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Applied Biochemistry and Microbiology (v.49, #2)

Fungal anthraquinones by N. N. Gessler; A. S. Egorova; T. A. Belozerskaya (pp. 85-99).

The review is devoted to the characteristics of anthraquinones—a group of pigments of quinoid nature often found in fungi. The distribution of anthraquinones in fungi, the routes of their biosynthesis, and their biological activity are considered.

Identification of the functional activity of synthetic polyamine analogues using a biotest system based on highly proliferating cultured human cells by K. V. Lisitskaya; N. A. Sokueva; Yu. G. Malysheva; A. V. Ivanov; S. S. Shishkin; S. P. Syatkin (pp. 100-105).

A new biotest system was developed based on highly proliferating human cell cultures (lines LNCaP and PC-3). With the help of this system, two known synthetic polyamines—α-difluoromethylornithine (DFMO) and methylglioxalbis(guanylhydrason) (MGBG)—as well as four new synthetic analogues difenylcontaining amines (DFCA-1-DFCA-4) with molecular weights of 725.5 (DFCA-1), 755.5 (DFCA-2), 655.5 (DFCA-3), and 681.5 Da (DFCA-4) were tested. In this biotest system, DFMO (0.1–400 μM) did not reveal functional activity, whereas for MGBG a cytotoxic effect was registered (100–200 μM). DFCA-1, DFCA-2, and DFCA-4 had a similar effect at concentrations of 10 μM and higher; DFCA-3, at a concentration of 50 μM and higher. Thus, DFCA-1 has a higher level of antiproliferating activity and may be considered as the most potent cytostatic agent.

Catalytic properties of enzymes from Erwinia carotovora involved in transamination of phenylpyruvate by A. M. Paloyan; L. A. Stepanyan; S. A. Dadayan; A. A. Hambardzumyan; Gh. P. Halebyan; A. S. Saghiyan (pp. 106-112).

K _m for L-phenylalanine, L-glutamic acid, L-aspartic acid, and the corresponding keto acids were calculated, as well as V _max was measured for the following pairs of substrates: L-phenylalanine-2-ketoglutarate, L-phenylalanine-oxaloacetate, L-glutamic acid-phenylpyruvate, and L-aspartic acid-phenylpyruvate for aminotransferases PAT1, PAT2, and PAT3 from Erwinia carotovora catalyzing transamination of phenylpyruvate. The ping-pong bi-bi mechanism was shown for the studied aminotransferases. The substrate inhibition (K _s) of PAT3 with 2-ketoglutarate and oxaloacetate was 10.23 ± 3.20 and 3.73 ± 1.99 mM, respectively.It was shown that L-β-(N-benzylamino)alanine was a competitive inhibitor with respect to L-phenylalanine for PAT1 (K _i = 0.32 ± 0.07 mM, K _m = 0.45 ± 0.1 mM, V _max = 11. 6 ± 0.4 U/mg) at 25 mM concentration of 2-ketoglutarate in the reaction medium. L-β-(N-methylamino)alanine is a noncompetitive inhibitor with respect to L-phenylalanine for PAT3 (K _I = 138.4 ± 95.4 mM, K _m = 13.7 ±3.9 mM, V _max = 18.6 ± 4.1 U/mg) at 2 mM concentration of 2-ketoglutarate in the reaction medium. L-stereo isomers of nonprotein analogues of aromatic amino acids were studied as substrates for PAT1, PAT2, and PAT3. L-β-(2-Br-phenyl)alanine, L-β-(4-Br-phenyl)alanine, L-β-(2-F-phenyl)alanine, and L-(2-F)tryptophan were good substrates for all three aminotransferases; L-α-methyl-β-(2-Br-phenyl)alanine and L-O-benzyltyrosine were substrates only for PAT3; L-β-(4-F-phenyl)alanine was a substrate for PAT1 and PAT3. Thus, these analogues of aromatic amino acids can be stereoselectively synthesized using the studied aminotransferases in the presence of the corresponding keto acids.

Recombinant Escherichia coli strains deficient in mixed acid fermentation pathways and capable of rapid aerobic growth on glucose with a reduced crabtree effect by A. A. Morzhakova; A. Yu. Skorokhodova; A. Yu. Gulevich; V. G. Debabov (pp. 113-119).

In this study, we constructed and characterized Escherichia coli strains deficient in mixed acid fermentation pathways, which are capable of rapid aerobic growth on glucose without pronounced bacterial Crabtree effect. The main pathways of production of acetic and lactic acids and ethanol in these strains were inactivated by a deletion of the ackA, pta, poxB, ldhA, and adhE genes. The phosphoenolpyruvate-dependent phosphotransferase system of glucose transport and phosphorylation was inactivated in the strains by a deletion of the ptsG gene. The possibility of alternative transport and phosphorylation of the carbohydrate substrate was ensured in recombinants by constitutive expression of the galP and glk genes, which encode the low-affinity H⁺-symporter of D-galactose and glucokinase, respectively. The resulting SGM1.0ΔptsG P_tacgalP and SGM1.0ΔptsG PLglk P_tacgalP strains were capable of rapid aerobic growth in a minimal medium containing 2.0 and 10.0 g/L of glucose and secreted only small amounts of acetic acid and trace amounts of pyruvic acid.

Study of regulation of some key enzymes of L-alanine biosynthesis by Brevibacterium Flavum producer strains by L. H. Melkonyan; G. Y. Avetisova; A. A. Hambardzumyan; A. Kh. Chakhalyan; A. S. Saghyan (pp. 120-124).

The mechanisms of L-alanine overproduction by Brevibacterium flavum strain-producers were studied. It was shown that β-Cl-L-alanine is an inhibitor of some key enzymes involved in the synthesis of L-alanine: alanine transaminase and valine-pyruvate transaminase. Two highly active B. flavum GL1 and GL18 strain-producers, which are resistant to the inhibitory effect of β-Cl-L-alanine, were obtained using a parental B. flavum AA5 strain-producer, characterized by a reduced activity of alanine racemase (≥98%). It was demonstrated that the increased L-alanine synthesis efficiency observed in the strain-producer developed in this work is associated with the absence of inhibition of alanine transaminase by the end product of the biosynthesis reaction, as well as with the effect of derepression of both alanine transaminase and valine-pyruvate transaminase synthesis by the studied compound.

Expression of the Escherichia Coli TdcB gene encoding threonine dehydratase in L-isoleucine-overproducing Corynebacterium Glutamicum Yilw by L. Xu; X. Xie; J. Shi; Q. Xu; N. Chen (pp. 125-129).

Threonine dehydratase (EC 4.3.1.19, TDH) catalyzing the degradation of Thr to α-ketobutyrate, is a rate-limiting enzyme in L-Ile pathway. The tdcB gene encoding TDH was obtained from Escherichia coli K12 by PCR and expressed at E. coli BL21 (DE3). Then the tdcB gene was inserted into the shuttle expression vector pXMJ19 and the recombinant plasmid was electroporated into the L-isoleucine-producing strain of Corynebacterium glutamicum YILW. Crude extracts of the microbial strain containing the plasmid pXMJ19tdcB retained 60% of the original TDH activity even in the presence of 300 mM L-Ile. The recombinant strain of bacteria showed 7.5% higher enzyme activity and 11.3% higher L-Ile production compared to the original strain.

The directed modification of Escherichia coli MG1655 to obtain histidine-producing mutants by V. G. Doroshenko; A. O. Lobanov; E. A. Fedorina (pp. 130-135).

Strain MG1655⁺ hisG ^r hisL′-ΔΔ, purR, which produces histidine with a weight yield of approximately 12% from glucose, was constructed through directed chromosomal modifications of the laboratory Escherichia coli strain MG1655⁺, which has a known genome sequence. A feedback-resistant ATP-phosphoribosyl transferase encoded by the mutant hisG ^r (E271K) was the main determinant of histidine production. A further increase in histidine production was achieved by the expression enhance of a mutant his operon containing hisG ^r through the deleting attenuator region (hisL′-Δ). An increase in the expression of the wildtype his operon did not result in histidine accumulation. Deletion of the transcriptional regulator gene purR increased the biomass produced and maintained the level of histidine production per cell under the fermentation conditions used.

Novel NADPH-dependent L-aspartate dehydrogenases from the mesophilic nitrogen-fixing bacteria Rhodopseudomonas palustris and Bradyrhizobium japonicum by T. M. Kuvaeva; J. I. Katashkina; A. D. Kivero; S. V. Smirnov (pp. 136-143).

The genes encoding putative L-aspartate dehydrogenases (EC 1.4.1.21, ADH) from the mesophilic nitrogen-fixing bacteria Rhodopseudomonas palustris and Bradyrhizobium japonicum were cloned and expressed in Escherichia coli. The respective enzymes in the form of hybrid proteins with N-terminal hexahistidine tags were purified to apparent homogeneity. Both enzymes catalyzed in vitro the reductive amination of oxaloacetate to L-aspartate by an order faster than the reverse reaction at a respective pH optimum of 8.0–9.0 and 9.8; also, the enzymes only catalyzed amination under physiological conditions (pH 7.0–8.0). Their specificity to NADPH was higher by 1–2 orders of magnitude than that to NADH. The apparent K M values of ADH from R. palustris for oxaloacetate, ammonium, and NADPH at pH 9.0 were 9.2, 11.3, and 0.21 mM, respectively, and the corresponding K M values of ADH from B. japonicum were 21, 4.3, and 0.032 mM, respectively. The amination activity of novel ADHs may be important for the fixation of inorganic nitrogen in vivo and used for the construction of a bacterial strain-producer of L-aspartate by metabolic engineering methods.

Effect of cold plasma on the E. coli cell wall and plasma membrane by E. N. Kobzev; G. V. Kireev; Yu. A. Rakitskii; I. I. Martovetskaya; V. A. Chugunov; V. P. Kholodenko; M. V. Khramov; Yu. S. Akishev; N. I. Trushkin; M. E. Grushin (pp. 144-149).

The effect of cold plasma on E. coli cells was studied. It was shown that the treatment of E. coli cells with cold plasma caused partial or total disruption of the plasma membrane integrity, which was accompanied by a release of intracellular substances into the extracellular environment. A quantitative assessment of the extent of the damage to the cell membrane showed that a loss of no more than 23.6% of intracellular substances (calculated by the proportion of the intracellular nucleotide release) is sufficient to lead to cell death. The use of media with different ionic strength levels to create osmotic shock showed that the treatment of E. coli cells with cold plasma significantly decreased the cell wall strength.

Biosynthesis of polyhydroxybutyrate/valerate with different molecular weights during the growth of Methylobacterium extorquens G-10 on a methanol-pentanol mixture by V. A. Ezhov; N. V. Doronina; Yu. A. Trotsenko (pp. 150-153).

The influence of the concentration and time of addition of cosubstrate (pentanol) on the molecular weight (MW) of the polyhydroxybutyrate/valerate (PHBV) copolymer synthesized by Methylobacterium extorquens G-10 during cultivation in a methanol-containing medium has been studied. It was shown that an increase in the pentanol concentration to 20% in a mixture with methanol stimulated the biosynthesis of PHBV with a MW of ∼1500 kDa and increased the content of valerate up to 50%, especially when pentanol was added to the log phase culture. High pentanol concentrations are toxic for the producer and reduce the total yield of PHBV. An MW increase to 1500 kDa lowers the melting temperature (from 172 to 162°C) and the crystallinity degree (from 63 to 8%) of the biopolymer but increases its elasticity. The revealed variability of PHBV properties extends considerably the potential application areas of synthetic bioplastics.

Optimization of cultural conditions for the production of antifungal chitinase by Streptomyces sporovirgulis by M. Swiontek Brzezinska; U. Jankiewicz; K. Lisiecki (pp. 154-159).

Actinomycetes were screened from soil in the centre of Poland on chitin medium. Amongst 30 isolated strains one with high activity of chitinase was selected. It was identified as Streptomyces sporovirgulis. Chitinase activity was detected from the second day of cultivation, then increased gradually and reached maximum after 4 days. The maximum chitinase production was observed at pH 8.0 and 25–30°C in the medium with sodium caseinate and asparagine as carbon and nitrogen sources and with shrimp shell waste as inducer of enzyme. Chitinase of S. sporovirgulis was purified from a culture medium by fractionation with ammonium sulphate as well as by chitin affinity chromatography. The molecular weight of the enzyme was 27 kDa. The optimum temperature and pH for the chitinase were 40°C and pH 8.0. The enzyme activity was characterised by high stability at the temperatures between 35 and 40°C after 240 min of preincubation. The activity of the enzyme was strongly inhibited in the presence of Pb²⁺, Hg²⁺ and stabilized by the ions Mg²⁺. Purified chitinase from S. sporovirgulis inhibited growth of fungal phytopathogen Alternaria alternata. Additionally, the crude chitinase inhibited the growth of potential phytopathogens such as Penicillium purpurogenum and Penillium sp.

Inhibitory effect of components from Streptomyces species on α-glucosidase and α-amilase of different origin by P. Meng; C. Xie; P. Geng; X. Qi; F. Zheng; F. Bai (pp. 160-168).

The search for the effective and safe α-glucosidase and α-amylase inhibitors from Actinomycetaceae being antidiabetic agents is actual problem. Twenty one Streptomyces spp. of soil samples collected from different places of China were screened for the ability to produce this kind of inhibitory activities. Fermentation broth of isolated strains had absorbance between 350–190 nm. The Streptomyces strains PW003, ZG636, and ZG731 were characterized by special absorption at 280, 275, and 400 nm, respectively. Ten of the collected actinomycete strains had the ability to inhibit α-glucosidase or/and α-amylase and the fermentation broth of the same strain had inhibitory activity varied greatly depending on the enzyme source. In the process to screen the leading compounds used as antidiabetic agents, human α-glucosidase and α-amylase were revealed as the best used in trail compared with the same enzymes from other sources. Active α-glucosidase inhibitor was isolated from Streptomyces strain PW638 fermentation broth and identified as acarviostatin I03 by MS and NMR spectrometry. Its IC₅₀ value was 1.25 and 12.23 μg/ml against human intestinal N-terminal maltase-glucoamylase and human pancreatic α-amylase, respectively.

Biochemical parameters of Saccharopolyspora Erythraea during feeding ammonium sulphate in erythromycin biosynthesis phase by X. Zou; W. -J. Li; W. Zeng; H. -F. Hang; J. Chu; Y. -P. Zhuang; S. L. Zhang (pp. 169-175).

The physiology of feeding ammonium sulphate in erythromycin biosynthesis phase of Saccharopolyspora erythraea on the regulation of erythromycin A (Er-A) biosynthesis was investigated in 50 L fermenter. At an optimal feeding ammonium sulphate rate of 0.03 g/L per h, the maximal Er-A production was 8281 U/mL at 174 h of growth, which was increased by 26.3% in comparison with the control (6557 U/mL at 173 h). Changes in cell metabolic response of actinomycete were observed, i.e. there was a drastic increase in the level of carbon dioxide evolution rate and oxygen consumption. Assays of the key enzyme activities and organic acids of S. erythraea and amino acids in culture broth revealed that cell metabolism was enhanced by ammonium assimilation, which might depend on the glutamate transamination pathway. The enhancement of cell metabolism induced an increase of the pool of TCA cycle and the metabolic flux of erythromycin biosynthesis. In general, ammonium assimilation in the erythromycin biosynthesis phase of S. erythraea exerted a significant impact on the carbon metabolism and formation of precursors of the process for dramatic regulation of secondary metabolites biosynthesis.

Fungicidal activity of yeast isolated from chal by W. I. Golubev (pp. 176-181).

A Kluyveromyces strain secreting a fungicidal proteinaceous toxin has been isolated. Its maximal activity is observed at pH 5.0 and an increased osmotic pressure. This agent has been identified as a mycocin; it is active towards species belonging to the genus Kluyveromyces and some representatives of taxonomically related taxa.

Water-soluble glucans from true cardamom (Elettaria cardamomum White at Maton) seeds by D. N. Olennikov; A. V. Rokhin (pp. 182-187).

Water-soluble polysaccharides from seeds of true cardamom (Elettaria cardamomum White at Maton, family Zingiberaceae) have been studied. The study has shown the presence of neutral and acidic components in these polysaccharides. Three polysaccharides (380, 166, and 27 kDa) have been isolated from the neutral fraction. According to the structural analysis data, they represent α-glucans with different degrees of branching (7.1–46.1%); α-(1→4)-D-glucopyranose residues of their backbone chains are substituted at the C6 position with single α-D-glucopyranose residues. Polysaccharides with such structures have a wide range of biological activity. The presence of branched α-glucans in E. cardamomum seeds has been demonstrated.

Aerobic methylobacteria as the basis for a biosensor for dichloromethane detection by Yu. V. Plekhanova; Yu. E. Firsova; N. V. Doronina; Yu. A. Trotsenko; A. N. Reshetilov (pp. 188-193).

Cells of dichloromethane (DChM) bacteria-destructors were immobilized by sorption on different types of membranes, which were fixed on the measuring surface of a pH-sensitive field transistor. The presence of DChM in the medium (0.6–8.8 mM) led to a change in the transistor’s output signal, which was determined by the appearance of H⁺ ions in the medium due to DChM utilization by methylobateria. Among four strains of methylobacteria—Methylobacterium dichloromethanicum DM4, Methylobacterium extorquens DM17, Methylopila helvetica DM6, and Ancylobacter dichloromethanicus DM16—the highest and most stable activity toward DChM degradation was observed in the strain M. dichloromethanicum DM4. Among 11 types of membranes for cell immobilization, Millipore nitrocellulose membranes and chromatographic fiber paper GF/A, which allow one to obtain stable biosensor signals for 2 weeks without a bioreceptor change, were chosen as optimal carriers.

Potentiometric glucose determination in human serum samples with glucose oxidase biosensor based on iodide electrode by E. Karakuş; Ş. Pekyardımcı; E. Kiliç (pp. 194-198).

Glucose potentiometric biosensor was prepared by immobilizing glucose oxidase on iodide-selective electrode. The hydrogen peroxide formed after the oxidation of glucose catalysed by glucose oxidase (GOD) was oxidized by sodium molybdate (SMo) at iodide electrode in the presence of dichlorometane. The glucose concentration was calculated from the decrease of iodide concentration determined by iodide-selective sensor. The sensitivity of glucose biosensor towards iodide ions and glucose was in the concentration ranges of 1.0 × 10⁻¹–1.0 × 10⁻⁶ M and 1.0 × 10⁻²−1.0 × 10⁻⁴ M, respectively. The characterization of proposed glucose biosensor and glucose assay in human serum were also investigated.

Antimicrobial activity of stable silver nanoparticles of a certain size by Iu. P. Mukha; A. M. Eremenko; N. P. Smirnova; A. I. Mikhienkova; G. I. Korchak; V. F. Gorchev; A. Yu. Chunikhin (pp. 199-206).

Conditions for obtaining stable silver nanoparticles smaller than 10 nm were developed using a binary stabilizer polyvinylpyrrolidone/sodium dodecylsulphate in optimal ratio. Optical spectra, morphology and dependence of size of the nanoparticles on the amount of reducing agent were studied. Colloidal solutions of nanosilver showed a high bactericidal activity against strains of Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa, and fungicidal activity against Candida albicans. The mechanism of action of nanosized silver on microbial cell was examined by laser scanning confocal microscope using fluorescent label. First step of antimicrobial effect on microorganisms was membrane damage and penetration of silver nanoparticles into the cell. Prolonged stability of nanoparticles and their antimicrobial activity over the past two years were showed.

Erratum to: “Antiradical Properties of Oregano, Thyme, and Savory Essential Oils” by E. S. Alinkina; T. A. Misharina; L. D. Fatkullina (pp. 207-207).

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Applied Biochemistry and Microbiology (v.49, #2)

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Applied Biochemistry and Microbiology (v.49, #2)