Lipopeptide antibiotics first isolated from Streptomyces fungicidicus No.B547 [1]

Enduracidin displays good inhibition activities against Gram‐positive bacteria, including a number of drug‐resistant pathogens, for example, vancomycin‐resistant Enterococcus faecium (VRE) and methicillin‐resistant Staphylococcus aureus (MRSA). They can block the synthesis of bacterial cell wall by competitively binding to lipid II and preventing the subsequent transglycosylation step of peptidoglycan installation, which is a totally different mechanism from that of practically used drugs like vancomycin and β‐lactam antibiotics, which inhibiting synthesis of the bacterial cell wall by covalently binding with nucleophilic active site serine residues in D, D‐transpeptidases. Ramoplanin A2 is now an FDA‐approved molecule entering phase III clinical trials for the treatment of VRE and Clostridium difficile infections [1].

Besides antibacterial activity, enduracidins also display effective growth‐promoting activity and have been extensively used in livestock farming[1].

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The TAN had greater weight gain in the nursery phase and final weight (p<0.05) than the CONTR (394 vs. 360 g/d, and 22.6 vs. 21.1 kg, respectively), with these values being intermediate for the ENR+ZnO and BUT (365 and 382 g/d, and 21.3 and 22.1 kg, respectively). There was no difference between treatments for semi-liquid diarrhea (score 2), but CONTR had more cases of severe diarrhea (score 3; p<0.05) than ENR+ZnO, BUT and TAN, with 42, 18, 29 and 21 cases, respectively. The treatments had no impact on rare taxa or the relative abundances of taxonomic groups (uniformity), but the use of TAN promoted an increase in the abundances of Brevibacillus spp. and Enterococcus spp. compared to the other treatments (p<0.05). Conclusion: The use of condensed tannin from black wattle as a performance-enhancing additive was effective, with effects on performance and intestinal health, demonstrating its potential as a substitute for zinc oxide and enramycin in the diets of piglets in nursery phase.[2]

Fermentation and production of enduracidins[1]
Streptomyces fungicidicus ATCC 31731 was grown on MS agar for 6–8 days for spore collection. An aliquot of approximately 1·0 × 107 spores was inoculated into 50 ml of seed medium. The seed cultures were grown at 28°C, 220 rev min−1 for 48 h. Subsequently, 5 ml of the above seed cultures were inoculated into 50 ml of fermentation medium in 250‐ml flasks at 28°C, 220 rev min−1 for 8 days. The mycelia were collected after centrifugation and subjected to lyophilization. The dried mycelia were washed with methanol and sonicated for 30 min. Subsequently, the mixture was shaken at 18°C for 3 h and centrifuged to remove pellet. The supernatant was evaporated at 30°C in vacuum and then dissolved in 2 ml of methanol for HPLC analysis. The condition used for the fermentation and antibiotic production of the knockout and overexpressed strains was the same as wild type.

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Spectroscopic analyses of enduracidins production[1]
HPLC analysis was performed with a reverse C18 column (5 μm, 4·6 mm × 250 mm, Alltech, Deerfield, IL) on a Shimadzu HPLC system using a linear gradient of acetonitrile/water (10–30% for 20 min, 30–40% for 20 min, 100% for 5 min, flow rate 0·8 ml min−1) containing 0·1% trifluoroacetic acid. The detection wavelength is 267 nm. LC‐MS analysis was performed on an Agilent 1260/6460 Triple‐Quadrupole LC/MS system with an electrospray ionization source. HR‐ESI‐MS was performed on an Agilent 1260 HPLC/6520 QTOF‐MS instrument.

A total of 200 PIC® piglets that were 22 days old and weighed 6.0±0.9 kg were subjected to four treatments in the nursery phase (22 to 64 days of age): CONTR (control diet); ENR+ZnO (control diet + 10 mg/kg of enramycin  + 2,500 mg/kg of zinc oxide during the first 21 days); BUT (control diet + 900 mg/kg of sodium butyrate) and TAN (control diet + 2,000 mg/kg of condensed tannin). The experimental design was a randomized block with 4 treatments and 10 replicates, with a pen of five animals each as the experimental unit. The zootechnical performance, diarrhea index score, dietary digestibility and metagenomics of the deep rectum microbiota were evaluated.[2]

Oral LD50 in rats >10 g/kg, Takeda Research Institute Annual Report, 28(76), 1969
Intraperitoneal LD50 in rats 830 mg/kg Behavior: Tremor; Behavior: Ataxia; Lung, pleural or respiratory: Respiratory depression, Takeda Research Institute Annual Report, 28(76), 1969
Subcutaneous LD50 in rats >5 g/kg, Takeda Research Institute Annual Report, 28(76), 1969
Intravenous LD50 in rats 66600 μg/kg Behavior: Tremor; Behavior: Ataxia; Lung, pleural or respiratory: Respiratory depression, Takeda Research Institute Annual Report, 28(76), 1969
Intramuscular LD50 in rats >5 gm/kg, Takeda Research Institute Annual Report, 28(76), 1969

[1]. Characterization of Three Regulatory Genes Involved in Enduracidin Biosynthesis and Improvement of Enduracidin Production in Streptomyces Fungicidicus. J Appl Microbiol. 2019 Dec;127(6):1698-1705.

[2]. Performance and intestinal health of piglets in the nursery phase subjected to diets with condensed black wattle (Acacia mearnsii) tannin. Anim Biosci . 2024 Aug 23. doi: 10.5713/ab.24.0112.

Objective: To increase the yield of endostatin in Streptomyces fungicidicus ATCC 31731 by overexpressing positive regulators of endostatin biosynthesis. [1] Methods and Results: The orf22 and orf42 genes were knocked out using the in-frame deletion method based on CRISPR/Cas9 technology. At the same time, the orf41 gene was replaced with the apramycin resistance gene cassette aac(3)IV using the rapid blue-white screening system to inactivate it. The orf22, orf41 and orf42 genes were overexpressed using the integrative plasmid pSET152ermE. The constructed plasmids were transformed into wild-type strain Streptomyces ATCC 31731. The three gene-inactivating mutants Δorf22, Δorf41 and Δorf42 and the three recombinant strains overexpressing orf22, orf41 and orf42 were fermented, and the endonuclease yield of each strain was detected and compared by HPLC analysis. Two engineered strains were constructed by overexpressing the orf22 and orf42 genes in Streptomyces fungicidicus, respectively. Compared with the wild-type strain, the endonuclease activities of the two strains were increased by approximately 4.0-fold and 2.3-fold, respectively. [1] Conclusion: This study investigated the functions of three regulatory genes, orf22, orf41, and orf42, in the endonuclease activity gene cluster of Streptomyces fungicidicus ATCC 31731. The orf22 gene encodes a SARP family protein and is considered to play a positive regulatory role. The proteins encoded by the orf41 and orf42 genes are considered to constitute a two-component regulatory system, in which the response protein Orf41 was identified as a repressor protein, while the kinase Orf42 was confirmed as an activator protein. The production of endolactic acid was significantly increased by overexpressing the two positive regulatory genes, orf22 and orf42. [1]
Significance and impact of the study: By regulating the regulatory genes involved in enturamic acid biosynthesis, the yield of enturamic acid was successfully increased, providing an effective method for further increasing the yield of enturamic acid in fermentation industry and synthetic biology research. [1]
Enramycin is a commonly used growth promoter for chickens and pigs, sensitive to Gram-positive bacteria, with a maximum residue limit (MRL) of 30 μg/kg. However, the reported methods for detecting enturamic acid have failed to meet the accuracy requirements, and the required limit of quantitation is higher than the maximum residue limit. To solve this problem, we developed a highly sensitive and stable analytical method based on ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) for determining enturamic acid residues in pig tissues (including liver, kidney, pork and fat). Samples extracted with a mixture of 55% methanol and 0.2 M hydrochloric acid were purified and enriched using an ENV solid-phase extraction column. After comprehensive validation, the method showed good linearity for enturamic acid in various tissues, with coefficients of variation all higher than 0.99. The recoveries at four different spiking levels were satisfactory (70.99%–101.40%), with relative standard deviations all below 9%. In this study, the limit of quantitation (LOQ) of enramycin in fat was 5 μg/kg, and in other tissues it was 10 μg/kg, meeting the requirements for conducting relevant safety evaluation studies. This method exhibits excellent specificity, stability, and high sensitivity. In conclusion, this novel method is highly sensitive and robust, sufficient for assessing the safety of enramycin in food. https://pubmed.ncbi.nlm.nih.gov/39290506/

C107H138N26O31CL2

2355.30224275589

2337.92

C, 54.15; H, 5.95; Cl, 2.99; N, 15.34; O, 21.57

11115-82-5

56842192

Light brown to brown solid powder

3.835

34

35

34

167

5280

0

CCC(C)CCCC/C=C/C=C/C(=O)NC(CC(=O)O)C(=O)NC1C(OC(=O)C(NC(=O)C(NC(=O)C(NC(=O)NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)C(NC(=O)N(C(=O)C(NC1=O)C2=CC=C(C=C2)O)CCCCN)C(C)O)C3=CC=C(C=C3)O)C4=CC=C(C=C4)O)C(C)O)CCCNC(=O)N)CC5CNC(=N5)N)C6=CC=C(C=C6)O)CO)C7=CC(=C(C(=C7)Cl)O)Cl)CC8CNC(=N8)N)C)C9=CC=C(C=C9)O)C.O

NJCUSQKMYNTYOW-MWUYRYRWSA-N

InChI=1S/C107H138Cl2N26O31.H2O/c1-7-51(2)17-12-10-8-9-11-13-19-76(144)120-74(47-77(145)146)92(152)126-80-55(6)166-102(162)86(60-28-38-68(143)39-29-60)132-88(148)52(3)117-90(150)73(46-63-49-116-104(112)119-63)124-106(164)134-100(160)84(61-43-69(108)87(147)70(109)44-61)128-93(153)75(50-136)123-97(157)81(56-20-30-64(139)31-21-56)127-91(151)72(45-62-48-115-103(111)118-62)122-89(149)71(18-16-41-114-105(113)163)121-94(154)78(53(4)137)125-98(158)82(57-22-32-65(140)33-23-57)130-99(159)83(58-24-34-66(141)35-25-58)129-95(155)79(54(5)138)133-107(165)135(42-15-14-40-110)101(161)85(131-96(80)156)59-26-36-67(142)37-27-59;/h9,11,13,19-39,43-44,51-55,62-63,71-75,78-86,136-143,147H,7-8,10,12,14-18,40-42,45-50,110H2,1-6H3,(H,117,150)(H,120,144)(H,121,154)(H,122,149)(H,123,157)(H,125,158)(H,126,152)(H,127,151)(H,128,153)(H,129,155)(H,130,159)(H,131,156)(H,132,148)(H,133,165)(H,145,146)(H3,111,115,118)(H3,112,116,119)(H3,113,114,163)(H2,124,134,160,164);1H2/b11-9+,19-13+;

4-[[41-(4-aminobutyl)-9,23-bis[(2-amino-4,5-dihydro-1H-imidazol-4-yl)methyl]-26-[3-(carbamoylamino)propyl]-14-(3,5-dichloro-4-hydroxyphenyl)-29,38-bis(1-hydroxyethyl)-17-(hydroxymethyl)-3,20,32,35,43-pentakis(4-hydroxyphenyl)-6,47-dimethyl-2,5,8,11,13,16,19,22,25,28,31,34,37,40,42,45-hexadecaoxo-1-oxa-4,7,10,12,15,18,21,24,27,30,33,36,39,41,44-pentadecazacycloheptatetracont-46-yl]amino]-3-[[(2E,4E)-10-methyldodeca-2,4-dienoyl]amino]-4-oxobutanoic acid;hydrate

Enramycin; 11115-82-5; Enramicina; ENDURACIDIN; Enramycinum; Enradin; Enramycin [INN]; 12772-37-1;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)