Comparison of the main siderophores produced by some species of Streptomyces

The production of siderophores by four Streptomyces strains, S. ambofaciens, S. coelicolor, S. lividans, and S. viridosporus, was studied under iron-limited conditions. S. viridosporus produced two different siderophores: the linear desferrioxamine B and the cyclic desferrioxamine E. The latter was produced by the other strains and was the main siderophore of S. ambofaciens. The linear desferrioxamine G was the major form of S. coelicolor and S. lividans. The uptake rates of ⁵⁵Fe-labeled ferrioxamines by S. lividans and S. viridosporus showed that the G form was incorporated less efficiently than the B and E forms.     

Candida albicans cell wall lytic enzyme produced by Streptomyces thermodiastaticus

The production of lytic enzyme by Streptomyces thermodiastaticus was found to be affected by some growth conditions and nutritional factors. The highest enzyme production was obtained after 18 h of incubation at pH 5.5 and at 50 degrees C. The carbon source influenced the lytic enzyme production. A higher enzyme yield was obtained when Candida albicans cell wall (1 g/100 ml) was used as the sole carbon source. NaNO3 at 0.1 g/100 ml was the best nitrogen source for enzyme production. From all phosphorous sources, microelements, and growth factors tested, KH2PO4 (1 g/l), ZnSO4 (1 mg/I) and Tween 80 (0.1%), respectively, were found to favour the highest production of lytic enzymes by S. thermodiastaticus. The lytic enzymes mainly produced chitinolytic and proteolytic activities.     

Enzymes of Candida albicans cell-wall lytic system produced by Streptomyces thermodiastaticus

The production of the enzymes of Candida albicans cell-wall lytic system by S. thermodiastaticus was found to be affected by some growth conditions and nutritional factors. The highest lytic activity was obtained after 18 h of incubation at pH 5.5 and an incubation temperature of 50 degrees C. The carbon source influenced the production of the enzymes of the yeast cell wall lytic system. Maximum lytic activity was obtained when Candida albicans cell-wall (1 g/100 ml) was used as the sole carbon source. NaNO3 at 0.1 g/100 ml level was the best nitrogen source for the biosynthesis of the enzymes of the yeast lytic system. From all phosphor sources, microelements, and growth factors tested, KH2PO4 (1 g/l), ZnSO4 (1 mg/l) and Tween 80 (0.1%), respectively were found to favour highest enzymes production of the lytic system. The Candida albicans cell-wall lytic system produced by S. thermodiastaticus mainly contained chitinolytic and proteolytic activities.     

Protease production by Streptomyces thermovulgaris grown on rapemeal-derived media

A range of actinomycete species was tested for their ability to grow on particulate and particle-free rapeseed meal-derived media. Streptomycetes grew on both types of medium and produced a number of extracellular enzymes. Highest activities of protease were produced by Streptomyces thermovulgaris and reflected the high available protein content of rapemeal. Enzyme production and growth were analysed in fermentor-grown batch cultures of S. thermovulgaris using the particle-free rapemeal broth termed medium B. Growth was biphasic and the majority of the protease was produced during the second slower phase. Analysis of the protease as azocaseinase activity revealed a high degree of thermostability in the presence of calcium such that approximately 20% of the activity remained after incubation at 70°C for 24 h. Gel filtration suggested that S. thermovulgaris synthesized more than one kind of protease and this was confirmed by using specific peptide substrates and inhibitors which revealed the presence of distinct serine and metallo-type enzymes.     

An ammonium sulfate sensitive endoxylanase produced by Streptomyces

Streptomyces sp. CSWu2 was newly isolated and identified from Korean soil. In culture medium, the strain produced a highly active endoxylanase (Xynwu2), which was purified to homogeneity by a single-step chromatography on Poros-HQ. The xylanase was ~38 kDa and its activity was maximal at 65 °C and pH 11.0. It was stable up to 60 °C and from pH 8.0 to 12.0, and its activity was slightly enhanced by nonionic detergents, but inhibited by EDTA, EGTA, and divalent metal ions. Intriguingly, Xynwu2 was highly sensitive to ammonium sulfate, but its completely suppressed activity was recovered by desalting out. Xynwu2 produced xylose and xylobiose as principal end products from xylan, suggesting an endoxylanase nature. Importantly, scanning electron microscopy showed Xynwu2 efficiently degraded corncobs, an agro-industrial waste material. We believe that Xynwu2 is a potential candidate for converting lignocellulosic waste material into simple sugars which could be used to produce bioethanol and other value-added products.     

Isolation and characterization of an antibiotic produced by the scab disease-suppressive Streptomyces diastatochromogenes strain PonSSII

An antibiotic produced by the scab disease-suppressive Streptomyces diastatochromogenes strain PonSSII has been isolated and partially characterized. The antibiotic is produced throughout culture growth, with maximum amounts accumulating in the broth when the culture is in the early stationary phase of growth. The activity declines within about 30 h after the culture enters stationary phase. Purification techniques included chromatography on Amberlite XAD-2, DEAE Sephadex and SP Sephadex in addition to C18 HPLC with an average yield of 75%. This antibiotic only inhibits pathogenic strains of S. scabies that cause scab disease on potato and other tuberous vegetables and does not affect S. griseus, S. venezuelae, Actinomyces bovis, Nocardia asteroides, Clostridium perfringens, Bacillus subtilis, Staphylococcus aureus, S. epidermidis, Enterococcus faecalis, Micrococcus luteus, Serratia marcescens and Escherichia coli. The antibiotic has a molecular weight of 500 or less, and is stable for weeks at acidic pH but is very labile at alkaline pH conditions. Received 18 February 1997/ Accepted in revised form 11 August 1997     

Isolation, purification and characterization of levorin produced by Streptomyces levoris 99/23

Levorin produced by Streptomyces levoris 99/23 was isolated, purified and characterized. It was established that 80% of the levorin was localized in the mycelium and only 20% was in the cell-free supernatant. Amorphous yellow levorin with activity of 24 000 IU/mg and 96% purity was obtained. The preparation exhibited three absorption maxima: at λ 362, 382 and 404 nm. The antibiotic contained seven components: A₀, A₁, A₂, A₃, A₄, and two unidentified ones. According to its composition, the preparation corresponded to the levorin used for medicinal purposes. However, the levorin produced by S. levoris 99/23 contained half as much levorin A₂ and a more than 100 times larger quantity of the more active and less toxic component levorin A₃.     

Molecular cloning and expression in Streptomyces lividans of a proteinous alpha-amylase inhibitor (HaimII) gene from Streptomyces griseosporeus

The gene encoding a proteinous alpha-amylase inhibitor (HaimII) of Streptomyces griseosporeus YM-25 has been cloned in Escherichia coli K12 using a deoxyinosine-containing synthetic oligonucleotide as the probe. A 1.6 kilobases BamHI fragment was confirmed to hybridize with the probe and subcloned in an E. coli-S. lividans shuttle vector. The plasmid clone was transferred into S. lividans by transformation. An appreciable amount of alpha-amylase inhibitor activity was found in the culture medium of S. lividans harboring the plasmid. As the specificity was indistinguishable from that of HaimII produced by the original S. griseosporeus strain, we concluded that the HaimII protein was synthesized in S. lividans and excreted into the medium.     

Characterization of a thermostable and solvent-tolerant laccase produced by Streptomyces sp. LAO

Objectives

Decaying wood samples were collected, and actinomycetes were isolated and screened for laccase production. The identity of the efficient laccase-producing isolate was confirmed by using a molecular approach. Fermentation conditions for laccase production were optimized, and laccase biochemical properties were studied.

Results

Based on the 16S rRNA gene sequencing and phylogenetic analysis, the isolate coded as HWP₃ was identified as Streptomyces sp. LAO. The time-course study showed that the isolate optimally produced laccase at 84 h with 40.58?±?2.35 U/mL activity. The optimized physicochemical conditions consisted of pH 5.0, ferulic acid (0.04%; v/v), pine back (0.2 g/L), urea (1.0 g/L), and lactose (1 g/L). Streptomyces sp. LAO laccase was optimally active at pH and temperature of 8.0 and 90 °C, respectively, with remarkable pH and thermal stability. Furthermore, the enzyme had a sufficient tolerance for organic solvents after 16 h of preincubation, with laccase activity?>?70%. Additionally, the laccase maintained considerable residual activity after pretreatment with 100 mM of chemical agents, including sodium dodecyl sulphate (69.93?±?0.89%), ethylenediaminetetraacetic acid (93.1?±?7.85%), NaN₃ (96.28?±?3.34%) and urea (106.03?±?10.72%).

Conclusion

The laccase's pH and thermal stability; and robust catalytic efficiency in the presence of organic solvents suggest its industrial and biotechnological application potentials for the sustainable development of green chemistry.

     

Production of Hydrogen Sulfide by Streptomyces odorifer

By use of various trapping systems, as well as lead acetate papers, Streptomyces odorifer was shown to produce hydrogen sulfide. Other sulfur-containing compounds may be produced by S. odorifer, but the amounts obtained were too small for detailed analysis. It was suggested that hydrogen sulfide might be a part of the earthy-odor complex produced by S. odorifer.     

Purification and characterization of lysozyme produced by Streptomyces erythraeus.

A species of lysozyme (SE lysozyme) was purified from culture filtrate of Streptomyces erythraeus. The enzyme has a molecular weight of 18,500 as determined by ultracentrifugation. Its isoelectric point is 9.5, and it shows optimal activity at pH 4.0 with an optimal ionic strength of 0.1. Investigation of the substrate specificity showed SE lysozyme to be an N-acetyl-muramidase. The simplest product in the digest of cell walls of Micrococcus lysodeikticus was identified as a disaccharide, [GlcNAcbeta(1 leads to 4) MurNAc]. While S. aureus as well as M. lysodeikticus was lysed by this lysozyme, chitin and its derivatives were not.     

A new nematicidal compound produced by Streptomyces albogriseolus HA10002

Strain HA10002 was isolated from mangrove sediment collected from Dongzhaigang Mangrove Reserve in Hainan, China. It was selected with potent nematicidal activity and was identified as Streptomyces albogriseolus. By bioassay-guided fractionation, a new active component A22-1(S1) against root-knot nematodes was separated from its fermentation broth. On the basis of spectroscopic analyses and comparison with the data from correlative literature, the structure of S1 was established to be 6′-methyl-fungichromin, named as fungichromin B in this paper. The LD50 values of fungichromin B to the 2-stage juveniles of Meloidogyne incognita and Meloidogyne javanica were 7.64 and 7.83 μg/ml, respectively. Further examination demonstrated fungichromin B still showed a wide antifungal spectrum, as with fungichromin.     

Dibutyl phthalate, the bioactive compound produced by Streptomyces albidoflavus 321.2

It was found that the bioactive compound, dibutyl phthalate, was produced by a new soil isolate Streptomyces albidoflavus 321.2. Once this active compound was recovered by ethyl acetate from the fermented broth, being possible to isolate 13.4 mg/l, it was purified by paper, silica gel column, thin layer and gas chromatography. Structure was determined by analysing UV, IR and GC-MS spectra. During analysis, such active compound showed strong activity against gram-positive and gram-negative bacteria, as well as unicellular and filamentous fungi. The antimicrobial activity of the compound was reversed by the amino acid proline. No acute toxicity was observed.     

Effect of amino acids on thaxtomin A biosynthesis by Streptomyces scabies

The regulatory effect of amino acids on the production of thaxtomin A, a phytotoxin produced by Streptomyces scabies, was investigated. Tryptophan had an important inhibitory effect on the toxin biosynthesis in all five strains of S. scabies tested. Two other aromatic amino acids (tyrosine and phenylalanine) also inhibited thaxtomin A biosynthesis, while aliphatic amino acids did not cause an important decline in thaxtomin A production. Methylation of tryptophan prevented or reduced the inhibitory effect on thaxtomin A biosynthesis. In spite of the inhibitory action of tryptophan and phenylalanine on thaxtomin A production, incorporation of these radiolabeled molecules into thaxtomin A confirmed that they are metabolic precursors for the biosynthesis of the phytotoxin.     

Determination of ionophore antibiotics nactins produced by fecal Streptomyces from sheep

To investigate the correlation between fecal actinobacteria and host animals, Streptomyces was isolated from fresh faeces of healthy sheep and secondary metabolites were analyzed. The most frequently isolated strain S161 with antibiotic activity against bacteria and fungi were analyzed. The S161 showed the highest 99 % similarity to Streptomyces canus DSB17 based on the 16S rRNA gene sequence analysis. Metabolite analysis based on MS and NMR spectra showed that S161 produces nactins, cyclotetralactones derived from nonactic acid and homononactic acid as building units of ionophoretic character. Due to ionophores are antimicrobial compounds that are commonly fed to ruminant animals to improve feed efficiency, stable beneficial interactions between Streptomyces bacteria and vertebrates have been demonstrated.     

A bleaching herbicidal activity of methoxyhygromycin (MHM) produced by an actinomycete strain Streptomyces sp. 8E-12

AIMS: To screen bioherbicidal isolates and evaluate herbicidal activity of methoxyhygromycin (MHM) produced by Streptomyces sp. 8E-12. METHODS AND RESULTS: Streptomyces sp. 8E-12 with herbicidal activity was selected through seed germination bioassay. An active metabolite, MHM was isolated from culture broth by carbon absorption, butanol extraction, silica gel, Sephadex LH-20 and G-10 chromatography, and preparative HPLC. The metabolite was identified by electrospray ionization mass spectra (ESI-MS) and 1H- and 13C-NMR spectral data analyses. In vivo herbicidal activity was examined against weeds and crops grown on pots. CONCLUSIONS: Streptomyces sp. 8E-12 produced a selective herbicidal metabolite which was identified as MHM. The metabolite showed stronger in vivo activity against monocotyledonous plants than dicotyledonous plants, and caused a bleaching (albino symptom) on some weeds including Digitaria sanguinalis and Echinochloa crus-galli. SIGNIFICANCE AND IMPACT OF THE STUDY: These results showed that Streptomyces sp. 8E-12 produced a bioherbicidal metabolite, MHM and can be developed as a biocontrol agent (BCA) for weed control.     

黑暗链霉菌中tbmA基因的功能研究

PCR获得tbmA基因内部863 bp片段,构建基因阻断穿梭载体pSPU112-1,经接合转移导入Strepto-myces tenebrariusH6,筛选单交换阻断变株,并用Southern blot验证阻断变株的tbmA已经被破坏。经发酵产物分析,阻断变株不再合成氨甲酰妥布霉素,只合成安普霉素。首次从分子水平证明了tbmA只参与氨甲酰妥布霉素生物合成,而不参与安普霉素的生物合成。     

A protease inhibitor produced by Streptomyces lividans 66 exhibits inhibitory activities toward both subtilisin BPN' and trypsin.

A proteinaceous protease inhibitor was isolated from the culture broth of Streptomyces lividans 66 by a series of purification steps (salting out by ammonium sulfate, ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on Phenyl-Sepharose, and gel-filtration on Sephacryl S-200), and was named S. lividans protease inhibitor (SLPI). The purified SLPI existed in a dimeric form consisting of two identical subunits, each of which was composed of 107 amino acids. SLPI exhibited strong inhibitory activity toward subtilisin BPN'. These features were similar to those of protein protease inhibitors produced by other Streptomyces (SSI family inhibitor). In addition, SLPI was capable of inhibiting trypsin with an inhibitor constant (Ki) of about 10(-9) M. The primary structure of SLPI and location of two disulfide bridges were homologous to those of the other serine protease inhibitors of Streptomyces. The reactive site of SLPI was found to be Arg67-Glu68 from the sequence analysis of cleaved SLPI which was produced by acidification of subtilisin-SLPI complex. An Arg residue at the P1 site was consistent with the trypsin-inhibitory property of SLPI. Sequence comparison with other members of the SSI family revealed that amino acid replacements in SLPI were mainly localized on the surface of the SLPI molecule, and many of the amino acid residues in beta-sheets and hydrophobic core were well conserved.     

大孔吸附树脂吸附链霉菌702抗真菌活性物质工艺

研究大孔吸附树脂吸附链霉菌702抗真菌活性物质的工艺条件。采用5种不同大孔吸附树脂吸附链霉菌702发酵液提取液中抗真菌活性物质,选择吸附效果较佳树脂进行吸附条件优化,以桔青霉为指示菌,纳他霉素为对照抗生素．采用“管碟法”测定抗真菌活性物质含量。结果发现,XAD18树脂吸附效果较好,获得优化吸附条件：上样液pH6,NaCl质量浓度10g/L,上样量22．63mg／g湿树脂,吸附流速2．5mL／min,水洗体积180mL,洗脱流速1．5mL／min,洗脱剂为体积分数10％、50％和90％的甲醇,洗脱方式为梯度洗脱。在确定的工艺条件下XAD18对链霉菌702抗真菌活性物质的吸附率可达90％,洗脱率可达75％,回收率可达80％。