Katarzyna Hupert-Kocurek

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].

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.

Genetic method to analyze essential genes of Escherichia coli

ABSTRACT The genetic analysis of essential genes has been generally restricted to the use of conditional mutations, or inactivating chromosomal mutations, which require a complementing plasmid that must either be counterselected or lost to measure a phenotype. These approaches are limited because they do not permit the analysis of mutations suspected to affect a specific function of a protein, nor do they take advantage of the increasing abundance of structural and bioinformatics data for proteins. Using the dnaC gene as an example, we developed a genetic method that should permit the mutational analysis of other essential genes of Escherichia coli and related enterobacteria. The method consists of using a strain carrying a large deletion of the dnaC gene, which is complemented by a wild-type copy expressed from a plasmid that requires isopropyl-β-d-thiogalactopyranoside for maintenance. Under conditions in which this resident plasmid is lost, the method measures the function of a dnaC mutation encoded by a second plasmid. This methodology should be widely applicable to the genetic analysis of other essential genes.

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.

Substitutions of Conserved Residues in the C-terminal Region of DnaC Cause Thermolability in Helicase Loading

The DnaB-DnaC complex binds to the unwound DNA within the Escherichia coli replication origin in the helicase loading process, but the biochemical events that lead to its stable binding are uncertain. This study characterizes the function of specific C-terminal residues of DnaC. Genetic and biochemical characterization of proteins bearing F231S and W233L substitutions of DnaC reveals that their activity is thermolabile. Because the mutants remain able to form a complex with DnaB at 30 and 37 °C, their thermolability is not explained by an impaired interaction with DnaB. Photo-cross-linking experiments and biosensor analysis show an altered affinity of these mutants compared with wild type DnaC for single-stranded DNA, suggesting that the substitutions affect DNA binding. Despite this difference, their activity in DNA binding is not thermolabile. The substitutions also drastically reduce the affinity of DnaC for ATP as measured by the binding of a fluorescent ATP analogue (MANT-ATP) and by UV cross-linking of radiolabeled ATP. Experiments show that an elevated temperature substantially inhibits both mutants in their ability to load the DnaB-DnaC complex at a DnaA box. Because a decreased ATP concentration exacerbates their thermolabile behavior, we suggest that the F231S and W233L substitutions are thermolabile in ATP binding, which correlates with defective helicase loading at an elevated temperature.

Protocatechuate 3,4-Dioxygenase: A Wide Substrate Specificity Enzyme Isolated from Stenotrophomonas maltophilia KB2 as a Useful Tool in Aromatic Acid Biodegradation

Protocatechuate 3,4-dioxygenases (P34Os) catalyze the reaction of the ring cleavage of aromatic acid derivatives. It is a key reaction in many xenobiotic metabolic pathways. P34Os characterize narrow substrate specificity. This property is an unfavorable feature in the biodegradation process because one type of pollution is rarely present in the environment. Thus, the following study aimed at the characterization of a P34O from Stenotrophomonas maltophilia KB2, being able to utilize a wide spectrum of aromatic carboxylic acids. A total of 3 mM vanillic acid and 4-hydroxybenzoate were completely degraded during 8 and 4.5 h, respectively. When cells of strain KB2 were grown on 9 mM 4-hydroxybenzoate, P34O was induced. Biochemical analysis revealed that the examined enzyme was similar to other known P34Os, but showed untypical wide substrate specificity. A high activity of P34O against 2,4- and 3,5-dihydroxybenzoate was observed. As these substrates do not possess ortho configuration hydroxyl groups, it is postulated that their cleavage could be connected with their monodentate binding of substrate to the active site. Since this enzyme characterizes untypical wide substrate specificity it makes it a useful tool in applications for environmental clean-up purposes.

Cloning and Mutagenesis of Catechol 2,3-Dioxygenase Gene from the Gram-Positive Planococcus sp. Strain S5

In this study, the catechol 2,3-dioxygenase gene that encodes a 307- amino-acid protein was cloned from Planococcus sp. S5. The protein was identified to be a member of the superfamily I, subfamily 2A of extradiol dioxygenases. In order to study residues and regions affecting the enzymes catalytic parameters, the c23o gene was randomly mutated by error-prone PCR. The wild-type enzyme and mutants containing substitutions within either the C-terminal or both domains were functionally produced in Escherichia coli and their activity towards catechol was characterized. The C23OB65 mutant with R296Q substitution showed significant tolerance to acidic pH with an optimum at pH 5.0. In addition, it showed activity more than 1.5 as high as that of the wild type enzyme and its Km was 2.5 times lower. It also showed altered sensitivity to substrate inhibition. The results indicate that residue at position 296 plays a role in determining pH dependence of the enzyme and its activity. Lower activity toward catechol was shown for mutants C23OB58 and C23OB81. Despite lower activity, these mutants showed higher affinity to catechol and were more sensitive to substrate concentration than nonmutated enzyme.