N. A. Zagustina

Organization of metabolic pathways and molecular-genetic mechanisms of xenobiotic degradation in microorganisms: A review

Contemporary data on the mechanism of biodegradation of aromatic hydrocarbons and biodegradation genes (genomic organization and pathways of evolution) in diverse groups of microorganisms have been reviewed. Studies of this problem are topical, in view of the need in identification and construction of new strains degrading xenobiotics, particularly those halogenated. For this reason, emphasis is placed on specific features of explored metabolic pathways that can be used for constructing new enzymatic systems not present in nature. Sections on the mechanisms of genomic rearrangements involving biodegradation determinants are presented from the same standpoint. Part of the review is devoted to analyzing methods used for studying the population dynamics of bacterial communities involved in xenobiotic degradation in natural biotopes or industrial waste disposal plants. Particular attention is given to methods of gene systematics.

Monitoring of microbial degraders in manned space stations

Samples of microorganisms from the surface of constructions of Mir Space Station (Mir SS) were taken and examined after 13 years of operation. The following microorganisms were isolated and identified: 12 fungal species belonging to the genera Penicillium, Aspergillus, Cladosporium, and Aureobasidium; 3 yeast species belonging to the genera Debaryomyces, Candida, and Rhodotorula; and 4 bacterial species belonging to the genera Bacillus, Myxococcus, and Rhodococcus. The predominant species in all samples was Penicillium chrisogenum. It was shown that the fungi isolated could damage polymers and induce corrosion of aluminum-magnesium alloys. We commenced a study of microbial degraders on constructions of the Russian section of the International Space Station (RS ISS). Twenty-six species of fungi, bacteria, yeasts, and actinomycetes, known as active biodegraders, were identified in three sample sets taken at intervals. We founded a collection of microorganisms surviving throughout space flights. This collection can be used to test spacecraft production materials, in order to determine their resistance to biodegradation.

Lipases in catalytic reactions of organic chemistry

Aspects of enzymatic catalysis in lipase-catalyzed reactions of organic synthesis are discussed in the review. The data on modern methods of protein engineering and enzyme modification allowing a broader range of used substrates are briefly summarized. The application of lipase in the preparation of pharmaceuticals and agrochemicals containing no inactive enantiomers and in the synthesis of secondary alcohol enantiomers and optically active amides is demonstrated. The subject of lipase involvement in the C-C bond formation in the Michael reaction is discussed. Data on the enzymatic synthesis of construction materials—polyesters, siloxanes, etc.—are presented. Examples demonstrating the application of lipase enzymatic catalysis in industry are given.

Enzymatic biocatalysis in chemical synthesis of pharmaceuticals (Review)

In this review we summarize the available research on enzymatic biocatalysis in the chemical synthesis of drugs. We focus on oxydoreductsases, particularly ketoreductases, that are widely used in biotechnological processes: alpha- and omega-transaminases, lipases, nitrile hydrolases, and aldolases. The potential for the extended use of novel enzymes produced via bioengineering is discussed.

Application of molecular systematics to study of bacterial cultures consuming volatile organic compounds

A range of species of four mixed bacterial cultures was studied by molecular systematics methods with the use of 16S rRNA genes. The cultures had been developed for application in minireactors, to degrade volatile organic compounds (VOCs): ethyl benzene, m-xylene, styrene, and o-xylene. A sample of 30 plasmid rDNA clones was obtained for each of the mixed cultures. The clones were analyzed by RFLP according to two restriction sites. Major variants of the 16S-rDNA sequences, corresponding to the most abundant species, were determined for each association. Sequencing of four clones of predominant 16S-rDNAs showed that the culture consuming ethyl benzene was dominated by Pseudomonas fluorescens; o-xylene, by Achromobacter xylosoxydans; styrene, by Pseudomonas veronii; and m-xylene, by Delftia acidovorans. Minor components of all four cultures were generally similar. They included species of the genera Sphingobacter, Rhizobium, Mesorhizobium, Pedobacter, and Paenibacillus. Sampling sequencing of genes for 16S rRNA cloned from total genomic DNA allowed quantitative determination of the composition of actual bacterial associations consuming VOCs in minireactors.

Isolation and purification of acetolactate synthase and acetolactate decarboxylase from the culture ofLactococcus lactis

Enzymes catalyzing the synthesis and subsequent transformation of α-acetolactate (AcL)—acetolactate synthase (AcLS) and acetolactate decarboxylase (AcLDC)—were isolated and partially purified from the cells of lactic acid bacteriaLactococcus lactis ssp.lactis biovar.diacetylactis, strain 4. The preparation of AcLS, purified 560-fold, had a specific activity of 358 300 U/mg protein (9% yield). The preparation of AcLDC., purified 4828-fold, had a specific activity of 140 U/mg protein (4.8% yield). The enzymes exhibited optimum activity at pH 6.5 and 6.0, respectively (medium, phosphate buffer). The values of apparentKm, determined for AcLS and AcLDC with pyruvate and AcL, respectively, were equal to 70 mM and 20 mM. AcLS appeared as an allosteric enzyme with low affinity for the substrate and a sigmoid dependence of the activity on the substrate concentration. In the case of AcLDC, this dependence was hyperbolic and the affinity of the enzyme for its substrate was high (Km = 20 mM). Leucine, valine, and isoleucine were shown to be activators of AcDLC.

Metabolic pathways responsible for consumption of aromatic hydrocarbons by microbial associations: Molecular-genetic characterization

Genes for catechol 1,2- and 2,3-dioxygenases were cloned. These enzymes hold important positions in the ortho and meta pathways of the metabolism of aromatic carbons by microbial associations that consume the following volatile organic compounds in pilot minireactors: toluene, styrene, ethyl benzene, o-xylene, m-xylene, and naphthalene. Genes of both pathways were found in an association consuming m-xylene; only genes of the ortho pathway were found in associations consuming o-xylene, styrene, and ethyl benzene, and only genes of the meta pathway were found in associations consuming naphthalene and toluene. Genes of the ortho pathway (C12O) cloned from associations consuming o-xylene and ethyl benzene were similar to corresponding genes located on the pND6 plasmid of Pseudomonas putida. Genes of the ortho pathway from associations consuming o-xylene and m-xylene were similar to chromosomal genes of P. putida. Genes of the meta pathway (C23O) from associations consuming toluene and naphthalene were similar to corresponding genes formerly found in plasmids pWWO and pTOL.

Xylitol Production by a Culture ofCandida guilliermondii2581

The yeast strain Candida guilliermondii2581 was chosen for its ability to produce xylitol in media with high concentrations of xylose. The rate of xylitol production at a xylose concentration of 150 g/l is 1.25 g/l per h; the concentration of xylitol after three days of cultivation is 90 g/l; and the relative xylitol yield is 0.6 g per g substrate consumed. The growth conditions were found that resulted in the maximum relative xylitol yield with complete consumption of the sugar: xylose concentration, 150 g/l; pH 6.0; and shaking at 60 rpm. It was shown that the growth under conditions of limited aeration favors the reduction of xylose.

Elimination of volatile compounds of leaf tobacco from air emissions using biofiltration

The composition of the volatile organic compounds (VOCs) of various leaf tobacco brands and their blends has been studied. The differences in the content of nicotine, solanone, tetramethyl hexadecenol, megastigmatrienones, and other compounds, determining the specific tobacco smell, have been revealed. A microbial consortium, which is able to deodorize simulated tobacco emissions and decompose nicotine, has been formed by long-term adaptation to the VOCs of tobacco leaves in a laboratory reactor, functioning as a trickle-bed biofilter. Such a biofilter eliminates 90% of the basic toxic compound (nicotine) and odor-active compounds; the filtration efficiency does not change for tobacco brands with different VOC concentrations or in the presence of foreign substances. The main strains, isolated from the formed consortium and participating in the nicotine decomposition process, belong to the genera Pseudomonas, Bacillus, and Rhodococcus. An examination of the biofilter trickling fluid has shown full decomposition of nicotine and odor-active VOCs. The compounds, revealed in the trickling fluid, did not have any odor and were nontoxic. The obtained results make it possible to conduct scaling of the biofiltration process to eliminate odor from air emissions in the tobacco industry.

Monitoring of initial stages of the biodamage of construction materials used in aerospace equipment using electron microscopy

The biocorrosion activity of microorganisms extracted from the Mir Space Station and the Russian segment of the International Space Station was estimated with respect to the aluminum-magnesium alloy AMg-6 used in space equipment. Thirty-six cultures belonging to 28 species were analyzed. As test objects, 180 AMg-6 samples with mechanically treated, anodized, and chemically polished surfaces were used. Almost all the studied microorganism species were technophiles capable of causing corrosion changes in the surface even protected by anodizing or chemical polishing. Mechanically treated samples were biocorroded to a greater extent. The most aggressive technophilic microorganisms caused stronger surface changes in comparison with chemical corrosion: surface oxidation, pitting, and cracking were observed.