Production of bioactive tryptamine derivatives by co-culture of marine Streptomyces with Bacillus mycoides

Tryptamine derivatives such as tryptamine and bacillamides were strong algicidal compounds promising in controlling harmful algae blooms, but their bioactivity and application researches were hindered by extremely low natural production rates. This study found an induced production of algicidal tryptamine derivatives by co-culture of marine Streptomyces with Bacillus mycoides, and optimised the culture method through changing important factors such as medium nutrition content, culture mode and pH value. The final established co-culture method used only 5 g yeast extracts and 5 g glycerol in 1 L 75% sea water, but got a yield of 14.9 mg/L N-acetyltryptamine, 2.8 mg/L N-propanoyltryptamine, 3.0 mg/L bacillamide A, 13.7 mg/L bacillamide B and 9.6 mg/L bacillamide C, which were all undetectable under normal culture conditions.     

Characterization of covalently inhibited extracellular lipase from Streptomyces rimosus by matrix-assisted laser desorption/ionization time-of-flight and matrix-assisted laser desorption/ionization quadrupole ion trap reflectron time-of-flight mass spectrometry: localization of the active site serine

A chemical modification approach combined with matrix-assisted laser desorption/ionization (MALDI) mass spectrometry was used to identify the active site serine residue of an extracellular lipase from Streptomyces rimosus R6-554W. The lipase, purified from a high-level overexpressing strain, was covalently modified by incubation with 3,4-dichloroisocoumarin, a general mechanism-based serine protease inhibitor. MALDI time-of-flight (TOF) mass spectrometry was used to probe the nature of the intact inhibitor-modified lipase and to clarify the mechanism of lipase inhibition by 3,4-dichloroisocoumarin. The stoichiometry of the inhibition reaction revealed that specifically one molecule of inhibitor was bound to the lipase. The MALDI matrix 2,6-dihydroxyacetophenone facilitated the formation of highly abundant [M + 2H](2+) ions with good resolution compared to other matrices in a linear TOF instrument. This allowed the detection of two different inhibitor-modified lipase species. Exact localization of the modified amino acid residue was accomplished by tryptic digestion followed by low-energy collision-induced dissociation peptide sequencing of the detected 2-(carboxychloromethyl)benzoylated peptide by means of a MALDI quadrupole ion trap reflectron TOF instrument. The high sequence coverage obtained by this approach allowed the confirmation of the site specificity of the inhibition reaction and the unambiguous identification of the serine at position 10 as the nucleophilic amino acid residue in the active site of the enzyme. This result is in agreement with the previously obtained data from multiple sequence alignment of S. rimosus lipase with different esterases, which indicated that this enzyme exhibits a characteristic Gly-Asp-Ser-(Leu) motif located close to the N-terminus and is harboring the catalytically active serine residue. Therefore, this study experimentally proves the classification of the S. rimosus lipase as GDS(L) lipolytic enzyme.     

间接竞争酶联适配体检测食品中土霉素

研究建立了一种间接竞争酶联适配体检测食品中土霉素(OTC)的分析方法。通过方阵滴定法和单因素实验优化了检测条件。在最佳实验条件下,方法的半抑制浓度(IC50)为6.3 ng/m L,对OTC的检测线性范围为0.5~50 ng/m L;与结构类似物有较低的交叉反应;对牛奶、奶粉、鸡肉、水产品和蜂蜜样品中土霉素的加标回收率为62.1%~102%,相对标准偏差(RSD)小于15%。将建立的方法用于实际样品检测,并与国标方法进行对比,两者获得较高的相关性(r2=0.979)。本方法可实现对实际样品中土霉素的快速、高灵敏和高通量检测。     

Analysis of Tetracycline,Oxytetracycline and Chlortetracycline in Plasma Extracts by Electrospray Tandem Mass-Spectrometry and by Liquid Chromatography

Abstract

Pure standards of tetracyclines (tetracycline, oxytetracycline and chlortetracycline) have been analyzed on a triple quadrupole mass spectrometer with Atmospheric Pressure Chemical Ionization (APCI) and Electrospray Ionization (ESI). ESI appeared to be considerably more sensitive than APCI. Collisional activation of the MH⁺ ions gave fragment ions at m/z values equal to MH⁺ - 35 which corresponds to loss of both H₂O and NH₃. The collisionally induced loss of 35 from MH⁺ was used in developing a mass spectrometric method based on loop injection and selected reaction monitoring (SRM) as the final analytical step. The method was tested on extracts from fortified plasma and the measurements from the MS-MS analysis were compared with results from High Performance Liquid Chromatography (HPLC) analysis of the same samples. The fortified plasma (from pig) samples were purified by chelate affinity chromatography (amberlite XAD columns). After filtration and evaporation of the solvent the redissolved residues were analyzed by HPLC and by MS-MS with ESI. The HPLC eluates (gradient of 0.01 M aqueous oxalic acid and CH₃CN) were monitored at 356 and 369 nm. The signal to noise ratio in the analyses of extracts from plasma fortified to 20 ppb suggests a detection limit for the MS-MS method below 10 ppb of tetracylines in plasma.     

毛细管电泳电化学发光法测定蜂蜜中土霉素残留量

基于土霉素对联吡啶钌(Ru(bpy)2+3)的电化学发光(ECL)信号的增强效应,建立了毛细管电泳-电化学发光法(CE-ECL)测定土霉素的新方法。利用场放大效应将检测灵敏度提高了3.5倍,最佳检测电位为1.22 V,运行缓冲液为8 mmol·L-1硼砂溶液(p H 8.0),进样时间及电压分别为6 s和10 k V。在优化条件下,土霉素可在9 min内检出,其线性范围为20~200μg·L-1,相关系数为0.995 3,仪器检出限(S/N=3)为5μg·L-1;5次测定100μg·L-1土霉素溶液的ECL信号和迁移时间的日内重现性分别为2.4%和2.1%,日间重现性分别为3.6%和2.8%。方法用于市售蜂蜜中土霉素残留的测定,在200,1 000,2 000μg·kg-1加标浓度下的回收率为91.5%~105.2%。方法检出限(LOD,S/N=3)为10μg·kg-1,低于食品中土霉素的最大残留限值(MRL)。结果表明该方法在食品分析中具有一定的应用价值。     

Production of C-terminal amidated recombinant salmon calcitonin in Streptomyces lividans

Salmon calcitonin (sCT) is one of the many bioactive peptides that require C-terminal amidation for full biologic activity. To produce fully bioactive sCT in large scale, we constructed Streptomyces lividans [pMSA], an engineering Streptomyces strain. In the expression vector, glycine-extended sCT, the substrate for amidation, and rat α-amidating enzyme cDNA were cloned under the control of the strong constitutive promoter from the Streptomyces fradiae aph gene in pIJ680. Both were expressed in a secretory manner by the recombinant strain using the expression and secretion signals of melC1. Extracellularly expressed recombinant sCT was purified to near homogeneity and characterized by enzyme immunoassay, followed by direct amino-terminal sequencing. High-performance liquid chromatography, matrixassisted laser desorption ionization-time-of-flight mass spectrometry, and bioassay in vivo demonstrated purified product to be equivalent to synthetic standard. Thus, the engineered Streptomyces strain can produce bioactive, C-terminal amidated recombinant sCT in the culture supernatant directly. The ease of the recombinant process, as well as its potential for scale-up, makes it adaptable to production demands for sCT, and it may be applied to other bioactive peptides that need C-terminal amidation.     

固相萃取–超高效液相色谱三重四级杆质谱联用法测定水中四环素、土霉素及罗红霉素

建立了固相萃取–超高效液相色谱三重四级杆质谱联用法同时测定水中的罗红霉素、四环素和土霉素残留。水样经过固相萃取纯化、富集,液质联用分析,采用甲酸溶液和乙腈作为流动相,在5 min内完成对3种目标化合物的分析,3种目标化合物的方法检出限介于0.08~0.35 ng/L之间,测定结果的相对标准偏差为1.4%~5.6%,空白样品和实际样品的加标回收率分别为82.5%~114%,71.5%~126%。     

Production of Siderophores by an Apple Root-Associated Streptomyces ciscaucasicus Strain GS2 Using Chemical and Biological OSMAC Approaches

Apple Replant Disease (ARD) is a significant problem in apple orchards that causes root tissue damage, stunted plant growth, and decline in fruit quality, size, and overall yield. Dysbiosis of apple root-associated microbiome and selective richness of Streptomyces species in the rhizosphere typically concurs root impairment associated with ARD. However, possible roles of Streptomyces secondary metabolites within these observations remain unstudied. Therefore, we employed the One Strain Many Compounds (OSMAC) approach coupled to high-performance liquid chromatography-high-resolution tandem mass spectrometry (HPLC-HRMSⁿ) to evaluate the chemical ecology of an apple root-associated Streptomyces ciscaucasicus strain GS2, temporally over 14 days. The chemical OSMAC approach comprised cultivation media alterations using six different media compositions, which led to the biosynthesis of the iron-chelated siderophores, ferrioxamines. The biological OSMAC approach was concomitantly applied by dual-culture cultivation for microorganismal interactions with an endophytic Streptomyces pulveraceus strain ES16 and the pathogen Cylindrocarpon olidum. This led to the modulation of ferrioxamines produced and further triggered biosynthesis of the unchelated siderophores, desferrioxamines. The structures of the compounds were elucidated using HRMSⁿ and by comparison with the literature. We evaluated the dynamics of siderophore production under the combined influence of chemical and biological OSMAC triggers, temporally over 3, 7, and 14 days, to discern the strain’s siderophore-mediated chemical ecology. We discuss our results based on the plausible chemical implications of S. ciscaucasicus strain GS2 in the rhizosphere.     

Biosynthesis of Gold Nanoparticles by Streptomyces sp. ERI-3 Supernatant and Process Optimization for Enhanced Production

In this study, we present a simple and eco-friendly method for extracellular biosynthesis of gold nanoparticles by Streptomyces sp. ERI-3 cell-free supernatant. The research was also aimed to evaluate the effects of different reaction parameters including incubation temperature, reaction time, HAuCl₄ concentration and pH on gold nanoparticles production. The UV?CVis spectroscopy was used to monitor the formation of gold nanoparticles. The synthesized gold nanoparticles were characterized with XRD, TEM, and SEM. The average particle size ranged from 10 to 30?nm with spherical shape at optimum conditions.     

Medium Optimization Based on Statistical Methodologies for Pristinamycins Production by <Emphasis Type="Italic">Streptomyces pristinaespiralis</Emphasis>

The optimization of nutrient levels for the production of pristinamycins by Streptomyces pristinaespiralis CGMCC 0957 in submerged fermentation was carried out using the statistical methodologies based on the Plackett–Burman design, the steepest ascent method, and the central composite design (CCD). First, the Plackett–Burman design was applied to evaluate the influence of related nutrients in the medium. Soluble starch and MgSO₄·7H₂O were then identified as the most significant nutrients with a confidence level of 99%. Subsequently, the concentrations of the two nutrients were further optimized using response surface methodology of CCD, together with the steepest ascent method. Accordingly, a second-order polynomial regression model was finally fitted to the experimental data. By solving the regression equation from the model and analyzing the response surface, the optimal levels for soluble starch and MgSO₄·7H₂O were determined as 20.95 and 5.67g/L, respectively. Under the optimized medium, the yield of pristinamycins in the shake flask and 5-L bioreactor could reach 1.30 and 1.01g/L, respectively, which is the highest yield reported in literature to date.     

Production of Hydrogen-Rich Synthesis Gas by Pulsed Atmospheric Plasma Submerged in Mixture of Water with Ethanol

Hydrogen-rich synthesis gas was produced by pulsed dc plasma submerged into ethanol–water mixtures using an original system with a coaxial geometry. The ignition of the discharge is immediately followed by production of hydrogen and after a short time necessary for filling the outlet tubing a flame can be ignited. No auxiliary gas was used for the reforming process. The synthesis gas containing up to 60% of hydrogen was formed, at the outflow rate of 250 sccm at the average power as low as 10 W. The hydrogen production efficiency corresponds to 12 kWh/kg H₂.     

Effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A

The effect of aeration on lignin peroxidase production by Streptomyces viridosporus T7A was studied in a bench-scale bioreactor using a previously optimized growth medium (0.65% yeast extract and 0.1% corn oil, pH7.0) at 37°C and natural pH. Airflow rates of 0.3, 1.0, and 1.5 vvm and a fixed agitation of 200 rpm were initially studied followed by 1.0 vvm and 200, 300, 400, and 500 rpm. The use of 1.0 vvm and 400 rpm increased enzyme concentration 1.8-fold (100–180 U/L) and process productivity 4.8-fold (1.4–6.7 U/[L·h]) in comparison with the use of 200 rpm and 0.3 vvm. The inexpensive corn oil, used as carbon source, besides its antifoam properties, proved to be nonrepressive for enzyme production.     

Istamycin aminoglycosides profiling and their characterization in Streptomyces tenjimariensis ATCC 31603 culture using high‐performance liquid chromatography with tandem mass spectrometry

A high‐performance liquid chromatography with electrospray ionization ion trap tandem mass spectrometry method was developed and validated for the robust profiling and characterization of biosynthetic congeners in the 2‐deoxy‐aminocyclitol istamycin pathway, from the fermentation broth of Streptomyces tenjimariensis ATCC 31603. Gradient elution on an Acquity CSH C₁₈ column was performed with a gradient of 5 mM aqueous pentafluoropropionic acid and 50% acetonitrile. Sixteen natural istamycin congeners were profiled and quantified in descending order; istamycin A, istamycin B, istamycin A₀, istamycin B₀, istamycin B₁, istamycin A₁, istamycin C, istamycin A₂, istamycin C₁, istamycin C₀, istamycin X₀, istamycin A₃, istamycin Y₀, istamycin B₃, and istamycin FU‐10 plus istamycin AP. In addition, a total of five sets of 1‐ or 3‐epimeric pairs were chromatographically separated using a macrocyclic glycopeptide‐bonded chiral column. The lower limit of quantification of istamycin‐A present in S. tenjimariensis fermentation was estimated to be 2.2 ng/mL. The simultaneous identification of a wide range of 2‐deoxy‐aminocyclitol‐type istamycin profiles from bacterial fermentation was determined for the first time by employing high‐performance liquid chromatography with tandem mass spectrometry analysis and the separation of istamycin epimers.     

Induced production of cytochalasans in co-culture of marine fungus Aspergillus flavipes and actinomycete Streptomyces sp.

Secondary metabolites profiles of co-culture of Aspergillus flavipes and Streptomyces sp. that isolated from the same habitat showed an induced production of a series of cytochalasans (five aspochalasins and rosellichalasin, determined by MS and NMR analysis). These cytochalasans were found to be produced by A. flavipes in LC–MS comparison analysis, and biological activity assays revealed that they were able to cause cytotoxic effects against Streptomyces sp. within a wide range of concentrations without causing any effect to the producer A. flavipes, which favoured the producer in competition. Further induction mechanism study applying membrane-separated culture and morphology study with scanning electron microscopy (SEM) suggested that the successful induction of active secondary metabolites required microbial physical contact.     

Improved Polysaccharide Production in Submerged Culture of Ganoderma lucidum by the Addition of Coixenolide

Polysaccharides from Ganoderma lucidum have various bioactivities and have been widely used as nutraceuticals and functional foods. Coixenolide was added into the media to enhance the production of mycelia biomass and polysaccharides in the submerged culture of G. lucidum in this work. The results showed that when a level of 0.2 % coixenolide was added at day 1, the biomass, exopolysaccharide, and intracellular polysaccharide reached 5.224, 0.222, and 0.399 g l^?1, respectively, which were 1.39-fold, 2.58-fold, and 2.24-fold compared to that of control. Analysis of the fermentation kinetics of G. lucidum suggested that glucose concentration in the coixenolide-added group decreased more quickly as compared to the control group from days 3 to 9 of the fermentation process, and the polysaccharides biosynthesis were promoted at the same culture period. However, the culture pH profile was not affected by the addition of coixenolide. Enzyme activities analysis indicated that coixenolide affected the synthesis level of phosphoglucose isomerase and α-phosphoglucomutase.     

Production of Cordycepin and Mycelia by Submerged Fermentation of Cordyceps militaris in Mixture Natural Culture

The submerged fermentation of Cordyceps militaris for cordycepin production and mycelial growth was investigated in this study. Three natural materials of brown rice paste (BRP), beerwort (B), and soybean meal juice (SMJ) were used for fermentation of C. militaris in shaking flasks. The effects of the ratio of three natural materials on dry mycelium weight (DMW) and on cordycepin yield (CY) were analyzed. D-Optional mixture design was used to optimize the ratio of these materials. Compared with the signal culture, the higher mycelial growth and cordycepin production were obtained in mixture. The analysis of Design Expert 6.0 indicated that BRP, B, and SMJ very significantly influenced (P < 0.001) DMW and CY of C. militaris, respectively. The highest DMW (18.96 g/l) and CY (2.17 mg/g) were both obtained at a ratio of 53:6:42. The experiments’ results indicated that the above mixture of these natural materials by D-optional mixture design can be used as a proper medium for the growth of mycelium and the production of cordycepin.     

高效液相色谱同时测定饲料中三种抗生素

建立了反相高效液相色谱法同时检测饲料中土霉素、四环素、金霉素的方法.色谱柱为AichromBond-AQ C18柱,150×4.6mm,粒度5μm;流动相:0.01mol/L磷酸二氢钠溶液(pH2.5)+乙氰,采用梯度洗脱,乙氰浓度在0～13min内由13%上升到40%;柱温35℃;流速为1.0mL/min;进样量101μL;检测波长375nm.方法检出限:土霉素和四环素0.25μg/mL、金霉素0.50μg/mL,回收率91.70%～101.19%,RSD<1.39%(n=7).     

Optimization of the Production Medium for Biosynthesis of Antifungal Antibiotic Ak-111-81 by Phosphate-Deregulated Mutant of <Emphasis Type="Italic">Streptomyces hygroscopicus</Emphasis>

Experimental mathematical designs were applied for optimization of a nutrient medium for biosynthesis of the antifungal antibiotic AK-111-81 by phosphate-deregulated mutant of Streptomyces hygroscopicus 111-81. Antifungal antibiotic AK-111-81 possesses well-expressed activity against Fusarium graminearum and other phytopathogenic fungi. The level of the production of the antibiotic AK-111-81 on this medium is more than three times higher than on the initial medium. The optimized quantitative composition of the nutrient culture media is (g/l): glucose, 20; soy meal, 18; ammonium succinate, 3; CaCO₃, 1.     

Secondary Metabolites with Cytotoxic Activities from Streptomyces sp. BM-8 Isolated from the Feces of Equus quagga

A new aliphatic acid, compound 1, together with six known metabolites, including nonactic acid (2), homononactic acid (3), ethyl homononactate (4), homononactylhomononactate (5), valinomycin (6), and cyclo-(Pro-Leu) (7), was isolated from the culture broth of Streptomyces sp. BM-8, an actinobacterial strain isolated from the feces of Equus quagga. The structures of these compounds were established by analyses of spectroscopic data, including 1D and 2D nuclear magnetic resonance spectra (NMR), as well as by HR-ESI-MS spectrometry and chemical derivative analyses. Additionally, a serial analogue of nonactic acid and homononacticacid (8–21) was synthesized. The cytotoxicity of 1–21 wastested against a panel of cancer cell lines, such as HT-29, MCF-7, A375 and K562, with MTT assay. In addition, the cytotoxicity tests revealed that 1 was less cytotoxic toward a panel of cancerous cells, as compared with valinomycin (6).