine

The interaction of a homologous series of saturated aliphatic n-alkanols (containing 1–13 carbon atoms) with dipalmitoylphosphatidylcholine was studied by a semi-empirical conformational analysis. The minimal conformational energy of the isolated molecule at the hydrocarbon-water interface was calculated as the sum of the contributions resulting from the Van der Waals, torsional, electrostatic and transfer energies. From the conformers of minimal energies were calculated the hydrophilic-hydrophobic balance, the distance between hydrophilic and hydrophobic centres and the energies of interaction between homologous alkanols and between alkanols and lipids. Using these parameters, different modes of conformation, orientation and interaction of n-alkanols and dipalmitoylphosphatidylcholine were described. For methanol, ethanol and n-propanol, the gauche conformers were the most probable interacting only with the lipid polar heads. Only all-trans conformers were obtained for alkanols with longer acyl chains. n-Butanol to n-octanol form clusters in the lipid matrix. Longer n-alkanols are randomly distributed in the lipid layer. However, due to the increase in hydrocarbon chain-length, n-nonanol and higher alkanols have an interaction energy identical or superior to that found in a pure lipid monolayer, leading to a more ordered alkanol-lipid organization.     

Enhancement of esculetin on arsenic trioxide-provoked apoptosis in human leukemia U937 cells

In order to overcome chemotherapy resistance, many laboratories are searching for agents that increase the sensitivity of cancer cells to anticancer drugs. Arsenic trioxide (As₂O₃) is widely used in treating human acute polymyelocytic leukemia (APL). However, solid tumors and other leukemia cells such as U937 promonocytic leukemia cells are insensitive to As₂O₃. Esculetin, a coumarin derivative, has previously induced cell cycle arrest and apoptosis of HL-60 cells as well as enhanced taxol-induced apoptosis in HepG2 cells, thereby displaying anticancer potential. In this study, esculetin inhibited proliferation and mitogen activated protein kinases (MAPKs) activation in human leukemia U937 cells. Since inhibitors of MAPKs have modulated the GSH-redox state and enhanced the sensitivity of leukemia cells to As₂O₃-provoked apoptosis, we monitored the effect of combining esculetin and As₂O₃ (2.5 μM) on the GSH level. Our study showed that esculetin, PD98059 (MEK/ERK inhibitor), and SP600125 (JNK inhibitor) similarly enhanced the As₂O₃-induced GSH depletion. We found that the As₂O₃ (2.5 μM) treatment slightly induced apoptosis and the pretreatment of esculetin enhanced the As₂O₃-provoked apoptosis significantly. In addition, esculetin enhanced the effect of As₂O₃ on caspase activation in U937 cells. We compared the combined esculetin and As₂O₃ treatment to the As₂O₃ treated alone. The combined esculetin and As₂O₃ treatment increased Bid cleavage, Bax conformation change and cytochrome C release. The study also indicated that esculetin enhanced the As₂O₃-induced lysosomal leakage and apoptosis. Furthermore, pretreatment with N-acetylcysteine (NAC) reduced these enhanced effects. Based on these studies, esculetin enhances the As₂O₃-provoked apoptosis by modulating the MEK/ERK and JNK pathways and reducing intracellular GSH levels. GSH depletion led to higher oxidative stress which activated lysosomal-mitochondrial pathway of apoptosis.     

Position specificity of an o-dihydroxycoumarin glucosyltransferase from tobacco cell suspension culture

A partially purified UDP-glucose: o-dihydroxycoumarin glucosyltransferase from tobacco cell culture required an intact coumarin ring system with o-dihydroxy groups for highest activity. The enzyme exhibited strict position specificity towards the 7-OH group of both esculetin and daphnetin with the formation of cichoriin and daphnin, respectively. The apparent K_m values were 111 and 95 μM, while the V_max for esculetin was 26.4 pkat/mg protein.     

Separation of glycoprotein-derived oligosaccharides by thin-layer chromatography.

A thin-layer chromatography (tlc) system has been developed for the separation of glycoprotein-derivedoligosaccharides. The method involves chromatography on silica gel using n-propanol/acetic acid/water (3:3:2 v/v) as the solvent. This tlc method was used to separate pathological oligosaccharides isolated from individuals with G_M1 gangliosidosis and with neuraminidase deficiency. The results indicate the potential usefulness of the system in the analysis of complex carbohydrates.