Identification of intermediates and enzymes involved in the early steps of artemisinin biosynthesis in Artemisia annua.

An important group of antimalarial drugs consists of the endoperoxide sesquiterpene lactone artemisinin and its derivatives. Only little is known about the biosynthesis of artemisinin in Artemisia annua L., particularly about the early enzymatic steps between amorpha-4,11-diene and dihydroartemisinic acid. Analyses of the terpenoids from A. annua leaves and gland secretory cells revealed the presence of the oxygenated amorpha-4,11-diene derivatives artemisinic alcohol, dihydroartemisinic alcohol, artemisinic aldehyde, dihydroartemisinic aldehyde and dihydroartemisinic acid. We also demonstrated the presence of a number of biosynthetic enzymes such as the amorpha-4,11-diene synthase and the--so far unknown--amorpha-4,11-diene hydroxylase as well as artemisinic alcohol and dihydroartemisinic aldehyde dehydrogenase activities in both leaves and glandular trichomes. From these results, we hypothesise that the early steps in artemisinin biosynthesis involve amorpha-4,11-diene hydroxylation to artemisinic alcohol, followed by oxidation to artemisinic aldehyde, reduction of the C11-C13 double bond to dihydroartemisinic aldehyde and oxidation to dihydroartemisinic acid.

The effect of ethanol on the kinetics of lipase-mediated enantioselective esterification of 4-methyloctanoic acid and the hydrolysis of its ethyl ester.

The Novozym 435(R) catalyzed esterification and hydrolysis reactions of 4-methyloctanoic acid (ethyl ester) were investigated. In both the hydrolysis and esterification reactions, the increase of ethanol concentration led to an increase in enantiomeric ratio (E). For hydrolysis of the ethyl ester, the E-value increased from 5.5 [0% (v/v) EtOH] up to 12 [20% (v/v) EtOH]. In case of esterification, the E-value was already 16 [14% (v/v) EtOH] and rose to 57 [73% (v/v) EtOH]. When combining these results of esterification and hydrolysis, an enantiomeric ratio of 350 can be estimated for the sequential kinetic resolution of 4-methyloctanoic acid. In this way, enantiopure 4-methyloctanoic acid could be obtained after two consecutive reaction steps.

Downstream processing of enzymatically produced geranyl glucoside.

Geraniol plays an important role in the fragrance and flavor industry. The corresponding glucoside has interesting properties as a "slow release" aroma compound. Therefore, the enzymatic production and downstream processing of geranyl glucoside were investigated. Geranyl glucoside was produced in a spray column reactor with an initial production rate of 0.58 mg x U(-1) x h(-1). A pretreated hydrophobic microfiltration membrane was used to prevent migration of the aqueous, enzyme-containing phase to the downstream process. No retention of the glucoside, which accumulated in the geraniol phase, was found. On the basis of examples from the literature, four downstream processes were tested on their viability for this system. Extraction with water and foaming were not suitable to recover geranyl glucoside from geraniol. In the first case, the glucoside selectivity for the geraniol phase was found to be high, which made extraction with water unsuccessful. In the second case it was possible to obtain a stable foam, but significant enrichment of the foam with glucoside did not occur. Adsorption on alumina and distillation under reduced pressure were applied successfully and tested in-line with the bioreactor. A maximum glucoside adsorption of 7.86 mg x g(-1) was achieved on alumina. After desorption and evaporation of the extractant the pure glucoside was obtained quantitatively. A pure product could not be obtained after distillation because a small amount of glucose was present in the permeate as well, which accumulated in the bottom fraction. It was shown that with this reactor system a production of 1 kg of geranyl glucoside in 2 days is possible using an initial amount of 50,000 units of enzyme.

Germacrenes from fresh costus roots.

Four germacrenes, previously shown to be intermediates in sesquiterpene lactone biosynthesis, were isolated from fresh costus roots (Saussurea lappa). The structures of (+)-germacrene A, germacra-1(10),4,11(13)-trien-12-ol, germacra-1(10),4,11(13)-trien-12-al, and germacra-1(10),4,11(13)-trien-12-oic acid were deduced by a combination of spectral data and chemical transformations. Heating of these compounds yields (-)-beta-elemene, (-)-elema-1,3,11(13)-trien-12-ol, (-)-elema-1,3,11(13)-trien-12-al, and elema-1,3,11(13)-trien-12-oic acid respectively, in addition to small amounts of their diastereomers. Acid induced cyclisation of the germacrenes yields selinene, costol, costal, and costic acid respectively. It is highly probable that the elemenes reported in literature for costus root oil are artefacts.

Optimization of production and downstream processing of the almond beta-glucosidase-mediated glucosylation of glycerol.

This article describes the synthesis of glyceryl glucoside from glycerol and glucose with almond beta-glucosidase as the catalyst. A yield of 54% (0.45 mmol/g) was obtained. The influence of the enzyme stability, the water concentration, and the water activity on the glucoside yield were determined. A molar fraction-based equilibrium constant of 2.4 +/- 0.6 was found, with which the glucoside yield could be calculated for all possible combinations of initial substrate and water fractions in the reaction mixture. A model was used to optimize the glucoside yield while minimizing one of the substrate concentrations at equilibrium. This straightforward model gives a good prediction of the measured glucoside yield, according to a parity plot.

Biosynthesis of germacrene A carboxylic acid in chicory roots. Demonstration of a cytochrome P450 (+)-germacrene a hydroxylase and NADP+-dependent sesquiterpenoid dehydrogenase(s) involved in sesquiterpene lactone biosynthesis.

Sprouts of chicory (Cichorium intybus), a vegetable grown in the dark, have a slightly bitter taste associated with the presence of guaianolides, eudesmanolides, and germacranolides. The committed step in the biosynthesis of these compounds is catalyzed by a (+)-germacrene A synthase. Formation of the lactone ring is the postulated next step in biosynthesis of the germacrene-derived sesquiterpene lactones. The present study confirms this hypothesis by isolation of enzyme activities from chicory roots that introduce a carboxylic acid function in the germacrene A isopropenyl side chain, which is necessary for lactone ring formation. (+)-germacrene A is hydroxylated to germacra-1(10),4,11(13)-trien-12-ol by a cytochrome P450 enzyme, and is subsequently oxidized to germacra-1(10),4,11(13)-trien-12-oic acid by NADP+-dependent dehydrogenase(s). Both oxidized germacrenes were detected as their Cope-rearrangement products elema-1,3,11(13)-trien-12-ol and elema-1,3,11(13)-trien-12-oic acid, respectively. The cyclization products of germacra-1(10),4,11(13)-trien-12-ol, i.e. costol, were also observed. The (+)-germacrene A hydroxylase is inhibited by carbon monoxide (blue-light reversible), has an optimum pH at 8.0, and hydroxylates beta-elemene with a modest degree of enantioselectivity.

On a revised mechanism of side product formation in the lignin peroxidase catalyzed oxidation of veratryl alcohol.

The O2-dependent formation of side products during the oxidation of veratryl alcohol (VA) by lignin peroxidase has previously been proposed to start with the attack of H2O on the VA radical cation (VA*+). This initial reaction is unlikely since it would also lead to side product formation in the absence of O2, which is not the case. In the current mechanism VA* reacts first with O2, whereafter H2O attacks. Furthermore, this paper describes an alternative explanation for the inhibitory effect of Mn2+ on VA side product formation. It is proposed that Mn2+ reduces reactive intermediates back to VA.

Lignin peroxidase initiates O2-dependent self-propagating chemical reactions which accelerate the consumption of 1-(3',4'-dimethoxyphenyl)propene.

Lignin peroxidase (LiP) has been used to study the C(alpha)-C(beta) cleavage of the propylene side chain in 1-(3',4'-dimethoxyphenyl)propene (DMPP) to 3,4-dimethoxybenzaldehyde (veratraldehyde, VAD). Under an air atmosphere, LiP oxidized DMPP to VAD (27.8%) and 1-(3',4'-dimethoxyphenyl)propan-2-one (DMPA, 8.7%), after 10 min of incubation. Dissolved O(2) was rapidly consumed during DMPP conversion, of which one-third was converted into superoxide. The remaining two-thirds of the consumed O(2) was involved in C(alpha)-C(beta) cleavage of DMPP to VAD and in self-propagating chemical reactions stimulating the consumption of DMPP. The involvement of peroxyl radicals, in the chemical consumption of DMPP, was confirmed by using the well-known peroxyl radical reductant Mn(2+). This metal ion severely inhibited the DMPP consumption rate under air, but did not affect the lower enzymic DMPP consumption rate under N(2). The substoichiometric requirement of LiP for H(2)O(2) during DMPP oxidation could be explained in part by dismutation of superoxide, but more importantly by direct chemical reactions of DMPP-derived peroxyl radicals with fresh DMPP. Another VAD-producing route was found by incubating the DMPP oxidation product, DMPA, with LiP. Under air the molar yield of VAD was 29.7%. In the absence of O(2), the C(alpha)-C(beta) cleavage of DMPA to VAD was strongly inhibited and side-chain coupling products (dimers) were formed instead. As a whole, the results suggest two new roles of O(2) in LiP-mediated oxidation of aromatic substrates. First, O(2) is responsible for the formation of reactive peroxyl intermediates, which can directly react with other substrate molecules and thereby accelerate consumption rates. Secondly, O(2) can prevent coupling reactions by lowering the pool of carbon-centred radicals accumulating during LiP catalysis.

Amorpha-4,11-diene synthase catalyses the first probable step in artemisinin biosynthesis.

The endoperoxide sesquiterpene lactone artemisinin and its derivatives are a promising new group of drugs against malaria. Artemisinin is a constituent of the annual herb Artemisia annua L. So far only the later steps in artemisinin biosynthesis--from artemisinic acid--have been elucidated and the expected olefinic sesquiterpene intermediate has never been demonstrated. In pentane extracts of A. annua leaves we detected a sesquiterpene with the mass spectrum of amorpha-4,11-diene. Synthesis of amorpha-4,11-diene from artemisinic acid confirmed the identity. In addition we identified several sesquiterpene synthases of which one of the major activities catalysed the formation of amorpha-4,11-diene from farnesyl diphosphate. This enzyme was partially purified and shows the typical characteristics of sesquiterpene synthases, such as a broad pH optimum around 6.5-7.0, a molecular mass of 56 kDa, and a K(m) of 0.6 microM. The structure and configuration of amorpha-4,11-diene, its low content in A. annua and the high activity of amorpha-4,11-diene synthase all support that amorpha-4,11-diene is the likely olefinic sesquiterpene intermediate in the biosynthesis of artemisinin.

Regioselectivity and fatty acid specificity of Chromobacterium viscosum lipase.

The fatty acid specificity of Chromobacterium viscosum lipase was studied by comparing the pseudo-first-order rate constants for the transesterification of different fatty acid methyl esters with 1-propanol in dry acetonitrile as solvent. It was found that this enzyme shows a significant preference towards long chain fatty acids and, for chains with the same length, towards saturated ones. The same enzyme was used to study the esterification of sorbitol and decanoic acid. A mixture of mono-, di-, tri- and tetraesters was obtained. The concentration of esters was strongly increased upon raising the temperature from 35 to 70 degrees C. The structures of the di-, tri- and tetraesters were determined using 13C NMR spectrometry. The diester appeared to be sorbitol 1,6-didecanoate, the triester was sorbitol 1,5,6-tridecanoate and the tetraester was the 1,2,5,6-tetradecanoate, which indicates that the C. viscosum lipase acylates sorbitol in a regioselective manner.

Prospects for the increased application of biocatalysts in organic transformations.

Predicting the future use of biocatalysts for the transformation of organic compounds--both natural substrates and other compounds--needs to take many factors into account. To date, relatively few biotransformations have been developed to the industrial scale, primarily because there has been little economic incentive to replace existing successful processes with biocatalysts, with their inherent problems of instability, lack of selectivity and narrow operational range. However, advances that improve biocatalyst performance, coupled with the increasing emphasis on 'chirotechnology', are driving the development of biocatalysis as a complementary, if not a rival technology to existing chemical approaches.

Lipase activity in vesicular systems: characterization of candida cylindracea lipase and its activity in polymerizable dialkylammonium surfactant vesicles.

Lipase from Candida cylindracea (CCL) was incorporated into polymerizable positively charged dialkylammonium bromide surfactant vesicles. The enzyme was incorporated by the use of the dehydration-rehydration method or by incubation. In the latter case, trapping efficiencies of up to 100% could be obtained. Activities of free and vesicle-incorporated CCL were tested for three triglycerides: triacetin, tributyrin, and tricaprylin. Enzyme activity was lowest in homogeneous mixtures (triacetin and small concentrations of tributyrin) and highest in heterogeneous mixtures (tricaprylin and high concentrations of tributyrin). Entrapment in vesicular systems is advantageous, especially in homogeneous reaction mixtures and in the case of the production of insoluble fatty acid (caproate), because inhibition by the acid can be suppressed. The influence of several surface-active additives, including vesicles, on the activity of lipase in triglyceride assays was tested. Vesicles have a positive influence on the activity, whereas other positively charged additives act as inhibitors. In the case of tricaprylin assays, the positively charged additives increase the activity. Finally, tryptic digestion for free and incorporated CCL were compared. Free CCL is readily inactivated, whereas incorporated enzyme is protected from proteolytic degradation.

Vanadium containing bromoperoxidase: An example of an oxidoreductase with high operational stability in aqueous and organic media.

The conversion is described of phenolsulphonephtalein (phenol red) to 3,3',5,5'-tetrabromophenolsulphonephthalein (bromophenol blue) by bromoper-oxidase from the brown alga Ascophyllum nodosum. This reaction provides a convenient assay for the detection of bromoperoxidase activity in vitro. Bromoperoxidase was shown to be stable under turnover conditions for three weeks at room temperature, catalyzing the bromination of phenol red into bromophenol blue. When stored at room temperature in organic sol vents such as acetone, methanol, ethanol [present up to 60% (v/v)], and 1-propanol [40% (v/v)], bromoperoxidase was stable for more than one month. As far as we know this is the first example of an oxidoreductase which displays such great stability. This enhances the applicability of the enzyme in organic synthesis.