Enduracidin (Enramycin) is a bioactive peptide antibiotic generated by Streptomyces sp. Enramycin, a common growth promoter utilized in chickens and pigs, is sensitive against Gram-positive bacteria, and the maximum residue limit (MRL) of enramycin set up by is 30 μg/kg.

Physicochemical Properties

Molecular Formula	C107H138N26O31CL2
Molecular Weight	2355.30224275589
Exact Mass	2337.92
Elemental Analysis	C, 54.15; H, 5.95; Cl, 2.99; N, 15.34; O, 21.57
CAS #	11115-82-5
PubChem CID	56842192
Appearance	Light brown to brown solid powder
LogP	3.835
Hydrogen Bond Donor Count	34
Hydrogen Bond Acceptor Count	35
Rotatable Bond Count	34
Heavy Atom Count	167
Complexity	5280
Defined Atom Stereocenter Count	0
SMILES	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
InChi Key	NJCUSQKMYNTYOW-MWUYRYRWSA-N
InChi Code	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+;
Chemical Name	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
Synonyms	Enramycin; 11115-82-5; Enramicina; ENDURACIDIN; Enramycinum; Enradin; Enramycin [INN]; 12772-37-1;
HS Tariff Code	2934.99.9001
Storage	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.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Lipopeptide antibiotics first isolated from Streptomyces fungicidicus No.B547 [1]
ln Vitro	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].
ln Vivo	Besides antibacterial activity, enduracidins also display effective growth‐promoting activity and have been extensively used in livestock farming[1]. 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]
Enzyme Assay	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. 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.
Animal Protocol	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]
Toxicity/Toxicokinetics	rat LD50 oral >10 gm/kg Takeda Kenkyusho Nempo. Annual Report of the Takeda Research Laboratories., 28(76), 1969 rat LD50 intraperitoneal 830 mg/kg BEHAVIORAL: TREMOR; BEHAVIORAL: ATAXIA; LUNGS, THORAX, OR RESPIRATION: RESPIRATORY DEPRESSION Takeda Kenkyusho Nempo. Annual Report of the Takeda Research Laboratories., 28(76), 1969 rat LD50 subcutaneous >5 gm/kg Takeda Kenkyusho Nempo. Annual Report of the Takeda Research Laboratories., 28(76), 1969 rat LD50 intravenous 66600 ug/kg BEHAVIORAL: TREMOR; BEHAVIORAL: ATAXIA; LUNGS, THORAX, OR RESPIRATION: RESPIRATORY DEPRESSION Takeda Kenkyusho Nempo. Annual Report of the Takeda Research Laboratories., 28(76), 1969 rat LD50 intramuscular >5 gm/kg Takeda Kenkyusho Nempo. Annual Report of the Takeda Research Laboratories., 28(76), 1969
References	[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.
Additional Infomation	Aims: To increase enduracidin production in Streptomyces fungicidicus ATCC 31731 by overexpressing positive regulators in enduracidin biosynthesis.[1] Methods and results: Genes orf22 and orf42 were knocked out by in-frame deletion based on CRISPR/Cas9 strategy, while the orf41 gene was inactivated by replacing it with the apramycin resistance gene cassette aac(3)IV using a fast screening blue/white system. The integrative plasmid pSET152ermE was used for the overexpression of orf22, orf41 and orf42 individually. The constructed plasmids were transformed into wild-type strain Streptomyces fungicidicus ATCC 31731. Three gene inactivation mutants Δorf22, Δorf41 and Δorf42 and three recombinant strains overexpressing orf22, orf41 and orf42 were all fermented and the enduracidin production of each strain was detected and compared by HPLC analysis. Two resulting engineered strains were generated through overexpression of gene orf22 and orf42 in Streptomyces fungicidicus, respectively, and in these strains the enduracidins titres were increased by approximately 4·0-fold and 2·3-fold higher than that of the wild-type strain.[1] Conclusions: The functions of three regulatory genes orf22, orf41 and orf42 in the enduracidin gene cluster in Streptomyces fungicidicus ATCC 31731 were examined. The orf22 gene, encoding a SARP family protein, was proposed to act in a positive manner. The proteins encoded by genes orf41 and orf42 were proposed to compose a two-component regulation system, in which the response protein Orf41 was characterized as a repressor, and the kinase Orf42 was shown to be an activator. The production of enduracidins was improved considerably by overexpression of the two positive regulatory genes orf22 and orf42 respectively.[1] Significance and impact of the study: The production of enduracidins was successfully improved by manipulating the regulatory genes involving in enduracidin biosynthesis, providing an efficient approach to improve enduracidin production further for fermentation industry and synthetic biological research.[1] Enramycin, a common growth promoter utilized in chickens and pigs, is sensitive against Gram-positive bacteria, and the maximum residue limit (MRL) of enramycin set up by is 30 μg/kg. However, the methods have been reported for detecting enramycin have failed to meet the accuracy requirements, with the required limit of quantification being higher than the MRL. To address this issue, we developed a high-sensitive and robust analytical method based on ultrahigh-performance liquid chromatography coupled with mass spectrometry (UHPLC-MS/MS), to determine enramycin residues in swine tissues, including liver, kidney, pork, and fat. The ENV cartridge was selected to cleanup and enrich analytes after being extracted using a mixture of 55% methanol containing 0.2 M hydrochloric acid. With comprehensively validation, this established method was found great linearity of enramycin in each tissue, with a coefficient of variation above 0.99. Satisfactory recoveries from four different spiking levels were acquired (70.99-101.40%) while the relative standard deviations were all below 9%. The limit of quantification of enramycin in the present study is 5 μg/kg in fat and 10 μg/kg in other tissues, meeting the requirements for conducting the corresponding safety evaluation study. This method was demonstrated with excellent specificity, stability, and high sensitivity. To conclude, this novel approach is sufficiently sensitive and robust for the safety evaluation of enramycin in food products. https://pubmed.ncbi.nlm.nih.gov/39290506/

Solubility Data

Solubility (In Vitro)

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

Solubility (In Vivo)

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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	0.4246 mL	2.1229 mL	4.2457 mL
	5 mM	0.0849 mL	0.4246 mL	0.8491 mL
	10 mM	0.0425 mL	0.2123 mL	0.4246 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.