Urszula Guzik

Immobilization as a Strategy for Improving Enzyme Properties-Application to Oxidoreductases

The main objective of the immobilization of enzymes is to enhance the economics of biocatalytic processes. Immobilization allows one to re-use the enzyme for an extended period of time and enables easier separation of the catalyst from the product. Additionally, immobilization improves many properties of enzymes such as performance in organic solvents, pH tolerance, heat stability or the functional stability. Increasing the structural rigidity of the protein and stabilization of multimeric enzymes which prevents dissociation-related inactivation. In the last decade, several papers about immobilization methods have been published. In our work, we present a relation between the influence of immobilization on the improvement of the properties of selected oxidoreductases and their commercial value. We also present our view on the role that different immobilization methods play in the reduction of enzyme inhibition during biotechnological processes.

Bacterial degradation of naproxen--undisclosed pollutant in the environment.

The presence of non-steroidal anti-inflammatory drugs (NSAIDs) in the environment is an emerging problem due to their potential influence on human health and biocenosis. This is the first report on the biotransformation of naproxen, a polycyclic NSAID, by a bacterial strain. Stenotrophomonas maltophilia KB2 transformed naproxen within 35 days with about 28% degradation efficiency. Under cometabolic conditions with glucose or phenol as a carbon source degradation efficiency was 78% and 40%, respectively. Moreover, in the presence of naproxen phenol monooxygenase, naphthalene dioxygenase, hydroxyquinol 1,2-dioxygenase and gentisate 1,2-dioxygenase were induced. This suggests that degradation of naproxen occurs by its hydroxylation to 5,7,8-trihydroxynaproxen, an intermediate that can be cleaved by hydroxyquinol 1,2-dioxygenase. The cleavage product is probably further oxidatively cleaved by gentisate 1,2-dioxygenase. The obtained results provide the basis for the use of cometabolic systems in the bioremediation of polycyclic NSAID-contaminated environments.

Intradiol Dioxygenases — The Key Enzymes in Xenobiotics Degradation

Aromatic compounds are derived from both natural and anthropogenic sources. Under natural conditions, arenes are formed as a result of the pyrolysis of organic materials at high temperatures during forest, steppe and peatland fires, and during volcanic eruptions. Biogenic aromatic compounds like aromatic amino acids and lignin, the second most abundant organic compound in the environment, are universally distributed in nature. Many species of plants, especially willow (Salix), thyme (Thymus vulgaris), camomile (Chamomilla recutita), bean (Phaesoli vulgaris) or strawberry (Fregaria ananasa), water plants as sweet flag (Acorus calamus) and many species of alga are known to produce aromatic compounds as secondary metabolites [1-4]. A lot of aromatic compounds are introduced to the environment as contaminating compounds from chemical, pharmaceutical, explosive, dyes, and agrochemicals industry. Chloro-, aminoand nitroaromatic derivatives, biphenyls, polycyclic aromatic hydrocarbons accumulate in the soil and water. They are toxic to living systems including humans, animals, and plants. Moreover, most of them may bioaccumulate in the food chain and have mutagenic or carcinogenic activity [5-8].

Biodegradation of alkyl derivatives of aromatic hydrocarbons and cell surface properties of a strain of Pseudomonas stutzeri

Pseudomonas stutzeri strain 9 was isolated from petroleum-contaminated soil. The main purpose of this study was to investigate how the long-term contact of this strain with diesel oil influences its surface and biodegradation properties. The experiments showed that the tested strain was able to degrade aromatic alkyl derivatives (butylbenzene, sec-butylbenzene, tert-butylbenzene and isobutylbenzene) and that the storage conditions had an influence on the cell surface properties. Also greater agglomeration of the cells was observed in the scanning electron microscope (SEM) micrographs and confirmed in particle size distribution results. The results also indicated that the addition of rhamnolipids to the hydrocarbons led to modification of the surface properties of P. stutzeri strain 9, which could be observed in the zeta potential and hydrophobicity values.

Biodegradation and biotransformation of polycyclic non-steroidal anti-inflammatory drugs

In recent years the increased use of polycyclic non-steroidal anti-inflammatory drugs has resulted in their presence in the environment. This in turn may cause potential negative effects on living organisms. While the biotransformation mechanisms of polycyclic non-steroidal anti-inflammatory drugs in the human body and in other mammals have been extensively studied, degradation of these drugs by microorganisms has seldom been investigated and is largely unknown. Biotransformation/biodegradation of polycyclic non-steroidal anti-inflammatory drugs is caused by fungal microorganisms, mainly white-rot fungi, and a few strains of bacteria. However, hitherto only complete degradation of olsazine was described. The first step of the transformation is most often hydroxylation catalyzed by cytochrom P-450 monooxygenases, or oxygenation by laccases and three peroxidases: lignin peroxidase, manganese-dependent peroxidase and versatile peroxidase manganese-dependent peroxidase. The aim of this work is to summarize the knowledge about the biotransformation and/or biodegradation of polycyclic non-steroidal anti-inflammatory drugs and to present their biotransformation pathways.

Cell surface properties and fatty acids composition of Stenotrophomonas maltophilia under the influence of hydrophobic compounds and surfactants.

Surface properties of newly isolated Stenotrophomonas maltophilia strain 6 were tested. The bacteria were stored in two different ways to determine the influence of hydrocarbons and surfactants on surface and enzymatic characteristics of the isolated strain. The influence of surface active agents, natural and synthetic, on membranes lipid composition and cell surface hydrophobicity (CSH) was investigated. Our results indicate that long-term contact with diesel oil as a hydrophobic sole carbon source leads to the increased enzymatic activity of S. maltophilia strain 6 as well as to modification of fatty acids profiles and its facility to adhere to hydrophobic compounds. Among surfactants there were saponins and Triton X-100 which changed the composition of fatty acids the most, increasing the amount of branched acids. The comparison of fatty acid profiles with CSH of systems with diesel oil, rhamnolipids, saponins and Triton X-100 points out that the growing amount of hydroxy fatty acids corresponds to lower hydrophobicity. Moreover, CSH is a dynamic parameter which can change during cultivation of microorganisms.

Degradation Potential of Protocatechuate 3,4-Dioxygenase from Crude Extract of Stenotrophomonas maltophilia Strain KB2 Immobilized in Calcium Alginate Hydrogels and on Glyoxyl Agarose

Microbial intradiol dioxygenases have been shown to have a great potential for bioremediation; however, their structure is sensitive to various environmental and chemical agents. Immobilization techniques allow for the improvement of enzyme properties. This is the first report on use of glyoxyl agarose and calcium alginate as matrixes for the immobilization of protocatechuate 3,4-dioxygenase. Multipoint attachment of the enzyme to the carrier caused maintenance of its initial activity during the 21 days. Immobilization of dioxygenase in calcium alginate or on glyoxyl agarose resulted in decrease in the optimum temperature by 5°C and 10°C, respectively. Entrapment of the enzyme in alginate gel shifted its optimum pH towards high-alkaline pH while immobilization of the enzyme on glyoxyl agarose did not influence pH profile of the enzyme. Protocatechuate 3,4-dioygenase immobilized in calcium alginate showed increased activity towards 2,5-dihydroxybenzoate, caffeic acid, 2,3-dihydroxybenzoate, and 3,5-dihydroxybenzoate. Slightly lower activity of the enzyme was observed after its immobilization on glyoxyl agarose. Entrapment of the enzyme in alginate gel protected it against chelators and aliphatic alcohols while its immobilization on glyoxyl agarose enhanced enzyme resistance to inactivation by metal ions.

Rahnella sp. strain EK12: Cell surface properties and diesel oil biodegradation after long-term contact with natural surfactants and diesel oil

The changes in cell surface properties of Rahnella sp. strain EK12 and modifications in genetic material after long-term contact with saponins and rhamnolipids, were investigated. Rhamnolipids caused a decrease of hydrophobicity in liquid cultures compared with saponins. On the other hand, in cultures with rhamnolipids, the addition of diesel oil results in a rapid rise of cell surface hydrophobicity. The similar effect was not so significant in the presence of saponins. For the bacteria grown in the presence of saponins or rhamnolipids, but without diesel oil, the ratio of unsaturated to saturated fatty acids decreased, in comparison to the control culture. The differences observed in hydrophobicity, zeta potential and fatty acids profiles, indicated various mechanisms of an interaction between a surfactant and a bacterial cells. The results have also shown an impact of the long-term contact on changes in genetic material of Rahnella sp. strain EK12 cells. Moreover, the presence of saponins led to significant increase of diesel oil biodegradation.

Factors affecting activity of catechol 2,3-dioxygenase from 2-chlorophenol-degrading Stenotrophomonas maltophilia strain KB2

Abstract The effect of phenol on 2-chloro- and 2,4-dichlorophenol degradation by Stenotrophomonas maltophilia KB2 has been studied. During this study, we observed induction of catechol 2,3-dioxygenase (C23O). Since, in the environment, compounds which inhibit C23O activity are frequently present together with the main dioxygenase substrates, the main aim of this work was to determine the influence of various inhibitors and activators on the enzyme activity. Hydrogen peroxide of 60 μM concentration caused total inhibition of the enzyme. Addition of ascorbic acid suppressed the inhibitory effect of hydrogen peroxide. In its presence, 60 μM hydrogen peroxide caused only 40% inhibition of C23O activity. A positive effect in preventing C23O activity was observed also in the presence of chelators (8-hydroxyquinoline, EDTA, and phenanthroline). Most metal ions and aliphatic and aromatic hydroxylated derivatives caused a 20–40% decrease in enzyme activity. The results obtained indicate that C23O from Stenotrophomonas maltophilia strain KB2 holds great potential for bioremediation.

Flavin-Dependent Enzymes in Cancer Prevention

Statistical studies have demonstrated that various agents may reduce the risk of cancer’s development. One of them is activity of flavin-dependent enzymes such as flavin-containing monooxygenase (FMO)GS-OX1, FAD-dependent 5,10-methylenetetrahydrofolate reductase and flavin-dependent monoamine oxidase. In the last decade, many papers concerning their structure, reaction mechanism and role in the cancer prevention were published. In our work, we provide a more in-depth analysis of flavin-dependent enzymes and their contribution to the cancer prevention. We present the actual knowledge about the glucosinolate synthesized by flavin-containing monooxygenase (FMO)GS-OX1 and its role in cancer prevention, discuss the influence of mutations in FAD-dependent 5,10-methylenetetrahydrofolate reductase on the cancer risk, and describe FAD as an important cofactor for the demethylation of histons. We also present our views on the role of riboflavin supplements in the prevention against cancer.