Proposed biosynthetic pathway for rosmarinic acid in cell cultures of Coleus blumei Benth

A biosynthetic pathway for rosmarinic acid is proposed. This pathway is deduced from studies of the enzymes detectable in preparations from suspension cells of Coleus blumei. Phenylalanine is transformed to 4-coumaroyl-CoA by the enzymes of the general phenylpropanoid pathway: phenylalanine ammonia-lyase (EC 4.3.1.5), cinnamic acid 4-hydroxylase (EC 1.14.13.11) and hydroxycinnamic acid:CoA ligase (EC 6.2.1.12). Tyrosine is metabolized to 4-hydroxyphenyllactate by tyrosine aminotransferase (EC 2.6.1.5) and hydroxyphenylpyruvate reductase. The ester can be formed from 4-coumaroyl-CoA and 4-hydroxyphenyllactate by the catalytic activity of rosmarinic acid synthase with concomitant release of CoA. Microsomal hydroxylase activities introduce the hydroxyl groups at positions 3 and 3 of the aromatic rings of the ester 4-coumaroyl-4-hydroxyphenyllactate giving rise to rosmarinic acid.Abbreviations Caf-pHPL caffeoyl-4-hydroxyphenyllactate - DHPL 3,4-dihydroxyphenyllactic acid - pC-DHPL 4-coumaryl-3,4-dihydroxyphenyllactate - pC-pHPL 4-coumaryl-4-hydroxyphenyllactate - pHPL 4-hydroxyphenyllactic acid - RA rosmarinic acid The financial support of the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie is gratefully acknowledged.     

Interactions between parallel pathways during biosynthesis of rosmarinic acid in cell suspension cultures of Coleus blumei

Phenylalanine ammonia-lyase from an over-producer strain of Coleus blumei Benth. cell cultures accumulating high levels of rosmarinic acid (RA) has been shown to possess no special feed-back sensitivity to RA or its precursors. No tyrosine-3-hydroxylase activity could be detected in culture extracts and no specific inhibitors of tyrosine incorporation into RA were found. L--aminooxy--phenyl propionic acid, however, was effective in specifically blocking phenylalanine incorporation. This block also led to an accumulation of label from tyrosine in 4-hydroxyphenyllactic acid rather than in 3,4-dihydroxyphenylalanine (DOPA) or 3,4-dihydroxyphenyllactic acid. These observations require a re-evaluation of the possible role of DOPA as a major biogenic precursor to RA.Abbreviations AOPP -aminooxy--phenylpropionic acid - DOPA 3,4-dihydroxyphenylalanine - RA rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyllactic acid) - PAL L-phenylalanine ammonia-lyase (EC 4.3.1.5)     

Rosmarinic acid production in Coleus cell cultures

Cell suspension cultures of Coleus blumei Benth. have been found to accumulate 8–11% of their dry weight as rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyl-lactic acid). Actively-growing tissue converts >20% of exogenously supplied phenylalanine and tyrosine to the caffeoyl ester and this high rate of synthesis coincides with an increase in phenylalanine ammonia-lyase specific activity. Administration to the cultures of known phenylpropanoid precursors of rosmarinic acid failed to enhance the latter's production and in some cases inhibited it.Abbreviations RA rosmarinic acid (-O-caffeoyl-3,4-dihydroxyphenyllactic acid - DOPA dihydroxyphenylalanine - PAL phenylalanine ammonialyase - DOPL dihydroxyphenyl-lactic acid     

The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei accumulate very high amounts of rosmarinic acid, an ester of caffeic acid and 3,4-dihydroxyphenyllactate, in medium with elevated sucrose concentrations. Since the synthesis of this high level of rosmarinic acid occurs in only five days of the culture period, the activities of the enzymes involved in the biosynthesis are very high. Therefore all the enzymes necessary for the formation of rosmarinic acid from the precursors phenylalanine and tyrosine could be isolated from cell cultures of Coleus blumei: phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, hydroxycinnamoyl:CoA ligase, tyrosine aminotransferase, hydroxyphenylpyruvate reductase, rosmarinic acid synthase and two microsomal 3- and 3-hydroxylases. The main characteristics of these enzymes of the proposed biosynthetic pathway of rosmarinic acid will be described.Abbreviations DHPL 3,4-dihydroxyphenyllactate - DHPP 3,4-dihydroxyphenylpyruvate - pHPL 4-hydroxyphenyllactate - pHPP 4-hydroxyphenylpyruvate - RA rosmarinic acid     

Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei

Suspension cultures of Coleus blumei were characterized with respect to growth and rosmarinic acid formation in media with different sugars and various sugar concentrations. Sucrose is the sugar with the highest stimulating effect on growth and rosmarinic acid accumulation, followed by glucose and fructose. The sugar alcohol mannitol cannot be metabolized by the plant cells. Sucrose is cleaved into glucose and fructose by the Coleus cells. Sucrose concentrations from 1 to 5% have an increasing positive effect on growth and rosmarinic acid synthesis in the cell cultures with a maximum rosmarinic acid content of 12% of the dry weight in medium with 5% sucrose; in medium with 6% sucrose rosmarinic acid accumulation obviously did not reach its highest level in the culture period of 14 days. A very high yield of rosmarinic acid (2 mg ml^-1 suspension) could also be achieved by maintaining a sucrose concentration of 2% during the whole culture period. The start of rosmarinic acid synthesis by the cell cultures seems to be regulated by the growth limitation when a nutrient, e.g. phosphate is depleted from the medium. The rate of rosmarinic acid accumulation is related to the amount of carbon left in the medium when growth ceases.Abbreviations RA rosmarinic acid     

Biosynthesis and accumulation of rosmarinic acid in suspension cultures ofColeus blumei

This communication reviews data on the accumulation and biosynthesis of rosmarinic acid in cell suspension cultures ofColeus blumei. The influence of the medium, mainly the carbohydrate source on growth and rosmarinic acid production in these cell cultures is described. The biosynthetic pathway of rosmarinic acid was elucidated inColeus blumei cell cultures: eight enzymatic activities are involved in the transformation of the precursors phenylalanine and tyrosine to the end product rosmarinic acid.Abbreviations CAH cinnamic acid 4-hydroxylase - 4CL 4-coumarate:CoA ligase - HPPR hydroxyphenylpyruvate reductase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - 3-H hydroxycinnamoyl-hydroxyphenyllactate 3-hydroxylase - PAL phenylalanine ammonia-lyase - RAS rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase) - TAT tyrosine aminotransferase     

Fungal elicitor preparations and methyl jasmonate enhance rosmarinic acid accumulation in suspension cultures of Coleus blumei

bstract Suspension cultures of Coleus blumei (Lamiaceae) treated with either an elicitor preparation from the culture medium of the phytopathogenic oomycete Pythium aphanidermatum or with methyl jasmonate enhanced accumulation of rosmarinic acid approximately threefold. The specific activities of phenylalanine ammonia lyase and rosmarinic acid synthase were also enhanced after addition of the fungal elicitor. The addition of methyl jasmonate transiently increased activities of phenylalanine ammonia lyase and hydroxyphenylpyruvate reductase, whereas the activity of rosmarinic acid synthase was not stimulated and the activity of tyrosine aminotransferase was slightly and constantly enhanced. Methyl jasmonate stimulated rosmarinic acid accumulation not only when added directly to the culture medium, but also when it could reach the cells only via the gas phase. Received: 2 April 1997 / Revision received:16 June 1997 / Accepted: 15 September 1997     

Photoassimilate-transport characteristics of nonchlorophyllous and green tissue in variegated leaves of Coleus blumei Benth

The nonchlorophyllous (albino) tissue of mature C. blumei leaves is a sink for photoassimilate. Transport from the green to the albino region of the same leaf was inhibited by cold and anoxia. When the green tissue of mature leaves was removed, the remaining albino portion imported labeled translocate from other mature leaves in the phloem. Photoassimilate unloading in the albino region of mature leaves was studied by quantitative autoradiography. The unloading was inhibited by cold but not by anoxia. No labeled photoassimilate could be detected in the free space of mature albino tissue by compartmental efflux analysis as phloem unloading proceeded in a N₂ atmosphere, indicating that unloading, may occur by a symplastic pathway as it apparently does in sink leaves of other species. The minor veins of mature albino leaf tissue did not accumulate exogenous [¹⁴C]sucrose. Minor veins of green tissue in the same leaves accumulated [¹⁴C]sucrose but, in contrast to other species studied to date, this accumulation was insensitive to the inhibitor p-chloromercuribenzensulfonic acid (PCMBS).In its capacity to import and unload photoassimilate, and in the inability, of the minor veins to accumulate exogenous sucrose, the albino region of the mature C. blumei lamina differs from mature albino tobacco leaves and darkened mature leaves of other species. This, together with evidence indicating that phloem loading in C. blumei and other species may occur by different routes and with different sensitivity to PCMBS, indicates that the mechanism of transfer of photoassimilates between veins and surrounding tissues, and the mechanism of the sink-source transition, may not be the same in the leaves of all species. It is speculated that the unusual properties of the C. blumei leaf may be a consequence of the presence, in the minor veins, of intermediary cells, large companion cells connected to the bundle sheath by abundant plasmodesmata.Abbreviation PCMBS p-chloromercuribenzenesulfonic acid     

Ultrastructure,plasmodesmatal frequency,and solute concentration in green areas of variegated Coleus blumei Benth. leaves

The photosynthetic tissue of green portions of variegated Coleus blumei leaves consists primarily of palisade and spongy parenchyma cells as well as bundle-sheath cells. The moderate numbers of plasmodesmata connecting these cells may be sufficient to provide a symplastic pathway for assimilates moving toward the minor veins. The minor veins, however, are unusual in having two sets of phloem-loading cells which have little symplastic continuity with one another: one consisting of large, peripherally located intermediary cells, and a second set made up of smaller, usually more internal companion cells, both sets having their associated sieve-tube members. The intermediary cells are connected to vascular-parenchyma and bundle-sheath cells by unique branched plasmodesmata which are particularly abundant at the bundle-sheath interface. In addition, numerous plasmodesmata-pore connections occur between the intermediary cells and their associated sieve-tube members. Neither the intermediary cells nor their sieve-tube members plasmolyze when treated with 1.4 M mannitol, whereas mesophyll and vascular-parenchyma cells plasmolyze at 0.5 M and bundle-sheath cells at 0.6 M mannitol. By contrast, the companion cells and their associated sieve-tube members are symplastically isolated from the bundle-sheath cells and the sieve-tube-intermediary-cell complexes, and share few plasmodesmata with the vascular-parenchyma cells. Moreover, the companion cells plasmolyze at 1.1 M mannitol and their sieve tubes at 1.3 M. The intermediary-cell-sieve-tube complex thus appears to be structurally equipped to load assimilates entirely via the symplast, while the sieve-tube-companion-cell complex is probably loaded from the apoplast.Abbreviation ER endoplasmic reticulum     

Methyl jasmonate-induced rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures

Summary A dramatic increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after their exposure to methyl jasmonate (MJ). Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) and 4-hydroxyphenylpyruvate reductase (HPR) activities increased rapidly and transiently, whereas tyrosine aminotransferase (TAT) activity showed only a slight increase. The elicitation activity of MJ was much higher than that of yeast extract (YE) in terms of the induction of PAL and HPR activities, RA accumulation and incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA. However, the response of the cultured cells to MJ-treatment was slower than that to YE-treatment.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - LS Linsmaier and Skoog - HPR 4-hydroxyphenylpyruvate reductase - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - MJ methyl jasmonate - YE yeast extract     

Cloning and characterisation of rosmarinic acid synthase from Melissa officinalis L

Lemon balm (Melissa officinalis L.; Lamiaceae) is a well-known medicinal plant mainly due to two groups of compounds, the essential oil and the phenylpropanoid derivatives. The prominent phenolic compound is rosmarinic acid (RA), an ester of caffeic acid and 3,4-dihydroxyphenyllactic acid. RA shows a number of interesting biological activities. Rosmarinic acid synthase (RAS; 4-coumaroyl-CoA:hydroxyphenyllactic acid hydroxycinnamoyltransferase) catalyses the ester formation. Cell cultures of M. officinalis have been established in order to characterise the formation of RA in an important diploid medicinal plant. RAS activity as well as the expression of the RAS gene are closely correlated with the accumulation of RA in suspension cultures of M. officinalis. The RAS cDNA and gene (MoRAS) were isolated. The RAS gene was shown to be intron-free. MoRAS belongs to the BAHD superfamily of acyltransferases. Southern-blot analysis suggests the presence of only one RAS gene copy in the M. officinalis genome. The enzyme was characterised with respect to enzyme properties, substrate preferences and kinetic data in crude plant extracts and as heterologously synthesised protein from Escherichia coli.     

Induction of rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures by yeast extract

Summary A transient increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after addition of yeast extract (YE) to the suspension cultures, reaching a maximum at 24 hr. The highest increase of the RA content (2.5-fold) was obtained when 6-day-old cells in the exponential growth phase were treated with YE. Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) activity increased rapidly, whereas tyrosine aminotransferase (TAT) activity was largely unaffected by the treatment. The incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA was enhanced in the YE-treated cells, consistent with increased synthesis of the ester.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - RA rosmarinic acid - YE yeast extract     

Selection of cell lines for the production of rosmarinic acid from cell suspension cultures of Orthosihon stamineus benth

Summary Cell suspension cultures of Orthosiphon stamineus were established from friable calluses produced from leaf pieces of in vitro plantlets that were derived from nodal segments of the mother plants collected from three different geographical locations. Eight lines were eventually selected after seven subculture cycles based on the growth characteristic (plant height) of the plantlets from the three locations: two fast-growing lines (>5.1 cm tall), three intermediate-growing lines (3.1–5.0 cm tall), and three slow-growing lines (<3.0 cm tall). All eight lines grew well in liquid Murashige and Skoog medium supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 5.4 μM 1-naphthaleneacetic acid (NAA). All cell lines exhibited the same growth pattern but produced different maximum cell biomass when cultured in this medium. The time of harvesting the plant cells from the culture medium and the geographical source of the original plant material were both found to affect the production of rosmarinic acid (RA) in cell cultures. Two cell lines were successfully selected and identified to produce high amounts of RA. These cell lines were a fast-growing cell line from Air Itam, Penang and an intermediate-growing cell line from Relau Agriculture Research Centre, Penang which could produce 5% [(w/w) dry weight] and 4.5% [(w/w) dry weight] of RA, respectively.     

Sugar synthesis and phloem loading in Coleus blumei leaves

Sugar-synthesis and -transport patterns were analyzed in Coleus blumei Benth. leaves to determine where galactinol, raffinose, and stachyose are made and whether phloem loading includes an apoplastic (extracellular) step or occurs entirely within the symplast (plasmodesmata-connected cytoplasm). To clarify the sequence of steps leading to stachyose synthesis, a pulse (15 s) of ¹⁴CO₂ was given to attached leaves followed by a 5-s to 20-min chase: sucrose was rapidly labeled while galactinol, raffinose and stachyose were labeled more slowly and, within the first few minutes, to approximately the same degree. Leaf tissue was exposed to either ¹⁴CO₂ or [¹⁴C]glucose to identify the sites of synthesis of the different sugars. A 2-min exposure of peeled leaf tissue to [¹⁴C]glucose resulted in preferential labeling of the minor veins, as opposed to the mesophyll; galactinol, raffinose and stachyose were more heavily labeled than sucrose in these preparations. In contrast, when leaf tissue was exposed to ¹⁴CO₂ for 2 min for preferential labeling of the mesophyll, sucrose was more heavily labeled than galactinol, raffinose or stachyose. We conclude that sucrose is synthesized in mesophyll cells while galactinol, raffinose and stachyose are made in the minorvein phloem. Competition experiments were performed to test the possibility that phloem loading involves monosaccharide uptake from the apoplast. Two saturable monosaccharide carriers were identified, one for glucose, galactose and 3-O-methyl glucose, and the other for fructose. Washing the apoplast of peeled leaf pieces with buffer or saturating levels of 3-O-methyl glucose, after providing a pulse of ¹⁴CO₂, did not inhibit vein loading or change the composition of labeled sugars, and less than 0.5% of the assimilated label was recovered in the incubation medium. These and previous results (Turgeon and Gowan, 1991, Plant Physiol. 94, 1244–1249) indicate that the phloem loading pathway in Coleus is probably symplastic.Abbreviations 3-OMG 3-O-methyl glucose - PCMBS p-chloromercuribenzenesulfonic acid - SE-CCC sieve-element-companion-cell complex This research was supported by National Science Foundation Grant DCB-9104159, U.S. Department of Agriculture Competetive Grant 90000854, and Hatch funds.     

Rosmarinic acid synthase is a new member of the superfamily of BAHD acyltransferases

Purification of rosmarinic acid synthase (hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyltransferase) from suspension cells of Coleus blumei Benth. (Lamiaceae) by fractionated ammonium sulphate precipitation, hydrophobic interaction chromatography and two affinity chromatography steps led to the identification of peptide sequences, which enabled a PCR-based approach to isolate the full-length cDNA encoding this enzyme. The open reading frame of the cDNA had a length of 1290 base pairs encoding a protein of 430 amino acid residues with a molecular mass of 47,932 Da with typical characteristics of an acyltransferase of the BAHD superfamily. The cDNA was heterologously expressed in Escherichia coli. The enzyme displayed the activity of rosmarinic acid synthase using 4-coumaroyl- and caffeoyl-coenzyme A and 4-hydroxyphenyllactate as well as 3.4-dihydroxyphenyllactate as substrates. Shikimic acid and quinic acid were not able to serve as hydroxycinnamoyl acceptors. This therefore is the first report of the cDNA-cloning of a rosmarinic acid synthase.     

Methyl jasmonate enhanced production of rosmarinic acid in cell cultures of Satureja khuzistanica in a bioreactor

The growing interest in rosmarinic acid (RA), an ester of caffeic acid and 3,4‐dihydroxyphenyl lactic acid, is due to its biological activities, which include cognitive‐enhancing effects, slowing the development of Alzheimer's disease, cancer chemoprotection, and anti‐inflammatory activity. Inspired by the challenge of meeting the growing demand for this plant secondary metabolite, we developed a biotechnological platform based on cell suspension cultures of Satureja khuzistanica. The high amounts of RA produced by this system accumulated mainly inside the cells. To further improve production, two elicitors, 100 μM methyl jasmonate (MeJA) and 40 mM cyclodextrin (CD), were tested, separately and together. MeJA increased RA productivity more than 3‐fold, the elicited cultures achieving an RA production of 3.9 g L^?1 without affecting biomass productivity. CD did not have a clear effect on RA production, and under the combined treatment of MeJA + CD only a small amount of RA was released to the medium. When the cell culture was transferred from a shake flask to a wave‐mixed bioreactor, a maximum RA production of 3.1 g L^?1 and biomass productivity of 18.7 g L^?1 d^?1 was achieved under MeJA elicitation, demonstrating the suitability of S. khuzistanica cell suspensions for the biotechnological production of this bioactive plant secondary metabolite.     

Growth and production characteristics of permeabilized Coleus blumei cells in immobilized fed-batch culture

Summary Permeabilized Coleus blumei cells were cultivated in an immobilized state to study the effect of dimethyl sulfoxide (DMSO) concentrations and growth regulators on cell growth and rosmarinic acid (RA) production characteristics. Luffa (the fibrous skeleton of mature fruit of Luffa cylindrica) was a good support matrix for cell immobilization because of its high void volume. Maximum cell loading capacity was 1.33 g dry cell weight (DCW)/g dry Luffa. The experiments were done in shake flasks with no free medium. The medium was supplied in a fed-batch mode to avoid the flotation of Luffa pieces. The sucrose in the medium was completely hydrolyzed to glucose and fructose without any sugar accumulation in the medium. The cell viability was slightly higher in the cells on top of the Luffa than those in the middle. Cell growth rate and rosmarinic acid (RA) production were approximately half that obtained in cell suspension cultures. Cell yield (g DCW/g glucose) was similar to that of cell suspension cultures. The absence of growth regulators did not promote an increase of RA production but did decrease the cell mass. The second step preconditioning with 0.5% DMSO did not improve the cell's adaptability to higher DMSO concentrations and the cell mass did not increase with 2.5% DMSO.     

Purification of hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase from cell-suspension cultures of Solanum tuberosum L. cv. Datura

A pathogen-elicitor-inducible acyltransferase [tyramine hydroxycinnamoyltransferase (THT); EC 2.3.1], which catalyzes the transfer of hydroxycinnamic acids from hydroxycinnamoyl-CoA esters to tyramine in the formation of N-hydroxycinnamoyltyramine, was purified to apparent homogeneity from cell-suspension cultures of potato (Solanum tuberosum L. cv. Datura), with a 1400-fold enrichment, a 5% recovery and a final specific activity of 208 mkat·(kg protein)^–1. Affinity chromatography on Reactive Yellow-3-Agarose using the acyl donor (feruloyl-CoA) as eluent was the decisive step in the purification sequence. The purified protein showed a native molecular mass of ca. 49 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and in the absence of a reducing agent (2-mercaptoethanol) indicated that THT is a heterodimer in which the protein subunits (ca. 25 kDa) are non-covalently associated. The enzyme was stimulated fivefold by 10 mM Ca²⁺. The apparent K _m value for tyramine was dependent on the nature of the hydroxycinnamoyl-CoA present. Thus, the K _m value for tyramine was about tenfold greater (174 M) in the presence of 4-coumaroyl-CoA than in the presence of feruloyl-CoA (20 M).Abbreviations PAL phenylalanine ammonia-lyase - THT hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase We thank the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie for financial support. Further support by a grant from the Studienstiftung des Deutschen Volkes to H.H. is gratefully acknowledged.     

Purification and molecular properties of ferredoxin-glutamate synthase from Chlamydomonas reinhardii

Ferredoxin-glutamate synthase (EC 1.4.7.1) from Chlamydomonas reinhardii has been purified to electrophoretic homogeneity, with a specific activity of 10.4 units mg^-1 protein, by a method which included chromatography on diethylaminoethyl sephacel and hydroxylapatite, and ferredoxin-sepharose affinity treatment. The enzyme is a single polypeptide chain of M _r 146000 dalton which shows an absorption spectrum with maxima at 278, 377 and 437 nm, and an A₂₇₆/A₄₃₇ absorptivity ratio of 7.0. The anaerobic addition of dithionite results in the loss of the absorption peak at 437 nm, which is restored upon reoxidation of the enzyme with an excess of 2-oxoglutarate, alone or in the presence of glutamine. This indicates the presence in the enzyme of a flavin prosthetic group, which is functional during the catalysis. The ferredoxin-glutamate synthase can be assayed with methyl viologen, chemically reduced with dithionite, but it is unable to use reduced pyridine nucleotide. Azaserine, 6-diazo-5-oxo-norleucine, bromocresol green and p-hydroxymercuribenzoate are potent inhibitors of this activity, which, on the other hand, is stable upon heating at 45°C for 10 min.Abbreviations DEAE-sephacel diethylaminoethyl sephacel - Fd ferredoxin - GOGAT glutaniate synthase (glutamine: -ketoglutarate aminotransferase) - SDS sodium dodecyl sulfate     

Enhanced release of rosmarinic acid from Coleus blumei permeabilized by dimethyl sulfoxide (DMSO) while preserving cell viability and growth

Continuous permeabilization of preconditioned Coleus blumei cells with dimethyl sulfoxide (DMSO) is shown to be an effective strategy for the enhanced release of rosmarinic acid (RA) while preserving cell viability. When nonpreconditioned cells were permeabilized with DMSO, they lost their viability at DMSO concentrations higher than a critical value located between 0.1% and 0.5% DMSO. Product release was low [0.49 g RA/100 g dry cell weight (DCW)] at 0.1% DMSO. Preconditioning cells at 0.1% DMSO ensured high viability at DMSO concentrations of 0.5%, 1.0%, and 1.5%. Product release reached a maximum of 2.85 g RA/100 g DCW at 0.5% DMSO, which was 66.4% of the total rosmarinic acid produced. (c) 1992 John Wiley & Sons, Inc.