Plant, mammalian, and microbial cells are commonly immobilized in calcium alginate gels for the production of valuable secondary metabolites. However, calcium ions are known to inhibit growth in various type of cells, and calcium is an integral part of such gels. Therefore, an investigation was conducted to evaluate the effect of calcium on the growth and alkaloid production of a model cell-line, Coffea arabica, in suspension culture before attempting to immobilize such cells in alginate. A kinetic model was then developed from the results to describe cell growth and alkaloid production and the mechanism by which calcium influences these variables. In addition, it was observed that there was a characteristic relationship between the concentration of calcium in the external medium and the concentration of extracellular and intracellular phosphate. The intracellular phosphate level was, in turn, related to the production of alkaloids. Using these results, a dynamic mathematical model of cell growth and alkaloid production was developed based on the proposed roles of calcium and phosphate. The model showed satisfactory agreement with three sets of experiments at different calcium concentrations. A possible linkage between the calcium and phosphate results is postulated based on the limited solubility of calcium phosphate.
A novel bioreactor using magnetically stabilized fluidized bed (MSFB) technology has been developed that has certain advantages for cultivating cells continuously. In this system, the cells are protected from shear and are constrained to move through the fermenter in lock-step fashion by being immobilized in calcium alginate beads. The MSFB permits good mass transfer, minimizes particle collisions, and allows for the production of cells while maintaining a controlled cell residence time. Details of the experimental system are described. In addition, the experimental performance of an MSFB used to grow plant cells in batch mode is compared to the results obtained in shake flask culture.
The past year or so has seen the development of new enzyme assays, as well as the improvement of existing ones. Assays are becoming more rapid and sensitive as a result of modifications such as amplification of the enzyme product(s). Recombinant DNA technology is now being recognized as a particularly useful tool in the search for improved assay systems.
The effects of various chemical substanceson the permeability of plasma membranesand tonoplasts of three suspension cultures (Catharanthusroseus, Thalictrum rugosum and Chenopodiumrubrum) have been studied. The permeabilityof the plasma membrane is monitoredby measuring the activity of the cytosolic enzymeisocitrate dehydrogenase and the permeability ofthe tonoplast is measured by determining the releaseof substances stored in the vacuoles (inorganicphosphate, berberine and betanin for thethree cell lines, respectively). The minimum concentrationrequired for quantitative release of vacuolarproducts have been established for five differentpermeabilization agents. Cell viability islost upon permeabilization except for treatmentof Catharanthus roseus with DMSO and Triton X-100.
It has long been hoped that the unique biosynthetic capacity of plants could be exploited in vitro using culture systems analogous to microbial fermentations. However, the characteristics of both the growth and metabolism of plant cells in vitro differ considerably from those of microbial cells and plant cell suspension culture systems have met with limited success. Immobilizing the cells creates a new set of options for the plant biotechnologist to explore. Improvements in some process criteria are apparent although evaluating the potential of immobilized plant cells for producing commercial compounds will only be possible when the biological problems have been overcome.
An experimental method for determination of the optimum chitosan concentration for elicitation of plant cell suspension cultures is presented. The procedure, which is based on measurements of the conductivity of the culture medium after addition of chitosan, has been applied to suspension cultures of Nicotiana tabacum and Eschscholtzia californica. Increased conductivity of the medium (due to permeabilization of the cells) results in decreased secondary product formation and cell growth. Maximum product formation is observed for cells elicited with the highest chitosan concentration which does not affect membrane permeability.
Plant cell suspension cultures producing secondary metabolites have been permeabilized for product release by electroporation. The two cell cultures studied, i.e. Thalictrum rugosum and Chenopodium rubrum, require about 5 and 10 kV cm–1, respectively, for complete permeabilization (release of all the intracellularly stored product). The number of electrical pulses and capacitance used had a relatively limited effect on product release while the viability of the cells was strongly influenced by the latter. Conditions for complete product release resulted in total loss of viability of the cells after treatment. The release of product from immobilized cells was also achieved by electroporation. Cells entrapped in alginate required less voltage for permeabilization than free or agarose entrapped cells.
Bovine liver glutamate dehydrogenase has been studied by analytical affinity chromatography on two immobilized AMP analogs, i.e., N6-(6-aminohexyl)-AMP and 8-(6-aminohexyl)-amino-AMP. The existence of various enzyme-coenzyme and enzyme-effector complexes has been verified. Also the cooperative formation of two ternary complexes, i.e., glutamic dehydrogenase (GHD)-NADP-glutamate and GDH-ADP-leucine, has been shown. The results of this study have been rationalized by the “ligand exclusion theory.” which has been proposed for the regulation of the glutamic dehydrogenase. It has been shown that the active site and the ADP-binding effector site are oriented close to each other on the enzyme. Furthermore, the data suggest that the adenylic site is not identical to the nonactive coenzyme binding site. A mechanism based on electrostatic interactions is suggested for the cooperative binding of oxidized coenzyme and substrate. Dissociation constants for complexes between the enzyme and two coenzyme fragments (P-ADPR and 2′,5′-ADP) have been estimated.
The synthesis of a new general ligand, i.e., 8-(6-aminohexyl)-amino-GMP, has been achieved by bromination of GMP and subsequent substitution of the bromine for hexamethylene diamine. The overall yield of the synthesis has been 60 to 70%. This new general ligand was immobilized on BrN-activated Sepharose and used as an affinity adsorbent for inosinic acid dehydrogenase (E.C. 1.2.1.14) from Aerobacter aerogenes. Various elution methods were investigated in order to increase the specific activity of the purified enzyme.
Phosphorylation of 6-chloropurine riboside with phosphorus oxychloride and phosphorus trichloride gave a mixture of the two isomers, 6-chloropurine-riboside 2’,5‘-bisphosphate and 6-chloropurine-riboside 3‘,5‘-bisphosphate. Reaction with Iy6-diaminohexane followed by resolution of the isomeric mixture on a Dowex 1-X2 column yielded N6-(6-aminohexyl)-adenosine 2’,5’-bisphosphate and N6-(6-aminohexyl)-adenosine 3’,5‘-bisphosphate.The inhibition of several NADP+-dependent and NAD+-dependent dehydrogenases by N6-(6-aminohexyl)-adenosine 2’,5‘-bisphosphate, N6-(6-aminohexyl)-adenosine 3’,5’-bisphosphate and N6-(6-aminohexyl)-adenosine 5’-monophosphate was examined.These three AMP-analogues were attached to Sepharose 4B by the cyanogen bromide method and the binding of several NAD(P)+-dependent enzymes were investigated. NADP+-dependent enzymes were bound to Sepharose substituted with N6-(6-aminohexyl)-adenosine 2’,5‘-bisphosphate, whereas NAD+-dependent enzymes were not bound under the same conditions. Conversely, when N6-(6-aminohexy1)-adenosine 5‘-monophosphate was used as the immobilised ligand only the NAD+- dependent enzymes were bound, as well as glucose-6-phosphate dehydrogenase showing weak affinity. These observations strongly suggest that these two immobilised analogues represent true biospecific and group-specific adsorbents. The enzymes were eluted with their complementary nucleotides, NAD(H) and NADP(H). These techniques were utilised to purify several NADPf-dependent enzymes from a crude Candida utilis extract by chromatography on the new biospecific adsorbent.
Various alcohol and lactate dehydrogenases were adsorbed to an affinity column of immobilized N6-(6-aminohexyl)-AMP and subsequently eluted with gradients of coenzymes or coenzyme fragments. A linear relation was observed between the eluting concentration of nucleotide and the reported dissociation constants for the corresponding binary enzyme-nucleotide complexes. This relation has been utilized to determine unknown dissociation constants by affinity chromatography. The method presented can also be utilized for the estimation of dissociation constants betweendehydrogenases and coenzyme analogues.
High resolution "P NMR spectra (103.2 MHz) ofoxygenated Catharanthus roseu8 and Daucus carotacells grown in suspension cultures were obtained usinga solenoidal perfusion probe. The spectra showed resonancesfor various phosphorylated metabolites suchas ATP, ADP, NAD(P)(H), nucleoside diphosphoglucose,and sugar phosphates. The relative levels ofthe phosphorylated metabolites remained constantthroughout the growth curve. No resonances for storagecompounds such as polyphosphates, pyrophosphate,or phytates were observed. Two resolved resonancesfor Pi indicated an intracellular pH of 7.3 and5.7 (or below) for the cytoplasm and vacuoles, respectively.The time course of Pi uptake and storage duringgrowth in fresh culture medium was followed by studyingthe level of vacuolar Pi with 31PN MR (145.7 MHz).Simultaneously, the level of Pi in the culture mediumwas followed with radioactive s2P. C. roseus quicklytakes up all the Pi from the culture medium (maximumrate 1.7 pmol min" g" (dry weight of cells)). The Pi isfirst stored in the vacuoles; subsequently, one part ofthis pool is used to keep a constant cytoplasmic Pi levelwhile another part is apparently accumulated as anNMR invisible Pi store, probably in another cell organelle.In contrast, D. carota does not accumulate Pi inthe vacuoles and consequently it takes up Pi from themedium at a much slower rate (0.05 pmol min" g"(dry weight of cells)).
A number of benzophenanthridine alkaloids are induced in suspension cultures of Eschscholtzia californica after treatment with an elicitor prepared from yeast extract. The formation of the alkaloids sanguinarine, chelerythrine and macarpine has been studied in relation to; elicitor concentration, incubation time after elicitation, and culture age. A significant portion of these alkaloids is released into the medium. Sanguinarine and chelerythrine reach maximum levels a few hours after the time of elicitation. Thereafter, their levels decline and the amount of macarpine increases. Viability of elicited cells, as determined by their subsequent growth, is not significantly reduced. There is a good correlation between induced tyrosine decarboxylase activity and alkaloid formation.
A cell suspension culture of Vanilla planifolia has been established in MS-medium. 2,4-D suppressed while NAA enhanced the formation of extractable phenolics. Cytokinins appeared to favour lignin biosynthesis. Treatment of the culture with chitosan resulted in the induction of various enzymes of phenylpropanoid metabolism, while the amount of extractable phenolics decreased due to their rapid incorporation into polymeric ligneous material.
Feeding of cinnamic acid and ferulic acid to non-treated andchitosan-treated cell suspension cultures of Vanilla planifoIiaresulted in the formation of trace amounts of p-hydroxy benzoicacid (5.2 micrograms per gram fresh weight of cells) and vanillicacid (6.4 micrograms per gram fresh weight of cells), respectively.Addition of a 4-hydroxycinnamate: CoA-ligase inhibitor, 3,4-(methylenedioxy)-cinnamic acid (MDCA), resulted in a reducedbiosynthesis of ligneous material with a simultaneous significantincreased vanillic acid formation (around 75 micrograms per gramfresh weight of cells). A K, of 100 micromolar for 4-hydroxycinnamate:CoA-ligase in a crude preparation was estimated for thisinhibitor. It is suggested that the conversion of cinnamic acidsinto benzoic acids does not involve cinnamoyl CoA esters asintermediates. Feeding of 14C-cinnamic acid and 14C-ferulic acidto cells treated with MDCA indicate that cinnamic acid, but notferulic acid, is a precursor of vanillic acid in these cultivated cellsof V. planifolia.
Feeding of 4-methoxycinnamic acid, 3,4-dimethoxycinnamicacid and 3,4,5-trimethoxycinnamic acid to cell suspension culturesof Vanilla planifolia resulted in the formation of 4-hydroxybenzoicacid, vanillic acid, and syringic acid, respectively. Thehomologous 4-methoxybenzoic acids were demethylated to thesame products. It is concluded that the side chain degradingenzyme system accepts the 4-methoxylated substrates while thedemethylation occurs at the benzoic acid level. The demethylatingenzyme is specific for the 4-position. Feeding of [10_4Cmethyl]-3,4-dimethoxycinnamic acid revealed that the first stepin the conversion is the glycosylation of the cinnamic acid to itsglucose ester. A partial purification of a UDP-glucose: transcinnamicacid glucosyltransferase is reported. 4-Methoxy substitutedcinnamic acids are better substrates for this enzyme than4-hydroxy substituted cinnamic acid. It is suggested that 4-methoxysubstituted cinnamic acids are intermediates in the biosyntheticconversion of cinnamic acids to benzoic acids in cells ofV. planifolia.
A carbohydrate fraction isolated from yeast extract by ethanolic precipitation was used as an elicitor to induce secondary product formation in plant cell suspension cultures. The elicitor preparation is effective in inducing glyceollin isomer synthesis (up to 200 μg glyceollin per g dry wt) in cells of Glycine max and enhancing berberine biosynthesis (up to four-fold) in cells of Thalictrum rugosum. The response of the cell cultures to the elicitor treatment is dependent on the amount of carbohydrate per unit of biomass and on the physiological state of the cells. Cells are optimally induced in late exponential or early stationary growth phases.
Plants have a long history as therapeutic tools in the treatment of human diseases and have been used as a source of medicines for ages. In search of new biologically active natural products, many plants and herbs used in traditional medicine are screened for natural products with pharmacological activity. In this paper, we present a group of natural products, the sesquiterpene coumarins isolated from plants, and describe their wide range of biological activity. Sesquiterpene coumarins are found in some plants of the families Apiaceae (Umbelliferae), Asteraceae (Compositae) and Rutaceae. The coumarin moiety is often umbelliferone (7-hydroxycoumarin) but scopo- letin (7-hydroxy-6-methoxycoumarin) and isofraxidin (7-hydroxy-6,8-dimethoxycoumarin) are also found. These coumarins are linked to a C15 terpene moiety through an ether linkage. Another group of sesquiter- pene coumarins is the prenylated 4-hydroxycoumarins where the link between the coumarin and the C15 terpene moiety is a C–C-bond at carbon 3 of the coumarin moiety. Finally, the prenylfurocoumarin-type sesquiterpenoids are a separate group of sesquiterpene coumarins based on the suggested biosynthetic pathway. Our relatively limited knowledge on the biosynthesis of sesquiterpene coumarins is reviewed.
Self-healing carbohydrate polymers were synthesized by Diels-Alder reaction. Intermediate products and the carbohydrate matrices were characterized by Fourier transform infrared spectroscopy (FT-IR) and H-1 NMR, while the thermally reversible properties were assessed by FT-IR and differential scanning calorimetry. The mechanical properties, water absorption, and enzymatic degradation of starch/PVA/modified carbohydrate films were examined, as well as the relationship of the properties to the DA and rDA reactions. These results showed that DA bonds were introduced into the carbohydrate polymers successfully and endow the material with self-healing thermal recyclability. The mixed films exhibited alternating strong and weak mechanical properties upon cycling through the DA and rDA reactions. Water absorption was limited and the films demonstrated good water resistance. The status of the DA bonds was found not to affect the enzymatic degradation rates of the various carbohydrate films.
New molecularly imprinted polymers (MIPs), which exhibit specific recognition of ethyl carbamate (EC) have been synthesized and studied. In this process, EC was the template molecule and -cyclodextrin derivatives were employed as functional monomers in the molecular imprinting technique (MIT). An EC molecularly imprinted sensor (EC-MIS) was prepared by using MIT surface modification. The EC-MIS was characterized by cyclic voltammetry, electrochemical impedance spectroscopy and differential pulse voltammetry. EC detection performance, binding parameters and dynamics mechanism were investigated. The result showed that the synthetic route designed was appropriate and that new MIP and EC-MIS were successfully prepared. The EC-MIS exhibited a good molecular recognition of EC. A linear relationship between current and EC concentration was observed using cyclic voltammetry and the detection limit was 5.86 g L-1. The binding constant (K = 4.75 x 10(6) L mol(-1)) between EC and the EC-MIS, as well as, the number of binding sites (n = 1.48) has been determined. The EC-MIS recognition mechanism for the EC is a two-step process. The sensor was applied for the determination of EC in Chinese yellow wines, and the results were in good agreement with the gas chromatography-mass spectrometry (GC-MS) method.
The binding properties of pentacyclic triterpenoid isomeric drugs, i.e. ursolic acid (UA) and oleanolic acid (OA), to bovine lactoferrin (BLF) have been studied by molecule modeling, fluorescence spectroscopy, UV-visible absorbance spectroscopy and infrared spectroscopy (IR). Molecular docking, performed to reveal the possible binding mode or mechanism, suggested that hydrophobic interaction and hydrogen bonding play important roles to stabilize the complex. The results of spectroscopic measurements showed that the two isomeric drugs both strongly quenched the intrinsic fluorescence of BLF through a static quenching procedure although some differences between UM and OA binding strength and non-radiation energy transfer occurred within the molecules. The number of binding sites was 3.44 and 3.10 for UA and OA, respectively, and the efficiency of Forster energy transfer provided a distance of 0.77 and 1.21 nm for UA and OA, respectively. The conformation transformation of BLF affected by the drugs conformed to the "all-or-none" pattern. In addition, the changes of the ratios of alpha-helices, beta-sheets and beta-turns of BLF during the process of the interaction were obtained. The results of the experiments in combination with the calculations showed that there are two modes of pentacyclic triterpenoid binding to BLF instead of one binding mode only governed by the principle of the lowest bonding energy.
In order to obtain the biological active compound, alpha-mangostin, from the traditional native mangosteen (Garcinia mangostana L.), an extraction method for industrial application was explored. A high yield of a-mangostin (5.2%) was obtained by extraction from dried mangosteen pericarps with subsequent purification on macroporous resin HPD-400. The chemical structure of alpha-mangostin was verified mass spectrometry (MS), nuclear magnetic resonance (H-1 NMR and C-13 NMR), infrared spectroscopy (IR) and UV-Vis spectroscopy. The purity of the obtained alpha-mangostin was 95.6% as determined by HPLC analysis. The binding of native alpha-mangostin to human serum albumin (HSA) or transferrin (TRF) was explored by combining spectral experiments with molecular modeling. The results showed that amangostin binds to HSA or TRF as static complexes but the binding affinities were different in different systems. The binding constants and thermodynamic parameters were measured by fluorescence spectroscopy and absorbance spectra. The association constant of HSA or TRF binding to alpha-mangostin is 6.4832x10(5) L/mol and 1.4652x10(5) L/mol at 298 K and 7.8619x10(5) L/mol and 1.1582x10(5) L/mol at 310 K, respectively. The binding distance, the energy transfer efficiency between alpha-mangostin and HSA or TRF were also obtained by virtue of the Forster theory of non-radiation energy transfer. The effect of alpha-mangostin on the HSA or TRF conformation was analyzed by synchronous spectrometry and fluorescence polarization studies. Molecular docking results reveal that the main interaction between amangostin and HSA is hydrophobic interactions, while the main interaction between alpha-mangostin and TRF is hydrogen bonding and Van der Waals forces. These results are consistent with spectral results.
Verbascoside (VER) is the enzymatic hydrolysis product of echinacoside (ECH). The molecular structures of ECH and VER have different glucosyl groups so they bind to serum albumin in different ways, resulting in different pharmacological actions. In this report, we have examined the binding characteristics between human serum albumin (HSA) and ECH/VER by molecular modeling and spectroscopic approaches. Molecular modeling revealed that VER bound to HSA mainly through hydrogen bonds, van der Waals forces and hydrophobic forces. The spectroscopic results showed that the interactions between HSA and VER/ECH involved a static binding process, and the bonding strength of the VER-HSA complex was stronger than that of the ECH-HSA complex. The value of the binding distances (r) was low, which indicated the occurrence of energy transfer. The reaction conformational pattern of HSA-VER and HSA-ECH gave a "two-state model" based on fluorescent phase diagram analysis. According to the thermodynamic model, the main forces between interaction of VER and HSA were hydrogen bonds and van der Waals forces, whereas the interaction between ECH and HSA was hydrophobic force. The fluorescence polarization analysis demonstrated that the interaction between HSA and VER or ECH generated a non-covalent complex. Compared with ECH, VER was more likely to bind with HSA because of its smaller molecular size and low polarity. The results of the spectral analysis concurred with the molecular modeling data, which provides a helpful reference for the study of the molecular reaction mechanism of VER/ECH binding to HSA.
Tyrosine decarboxylase (EC 4.1.1.25) was induced in suspension cultures of Thalictrum rugosum by treatment with a yeast glucan elicitor. Maximum induction was observed at a carbohydrate concentration of 0.4 mg/g fresh weight of cells and maximum enzyme activity was reached 20 h after addition of elicitor. The enzyme was inducible in late exponential and early stationary growth phases. A good correlation between induced tyrosine decarboxylase activity and berberine biosynthesis has been established. It is suggested that tyrosine decarboxylase may be a key enzyme between primary and secondary metabolisms in the biosynthesis of norlaudanosoline-derived alkaloids.
Suspension-cultured cells of Coffea arabica have been immobilized by entrapment in calcium alginate gels to mimic natural aggregation. The production of methylxanthine alkaloid was increased up to 13-fold by the immobilization. This increased production has been ascribed to organization of the entrapped cells through physicochemical interactions between the polymer (alginate) and the plant cell wall. It has been shown that the metabolic changes induced by the immobilization are reversible.