Mariusz Trytek

Terpenes: substances useful in human healthcare

Terpenes are naturally occurring substances produced by a wide variety of plants and animals. A broad range of the biological properties of terpenoids is described, including cancer chemopreventive effects, antimicrobial, antifungal, antiviral, antihyperglycemic, anti-inflammatory, and antiparasitic activities. Terpenes are also presented as skin penetration enhancers and agents involved in the prevention and therapy of several inflammatory diseases. Moreover, a potential mechanism of their action against pathogens and their influence on skin permeability are discussed. The major conclusion is that larger-scale use of terpenoids in modern medicine should be taken into consideration.

A novel psychrotrophic fungus, Mortierella minutissima, for D-limonene biotransformation

Of 98 strains of moulds, isolated from arctic soils, Mortierella minutissima 01, grew the best on agar plates with limonene vapor. Perillyl alcohol and perillic acid were the main products of limonene biotransformation. Maximal yield of perillyl alcohol (125 mg l−1) occurred in medium containing 0.8% substrate, at 15 °C, pH 6 and after 4–5 d.

A Novel Porphyrin-Based Photocatalytic System for Terpenoids Production from (R)-(+)-Limonene

A porphyrin‐based photoexcited system has been revealed to be an efficient catalyst for d‐limonene biotransformation under mild conditions and using molecular oxygen or/and H2O2 as oxidants. The influence of the oxidant, the wavelength of visible light, and the photoexcitation time on the catalytic system were studied for limonene oxidation with 5,10,15,20‐tetraphenylporphyrin (H2TPP) as a catalyst. This porphyrin‐catalyzed oxidation of limonene to three main products identified as carvone, an unknown product with a verbenone‐like mass spectrum (1), and a (1 S,4 R)‐ p‐mentha‐2,8‐diene 1‐hydroperoxide (2). The highest conversion yield of these products was achieved at a very high molar ratio of H2TPP to limonene. The dependence of the biotransformation yield on the kind of solvent with different acceptor/donor electron properties was also investigated. Ethyl alcohol proved to be the best among the considered additives used for the reaction. Limonene photooxidation was not significantly dependent on wavelengths of visible light. It was concluded by UV‐vis experiments that the reaction proceeds via a free‐radical or/and molecular mechanism. Additional evidence for its radical nature was obtained from reactivity investigations. Maximal yield of carvone was obtained in the medium containing 90% of the substrate, within the period of 18 to 36 h of exposition to sunlight.

Biological activity of terpene compounds produced by biotechnological methods

Abstract Context Biotransformation systems are profitable tools for structural modification of bioactive natural compounds into valuable biologically active terpenoids. Objective This study determines the biological effect of (R)-(+)-limonene and (−)-α-pinene, and their oxygenated derivatives, (a) perillyl alcohol and (S)-(+)- and (R)-(−)-carvone enantiomers and (b) linalool, trans-verbenol and verbenone, respectively, on human colon tumour cells and normal colonic epithelium. Materials and methods Biotransformation procedures and in vitro cell culture tests were used in this work. Cells were incubated for 24 h with terpenes at concentrations of 5–500 μg/mL for NR, MTT, DPPH, and NO assays. IL-6 was determined by ELISA with/without 2 h pre-activation with 10 μg/mL LPS. Results trans-Verbenol and perillyl alcohol, obtained via biotransformation, produced in vitro effect against tumour cells at lower concentrations (IC50 value = 77.8 and 98.8 μg/mL, respectively) than their monoterpene precursors, (R)-(+)-limonene (IC50 value = 171.4 μg/mL) and (−)-α-pinene (IC50 value = 206.3 μg/mL). They also showed lower cytotoxicity against normal cells (IC50 > 500 and > 200 μg/mL, respectively). (S)-(+)-Carvone was 59.4% and 27.1% more toxic to tumour and normal cells, respectively, than the (R)-(−)-enantiomer. (R)-(+)-limonene derivatives decreased IL-6 production from normal cells in media with or without LPS (30.2% and 13.9%, respectively), while (−)-α-pinene derivatives induced IL-6 (verbenone had the strongest effect, 60.2% and 29.1% above control, respectively). None of the terpenes had antioxidative activity below 500 μg/mL. Discussion and conclusions Bioactivity against tumour cells decreased in the following order: alcohols > ketones > hydrocarbons. (R)-(+)-limonene, (−)-α-pinene, and their derivatives expressed diverse activity towards normal and tumour cells with noticeable enantiomeric differences.

INULINASE PRODUCTION BY ASPERGILLUS NIGER MYCELIUM IMMOBILIZED ON POLYURETHANE FOAM IN A BIOREACTOR WITH ALTERNATIVE OXYGENATION

A system for the production of extracellular inulinase with the use of Aspergillus niger 20 Osm mycelium immobilized on several polyurethane foam disks in a 2-liter bioreactor was developed. Because the application of the disks reduced the efficiency of the conventional aeration system initially employed in the study and caused difficulties in maintaining sufficient concentrations of dissolved oxygen, an alternative culture oxygenation method based on O2 generation by decomposition of sequential doses of hydrogen peroxide introduced into a bioreactor medium was used. The application of the alternative oxygenation system during repeated batch culture made it possible to maintain adequate concentrations of dissolved oxygen (50 – 99%) and a proper level of inulinase biosynthesis for a long time (up to 22 days). In the optimized conditions, the immobilized mycelium produced 86 321 units of inulinase activity during a 7-cycle process.

Efficiency of indoleacetic acid, gibberellic acid and ethylene synthesized in vitro by Fusarium culmorum strains with different effects on cereal growth

Three different Fusarium culmorum strains having a pathogenic, a deleterious (deleterious rhizosphere microorganism), or a promoting (plant growth promoting fungus) effect on plant growth were studied for their ability to synthesize in vitro the phytohormones indoleacetic acid (IAA), gibberellic acid (GA), and ethylene. All the phytohormones tested were synthesized in cultures supplemented with wide concentration ranges of glucose and tryptophan or methionine (precursors of phytohormone synthesis). The amounts of these secondary metabolites synthesized by the particular strains were found to be significantly different. The non-pathogenic PGPF strain (DEMFc2) synthesized the highest amounts of IAA and GA, a fact that could be responsible for the growth-promoting properties of this strain. A pathogenic strain synthesized the highest amount of ethylene, which could be responsible for the negative effect of this strain on plant growth. F. culmorum isolates with a high capacity for IAA synthesis also have a high capacity for GA synthesis and irrespective of the growth conditions, a high positive correlation (R > 0.9) between the concentrations of synthesized IAA and GA in F. culmorum cultures was found. It is worth mentioning that the optimal conditions for the growth of F. culmorum isolates and the synthesis of the individual phytohormones differed from one another. The optimal growth conditions were 1.0% of glucose and 9.9 mM of methionine or 6.0 mM of tryptophan. The optimal conditions for ethylene synthesis were 0.5% of glucose and 6.6 mM of methionine, whereas 1.0% of glucose and 9.0 mM of tryptophan were optimal for IAA and GA synthesis.

Homogeneous and Heterogeneous Free-Based Porphyrins Incorporated to Silica Gel as Fluorescent Materials and Visible Light Catalysts Mimic Monooxygenases

The use of enzymes and whole cells as biocatalysts in biotechnology and organic chemistry is recognized as being of great economic potential in the increasing production of unique and valuable compounds present in nature in an insufficient amount. Bioconversion with living microbial cells, which have the ability to regenerate their own respective cofactors and exhibit a spectrum of enzymatic activity, is a very useful tool in bioorganic synthesis. There is no doubt that the advantage of microbial biotransformations is the possibility to induce enzymes of defined, desired activity, simply by the suitable preincubation methods (Griffin et al., 2001). However, this kind of biocatalysts has several limitations in application to oxidative biotransformations, including stability and activities, especially to those substrates and products that are toxic and require being performed in reaction media other than water. A disadvantage might be the membrane barrier and diffusion problems. Recent advances in addressing these problems include molecular and reaction engineering approaches (Burton, 2003). A broad field of activity of biological heme-containing oxygenases like cytochromes P450 has provoked the development of the invention of artificial catalytic systems based on porphyrins mimicking the controlled and selective oxidation reactions of these hemoproteins. The concept of artificial enzymes and biomimetic chemistry was introduced by Breslow in 1972 (Breslow, 1972), that started the field of bioinspired or biomimetic catalysis, wherein researchers try to copy Nature in designing the catalyst with the structure and properties similar to those of the active sites of enzymes. Such biomimetic systems are expected to work in organic media and have high stability toward toxic organic compounds in comparison with traditional catalysis employing enzymes or microorganisms. The porphyrins, due to their synthetic versatility and reactivity, are especially attractive in the construction of biomimetic analogues of monooxygenases through elaboration of the

Porphyrins inactivate Nosema spp. microsporidia

The study of organic/inorganic molecules with activity against intracellular fungi of the phylum Microsporidia is of critical importance. Here, for the first time, the inactivation of these parasitic fungi by porphyrins is reported. The biological effects of porphyrins (10 µM and 100 µM) on the microsporidian Nosema ceranae was investigated in honeybee hosts using cage experiments. A significant reduction in the number of spores (from 2.6 to 5 fold) was observed in Nosema-infected honeybees with a sucrose-protoporphyrin amide [PP(Asp)2] syrup diet compared to the control honeybees. PP(Asp)2 and the other porphyrin examined in vitro, TMePyP, had a direct impact on the microsporidia. Notably, neither porphyrin requires light excitation to be active against microsporidia. Moreover, microsporidia preincubated with these porphyrins exhibited decreased ability to infect honeybees. In particular, PP(Asp)2, possessing amphiphilic characteristics, exhibited significant inactivation of microsporidia, preventing the development of the microsporidia and diminishing the mortality of infected honeybees. In addition, the porphyrin-treated spores examined by scanning electron microscopy (SEM) showed morphological changes in their exosporium layers, which were distinctly deformed. Thus, we postulate that the mechanism of action of porphyrins on microsporidia is not based on photodynamic inactivation but on the destruction of the cell walls of the spores.

Strain improvement of industrially important microorganisms based on resistance to toxic metabolites and abiotic stress

Improvement of the biosynthetic capabilities of industrially relevant microbes to produce desired metabolites in higher quantities is one of the important topics of modern biotechnology. In this article, different strategies of improvement of mutated microbial strains are briefly described. This is followed by the first comprehensive review of the literature on obtaining high yielding microorganisms, that is, mutants exhibiting resistance to antimetabolites, nutritional repression, and abiotic stresses as well as tolerance to solvents and toxic substrates or products. Furthermore, the efficiency of the microbial metabolites produced by improved microbial strains, advantages, and limitations, as well as future prospects for strategies of strain development are discussed.

Biology and defensive secretion of myrmecophilous Thiasophila spp. (Coleoptera: Staphylinidae: Aleocharinae) associated with the Formica rufa species group

ABSTRACT Various aspects of the biology of the myrmecophilous Thiasophila angulata and T. szujeckii (Staphylinidae; Aleocharinae), mainly associated with the Formica rufa species group, are studied. The composition of defensive gland secretions from adults of T. angulata has been analysed. The complete development (egg-L1-3-pupa-adult form), including adult overwintering, of both species takes place solely in ant nests. Under laboratory conditions, this lasts for about 22 days in T. angulata, and the period elapsing between the old and new generations of insects is approximately two months. The mean fecundity of adults of this species (28 eggs) was determined, as well as the duration of its reproductive period (mean 28.5 days), and lifespan (mean 67 days). In natural conditions T. angulata, found within nests of Formica polyctena and F. rufa, produces three generations, and the sibling species T. szujeckii one generation per year. These two species differ in their phenologies and abundance dynamics of adults and larvae, which is linked to the size and temperature regime of the host nest. The results of this study uphold the recent separation of T. angulata and T. szujeckii based on morphological features of adults and selected molecular markers. Adults and larvae of T. angulata forage on both live and dead food items accumulated by the host, as well as on the host’s eggs and larvae. Both the beetle larvae and adults remain unmolested among the host workers. The adults use their defensive gland secretions, which contains substantial quantities of toxic quinones, when necessary. According to the current categories of myrmecophiles, T. angulata (and by analogy, T. szujeckii as well) should be classified as a species wholly integrated with the host.