Streptothiazolidine B, a new cytotoxic alkaloid produced by Streptomyces violaceoruber.

A new cytotoxic alkaloid, named streptothiazolidine B (1), together with three known compounds (2-4), were isolated from Streptomyces violaceoruber. The structure of the undescribed compound was established using 1D and 2D NMR, and HRESIMS. Streptothiazolidine B was isolated and identified as an amide alkaloid with a unique thiazolidine side chain and its absolute configuration was determined by a combination of NOESY experiment and ECD analysis. Streptothiazolidine B exhibited significant cytotoxic activities against two human tumor cell lines, Li-7 and A2780, with IC50 values of 7.8, and 9.1 µM. Meanwhile, compound 4 showed obvious cytotoxic activities against four human tumor cell lines, THP-1, HT29, Li-7 and A2780, with IC50 values ranging from 3.1 to 10.2 µM.

Safety evaluation of collagenase from Streptomyces violaceoruber.

A safety assessment was conducted for microbial collagenase (COL) enzyme expressed in Streptomyces violaceoruber. The acute oral toxicity of COL was examined in Sprague-Dawley rats and the LD50 of COL via single oral administration to rats was higher than 2000 mg/kg body weight. A 13-week oral gavage study of COL showed no adverse effects due to the enzyme up to a dose of 234.9 mg total organic solids (TOS)/kg body weight per day (NOAEL). A bacterial reverse mutation test showed no mutagenic activity at the highest dose (4698 µg TOS per plate). In the mouse lymphoma TK assay, a positive result was observed at the highest dose of 4698 µg TOS/mL although it had low reproducibility. To confirm the chromosome aberration potential, an in vivo micronucleus test was conducted that demonstrated the lack of mutagenic potential on the bone marrow of rats at doses up to 1879 mg TOS/kg body weight per day. The results of the genotoxicity studies and acute and subchronic rat studies support the safe use in food production of collagenase produced from S. violaceoruber.

Plant growth promotion and chilli anthracnose disease suppression ability of rhizosphere soil actinobacteria.

AIM: The aim of this study was to screen potential plant growth promoting rhizobacterial (PGPR) actinobacterial isolate with effective inhibition against anthracnose causing fungal pathogen Colletotrichum capsici. METHODS AND RESULTS: In this study, actinobacterias were isolated from rhizosphere soil using dilution plate method and tested for antagonistic potential against pathogenic fungi C. capsici. In primary and secondary screening tests, the actinobacterial isolate BS-26 displayed high antagonistic activity against the fungal pathogen. Isolate BS-26 was identified as Streptomyces violaceoruber based on 16S rDNA sequencing. Furthermore, indole acetic acid production, phosphate solubilization and ammonia production have been confirmed in the S. violaceoruber that suggest their potential to be used as PGPR bacteria. A green house experiment showed that application of S. violaceoruber fermentation broth reduced the incidence of the chilli anthracnose and promoted the growth of chilli seedlings with a significant increase in germination %, total plant height, fresh weight and chlorophyll content when compared to controls. CONCLUSION: Streptomyces violaceoruber can be applied as a biofertilizer and biocontrol agent for growing chillies against the attack of fungal pathogen C. capsici. SIGNIFICANCE OF IMPACT OF THE STUDY: The damage caused by anthracnose disease is an issue of concern, affecting negatively the economy involved in chilli cultivation. As chemical methods of control have serious disadvantages, biocontrol approach using beneficial (PGPR) micro-organisms shall be a better alternative to control crop diseases.

Violacin A, a new chromanone produced by Streptomyces violaceoruber and its anti-inflammatory activity.

A new chromanone derivative, named violacin A (1), was isolated from the fermentation broth of Streptomyces violaceoruber as a potential anti-inflammatory compound. The structure of violacin A was established using comprehensive NMR spectroscopic data analysis together with UV, IR, and MS data. The anti-inflammatory effects and action mechanisms of violacin A were investigated in vitro. The results demonstrated that violacin A attenuated the production of NO, IL-1ß, IL-6, and TNF-α as well as inhibited the expression of iNOS in LPS-induced RAW 264.7 cells. Additionally, Western blot and qRT-PCR results revealed that 1 down-regulated pro-inflammatory cytokines expression correlated with the suppression of NF-κB signaling pathway.

Safety evaluation of the food enzyme microbial collagenase from the genetically modified Streptomyces violaceoruber strain pCol.

The food enzyme microbial collagenase (EC 3.4.24.3) is produced with the genetically modified Streptomyces violaceoruber strain pCol by Nagase (Europa) GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in two food manufacturing processes: the production of modified meat and fish products and the production of protein hydrolysates from meat and fish proteins. The dietary exposure to the food enzyme-total organic solids (TOS) was estimated to be up to 1.098 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 940 mg TOS/kg bw per day, the highest dose tested, which, when compared with the estimated dietary exposure, resulted in a margin of exposure of at least 856. A search for the similarity of the amino acid sequence of the food enzyme to known allergens was made and no match was found. The Panel considered that the risk of allergic reactions by dietary exposure cannot be excluded, but the likelihood is low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns, under the intended conditions of use.

Safety evaluation of the food enzyme phospholipase A2 from the genetically modified Streptomyces violaceoruber strain AS-10.

The food enzyme phospholipase A2 (phosphatidylcholine 2-acylhydrolase EC 3.1.1.4) is produced with the genetically modified Streptomyces violaceoruber strain AS-10 by Nagase (Europa) GmbH. The genetic modifications do not give rise to safety concerns. The food enzyme is free from viable cells of the production organism and its DNA. It is intended to be used in four food manufacturing processes, i.e. egg processing, baking processes, degumming of fats and oils and milk processing for cheese production. Since residual amounts of total organic solids (TOS) are removed in degumming of fats and oils, dietary exposure was calculated only for the remaining three food manufacturing processes. Dietary exposure to the food enzyme-TOS was estimated to be up to 0.41 mg TOS/kg body weight (bw) per day in European populations. Genotoxicity tests did not indicate a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-day oral toxicity study in rats. The Panel identified a no observed adverse effect level of 191.2 mg TOS/kg bw per day, the mid-dose tested, which, when compared with the estimated dietary exposure, results in a margin of exposure above 460. A search for similarity of the amino acid sequence of the food enzyme to known allergens was made and no matches were found. The Panel considered that, under the intended conditions of use, the risk of allergic sensitisation and elicitation reactions by dietary exposure cannot be excluded, but the likelihood for this to occur is considered to be low. Based on the data provided, the Panel concluded that this food enzyme does not give rise to safety concerns under the intended conditions of use.

Expression of Highly Active Bacterial Phospholipase A2 in Yeast Using Intein-Mediated Delayed Protein Autoactivation.

Phospholipase A2 (PLA2) has found extensive use in industry. However, recombinant PLA2 production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA2 from Streptomyces violaceoruber strain A-2688 in the yeast Saccharomyces cerevisiae. The method is based on the use of the PRP8 mini-intein (from Penicillium chrysogenum) inserted into the phospholipase sequence with the purpose of temporal inactivation of the enzyme and its subsequent delayed autoactivation. We demonstrate that the most effective site for intein insertion is Ser76 of the mature phospholipase. As a result of intein-containing precursor secretion from yeast cells and its subsequent autocatalytic splicing, highly active enzyme accumulated in the yeast culture fluid. The properties of the obtained recombinant phospholipase A2 protein were similar to those of the native Streptomyces violaceoruber PLA2 protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed.

Safety evaluation of the food enzyme chitinase from Streptomyces violaceoruber (strain pChi).

The food enzyme, a chitinase (EC 3.2.1.14), is produced with the genetically modified Streptomyces violaceoruber strain pChi by Nagase. No information was provided regarding the presence of antimicrobial resistance genes in the production strain, other than that used in the genetic modification. The chitinase is intended to be used in baking processes. Based on the maximum use levels recommended, dietary exposure to the food enzyme-total organic solids (TOS) was estimated on the basis of individual data from the EFSA Comprehensive European Food Consumption Database. The exposure estimate is up to 0.829 mg TOS/kg body weight per day in European populations. Genotoxicity tests did not raise a safety concern. The systemic toxicity was assessed by means of a repeated dose 90-days oral toxicity study in rats. The Panel identified a no observed adverse effect level at the highest dose tested of 791 mg TOS/kg body weight, which, compared with the dietary exposure, results in margin of exposure of at least 1,171. Similarity of the amino acid sequence to those of known allergens was searched and no matches were found. The Panel considered that there are no indications for food allergic reactions to this chitinase. Based on the data provided and the derived margin of exposure, the Panel concluded that the food enzyme chitinase produced with the genetically modified S. violaceoruber strain pChi does not give rise to safety concerns arising from the toxicological studies and the production process under the intended conditions of use. The CEP Panel was unable to conclude on the absence of viable cells and DNA from the genetically modified production strain in the food enzyme, for which uncertainty remains on the possible presence of gene(s) conferring antimicrobial resistance.

Statistical optimization of alkaline xylanase production from Streptomyces violaceoruber under submerged fermentation using response surface methodology.

Response surface methodology employing central composite design (CCD) was used to optimize fermentation medium for the production of cellulase-free, alkaline xylanase from Streptomyces violaceoruber under submerged fermentation. The design was employed by selecting wheat bran, peptone, beef extract, incubation time and agitation as model factors. A second-order quadratic model and response surface method showed that the optimum conditions for xylanase production (wheat bran 3.5 % (w/v), peptone 0.8 % (w/v), beef extract 0.8 % (w/v), incubation time 36 h and agitation 250 rpm) results in 3.0-fold improvement in alkaline xylanase production (1500.0 IUml(-1)) as compared to initial level (500.0 IUml(-1)) after 36 h of fermentation, whereas its value predicted by the quadratic model was 1347 IUml(-1). Analysis of variance (ANOVA) showed a high coefficient of determination (R(2)) value of 0.9718, ensuring a satisfactory adjustment of the quadratic model with the experimental data.The economical and cellulase-free nature of xylanase would enhance its applicability in pulp and paper industry.