Enduracidin (Enramycin; Enramycin)   中文名称: 恩拉霉素

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

Endurocidn对革兰氏阳性菌显示出良好的抑制活性，包括许多耐药病原体，例如耐万古霉素的屎肠球菌（VRE）和耐甲氧西林的金黄色葡萄球菌（MRSA）。它们可以通过与脂质II竞争性结合来阻断细菌细胞壁的合成，并阻止随后肽聚糖安装的转糖基化步骤，这与万古霉素和β-内酰胺抗生素等实际使用的药物完全不同，后者通过与D，D转肽酶中的亲核活性位点丝氨酸残基共价结合来抑制细菌细胞壁合成。Ramoplanin A2现在是一种经美国食品药品监督管理局批准的分子，正在进入治疗VRE和艰难梭菌感染的III期临床试验[1]。

除了抗菌活性外，enduracidins还显示出有效的促生长活性，并已广泛应用于畜牧业[1]。

---

共有200头22日龄、体重6.0±0.9kg的PIC®仔猪在育苗阶段（22至64日龄）接受了四种处理：CONTR（对照饮食）；ENR+ZnO（对照饮食+10mg/kg恩拉霉素+2500 mg/kg氧化锌，前21天）；BUT（对照饮食+900 mg/kg丁酸钠）和TAN（对照饮食+2000 mg/kg缩合单宁）。实验设计是一个随机区组，有4个治疗组和10个重复组，每组5只动物作为实验单位。对直肠深层微生物群的动物技术性能、腹泻指数评分、日粮消化率和宏基因组学进行了评估。[2]

---

TAN在育苗阶段和最终体重的增加（p<0.05）大于CONTR（分别为394和360 g/d，22.6和21.1 kg），这些值在ENR+ZnO和BUT中处于中间水平（分别为365和382 g/d，21.3和22.1 kg）。半液体性腹泻的治疗方法之间没有差异（评分2），但CONTR的严重腹泻病例（评分3；p<0.05）多于ENR+ZnO、but和TAN，分别为42、18、29和21例。这些处理对稀有类群或分类群的相对丰度（一致性）没有影响，但与其他处理相比，TAN的使用促进了短杆菌属和肠球菌属丰度的增加（p<0.05）。 结论：使用黑荆树浓缩单宁作为性能增强添加剂是有效的，对性能和肠道健康有影响，表明其在仔猪育雏期日粮中替代氧化锌和恩拉霉素的潜力。

Enduracidins的发酵和生产[1]
真菌链霉菌ATCC 31731在MS琼脂上生长6-8天以收集孢子。将约1.0×107个孢子的等分试样接种到50ml种子培养基中。将种子培养物在28°C、220转/分-1的温度下生长48小时。随后，将5毫升上述种子培养物接种到250毫升烧瓶中的50毫升发酵培养基中，温度为28°C，220转/分钟-1，持续8天。离心后收集菌丝体并冷冻干燥。用甲醇洗涤干燥的菌丝体并超声处理30分钟。随后，将混合物在18°C下摇动3小时并离心以去除颗粒。将上清液在30°C下真空蒸发，然后溶解在2ml甲醇中进行HPLC分析。敲除和过表达菌株的发酵和抗生素生产条件与野生型相同。

---

enduracidins产生的光谱分析[1]
在Shimadzu HPLC系统上使用反向C18柱（5μm，4.6 mm×250 mm，Alltech，Deerfield，IL）进行HPLC分析，使用含有0.1%三氟乙酸的乙腈/水（10-30%20分钟，30-40%20分钟，100%5分钟，流速0.8 ml min-1）的线性梯度。检测波长为267nm。使用安捷伦1260/6460三四极LC/MS系统和电喷雾电离源进行LC-MS分析。HR-ESI-MS在安捷伦1260 HPLC/6520 QTOF-MS仪器上进行。

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]

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

[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.

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/

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

922.33

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

注意： 本产品在运输和储存过程中需避光。

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

---

注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

View More

注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

---

口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

View More

口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

---

注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。