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Applied Biochemistry and Microbiology (v.49, #1)

Fungi of the genus Penicillium as producers of physiologically active compounds (Review) by A. G. Kozlovskii; V. P. Zhelifonova; T. V. Antipova (pp. 1-10).

Fungi of the genus Penicillium isolated from little studied habitats are able to synthesize both previously known and new physiologically active compounds with diverse structures. They include secondary metabolites of alkaloid nature, i.e., ergot alkaloids, diketopiperazines, quinolines, quinazolines, benzodiazepines, and polyketides. We discuss the use of profiles of secondary metabolites for taxonomy purposes. Studying the physicochemical characteristics of producers of biologically active compounds showed that the biosynthesis of alkaloids is initiated on the first days of cultivation and proceeds simultaneously with growth. The cyclic character of alkaloid accumulation was recorded related to the processes of alkaloid biosynthesis, excretion from cells, degradation in culture fluid, and consumption by cells. Synchronic variations in the concentrations of intracellular tryptophan and alkaloids are necessary for the regulation of the optimal quantity of tryptophan necessary for the culture.

Refolding of recombinant human interferon α-2a from Escherichia coli by urea gradient size exclusion chromatography by F. Gao; L. Shi; L. -X. Xu (pp. 11-17).

Protein refolding is still a puzzle in the production of recombinant proteins expressed as inclusion bodies (IBs) in Escherichia coli. Gradient size exclusion chromatography (SEC) is a recently developed method for refolding of recombinant proteins in IBs. In this study, we used a decreasing urea gradient SEC for the refolding of recombinant human interferon α-2a (rhIFNα-2a) which was overexpressed as IBs in E. coli. In chromatographic process, the denatured rhIFNα-2a would pass along the 8.0–3.0 M urea gradient and refold gradually. Several operating conditions, such as final concentration of urea along the column, gradient length, the ratio of reduced to oxidized glutathione and flow rate were investigated, respectively. Under the optimum conditions, 1.2 × 10⁸ IU/mg of specific activity and 82% mass recovery were obtained from the loaded 10 ml of 1.75 mg/ml denatured protein, and rhIFNα-2a was also purified during this process with the purity of higher than 92%. Compared with dilution method, urea gradient SEC was more efficient for the rhIFNα-2a refolding in terms of specific activity and mass recovery.

Physicochemical properties and antiproliferative activity of recombinant Yersinia pseudotuberculosis L-asparaginase by V. S. Pokrovskii; M. V. Pokrovskaya; S. S. Aleksandrova; R. M. Anrianov; D. D. Zhdanov; N. M. Osmel’yanyuk; E. M. Treshchalina; N. N. Sokolov (pp. 18-22).

The physicochemical, catalytic, and antiproliferative activity of a recombinant L-asparaginase from Yersinia pseudotuberculosis (YpA) have been studied. The following results were obtained: the K _M value for L-asparagine is 17 ± 0.9 μM, the optimal temperature is 60°C, pH is 8.0, pI is 5.4 ± 0.3, the L-glutaminase activity is no more than 5–6% of the L-asparaginase activity, and the antiproliferative activity on the Fisher L5178y lymphadenosis cell line comprised T/C = 136% (p < 0.001) at a 15% recovery rate. The described characteristic allows one to regard YpA as an antitumor enzyme with biological features similar to the L-asparaginase of E. coli.

An amylase from fresh fruiting bodies of the monkey head mushroom Hericium Erinaceum by F. Du; H. X. Wang; T. B. Ng (pp. 23-27).

An amylase with a molecular mass of 55 kDa and an N-terminal sequence exhibiting similarity to enzyme from Bacteroides thetaitaomicron was isolated from fruiting bodies of the monkey head mushroom Hericium erinaceum. The purification scheme included extraction with distilled water, ion exchange chromatography on DEAE-cellulose and SP-sepharose, and gel filtration by FPLC on Superdex 75. The amylase of H. erinaceum was adsorbed on DEAE-cellulose in 10 mM Tris-HCl buffer (pH 7.4) and eluted with 0.2 M NaCl in the same buffer. The enzyme was subsequently adsorbed on SP-Sepharose in 10 mM ammonium acetate buffer (pH 4.5) and eluted with 0.3 M NaCl in the same buffer. This fraction was subsequently subjected to gel filtration on Superdex 75. The first peak eluted had a molecular mass of 55 kDa in SDS-PAGE. The amylase of H. erinaceum exhibited a pH optimum of 4.6 and a temperature optimum of 40°C. The enzyme activity was enhanced by Mn²⁺ and Fe³⁺ ions, but inhibited by Hg²⁺ ions.

Heterologous expression, purification, and properties of a chymotrypsin inhibitor isolated from potatoes by I. A. Parfenov; T. A. Revina; N. G. Gerasimova; G. V. Kladnitskaya; T. A. Valueva (pp. 28-33).

The PKPIJ-B gene encoding a chymotrypsin inhibitor from a subfamily of potato Kunitz-type proteinase inhibitors (PKPI) in potatoes (Solanum tuberosum L. cv. Yubilei Zhukova) was cloned into a pET23a vector and then expressed in Escherichia coli. The recombinant PKPIJ-B protein obtained in the inclusion bodies was denatured, purified by high-performance liquid chromatography (HPLC) on Mono Q under denaturing conditions, and renaturated. The renaturated protein was additionally purified using HPLC on DEAE-ToyoPearl. The PKPIJ-B protein efficiently suppressed chymotrypsin activity, had a weaker effect on trypsin, and inhibited the growth and development of phytopathogenic microorganisms affecting potato plants.

Crystal formation peculiarities in pigmented cultures of Bacillus thuringiensis by L. A. Chil-Hakobyan; A. A. Hambardzumyan; A. Kh. Chakhalyan (pp. 34-40).

A direct correlation has been established between pink-colored pigmentation and the production of insecticide crystals (toxins) for some Bacillus thuringiensis (BT) pigmented cultures. This regularity was for the first time determined by us for BT strains of the H3, H10, and H16 serotype. Pigment-free clones of these serotypes do not produce crystals. A correlation was not observed in the case of H14 serotype strains with oval inclusions. The revealed correlation makes it possible to distinguish crystal-yielding colonies in cultures of the above-mentioned serotypes by the availability of pigmentation. This method can serve as an effective express method for the detection of virulent clones, which is especially important if these strains are used for obtaining insecticide preparations.

β-Glucan content and hydration properties of filamentous fungi by M. V. Kyanko; R. S. Canel; V. Ludemann; G. Pose; J. R. Wagner (pp. 41-45).

The aim of this work was to isolate and identify filamentous fungi from several sources to study the dietary fiber and β-glucan content. The fungal hydration properties such as water absorption and water holding capacities were also evaluated. Total dietary fiber of isolates exhibited a noticeable variability from 16 to 53% and the highest values were obtained for the genera Paecilomyces and Penicillium, a fact consistent with a higher content of β-glucans (24 and 17%, respectively), higher than previously reported for Basidiomycetes and yeast. We observed a large decrease (75%) in the water holding capacity when the mycelia were dried. Isolates of filamentous fungi with greater water holding capacity also exhibited greater absorption capacity. Paecilomyces variotii and Penicillium nalgiovense had the best hydration properties. Our results contribute to the search for new unconventional ingredients providing a high protein and β-glucans content. The addition of these dried mycelia could change the hydration properties in the food system.

Biodiesel fuel production from lipids of filamentous fungi by V. V. Lunin; Ya. E. Sergeeva; L. A. Galanina; I. S. Mysyakina; A. A. Ivashechkin; V. I. Bogdan; E. P. Feofilova (pp. 46-52).

The main stages in the production of biodiesel fuel from lipids of filamentous fungi belonging to the order Mucorales are described. Fungi of the family Cunninghamellaceae have been screened; the lipogenic activity of the examined strains has been assessed; and a producer generating up to 50% of lipids, represented by triacylglycerols, has been found. The substitution effect of a source of carbon and nitrogen with less expensive components (in particular, various industrial wastes) has been studied, as well as their influence on the quantity and major characteristics of the final product. An ecologically friendly method for extracting lipids from fungal mycelia, utilizing supercritical technologies, has been used. A correlation between the lipid content in the spore inoculum and the maximal lipid content in biomass has been discovered; this correlation is proposed for optimizing the biotechnology and increasing the yield of final products.

Effect of plant stilbene precursors on the biosynthesis of resveratrol in Vitis amurensis Rupr. Cell cultures by K. V. Kiselev; O. A. Shumakova; A. Yu. Manyakhin (pp. 53-58).

The biosynthesis of resveratrol after the application of a precursor for biosynthesis, i.e., phenylalanine (Phe), has been studied. The application of Phe has been shown to increase significantly the expression of the phenylalanine-ammonia-lyase (PAL) and stilbene synthase (STS) genes and enhance the production of resveratrol by 8.5 times. Data on resveratrol production after the addition of Phe and coumaric acid (CA) were compared with known analogs.

Effect of cyclopentene β,β′-triketone disodium salt on the activity of hydrolases and the state of tobacco mosaic virus particles in tobacco leaves by L. A. Lapshina; A. V. Reunov; V. P. Nagorskaya; O. P. Shestak; V. L. Novikov (pp. 59-63).

The activity of hydrolases (protease, RNase) in uninfected and tobacco mosaic virus-infected tobacco leaves of the Samsun variety, untreated and treated with disodium salt of 2-acetyl-4-hydroxycarbonyl-methylthio-5-chlorocyclopent-4-en-1,3-dione (DS), was determined. It was shown that treatment of leaves with this compound significantly increased the activity of hydrolases in them compared to untreated leaves. In infected leaves treated with DS one day before infection, along with an increased level of hydrolases, one revealed more viral particles exposed to destructive changes in infected, rather than untreated, leaves. It is assumed that the DS-caused activation of hydrolases promotes the destruction of viral particles and is therefore one of the cell defense mechanisms induced by this compound that prevents the intracellular accumulation of virus.

Biochemical markers of virus cytopathogenicity in macrophages by N. G. Plekhova; L. M. Somova; N. V. Krylova; G. N. Leonova; I. N. Lyapun; I. S. Smirnov (pp. 64-72).

The results of macrophage metabolism studies at their infection by viruses differing in the level of virulence are presented. With the purpose of optimizing the estimation of viral cytopathogenic effects on macrophages, an index of cell reactions, which allows one to reveal the degree of virus influence in standard units, is offered. Generally, the application of high-sensitivity methods for functional activity determination and identification of the correlative communication between its changes and morphological features of cells can be prescribed to objective identification methods of not only viral reproduction, but also differentiation of types and the degree of their cytopathogenic effects.

Antiradical properties of oregano, thyme, and savory essential oils by E. S. Alinkina; T. A. Misharina; L. D. Fatkullina (pp. 73-78).

In model reactions with the stable free 2,2-diphenyl-1-picrylhydrazyl radical, the antiradical properties of essential oils of thyme (Thymus vulgare), oregano (Origanum vulgare), and savory (Satureja hortensis) that are similar in the qualitative composition, but differ in the quantitative content of the main components, were studied and compared with the properties of synthetic antioxidant ionol. The reaction rates of components of essential oils with the radical were almost identical for all essential oils and were twice the reaction rate of ionol. The antiradical efficiency values were close to each other for all essential oils and by an order of magnitude smaller than for ionol.

Antiradical properties of oregano, thyme, and savory essential oils by E. S. Alinkina; T. A. Misharina; L. D. Fatkullina (pp. 73-78).

Development of multiplex PCR for fast detection of Paenibacillus Larvae in putrid masses and in isolated bacterial colonies by N. V. Rusenova; P. Parvanov; S. Stanilova (pp. 79-84).

The present study was performed to develop a fast and sensitive multiplex polymerase chain reaction protocol for routine diagnostics of American foulbrood. A new approach for detection of Paenibacillus larvae in putrid masses was described. Forty five samples of putrid masses obtained from bee combs suspicious for American foulbrood, a reference strain Paenibacillus larvae (NBIMCC 8478), clinical isolates and 4 strains of closely related bacterial species were included in experiments. Bacterial colonies′ DNA was isolated by heat and centrifugation method (standard procedure) and with prepGem commercial kit. DNA from putrid masses was isolated by standard and modified procedure. Three pairs of primers specific for 16S rRNA and one pair specific for 35 kDa metalloproteinase genes of Paenibacillus larvae were tested as single pair and in different combinations as multiplex PCR. The sensitivity of the multiplex PCR protocol for putrid masses, developed in study was 100%, versus 45.2% for the standard protocol. The developed multiplex PCR protocol could be successfully used for rapid and specific detection of Paenibacillus larvae in both putrid masses and isolated bacterial colonies.

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Applied Biochemistry and Microbiology (v.49, #1)

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Applied Biochemistry and Microbiology (v.49, #1)