Characterization of Cyclodextrin Glucanotransferase Produced by <Emphasis Type="Italic">Bacillus megaterium</Emphasis>

Cyclodextrin glucanotransferase, produced by Bacillus megaterium, was characterized, and the biochemical properties of the purified enzyme were determined. The substrate specificity of the enzyme was tested with different α-1,4-glucans. Cyclodextrin glucanotransferase displayed maximum activity in the case of soluble starch, with a K _m value of 3.4 g/L. The optimal pH and temperature values for the cyclization reaction were 7.2 and 60 °C, respectively. The enzyme was stable at pH 6.0–10.5 and 30 °C. The enzyme activity was activated by Sr²⁺, Mg²⁺, Co²⁺, Mn²⁺, and Cu²⁺, and it was inhibited by Zn²⁺and Ag⁺. The molecular mass of cyclodextrin glucanotransferase was established to be 73,400 Da by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, 68,200 Da by gel chromatography, and 75,000 Da by mass spectrometry. The monomer form of the enzyme was confirmed by the analysis of the N-terminal amino acid sequence. Cyclodextrin glucanotransferase formed all three types of cyclodextrins, but the predominant product was β-cyclodextrin.     

<Emphasis Type="Italic">Z</Emphasis>-and <Emphasis Type="Italic">E</Emphasis>-conformers of <Emphasis Type="Italic">N</Emphasis>-chloroacetylcytisine

N-Chloroacetylcytisine was synthesized by acylation of (–)-cytisine. Stable Z- and E-conformers with respect to rotational isomerism around the N-12–CO bond were found in PMR spectra at room temperature. The point at which PMR resonances of the Z- and E-conformers coalesced upon heating was measured. The transition barrier between the conformers was estimated.     

Antioxidant flavonoids from <Emphasis Type="Italic">Tamus communis</Emphasis> ssp. <Emphasis Type="Italic">cretica</Emphasis>

A new flavonoid, kaempferol-3,4′-di-O-α-L-rhamnopyranoside (1), and three known flavonoids (2–4) were isolated from the aerial parts of T. communis L. The structure of the new compound was elucidated on the basis of spectroscopic data. Compounds 1 and 2 showed significant antioxidant activity (IC₅₀ 187.151 ± 0.821 μM, and 92.079±0.513 μM, respectively), whereas compounds 3 and 4 showed moderate activity in DPPH free radical scavenging assays. Published in Khimiya Prirodnykh Soedinenii, No. 3, pp. 295–297, May–June, 2009.     

Purification and Characterization of Bacteriocin Produced by a Strain of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> TG2

The aims of this study were to purify and characterize a bacteriocin produced by a strain of Enterococcus faecalis TG2 and to test the safety of the strain. In this work, the active peptide was purified through precipitation with 70% saturated ammonium sulfate, cation-exchange chromatography, and gel filtration. The specific activity of purified bacteriocin was 30,073.42 AU/mg of protein, which corresponded to a 33.34-fold increase. The molecular mass of the purified bacteriocin was 6.3362 kDa determined by LC-MS/MS. The ten amino acid of N-terminal was MTRSKKLNLR and the ten amino acid of C-terminal was ATGGAAGWKS. The activity of the bacteriocin was unaffected by pH 2–10 and thermostable but was sensitive to proteolytic enzymes. The antimicrobial activity of the bacteriocin was not affected by metal ions. Tween-20, Tween-80, Triton X-100, and EDTA did not affect the bacteriocin activity and SDS was able to increase the activity of bacteriocin. Bacteriocin activity was not lost after treatment by < 8% NaCl. Inhibitory spectrum of the bacteriocin showed a wide range of activities against other lactic acid bacteria, food-spoilage, and food-borne pathogens. Ent. faecalis TG2 was sensitive to tetracycline and erythromycin but resistant to ampicillin, gentamicin, kanamycin, and chloramphenicol. Results from PCR indicated that Ent. faecalis TG2 did not harbor any virulence genes. The study suggests that Ent. faecalis TG2 and its bacteriocin might be used as bio-preservatives in food products.     

Biosynthesis of Heliotropin by a Novel Strain of <Emphasis Type="Italic">Serratia liquefaciens</Emphasis>

The present study has been conducted towards isolation of bacteria capable of producing heliotropin via microbial conversion. Strain ZMT-1 capable of synthesizing heliotropin efficiently was obtained by enrichment culture of soil samples and a high-throughput screening method, and identified as Serratia liquefaciens. Heliotropin was identified by gas chromatography and gas chromatography–mass spectrometry analysis. In addition, the culture medium was optimized to improve heliotropin yield by experimental designs. The application of a Plackett–Burman design found that NH₄NO₃ and K₂HPO₄?3H₂O have significant effects on heliotropin production. Central composite design experiments were further used to predict the optimal concentrations of NH₄NO₃ and K₂HPO₄?3H₂O, which were 1.0 and 0.5 g/l, respectively. After the optimization of cultural medium, heliotropin yield was increased by 4.52-fold when compared with the unoptimized minimal medium. This study is the first to report the biosynthesis of heliotropin by S. liquefaciens. S. liquefaciens ZMT-1 can produce heliotropin efficiently, indicating its potential as one heliotropin-producing strain.     

Synthesis of Novel <Emphasis Type="Italic">D</Emphasis>-<Emphasis Type="Italic">Seco</Emphasis>-Pregnenes

Summary. A new synthetic route was developed for the preparation of trans-3-hydroxy-16,17-seco-pregna-5,17(20)-dien-16-al, using Grob fragmentation as the key step. This seco-steroid contains a formyl group and an unsaturated side-chain in a sterically favourable position, and is therefore a promising starting material for the synthesis of novel condensed steroid heterocycles.Received March 22, 2003; accepted April 22, 2003 Published online September 25, 2003     

Molecular properties of the copolymers of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride and maleic acid

The hydrodynamic and conformational properties of molecules of poly(N,N-diallyl-N,N-dimethylammonium chloride) and N,N-diallyl-N,N-dimethylammonium chloride-maleic acid copolymers of different compositions in solutions with various ionic-strength and pH values, as well as of the polyelectrolyte complex based on the copolymer with dodecyl sulfate anions in chloroform, are studied. For poly(N,N-diallyl-N,N-dimethylammonium chloride) molecules in a 1 M NaCl solution, the Kuhn segment length and the hydrodynamic diameter of the chain are estimated as A = 3.9 nm and d = 0.48 nm, respectively. In acidic solutions with pH 3.5, the copolymers demonstrate behavior typical for polyelectrolytes. In an alkaline solution with pH 13, when 1 M NaCl is added to the solution of the copolymer containing 29 mol % maleic acid units, there is an antipolyelectrolyte effect that manifests itself as an increase in the intrinsic viscosity of the copolymer and in the hydrodynamic radius of its molecules. It is found that an increase in the fraction of maleic acid units in the copolymer from 12 to 42 mol % brings about a reduction in the equilibrium rigidity of its macromolecules from 4.1 to 2.2 nm. The equilibrium rigidity of polyelectrolyte-complex molecules is higher than that of initial copolymer molecules owing to steric interactions arising between the aliphatic chains of dodecyl sulfate anions. In an electric field, the molecules of the complex are oriented owing to the induced dipole moment resulting from the displacement of dodecyl sulfate anions along the chain contour.     

Characterization of Rhamnolipid Produced by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> Isolate Bs20

Rhamnolipid produced by Pseudomonas aeruginosa isolate Bs20 is viscous sticky oily yellowish brown liquid with a fruity odor. It showed solubility at aqueous pH > 4 with optimum solubility at pH 7–7.5 and freely soluble in ethyl acetate. This biosurfactant has a very high surface activity as it could lower the surface tension of water to 30 mN/m at about 13.4 mg/L, and it exhibited excellent stabilities at high temperatures (heating at 100°C for 1 h and autoclaving at 121°C for 10 min), salinities (up to 6% NaCl), and pH values (up to pH 13). The produced biosurfactant can be used in the crude form either as cell-free or cell-containing culture broth of the grown bacteria, since both preparations showed high emulsification indices ranged between 59% and 66% against kerosene, diesel, and motor oil. These characters make the test rhamnolipid a potential candidate for use in bioremediation of hydrocarbon-contaminated sites or in the petroleum industry. High-performance thin-layer chromatography densitometry revealed that the extracted rhamnolipid contained the two most active rhamnolipid homologues dirhamno dilipidic rhamnolipid and monorhamno dilipidic rhamnolipid at 44% and 56%, respectively, as compared to 51% and 29.5%, respectively, in a standard rhamnolipid preparation. The nature and ratio of these two rhamnolipid homologues showed to be strain dependent rather than medium-component dependent.     

Copolymer of <Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-diallyl-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylammonium chloride with maleic acid as drug carrier

A copolymer of N,N-diallyl-N,N-dimethylammonium chloride with maleic acid of constant composition was prepared under the conditions of radical initiation. The possibility of the functionalization of the copolymer with drugs containing amino groups by polymer-analogous transformations was examined. Conditions were found for preparing conjugates of the copolymer with isoniazid. The structures and the quantitative compositions of the conjugates were determined by ¹³С NMR spectroscopy, and the possibility of preparing conjugates with controlled drug content was demonstrated.     

Characterization of Surfactin Produced by <Emphasis Type="Italic">Bacillus subtilis</Emphasis> Isolate BS5

Physical and chromatographic characterization of the surfactin biosurfactant produced by Bacillus subtilis isolate BS5 has been conducted to study its potentiality for industrial application. The crude extract of test surfactin appeared as off-white to buff flake-like amorphous residue with bad odor similar to sour pomegranate. Test surfactin showed solubility in aqueous solution at pH>5 with optimum solubility at pH 8-8.5. It was also soluble in organic solvents like ethanol, acetone, methanol, butanol, chloroform, and dichloromethane. Surfactin crystals appeared rectangular with blunt corners and were arranged perpendicular to each other making a plus sign. Extracted surfactin showed high surface activity, as it could lower the surface tension of water from about 70 to 36 mN/m at approximately 15.6 mg/l. Moreover, test surfactin exhibited excellent stabilities at high temperatures (100 degrees C for up to 1 h at and autoclaving at 121 degrees C for 10 min), salinities (up to 6% NaCl), and over a wide range of pH (5-13). Test surfactin in the cell-free supernatant or crude culture broth forms showed high emulsification indices against kerosene (62.5% and 59%, respectively), diesel (62.5% and 66%, respectively), and motor oil (62% and 66%, respectively). These characters can effectively make test surfactin, in its crude forms, a potential candidate for the use in bioremediation of hydrocarbon-contaminated sites or in the petroleum industry. Chromatographic characterization of test surfactin, using high-performance liquid chromatography technique, revealed that the extracted surfactin contained numerous isoforms, of which six were found in the standard surfactin preparation (Fluka). Additional peaks appeared in the test surfactin and not in the standard one. These peaks may correspond to new surfactin isoforms that may be present in the test surfactin produced by B. subtilis isolate BS5.     

Enhanced Production of <Emphasis Type="SmallCaps">l</Emphasis>-Arginine by Expression of <Emphasis Type="Italic">Vitreoscilla</Emphasis> Hemoglobin Using a Novel Expression System in <Emphasis Type="Italic">Corynebacterium crenatum</Emphasis>

Corynebacterium crenatum SYPA 5-5 is an aerobic and industrial l-arginine producer. It was proved that the Corynebacterium glutamicum/Escherichia coli shuttle vector pJC1 could be extended in C. crenatum efficiently when using the chloramphenicol acetyltransferase gene (cat) as a reporter under the control of promoter tac. The expression system was applied to over-express the gene vgb coding Vitreoscilla hemoglobin (VHb) to further increase the dissolved oxygen in C. crenatum. As a result, the recombinant C. crenatum containing the pJC-tac-vgb plasmid expressed VHb at a level of 3.4 nmol g⁻¹, and the oxygen uptake rates reached 0.25 mg A₅₆₂⁻¹ h⁻¹ which enhanced 38.8% compared to the wild-type strain. Thus, the final l-arginine concentration of the batch fermentation reached a high level of 35.9 g L⁻¹, and the biomass was largely increased to 6.45 g L⁻¹, which were 17.3% and 10.5% higher than those obtained by the wild-type strain, respectively. To our knowledge, this is the first report that the efficient expression system was constructed to introduce vgb gene increasing the oxygen and energy supply for l-arginine production in C. crenatum, which supplies a good strategy for the improvement of amino acid products.     

Enhanced <Emphasis Type="SmallCaps">l</Emphasis><Emphasis Type="Italic">-</Emphasis>(+)-Lactic Acid Production by an Adapted Strain of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using Corncob Hydrolysate

Corncob is an economic feedstock and more than 20 million tons of corncobs are produced annually in China. Abundant xylose can be potentially converted from the large amount of hemicellulosic materials in corncobs, which makes the crop residue an attractive alternative substrate for a value-added production of a variety of bioproducts. Lactic acid can be used as a precursor for poly-lactic acid production. Although current industrial lactic acid is produced by lactic acid bacteria using enriched medium, production by Rhizopus oryzae is preferred due to its exclusive formation of the l-isomer and a simple nutrition requirement by the fungus. Production of l-(+)-lactic acid by R. oryzae using xylose has been reported; however, its yield and conversion rate are poor compared with that of using glucose. In this study, we report an adapted R. oryzae strain HZS6 that significantly improved efficiency of substrate utilization and enhanced production of l-(+)-lactic acid from corncob hydrolysate. It increased l-(+)-lactic acid final concentration, yield, and volumetric productivity more than twofold compared with its parental strain. The optimized growth and fermentation conditions for Strain HZS6 were defined.     

Cloning,Expression, and Characterization of a Novel (<Emphasis Type="Italic">S</Emphasis>)-Specific Alcohol Dehydrogenase from <Emphasis Type="Italic">Lactobacillus kefir</Emphasis>

A gene encoding a novel (S)-specific NADH-dependent alcohol dehydrogenase (LK-ADH) was isolated from the genomic DNA of Lactobacillus kefir DSM 20587 by thermal asymmetric interlaced-polymerase chain reaction. The nucleotide sequence of (S)-LK-ADH gene (adhS) was determined, which consists of an open reading frame of 1,044 bp, coding for 347 amino acids with a molecular mass of 37.065 kDa. After a BLAST similarity search in GenBank database, the amino acid sequence of (S)-LK-ADH showed some homologies to several zinc containing medium-chain alcohol dehydrogenases. This novel gene was deposited into GenBank with the accession number of EU877965. adhS gene was subcloned into plasmid pET-28a(+), and recombinant (S)-LK-ADH was successfully expressed in E. coli BL21(DE3) by isopropyl-β-d-1-thiogalactopyranoside induction. Purified enzyme showed a high enantioselectivity in the reduction of acetophenone to (S)-phenylethanol with an ee value of 99.4%. The substrate specificity and cofactor preference of recombinant (S)-LK-ADH were also tested.     

Conformations and properties of <Emphasis Type="Italic">O</Emphasis>-Alkyl-<Emphasis Type="Italic">S</Emphasis>-(2-<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dialkylamino)-ethylmethylthiophosphonates

Conformers of the biologically active compounds CH₃P(O)(OR)(SCH₂CH₂NR ₂ ^′ ), where (I) R = i-C₄H₉, R′ = C₂H₅ and (II) R = C₂H₅, R′ = i-C₃H₇, are calculated within the AM1 level of theory. The elongated and twisted forms with maximum and minimum distances between a nitrogen atom and those of a phosphorus tetrahedron, respectively, and bearing a syn and anti oriented alkoxy group relative to a phosphoryl oxygen, are studied. It is found that the differences between the energy, electronic, and geometric parameters of these forms are apparent in differences between their properties, e.g., the ability to participate in complexation and protonation, reactions that to some extent simulate the interaction between a substance and a biological object.     

Molecular structure of <Emphasis Type="Italic">N</Emphasis>-(<Emphasis Type="Italic">o</Emphasis>-and <Emphasis Type="Italic">p</Emphasis>-hydroxybenzyl)cytisine

The molecular structures of N-(o-and p-hydroxybenzyl)cytisine were investigated by NMR spectroscopy, x-ray structure analysis, and molecular modeling. It was found that NMR resonances of the OH and aromatic protons in N-(o-hydroxybenzyl)cytisine were doubled because of the presence of two conformers in solution. __________ Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 165–168, March–April, 2008.     

Isolation and Characterization of a Novel Plasma Membrane Intrinsic Protein Gene, <Emphasis Type="Italic">LcPIP1</Emphasis>, in <Emphasis Type="Italic">Leymus chinensis</Emphasis> that Enhances Salt Stress Tolerance in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A novel plasma membrane intrinsic, LcPIP1, was isolated from Leymus chinensis using RACE method. The LcPIP1 has 288 amino acids with an estimated molecular mass of 30.6 kDa. Semi RT-PCR analysis indicated that the expression level of LcPIP1 was obviously higher in leaf than root. The LcPIP1 was also found to be induced by salt stress. In addition, transformed with the LcPIP1, Saccharomyces cerevisiae could increase tolerance to salt stress. These results indicate that the LcPIP1 gene seems to play a role in resistance against salt stress.