Characterization and application of several lysis cassettes / 生物工程学报

Lysis is a common functional module in synthetic biology and is widely used in genetic circuit design. Lysis could be achieved by inducing expression of lysis cassettes originated from phages. However, detailed characterization of lysis cassettes hasn't been reported yet. Here, we first adopted arabinose- and rhamnose-inducible systems to develop inducible expression of five lysis cassettes (S105, A52G, C51S S76C, LKD, LUZ) in Escherichia coli Top10. By measuring OD600, we characterized the lysis behavior of strains harboring different lysis cassettes. These strains were harvested at different growth stages, induced with different concentrations of chemical inducers, or contained plasmids with different copy numbers. We found that although all five lysis cassettes could induce bacterial lysis in Top10, lysis behaviors differed a lot at various conditions. We further found that due to the difference in background expression levels between strain Top10 and Pseudomonas aeruginosa PAO1, it was hard to construct inducible lysis systems in strain PAO1. The lysis cassette controlled by rhamnose-inducible system was finally inserted into the chromosome of strain PAO1 to construct lysis strains after careful screen. The results indicated that LUZ and LKD were more effective in strain PAO1 than S105, A52G and C51S S76C. At last, we constructed an engineered bacteria Q16 using an optogenetic module BphS and the lysis cassette LUZ. The engineered strain was capable of adhering to target surface and achieving light-induced lysis by tuning the strength of ribosome binding sites (RBSs), showing great potential in surface modification.

Enzymatic properties of α-L-rhamnosidase and the factors affecting its activity: a review / 生物工程学报

α-L-rhamnosidase is a very important industrial enzyme that is widely distributed in a variety of organisms. α-L-rhamnosidase of different origins show functional diversity. For example, the optimal pH of α-L-rhamnosidase from bacteria is close to neutral or alkaline, while the optimal pH of α-L-rhamnosidase from fungi is in the acidic range. Furthermore, the enzymatic properties of α-L-rhamnosidases of different origins differ in terms of the optimal temperature, the thermal stability, and the substrate specificity, which determine the different applications of these enzymes. In this connection, it is crucial to elucidate the similarities and differences in the catalytic mechanism and substrate specificity of α-L-rhamnosidase of different origins through analyzing its enzymatic properties. Moreover, it is important to explore and understand the effects of aglycon and metal cations on enzyme activity and the competitive inhibition of L-rhamnose and glucose on enzymes. These knowledge can help discover α-L-rhamnosidase of industrial significance and promote its industrial application.

Construction and optimization of Escherichia coli for producing rhamnolipid biosurfactant / 生物工程学报

Rhamnolipid biosurfactant is mainly produced by Pseudomonas aeruginosa that is the opportunistic pathogenic strain and not suitable for future industrial development. In order to develop a relatively safe microbial strain for the production of rhamnolipid biosurfactant, we constructed engineered Escherichia coli strains for rhamnolipid production by expressing different copy numbers of rhamnosyltransferase (rhlAB) gene with the constitutive synthetic promoters of different strengths in E. coli ATCC 8739. We further studied the combinatorial regulation of rhlAB gene and rhaBDAC gene cluster for dTDP-1-rhamnose biosynthesis with different synthetic promoters, and obtained the best engineered strain-E. coli TIB-RAB226. Through the optimization of culture temperature, the titer of rhamnolipd reached 124.3 mg/L, 1.17 fold higher than that under the original condition. Fed-batch fermentation further improved the production of rhamnolipid and the titer reached the highest 209.2 mg/L within 12 h. High performance liquid chromatography-mass spectrometry (LC-MS) analysis showed that there are total 5 mono-rhamnolipid congeners with different nuclear mass ratio and relative abundance. This study laid foundation for heterologous biosynthesis of rhanomilipd.

Applicability of a natural swelling matrix as the propellant of osmotic pump tablets / 药学学报

The purpose of this study is to investigate the applicability of a natural swelling matrix derived from boat-fruited sterculia seed (SMS) as the propellant of osmotic pump tablets. The sugar components, static swelling, water uptake and viscosity of SMS were determined and compared with that of polythylene oxide (WSR-N10 and WSR-303). Both ribavirin and glipizide were used as water-soluble and water-insoluble model drugs. Then, the monolayer osmotic pump tablets of ribavirin and the bilayer osmotic pump tablets of glipizide were prepared using SMS as the osmotically active substance and propellant. SMS was mainly composed of rhamnose, arabinose, xylose and galactose and exhibited relatively high swelling ability. The area of the disintegrated matrix tablet was 20.1 times as that at initial after swelling for 600 s. SMS swelled rapidly and was fully swelled (0.5%) in aqueous solution with relative low viscosity (3.66 +/- 0.03) mPa x s at 25 degrees C. The monolayer osmotic pump tablets of ribavirin and the bilayer osmotic pump tablets of glipizide using SMS as propellant exhibited typical drug release features of osmotic pumps. In conclusion, the swelling matrix derived from boat-fruited sterculia seed, with low viscosity and high swelling, is a potential propellant in the application of osmotic pump tablets.

Development of a new HPLC technique for analyzing monosaccharide composition and its application in the quality control of Silybum marianum polysaccharide / 药学学报

A new HPLC-UV technique for the separation and analysis of 10 monosaccharides achieved within 13.5 min using 1-phenyl-3-methyl-5-pyrazolone (PMP) as the labelling molecule of the reductive monosaccharides has been established by combining common high performance liquid chromatography-UV and C18 column. The established technique was applied to the quantification of the monosaccharide components in extract of Silybum marianum. The results showed that the tested 10 monosaccharides as PMP derivatives were baseline separated under the HPLC conditions proposed. It was confirmed that Silybum marianum extract was composed of mannose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, galactose and arabinose with the molar ratio of 0.66:0.84:0.58:1.0:1.6:0.69:2.7:4.8. Quantitative recoveries of the compositional monosaccharides separated from the extract were in the range of 92.4%-104.0%, and the RSD values fell within 0.68%-3.81%. The results demonstrated that the proposed HPLC method was simple, rapid, convenient, and precise, and it was applicable to the analysis of the compositional monosaccharides of Silybum marianum extract.

Activation of Macrophages by Exopolysaccharide Produced by MK1 Bacterial Strain Isolated from Neungee Mushroom, Sarcodon aspratus

BACKGROUND: The MK1 strain, a novel bacterial isolate from soft-rotten tissue of the Neungee mushroom, produces copious amounts of exopolysaccharide (EPS) in a dextrose minimal medium. This study examined the molecular characteristics and immunomodulatory activity of MK1 EPS. METHODS: The EPS in the culture supernatant was purified by cold ethanol precipitation, and characterized by SDS-PAGE/silver staining and Bio-HPLC. The immunomodulatory activities of the EPS were examined using the mouse monocytic cell line, RAW 264.7 cells. RESULTS: The molecular weights of the purified EPS were rather heterogeneous, ranging from 10.6 to 55 kDa. The EPS was composed of glucose, rhamnose, mannose, galactose, and glucosamine at an approximate molar ratio of 1.00:0.8:0.71:0.29:0.21. EPS activated the RAW cells to produce cytokines, such as TNF-alpha and IL-1beta, and nitric oxide (NO). EPS also induced the expression of co-stimulatory molecules, such as B7-1, B7-2 and ICAM-1, and increased the phagocytic activity. The macrophage-activating activity of EPS was not due to endotoxin contamination because the treatment of EPS with polymyin B did not reduce the macrophage-activating activity. CONCLUSION: The EPS produced from the MK1 strain exerts macrophage-activating activity.

Analysis of monosaccharides in Radix Rehmanniae by GC / 中国中药杂志

<p><b>OBJECTIVE</b>To isolate and purify the polysaccharides from Radix Rehmanniae and analysis the monosaccharides composition.</p><p><b>METHOD</b>The polysaccharides were extracted with hot water and precipitated by alcohol. Proteins in the precipitates were removed by TCA method. The products were further purified with column chromatography on Superdex 200 and Sephadex G100. The SRP I and SRP II were identified as homogeneous polysaccharide by HPLC, respectively, and then analyzed by GC after being hydrolysised.</p><p><b>RESULT</b>Two homogeneous polysaccharides (SRP I and SRP II) were obtained from Radix Rehmanniae.</p><p><b>CONCLUSION</b>SRP I contained rhamnose, arabinose, glucose and galactose in the percentage of 6.11%, 66.46%, 3.93% and 21.50%. SRP I was composed of rhamnose, fucose, mannose, galactose and fructose in the percentage of 21.82%, 24.47%, 10.48%, 29.94% and 13.29%.</p>

Studies on chemical constituents of Illicium simonsii / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical constituents from the active fractions against HIV in vitro, a crude ethanolic extract of Illicium simonsii.</p><p><b>METHOD</b>The compounds were isolated with column chromatography methods. MS and NMR spectroscopic methods were used to determine the structures of the compounds.</p><p><b>RESULT</b>Seven compounds were isolated from the active fractions against HIV in vitro of the 90% ethanol extract and their structures were elucidated as (+)-catechin (1), (-)-epicatechin (2), (+)-catechin 3-O-alpha-L-rhamnopyranoside (3), kaempferol 3-O-alpha-L-rhamnopyranoside (4), quercetin 3-O-alpha-L-rhamnopyranoside (5), erigeside C (6) and daucosterol (7).</p><p><b>CONCLUSION</b>Seven compounds were isolated from this plant for the first time, but none of them exhibited active against HIV in vitro. Compounds 3 and 6 were isolated from this genus for the first time.</p>

Isolation, purification and structural analysis of GL-PP-3A, an active polysaccharide peptide from Ganoderma lucidum / 药学学报

GL-PP-3A, an active polysaccharide peptide, was isolated and purified from Ganoderma lucidum, and then its structure was analyzed. Crude polysaccharide peptides were extracted from Ganoderma lucidum with hot water, precipitated with ethanol and then dialyzed from Ganoderma lucidum. Subsequently GL-PP-3A was isolated and purified from the crude polysaccharide peptides by fractional precipitation and chromatography of Bio-Gel P-10 column. The repetitive unit of GL-PP-3A was analyzed by high performance gel permeation chromatography (HPGPC), monosaccharide composition and methylation analysis, 1H NMR and 13C NMR. GL-PP-3A is a heteropolysaccharide which is composed mainly of glucose (Glc), and also contains saccharide residues such as rhamnose (Rha), xylose (Xyl), mannose (Man) and galactose (Gal) and 17 kinds of amino acids. Its weight-average molecular weight (Mw) and number-average molecular weight (Mn) were 1.7 x 10(4) and 1.1 x 10(4), respectively, with the ratio of Mw/Mn ( molecular weight distribution) being of 1.49. Its backbone chain is composed of 1,6-linked beta-D-Glcp and 1,3-liked beta-D-Glcp at a ratio of 2:1. Some of 1,6-linked glucose residuals of the backbone chain are substituted at 2-0 or 3-0, and there are 1 to 3 1,6-linked beta-D-Galp or 1,3-linked alpha-D-Manp in the branched chains, the nonreducing ends of which consist mainly of beta-D-Glcp and a few Rha.

Isolation and production of biosurfactant from pseudomonas aeruginosa isolated from Iranian southern wells oil

In this study one hundred and fifty two bacterial strains were isolated from oil contaminated. Hemolysis was used as a criterion for the primary isolation of biosurfactant producing-bacteria. Fifty five strains had haemolytic activity, among them twelve strains were good biosurfactant producers by measuring surface tension and emulsification activity. Two microorganisms showed the highest biosurfactant production when grown on paraffin and glycerol as sole carbon source. As a result of biosurfactant synthesis the surface tension of the medium were reduced from 73 mN/m to values below 32 mN/m. A rhamnolipid producing bacterium, P.aeruginosa isolate from oil wells in the southern of Iran. Isolated strain was identified by morphological, biochemical, physiological. The identified Pseudomonas aeruginosa confirmed by Persian type culture collection. Glycolipid production by isolated bacterium using different carbon [gasolin, paraffin oil, glycerol, whey] and nitrogen sources [NaNO[3], [NH[4]][2]SO[4] and CH[4]N[2]O] was studied. Biosurfactant production was quantified by surface tension reduction, critical micelle dilution [CMD], emulsification capacity [EC], and ThinLayerChromatogeraphy. The best result were obtained when using glycerol as a C/N ratio of 55/1 and use of sodium nitrate as nitrogen source resulted in higher production of the rhamnolipid, expressed by rhamnose [4.2 g/l] and by the yield in relation to biomass [Yp/x = 0.65 g/g]. Additionally, physical-chemical characteristics of the spent broth with and without cells were studied, providing a low critical micelle concentration of 19 mg/l and surface tension was reduced to 20 mN/m [%]

A new lignan glycoside from the flower of Castanea mollissima Blume / 药学学报

<p><b>AIM</b>To study the bioactive constituents of the flower of Castanea mollissima Blume.</p><p><b>METHODS</b>Compounds were isolated and purified by column chromatography of silica gel and TLC. Structures were determined by various spectroscopic data, including IR, 1HNMR and 13CNMR, EIMS, FABMS and HMBC as well as comparison of the data with those reported in literatures.</p><p><b>RESULTS</b>Five compounds were isolated and elucidated as myricetin (I), quercetin (II), gallic acid (III), 4-quinolinone-2-caboxylic acid (IV), (+) -isolariciresinol-9'-O-alpha-L-rhamnoside (V).</p><p><b>CONCLUSION</b>These compounds were separated from the flower for the first time and compound V is a new compounds, named chestnutlignansoide.</p>

Chemical studies on SC3, a polysaccharide from Salvia chinensis / 药学学报

<p><b>AIM</b>To study the chemical structure of SC3, an acidic polysaccharide from Salvia chinesis.</p><p><b>METHODS</b>Based on chemical (including sugar composition analysis, methylation analysis, uronic acid reduction and partial acid hydrolysis) and spectral analysis (IR, NMR, ESI-MS), the structural characterization of SC3 was investigated.</p><p><b>RESULTS</b>SC3 was composed Rha, Ara, Gal and GalA, with its mean molecular weight of 7.7 x 10(4). By means of methylation analysis, partial acid hydrolysis, NMR and ESI-MS spectrum, the linkages and sequence information of SC3 were obtained.</p><p><b>CONCLUSION</b>SC3 is an complicated acidic polysaccharide, obtained for the first time from the plant.</p>

Development of a Mycobacterial Gene Knock-out System using Sequence-specific Recombinase FLP/FRT and its Application to the Construction of a Rhamnose Biosynthetic Gene rmlD Deletion Mutant

Development of a new and effecient tuberculosis vaccine is very important since the efficacy of the only available vaccine against tuberculosis, BCG, is variable among races and different ages. Attempts to develop attenuated vaccines by disrupting virulence gene(s) specifically in Mycobacterium tuberculosis are now actively being tried after the release of whole genome sequence of M. tuberculosis in 1998. However, disruption of specific genes in M. tuberculosis is still very difficult due to the lack of effective gene knock-out system(s) in mycobacteria. In this study, we developed a novel method to delete specific genes in both Escherichia coli and mycobacteria. This knock-out system is operated by a sequence-specific recombinase FLP and its recognition sequence FRT (FLP/FRT system). Two shuttle vectors (an FLP expressing vector and a gene targeting vector) between Escherichia coli and Mycobacteria were developed. The gene targeting vector contains a kanamycin resistance gene (KmR) flanked by two neighboring genes and two FRTs (FLPrecognition targets). We applied this system to knock-out the rhamnose biosynthetic gene rmlD of Escherichia coli. The upstream and downstream genes of rmlD, rmlB and rmlA, were cloned into the gene targeting vector. After and allelic exchange of E. coli chromosomal rmlB, rmlD, rmlA with vectoral rmlB, FRT-KmR-FRT, rmlA by homologous recombination, FLP-expressing plasmid was introduced to induce the excision of KmR cassette remaining one FRT sequence between rmlB and rmlA. We also demonstrated our shuttle vector could disrupt a target gene (kanamycin resistance gene) in M. smegmatis. These results suggest that our gene knock-out system can be used for the development of an attenuated tuberculosis vaccines and for the functional genomic study of mycobacteria.

Structural features of a neutral heteropolysaccharide CPB-4 from Cynanchum paniculatum / 中国中药杂志

<p><b>OBJECTIVE</b>To study the chemical features of CPB-4, a heteropolysaccharide obtained from Cynanchum paniculatum.</p><p><b>METHOD</b>Sugar composition analysis, methylation analysis, partial hydrolysis and carbon-13 nuclear magnetic resonance were used to determine the sugar composition, linkages, main chain, branch chains and branching points.</p><p><b>RESULT</b>CPB-4 is composed of L-arabinose, L-xylose, L-rhamnose and D-galactose in closely molar ratios of 0.8:0.2:0.2:1.0. Its main chain is comprised of 1, 5 linked galactose and side chains are comprised of terminal xylose, terminal arabinose, oligosaccharide of arabinose and oligosaccharide of arabinose, rhamnose and galactose. The branching points are located at C-6 and C-2 of galactose.</p><p><b>CONCLUSION</b>CPB-4 is a new heteropolysaccharide from C. paniculatum.</p>

Biochemical Tests for Differential Identification of Enterococci with VanC phenotype / 대한임상미생물학회지

BACKGROUND: Pigment production and acidification of ribose are most frequently used biochemical tests for the differentiation of three enterococcal species carrying vanC genes such as Enterococcus gallinarum, Enterococcus casseliflavus, and Enterococcus flavescens. However, pigment production may occasionally be negative in E. casseliflavus, and some of E. casseliflavus may be negative or delayed reaction with ribose fermentation test. So, we performed this study to find out biochemical tests capable of distinguishing the strains possessing vanC genotypes. METHOD: A total of 17 enterococci composed of 14 clinical isolates with motility or pigment positive strains and three ATCC strains(E. gallinarum ATCC 49573, E. casseliflavus ATCC 25788, and E. flavescens ATCC 49997) Were tested by multiplex PCR of the vanC genes(vanC-1, vanC-2 and vanC-3)and various biochemical tests. RESULTS: Among the 17 isolates including three ATCC control strains, four were genotyped as VanC-1, 11 were VanC-2, one were vanC-2/3, and any of vanC genes were not detected in one clinical isolate, respectively, Among the enterococci with vanC genotype, acid production from alphaD-cyclodextrin and hippurate hydrolysis were positive only in VanC-1 gneotype(E. gallinarum), acid production from glycerol and methyl-alpha-D-mannopyranoside were positive only in vanC-2 genotype(E. casseliflavus), and acid production from rhamnose and pigment production were negative only in VanC-1 genotype. Acid production from alphaD-cyclodextrin was negative only in vanC-2 genotype. The positive rate of ribose fermentation of VanC-1, VanC-2, and VanC-2/3(E. flavescens) genotype were 100%, 82%, and 0%, respectively. CONCLUSION: Acid production from rhamnose, alphaD-cyclodextrin, betaD-cyclodextrin, glycerol and methly-alphaD-mannopyranoside, pigment production, and hippurate hydrolysis test were useful biochemical tests for differentitating E. gallinarum form E. casseliflavus. The production of acid from alphaD-cyclodextrin, glycerol, methyl-alpha-D-mannopyranoside and were suitable biochemical tests for differentiating E. casseliflavus from E. flavescens.

Presença de cepas de Listeria patogênica em linguiça frescal de carne suína, comercializada em supermercados de Goiânia, Goiás / Presence of pathogenic strains of Listeria in fresh pork sausages, vended in supermarktes of Goiânia, Goiás, Brasil

Observou-se a presença de 5 (11,9 pôr cento) amostras contendo cepas de Listeria monocytogenes pertencentes ao sorotipo 4b, em 42 amostras de linguiça fescal de carne suína, comercializadas em supermercados da cidade de Goiânia. A patogenicidade foi verificada e confirmada, nas amostras positivas, quando foram submetidas ao teste de Anton. Este percentual de amostras patogênicas evidencia o problema da possível veiculaçäo de Listeria ao consumidor, por alimentos