Biotransformation of d-limonene to carvone by means of glucose oxidase and peroxidase

A novel method for enzymatic biotransformation of limonene to carvone has been developed. It involves addition glucose oxidase and peroxidase to the biotransformation medium. Some factors affecting biotransformation yield were investigated. Maximal yield of carvone occurred in the medium containing 1.5% substrate, at 50 degrees C and pH 7.0.     

Biotransformation of monoterpenes by immobilized microalgae

This paper reports the biotransformation of carvone, limonene, β-pinene, thymol, and linalool using whole-cell-immobilized microalgal strains isolated from paddy fields of Iran. The strains was recognized by morphological characterization and assigned according to amplified 16S/18S rRNA genes by PCR. Ten unialgal strains including Chlorella, Oocystis, Chlamydomonas, and Synechococcus were immobilized in calcium alginate beads. After a 24-h incubation with substrates, characterization and identification of biotransformation products were done by GC/MS. None of the isolated immobilized microalgae converted β-pinene. In contrast, most of these strains biotransformed carvone and limonene to the related compounds. Some strains only reduced the C = C double bond to yield the dihydrocarvone isomers while others reduced the ketone to give the dihydrocarveol. The transformation ratio showed that Oocystis sp. MCCS 033 and Synechococcus sp. MCCS 035 produced dihydrocarvone isomers with the highest efficiency. Furthermore, limonene was converted into a mixture of five corresponding products and the maximum yield was 52.1% for carvone, the bioconverted product. Only one strain, Synechococcus sp. MCCS 034, oxidized thymol, and the product obtained from thymol was thymoquinone. Also, linalooloxide isomers and dihydrolinalool were obtained from linalool, and finally dihydrolinalool was the main product. These results showed a novel conversion pathway of linalool-forming dihydrolinalool.     

Effects of R-(+)-limonene on submerged cultures of the terpene transforming basidiomycete Pleurotus sapidus.

The biotransformation of limonene by the basidiomycete Pleurotus sapidus yielded cis/trans-carveol and carvone as the main products. The transformation period was extended from 4 days after direct addition to 12 days by gas phase addition of the substrate. After 2 days of transformation, 97% of the substrate had accumulated in the mycelium, while only 3% were present in the culture medium. Substrate toxicity led to a decrease of dry matter. Adaptation of the precultures with small amounts of substrate doubled the concentration of carveol and increased the concentration of carvone by a factor of 3-4. Total product concentrations of > 100 mg l-1 were reached.     

The synthesis of L-carvone and limonene derivatives with increased antiproliferative effect and activation of ERK pathway in prostate cancer cells

Thirty-one novel derivatives of carvone, carveol, and limonene were designed and synthesized using L-carvone as a starting material via chlorination, nucleophilic substitution, and reduction. The structures of these derivatives were characterized by MS and 1H NMR. The antiproliferative effect was evaluated in human prostate cancer LNCaP cells. L-carvone, L-carveol, and L-limonene were weak cell growth inhibitors and introduction of 4-(2-methoxyphenyl)piperazine to carvone, carveol or limonene significantly increased their antiproliferative effect. The antiproliferative effect was correlated with ERK activation and p21(waf1) induction.     

Micropropagation and in vitro production of secondary metabolites of Croton floribundus Spreng.

Croton floribundus Spreng., a native plant from South America, was utilized for in vitro micropropagation and phytochemical analyses. The effects of the addition of naphthaleneacetic acid and indole butyric acid, on the production of shoots and leaves, as well as volatile constituent production, were determined. The combination of naphthaleneacetic acid and indole butyric acid at a ratio of 1:1 led to the production of the maximum number of leaves and longest shoots after a 60-d subculture period. Analyses of leaf dichloromethane extracts using gas chromatography–mass spectrometry showed that monoterpenes and sesquiterpenes were the main chemical classes present in both in vivo and in vitro conditions. Use of these plant growth regulators in the medium-induced quantitative changes in the major monoterpenes (neral, geranial, limonene, and carvone). In vitro leaf extracts produced compounds such as carvone as well as a large amount of trans-β-farnesene, with the highest production of carvone (16.8%) being produced on medium supplemented with 1.0 mg?L^?1 naphthaleneacetic acid. These results suggested the occurrence of biotransformation reactions of limonene and farnesyl cations in culture.     

Biotransformation of limonene by bacteria,fungi, yeasts,and plants

The past 5 years have seen significant progress in the field of limonene biotransformation, especially with regard to the regiospecificity of microbial biocatalysts. Whereas earlier only regiospecific biocatalysts for the 1,2 position (limonene-1,2-diol) and the 8-position (alpha-terpineol) were available, recent reports describe microbial biocatalysts specifically hydroxylating the 3-position (isopiperitenol), 6-position (carveol and carvone), and 7-position (perillyl alcohol, perillylaaldehyde, and perillic acid). The present review also includes the considerable progress made in the characterization of plant P-450 limonene hydroxylases and the cloning of the encoding genes.     

Changes in monoterpene composition of Mentha aquatica produced by gene substitution from a high limonene strain of M. Citrata

The dominant gene Lm that causes 60–90% limonene/cineole was substituted into M. aquatica by four convergent backcrosses. The natural strain of M. aquatica has 7·7% cineole, 4·9% limonene, traces of terpinolene and pulegone, 0·1% menthone, 0.2% menthol, and 66·4% menthofuran. The two modified hybrid strains with dominant gene Lm have 53·8 and 78·7% limonene/cineole and a total of only 1·0-3·8% 3-oxygenated compounds in contrast to a total of 66·7% found in the natural strain. The postulate is made that the Lm gene largely prevents either the conversion of a-terpineol → terpinolene or of limonene → isopiperitenone and that in these strains the recessive cc genotype largely but not completely prevents the conversion of limonene → carvone resulting in limonene accumulation. Mentha species almost invariably have either 2-oxygenated or 3-oxygenated compounds, not both. Close coupling phase linkage of the genes Lm and C explains why the self-pollinated progeny of M. spicata or M. crispa C-Lm/c-lm have a ratio of 3 carvone/dihydrocarvone: 1 pulegone/menthone rather than a ratio of 9 carvone : 3 limonene : 3 carvone and menthone: 1 menthone which would be expected if the genes Lm and C were independently inherited     

Enantioselective Dynamic Process Reduction of α- and β-Tetralone and Stereoinversion of Resulting Alcohols in a Selected Strain Culture

α-Tetralone and β-tetralone were subjected to biotransformation by 14 fungal strains. Enantiomeric purity of the products depended on the reaction time. 3-Day transformation of α-tetralone in Absidia cylindrospora culture gave S-(+)-1,2,3,4-tetrahydro-1-naftol of 92 % ee, whereas longer biotransformation time resulted in decrease of ee value. 3-Day transformation of β-tetralone by the same strain gave predominantly S-(-)-1,2,3,4-tetrahydro-2-naftol, whereas after 9 days of the reaction, the R-enantiomer with 85 % ee was isolated. Transformation of β-tetralone by Chaetomium sp. KCh 6651 gave pure (S)-(-)-1,2,3,4-tetrahydro-2-naftol in high yield at the concentration of 1 g/l. In this process, a non-selective carbonyl reduction was observed, followed by a selective oxidation of the R-alcohol.     

Highly efficient selective oxidation of alcohols to carbonyl compounds catalyzed by ruthenium (III) meso-tetraphenylporphyrin chloride in the presence of molecular oxygen

Efficient selective oxidation of alcohols to carbonyl compounds by molecular oxygen with isobutyraldehyde as oxygen acceptor in the presence of metalloporphyrins has been reported. Ruthenium (III) meso-tetraphenylporphyrin chloride (Ru(TPP)Cl) showed excellent activity and selectivity for oxidation of various alcohols under mild conditions. Moreover, different factors influencing alcohols oxidation, for example, catalyst, solvent, temperature, and oxidant, have been investigated. In large-scale oxidation of benzyl alcohol, the isolated yield of benzaldehyde of 89% was observed.     

柠檬烯和红没药烯的微生物代谢工程

柠檬烯和红没药烯均为植物天然产物,分别属于单萜类和倍半萜类化合物,能够预防和治疗癌症等多种疾病。以其作为前体物,还可以转化合成多种具有高附加值的工业产品,例如药品、保健品、化妆品及生物燃料等。目前柠檬烯和红没药烯的工业生产主要是通过植物提取法实现的,但从植物组织中提取柠檬烯和红没药烯存在着产物含量低和分离纯化困难等缺点。微生物代谢工程的快速发展为这些植物天然产物的生产提供了一条更具潜力的生物合成路线。利用微生物代谢工程技术构建生产这些有价值的植物天然产物的微生物细胞工厂具有绿色清洁、可持续发展和经济效益好等独特优势。文中系统综述了近年来代谢工程技术在微生物合成柠檬烯和红没药烯过程中的应用进展,包括所涉及的宿主菌株、关键酶、代谢途径及其改造等,并探讨了其未来发展方向。     

Cell adaptation to solvent, substrate and product: a successful strategy to overcome product inhibition in a bioconversion system

Carvone has previously been found to highly inhibit its own production at concentrations above 50 mM during conversion of a diastereomeric mixture of (−)-carveol by whole cells of Rhodococcus erythropolis. Adaptation of the cells to the presence of increasing concentrations of carveol and carvone in n-dodecane prior to biotransformation proved successful in overcoming carvone inhibition. By adapting R. erythropolis cells for 197 h, an 8.3-fold increase in carvone production rate compared to non-adapted cells was achieved in an air-driven column reactor. After an incubation period of 268 h, a final carvone concentration of 1.03 M could be attained, together with high productivity [0.19 mg carvone h⁻¹ (ml organic phase)⁻¹] and high yield (0.96 g carvone g carveol⁻¹).     

Oxidation of biological electron donors and antioxidants by a reactive lactoperoxidase metabolite from nitrite (NO2-): an EPR and spin trapping study

We report that a lactoperoxidase (LPO) metabolite derived from nitrite (NO2-) catalyses one-electron oxidation of biological electron donors and antioxidants such as NADH, NADPH, cysteine, glutathione, ascorbate, and Trolox C. The radical products of the reaction have been detected and identified using either direct EPR or EPR combined with spin trapping. While LPO/H2O2 alone generated only minute amounts of radicals from these compounds, the yield of radicals increased sharply when nitrite was also present. In aerated buffer (pH 7) the nitrite-dependent oxidation of NAD(P)H by LPO/H2O2 produced superoxide radical, O2*-, which was detected as a DMPO/*O2H adduct. We propose that in the LPO/H2O2/NO2-/biological electron donor systems the nitrite functions as a catalyst because of its preferential oxidation by LPO to a strongly oxidizing metabolite, most likely a nitrogen dioxide radical *NO2, which then reacts with the biological substrates more efficiently than does LPO/H2O2 alone. Because both nitrite and peroxidase enzymes are ubiquitous our observations point at a possible mechanism through which nitrite might exert its biological and cytotoxic action in vivo, and identify some of the physiological targets which might be affected by the peroxidase/H2O2/nitrite systems.     

Photosensitized oxidation of 2',7'-dichlorofluorescin: singlet oxygen does not contribute to the formation of fluorescent oxidation product 2',7'-dichlorofluorescein

2',7'-Dichlorofluorescin (DCFH) is often employed to assess oxidative stress in cells by monitoring the appearance of 2',7'-dichlorofluorescein (DCF), its highly fluorescent oxidation product. We have investigated the photosensitized oxidation of DCFH in solution and elucidated the role played by singlet molecular oxygen (1O(2)) in this reaction. We used rose bengal (RB), protoporphyrin, and DCF as photosensitizers. Irradiation (550 nm) of RB (20 microM) in 50 mM phosphate (pH 7.4) in the presence of DCFH (50 microM) resulted in the rapid formation of DCF, measured as an increase in its characteristic absorbance and fluorescence. The oxidation rate was faster in deoxygenated solution, did not increase in D(2)O, and even increased in the presence of sodium azide. The presence of antioxidants that react with 1O(2), thus removing oxygen, accelerated DCF formation. Such results eliminate any potential direct involvement of 1O(2) in DCF formation, even though DCFH is an efficient (physical) quencher of 1O(2) (k(q) = 1.4 x 10(8) M(-1)s(-1) in methanol). DCF is also a moderate photosensitizer of 1O(2) with a quantum yield of circa phi = 0.06 in D(2)O and phi = 0.08 in propylene carbonate, which unequivocally indicates that DCF can exist in a triplet state upon excitation with UV and visible light. This triplet can initiate photo-oxidization of DCFH via redox-and-radical mechanism(s) similar to those involving RB (vide supra). Our results show that, upon illumination, DCF can function as a moderate photosensitizer initiating DCFH oxidation, which may prime and accelerate the formation of DCF. We have also shown that, while 1O(2) does not contribute directly to DCF production, it can do so indirectly via reaction with cellular substrates yielding peroxy products and peroxyl radicals, which are able to oxidize DCFH in subsequent dark reactions. These findings suggest that DCFH should not be regarded as a probe sensitive to singlet molecular oxygen, and that care must be taken when using DCFH to measure oxidative stress in cells as a result of both visible and UV light exposure.     

Biotransformation of (R)-(+)- and (S)-(-)-limonene by fungi and the use of solid phase microextraction for screening

The biotransformation of (R)-(-)- and (S)-(-)-limonene by fungi was investigated. More than 60 fungal cultures were screened for their ability to bioconvert the substrate, using solid phase microextraction as the monitoring technique. After screening, the best fungal strains were selected for further study and were grown as sporulated surface cultures in conical flasks and as submerged liquid cultures. It was found that (+)- and (-)-limonene were converted by Penicillium digitatum to alpha-terpineol (main metabolite), cis- and trans-p-menth-2-en-1-ol, neodihydrocarveol and limonene oxide (minor metabolites) using liquid cultures. The bioconversion of (R)-(-)- and (S)-(-)-limonene by Corwespora cassiicola yielded (1S,2S,4R)- and (1R,2R,4S)-limonene-1,2-diol respectively. The bioconversions by liquid cultures were also monitored by solid phase microextraction as a function of time. The optimum conversion of limonene to alpha-terpineol by Penicillium digitatum was obtained after 8 hours (yield up to 100%). Since an important pH-decrease was noticed in some liquid broths, the stability of limonene under acidic conditions was investigated. No acid catalysed conversion products were recovered after 8 days from control flasks at pH 3.5 containing limonene.     

Inhibitory effects of substrate and product on the carvone biotransformation activity of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The aqueous substrate and product toxicity thresholds in the microbial biotransformation of (-)-trans-carveol to the fragrance/flavor compound (R)-(-)-carvone by Rhodococcus erythropolis were determined. Above aqueous phase concentrations of approx. 500 mg carveol/l and 200-600 mg carvone/l, the biotransformation activity of the biocatalyst was inhibited. This biotransformation was undertaken in a single aqueous phase 3 l [corrected] reactor in which a total of 5 ml carveol (mixture of isomers) was added before the biotransformation rate decreased significantly. The carvone volumetric productivity was 31 mg/lh. Although the growth of the organism post-exposure was not affected, dramatic morphological changes in response to the accumulation of the inhibitory substrate and product were observed.     

Biotransformation of benzene and toluene to catechols by phenol hydroxylase from Arthrobacter sp. W1

Phenol hydroxylase gene engineered microorganism (PH_IND) was used to synthesize catechols from benzene and toluene by successive hydroxylation reaction. HPLC-MS and ¹H NMR analysis proved that the products of biotransformation were the corresponding catechols via the intermediate production of phenols. It was indicated that the main products of toluene oxidation were o-cresol and p-cresol. 3-Methylcatechol was the predominant product for m-cresol biotransformation. Formation rate of catechol (25 μM/min/g cell dry weight) was 1.43-fold higher than that of methylcatechols. It was suggested that phenol hydroxylase could be successfully used to transform both benzene and toluene to catechols by successive hydroxylation.     

Design, synthesis, and biological evaluation of folic acid targeted tetraphenylporphyrin as novel photosensitizers for selective photodynamic therapy

Photodynamic therapy (PDT) is a cancer treatment involving systemic administration of a tumor-localizing photosensitizer; this, when activated by the appropriate light wavelength, interacts with molecular oxygen to form a toxic, short-lived species known as singlet oxygen, which is thought to mediate cellular death. Targeted PDT offers the opportunity of enhancing photodynamic efficiency by directly targeting diseased cells and tissues. Two new conjugates of three components, folic acid/hexane-1,6-diamine/4-carboxyphenylporphyrine 1 and folic acid/2,2'-(ethylenedioxy)-bis-ethylamine/4-carboxyphenylporphyrine 2 were synthesized. The conjugates were characterized by 1H NMR, MALDI, UV-visible spectroscopy, and fluorescence quantum yield. The targeted delivery of these photoactive compounds to KB nasopharyngeal cell line, which is one of the numerous tumor cell types that overexpress folate receptors was studied. It was found that after 24 h incubation, conjugates 1 and 2 cellular uptake was on average 7-fold higher than tetraphenylporphyrin (TPP) used as reference and that 1 and 2 cellular uptake kinetics increased steadily over the 24 h period, suggesting an active transport via receptor-mediated endocytosis. In corresponding results, conjugates 1 and 2 accumulation displayed a reduction of 70% in the presence of a competitive concentration of folic acid. Survival measurements demonstrated that KB cells were significantly more sensitive to conjugated porphyrins-mediated PDT. Under the same experimental conditions and the same photosensitizer concentration, TPP displayed no photocytotoxicity while conjugates 1 and 2 showed photodynamic activity with light dose values yielding 50% growth inhibition of 22.6 and 6.7 J/cm2, respectively.     

Factors that influence the deoxyribose oxidation assay for Fenton reaction products.

The mechanism of oxidation of deoxyribose to thiobarbituric acid-reactive products by Fenton systems consisting of H2O2 and either Fe2+ or Fe2+ (EDTA) has been studied. With Fe2+ (EDTA), dependences of product yield on reactant concentrations are consistent with a reaction involving OH.. With Fe2+ in 5-50 mM phosphate buffer, yields of oxidation products were much higher and increased with increasing deoxyribose concentration up to 30 mM. The product yield varied with H2O2 and Fe2+ concentrations in a way to suggest competition between deoxyribose and both reactants. Deoxyribose oxidation by Fe2+ and H2O2 was enhanced 1.5-fold by adding superoxide dismutase, even though superoxide generated by xanthine oxidase increased deoxyribose oxidation. These results are not as expected for a reaction involving free OH. or site localized OH. product on the deoxyribose. They can be accommodated by a mechanism of deoxyribose oxidation involving an iron(IV) species formed from H2O2 and Fe2+, but the overall conclusion is that the system is too complex for definitive identification of the Fenton oxidant.     

Chemical composition and antigenotoxic properties of Lippia alba essential oils

The present work evaluated the chemical composition and the DNA protective effect of the essential oils (EOs) from Lippia alba against bleomycin-induced genotoxicity. EO constituents were determined by Gas Chromatography/Mass Spectrometric (GC-MS) analysis. The major compounds encountered being citral (33% geranial and 25% neral), geraniol (7%) and trans-β-caryophyllene (7%) for L. alba specimen COL512077, and carvone (38%), limonene (33%) and bicyclosesquiphellandrene (8%) for the other, COL512078. The genotoxicity and antigenotoxicity of EO and the compounds citral, carvone and limonene, were assayed using the SOS Chromotest in Escherichia coli. The EOs were not genotoxic in the SOS chromotest, but one of the major compound (limonene) showed genotoxicity at doses between 97 and 1549 mM. Both EOs protected bacterial cells against bleomycin-induced genotoxicity. Antigenotoxicity in the two L. alba chemotypes was related to the major compounds, citral and carvone, respectively. The results were discussed in relation to the chemopreventive potential of L. alba EOs and its major compounds.     

Preparation of enantiopure (R)-hydroxy metabolite of denbufylline using immobilized Lactobacillus kefiri DSM 20587 as a catalyst

Lactobacillus kefiri DSM 20587 cells were immobilized in calcium alginate and carrageenan. The immobilized cells were used as biocatalysts for the enantioselective reduction of the methyl ketone group of denbufylline to synthesize the enantiopure (R)-hydroxy metabolite: (-)-1,3-dibutyl-7-((2'R)-hydroxypropyl)-1H-purine-2,6(3H,7H)-dione (1). The experimental conditions for the biotransformation were optimized. As denbufylline is insoluble in aqueous media, the influence of cosolvents (dimethylsulfoxide (DMSO), acetonitrile) and different concentrations of each solvent in the reaction mixture on the yield and enantiomeric excess of the final biotransformation product was studied. The maximum biotransformation yield (96-98%) and highest enantioselectivity (96% ee) for the obtained metabolite were reached using DMSO as a cosolvent at a concentration of 7.5% (v/v) in the presence of L. kefiri immobilized either in calcium alginate or in carrageenan. The absolute configuration of the stereogenic center of 1 was determined by applying Mosher's method.