CN114907367B - 大环内酯类化合物fw-z、其发酵菌株、发酵方法及应用 - Google Patents
大环内酯类化合物fw-z、其发酵菌株、发酵方法及应用 Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
- C07D493/20—Spiro-condensed systems
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
- A01N43/90—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having two or more relevant hetero rings, condensed among themselves or with a common carbocyclic ring system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/10—Antimycotics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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- Life Sciences & Earth Sciences (AREA)
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- Veterinary Medicine (AREA)
- Agronomy & Crop Science (AREA)
- Pest Control & Pesticides (AREA)
- Plant Pathology (AREA)
- Medicinal Chemistry (AREA)
- Dentistry (AREA)
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- Animal Behavior & Ethology (AREA)
- Environmental Sciences (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
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Abstract
本发明属于微生物及新型医药农药技术领域,具体设计一种新型大环内酯类化合物FW‑Z及其应用,并进一步公开了一种可发酵所述化合物的海洋小单孢菌,以及基于所述菌株进行发酵制备所述大环内酯类化合物FW‑Z的方法。本发明所述大环内酯类化合物FW‑Z具有类似于寡霉素的抗真菌性能,对真菌中黑曲霉具有细胞毒活性。本发明分离并筛选的具有抗真菌活性的海洋小单孢菌(Micromonospora sp.)FIMYZ51可用于发酵制备具有显著的抗真菌‑黑曲霉抑制活性的一系列大环内酯类化合物FW‑Z2与FW‑Z5。
Description
技术领域
本发明属于微生物及新型医药农药技术领域,具体涉及一种新型大环内酯类化合物FW-Z及其应用,并进一步公开了一种可发酵所述化合物的海洋小单孢菌,以及基于所述菌株进行发酵制备所述大环内酯类化合物FW-Z的方法。
背景技术
寡霉素类Oligomycins是由链霉菌产生的一种大环内酯类抗生素,由典型的Ⅰ型聚酮合酶催化合成,它们具有多样的生物活性,包括抑制线粒体ATP酶活性、抗肿瘤活性、抗真菌活性及杀虫活性等。目前,寡霉素类化合物作为ATP合酶抑制剂,已被广泛应用于各项科学研究中,且作为化学试剂,其价格昂贵,具有广阔的经济价值。
2000年Librada M.Canedo等人从海洋小单孢菌Micromonospora sp.中发现了新型螺环类大环内酯化合物IB-96212,并验证其具有抗细菌活性,尤其对Micrococcusluteus(藤黄微球菌)MIC值达到0.4ug/ml[1-2];同时对多种肿瘤细胞有很强的细胞毒作用,尤其对P388细胞株的IC50值达0.1ng/ml,活性高于对照品紫杉醇(200ng/ml)以及依托泊苷(100ng/ml),甚至是阿霉素(20ng/ml);而且,IB-96212类化合物结构与寡霉素类化合物相比,除了特征性的螺环结构外,多出了一个糖环L-rhodinose(L-玫红糖)。目前,该类化合物仅发现唯一的IB-96212化合物,因此其抗菌机理和生物合成机理尚未报道,期待开发更多类似结构的化合物,进而完善其机理研究及产品组成。
小单孢菌属(Micromonospora)属于放线菌目小单孢菌科(Micromonosporaceae),其菌株来源广泛,有来自淡水湖泊,也有分离来自海洋环境的。截至2016年Talukdar等人的统计,近700种抗生素来自于小单孢菌属的发酵代谢。因此,小单胞菌是生物活性次级代谢产物的一个重要的宝库(Qi et al.,2020),对于开发多种新型抗生素药物具有重要的意义。
发明内容
为此,本发明所要解决的技术问题在于提供一种大环内酯类化合物FW-Z,所述化合物FW-Z具有真菌黑曲霉抑制活性;
本发明所要解决的第二个技术问题在于提供一种海洋小单孢菌株,所述菌株可用于发酵产所述大环内酯类化合物FW-Z;
本发明所要解决的第三个技术问题在于提供一种基于所述海洋小单孢菌株发酵制备所述大环内酯类化合物FW-Z的方法;
本发明所要解决的第四个技术问题在于提供上述大环内酯类化合物FW-Z的用途。
为解决上述技术问题,本发明所述的一种大环内酯类化合物FW-Z,其特征在于,所述大环内酯类化合物FW-Z具有如下式(Ⅰ)所示的结构:
其中,R1=O时,R2=CH3;R1=OH时,R2=CH2CH3。
具体的,所述大环内酯类化合物FW-Z包括化合物FW-Z2和/或化合物FW-Z5;其中,
所述化合物FW-Z2中,R1为=O,R2为-CH3;
所述化合物FW-Z5中,R1为-OH,R2为-CH2CH3。
本发明还提供了一种海洋小单孢菌株,所述菌株具有抑制细菌与真菌活性的放线菌,且通过传统分类和16S rDNA基因分析,其分类命名为放线菌小单孢菌属(Micromonospora sp.)FIMYZ51,于2021年12月9日保藏于中国科学院微生物研究所,保藏编号为CGMCC No.24067。
本发明还提供了所述海洋小单孢菌株用于发酵制备所述大环内酯类化合物FW-Z的用途。
本发明还提供了一种发酵制备所述大环内酯类化合物FW-Z的方法,包括将所述的海洋小单孢菌株接种于适宜发酵培养基中进行发酵培养的步骤。
具体的,所述发酵制备所述大环内酯类化合物FW-Z的方法,包括如下步骤:
(1)取斜面保存的所述海洋小单孢菌FIMYZ51接入液体种子培养基中进行恒温培养,收集种子液,备用;
所述液体种子培养基的组分包括:可溶性淀粉1-2wt%、葡萄糖0.1-1wt%、蛋白胨0.1-1wt%、酵母提取物0.1-1wt%、MgSO4·7H2O 0.01-0.1wt%、NaCl0.01-0.1wt%、(NH4)2SO4 0.01-0.1wt%、CaCO3 0.05-0.2wt%,pH 6.0-8.5;
优选的,所述液体种子培养基的组分包括:可溶性淀粉1.5wt%、葡萄糖0.5wt%、蛋白胨0.5wt%、酵母提取物0.5wt%、MgSO4·7H2O 0.05wt%、NaCl 0.05wt%、(NH4)2SO40.05wt%、CaCO3 0.1wt%,pH 6.0-8.5;
(2)将所述种子液转接至发酵培养基中进行恒温培养,即得含有所需化合物FW-Z的发酵液;
所述发酵培养基的组分包括:可溶性淀粉3-5wt%、葡萄糖0.1-1wt%、黄豆饼粉2-3wt%、酵母粉0.1-1wt%、MgSO4·7H2O 0.01-0.1wt%、K2HPO40.01-0.1wt%、CaCO3 0.05-0.2wt%,pH 6.0-8.5;
优选的,所述发酵培养基的组分包括:可溶性淀粉4wt%、葡萄糖0.5wt%、黄豆饼粉2.5wt%、酵母粉0.5wt%、MgSO4·7H2O 0.05wt%、K2HPO4 0.05wt%、CaCO3 0.1wt%,pH6.0-8.5。
具体的,所述发酵制备所述大环内酯类化合物FW-Z的方法:
所述步骤(1)中,所述培养步骤的温度为25-35℃,培养时间1-3天;
所述步骤(2)中,所述培养步骤的温度为25-35℃,培养时间3-6天。
具体的,所述发酵制备所述大环内酯类化合物FW-Z的方法,所述方法还包括对所述大环内酯类化合物FW-Z进行提取及纯化的步骤,具体包括:
提取:将收集的发酵液和菌丝体进行固液分离,其中,所述发酵液用大孔树脂HP20进行吸附,吸附后经乙醇解吸回收并浓缩后得到粗提物A;所述菌丝体经醇溶剂浸提,收集提取液减压浓缩得到粗提物B;优选的,所述大孔树脂HP20与发酵液比例为1:10-1:30,混合后上树脂柱吸附,吸附后用蒸馏水洗涤,以10-20%乙醇除去杂质,再用100%乙醇解吸,回收乙醇溶剂后的FW-Z的粗提物A;菌丝体经乙醇或甲醇浸提1-3次,将浸泡液减压浓缩为浸膏得粗提物B。
纯化:将上述粗提物A和粗提物B进行合并,采用正相硅胶柱层析以石油醚:乙酸乙酯溶剂(体积比10:0-0:10)梯度洗脱,薄层层析检测,合并浓缩10:1-1:1洗脱液;反向C18柱层析色谱法以甲醇:水溶剂(体积比50%-100%)梯度洗脱分离,合并收集70%-90%区段含大环内酯化合物FW-Z的洗脱液;经过制备型C18反相高压液相色谱的制备(甲醇水60-100%)梯度洗脱,得到抗真菌活性的大环内酯化合物FW-Z2与FW-Z5纯品。
本发明还公开了所述大环内酯类化合物FW-Z用于制备医用、兽用或农用非治疗目的抗真菌制剂的用途。
具体的,所述抗真菌制剂包括黑曲霉抑菌剂。
本发明所述大环内酯类化合物FW-Z为与IB-96212具有相似的具有26元环、螺环结构、L-玫红糖的大环内酯类抗生素,所述化合物FW-Z具有类似于寡霉素的抗真菌性能,该大环内酯化合物FW-Z对真菌中黑曲霉具有细胞毒活性,为研究开发新的抗真菌药物提供了先导化合物,对开发利用中国的海洋药物资源具有重要价值。
本发明从海洋放线菌中分离并筛选到一株具有抗真菌活性的海洋小单孢菌(Micromonospora sp.)FIMYZ51,所述海洋小单孢菌可用于发酵制备具有显著的抗真菌-黑曲霉抑制活性的一系列大环内酯化合物FW-Z2与FW-Z5。
本发明所述大环内酯类化合物FW-Z的制备方法,基于所述海洋小单孢菌(Micromonospora sp.)FIMYZ51进行发酵,经发酵液提取及纯化获得化合物FW-Z纯品,发酵效率及提取效率均较为理想。
附图说明
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明,其中,
图1是本发明所述海洋小单孢菌菌株基于16S rRNA基因序列的系统发育树的示意图;
图2是本发明中大环内酯化合物FW-Z2高分辨质谱图;
图3是本发明中大环内酯化合物FW-Z2的氢核磁共振图1H谱;
图4是本发明中大环内酯化合物FW-Z2的碳核磁共振图13C谱;
图5是本发明中大环内酯化合物FW-Z2的1H-1HCOSY图谱;
图6是本发明中大环内酯化合物FW-Z2的HSQC相关图谱;
图7是本发明中大环内酯化合物FW-Z2的HMBC相关图谱;
图8是本发明中大环内酯化合物FW-Z5高分辨质谱图;
图9是本发明中大环内酯化合物FW-Z5的氢核磁共振图1H谱;
图10是本发明中大环内酯化合物FW-Z5的碳核磁共振图13C谱;
图11是本发明中大环内酯化合物FW-Z5的1H-1HCOSY图谱;
图12是本发明中大环内酯化合物FW-Z5的HSQC相关图谱;
图13是本发明中大环内酯化合物FW-Z5的HMBC相关图谱。
具体实施方式
本发明筛选获得一株来自海洋小单孢菌菌株(Micromonospora sp.)FIMYZ51,通过16S rRNA基因分析确定其属于放线菌中的小单孢菌属,另外,从该小单孢菌菌株(Micromonospora sp.)FIMYZ51发酵液中提取分离获得两种对真菌中的黑曲霉具有细胞毒活性的大环内酯化合物FW-Z2与FW-Z5。
实施例1菌株FIMYZ51的鉴定
对筛选的菌株Micromonospora sp.FIMYZ51基因组DNA采用酶解法提取,得到的DNA样品储存于-20℃,将该DNA样品作为DNA模板进行16S rRNA基因PCR扩增,PCR产物送外测序。
所述海洋小单孢菌菌株基于16S rRNA基因序列的系统发育树的示意图如图1所示。将所测的菌株的16S rDNA序列与GenBank数据库中已有序列进行blast比对,并进行同源性分析。结果与小单孢菌Micromonospora eburnea相似性高达99.86%;登录号为:EU274362.1,16S部分序列总长1422bp,具体序列如SEQ ID No.1所示。
所述菌株FIMYZ51判定为小单孢菌属,于2021年12月9日保藏于中国科学院微生物研究所,保藏编号为CGMCC No.24067。
实施例2发酵制备新结构大环内酯化合物FW-Z2与FW-Z5
将筛选的海洋小单孢菌Micromonospora sp.FIMYZ51接种于淀粉天冬素琼脂斜面培养物接入液体种子培养基,温度30℃,培养时间2天后,将种子与人工种子培养基按照体积比1:10的比例混合于温度30℃,振荡培养5天,收集发酵产物。
选用的种子培养基的组分为:可溶性淀粉1.5wt%、葡萄糖0.5wt%、蛋白胨0.5wt%、酵母提取物0.5wt%、MgSO4·7H2O 0.05wt%、NaCl 0.05wt%、(NH4)2SO40.05wt%、CaCO3 0.1wt%,蒸馏水配制,pH 6.0-8.5;
选用的发酵培养基的组分为:可溶性淀粉4wt%、葡萄糖0.5wt%、黄豆饼粉2.5wt%、酵母粉0.5wt%、MgSO4·7H2O 0.05wt%、K2HPO4 0.05wt%、CaCO3 0.1wt%,蒸馏水配制,pH 6.0-8.5。
将收集的发酵产物中的发酵液和菌丝体进行固液分离,发酵液用大孔树脂HP20吸附,树脂与发酵液按照质量比1:20进行混合,混合后上树脂柱吸附,吸附后用蒸馏水洗涤,10-20%乙醇除去杂质,再用100%乙醇解吸,回收乙醇溶剂后的FW-Z的粗提物A;菌丝体经乙醇或甲醇浸提3次,将浸泡液减压浓缩为浸膏得粗提物B。
将上述粗提物A和粗提物B进行合并,采用正相硅胶柱层析以石油醚:乙酸乙酯溶剂(体积比10:0-0:10)梯度洗脱,薄层层析检测,合并浓缩10:1-1:1洗脱液;反向C18柱层析色谱法以甲醇:水溶剂(体积比50%-100%)梯度洗脱分离,合并收集70%-90%区段含大环内酯化合物FW-Z的洗脱液;经过制备型C18反相高压液相色谱的制备(甲醇水60-100%)梯度洗脱,得到抗真菌活性的大环内酯化合物FW-Z2与FW-Z5纯品。
实施例3化合物FW-Z2与FW-Z5的结构解析
从该新菌株的发酵液中分离到两个具有抗真菌活性的结构新颖的大环内酯化合物,通过MS和NMR技术鉴定大环内酯化合物FW-Z2与FW-Z5的结构。
FW-Z2:物理化学性状为棕色无定型固体,高分辨质谱(HR-ESI-MS):测量值:m/z1019.6293[M+Na]+,理论值m/z 1019.6278[M+Na]+,分子式:C54H92O16,其不饱和度为9。溶解度:可溶于甲醇、丙酮、乙腈、乙酸乙酯和二甲亚砜等有机溶剂,不溶于水。
FW-Z2的1H核磁共振波谱(DMSO-d6,600MHz):δ6.66(dd,J=15.5,10.3Hz,1H),5.96(dd,J=6.0,2.8Hz,1H),5.94(d,J=2.8Hz,1H),5.81(d,J=15.5Hz,1H),5.43(m,1H),5.14(m,1H),4.87(d,J=5.5Hz,1H),4.84(d,J=7.0Hz,2H),4.75(d,J=4.5Hz,1H),4.67(dd,J=11.2,5.1Hz,1H),4.38(d,J=7.0Hz,1H),4.23(s,1H),4.10(q,J=5.2Hz,1H),4.03(m,2H),4.00(s,1H),3.98(d,J=7.7Hz,1H),3.87(d,J=8.5Hz,1H),3.85(m,1H),3.79(m,1H),3.64(d,J=11.9Hz,1H),3.60(d,J=7.1Hz,1H),3.37(m,1H),3.55(d,J=10.3Hz,1H),3.44(m,1H),3.32(m,1H),3.24(m,1H),3.20(dd,J=8.8,6.2Hz,1H),3.00(m,1H),2.37(d,J=5.0Hz,2H),2.32(m,1H),2.11(m,1H),2.08–2.05(m,1H),2.03(s,3H),1.93(d,J=12.8Hz,1H),1.90(m,1H),1.84(d,J=7.5Hz,1H),1.77(dd,J=11.2,6.6Hz,1H),1.73(m,1H),1.67(dd,J=12.8,4.7Hz,1H),1.62(ddd,J=14.0,7.6,3.1Hz,2H),1.56(m,4H),1.49(m,1H),1.47(d,J=4.6Hz,1H),1.45(d,J=11.5Hz,2H),1.41(m,3H),1.39(d,J=3.0Hz,1H),1.36-1.33(m,3H),1.26(dd,J=13.9,7.5Hz,2H),1.16(d,J=6.2Hz,3H),1.05(d,J=6.5Hz,3H),0.91-0.90(m,4H),0.87-0.85(m,9H),0.75-0.73(m,9H),0.63(d,J=7.0Hz,3H)。
FW-Z2的13C核磁共振波谱(DMSO-D6,150MHz):δ207,164.3,150.0,134.4,131.9,131.2,131.0,121.4,102.6,97.7,81.7,78.8,76.3,75.7,75.6,72.671.5,69.9,67.6,67.3,50.0,41.2,38.6,38.3,37.2,37.0,35.2,32.9,31.7,30.9,30.4,30.4,29.9,29.8,27.6,27.4,18.4,18.1,17.8,15.9,15.0,14.9,11.8,9.3,9.1,6.1,4.5。
同时,本发明还测定了该化合物FW-Z2多种核磁共振图谱分别见图2-7,从而确定了该化合物所有的碳原子和氢原子的归属及该化合物的化学结构,确定其为结构新颖的大环内酯化合物,结构表征如下表1。
表1 FW-Z2化合物的1H和13C(DMSO-d6)归属
FW-Z5:物理化学性状为棕色无定型固体,分子式:C55H96O16,其不饱和度为8。高分辨质谱:测量值:m/z 1035.6597[M+Na]+,理论值m/z1035.6591[M+Na]+。溶解度:可溶于甲醇、丙酮、乙腈、乙酸乙酯和二甲亚砜等有机溶剂,不溶于水。
FW-Z5的1H核磁共振波谱(DMSO-d6,600MHz)1H NMR(600MHz,DMSO-D6)δ6.64(dd,J=15.5,10.4Hz,1H),5.97(dq,J=14.5,10.7Hz,2H),5.82(d,J=15.4Hz,1H),5.40(d,J=3.7Hz,1H),5.11(dd,J=14.0,9.9Hz,1H),4.86(dd,J=11.2,5.2Hz,4H),4.80(s,1H),4.38(d,J=7.3Hz,1H),4.25(s,1H),4.24(s,1H),4.10(br,1H),4.07(s,1H),4.06(s,1H),4.02(s,3H),3.87(m,2H),3.79(dt,J=10.8,2.5Hz,1H),3.67(dd,J=10.3,1.5Hz,1H),3.62(s,1H),3.55(d,J=9.6Hz,1H),3.52(m,1H),3.47(dd,J=13.5,4.7Hz,1H),3.37(dd,J=8.2,2.7Hz,1H),3.30(d,J=2.9Hz,1H),3.25(d,J=2.7Hz,1H),3.20(dd,J=8.9,6.1Hz,1H),3.00(m,1H),2.33(td,J=9.8,6.4Hz,1H),2.15(dd,J=14.5,3.9Hz,1H),2.11(dd,J=13.5,2.3Hz,1H),2.07(d,J=10.8Hz,1H),1.93(dd,J=13.5,3.9Hz,1H),1.87(m,1H),1.84(m,1H),1.72(d,J=5.2Hz,1H),1.70(d,J=4.7Hz,1H),1.62(qd,J=13.8,7.3,2.8Hz,1H),1.55(m,7H),1.48(d,J=8.5,4.2Hz,2H),1.43(d,J=4.3Hz,2H),1.41(t,J=3.0Hz,1H),1.39(d,J=2.7Hz,1H),1.35(m,1H),1.34(d,J=2.5Hz,2H),1.32(d,J=3.9Hz,1H),1.24(m,2H),1.21(dd,J=7.3Hz,1H),1.18(d,J=3.6Hz,1H),1.16(d,J=6.1Hz,3H),1.04(d,J=6.6Hz,3H),1.00(d,J=6.2Hz,3H),0.91(d,J=6.6Hz,3H),0.88(m,11H),0.72(m,9H),0.64(d,J=7.0Hz,3H)。
FW-Z5的13C核磁共振波谱(DMSO-D6,150MHz):δ164.2,150.1,136.4,131.6,130.9,130.8,121.5,102.9,98.7,81.7,79.9,79.0,76.3,75.6,75.5,72.8,71.5,70.2,69.9,67.8,67.4,63.6,45.7,43.3,41.3,40.4,38.5,38.3,37.0,35.4,35.3,32.9,32.1,31.6,31.0,30.7,30.3,29.9,27.7,27.3,24.6,21.1,18.4,18.1,17.9,15.9,15.0,14.9,13.7,9.3,9.1,6.1,4.6。
同时,本发明还测定了该化合物FW-Z5的多种核磁共振图谱,分别见图8-13所示,从而确定了该化合物所有的碳原子和氢原子的归属及该化合物的化学结构,确定其为结构新颖的大环内酯化合物,结构表征如下表2。
表2.FW-Z5化合物的1H和13C(DMSO-d6)归属
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综上,本发明经提取纯化得到的化合物FW-Z2和化合物FW-5的结构式如下:
所述化合物FW-Z2中,R1为=O,R2为-CH3;
所述化合物FW-Z5中,R1为-OH,R2为-CH2CH3。
实施例4大环内酯化合物FW-Z2与FW-Z5生物活性测定
本实施例对大环内酯化合物FW-Z2与FW-Z5进行了体外抑制真菌试验,采用纸片-琼脂扩散实验(paper-agar disk diffusion assay)测定了化合物FW-Z2与FW-Z5对细菌与真菌的抑制活性,结果表明其具有显著的抑制黑曲霉的作用。
首先将大肠杆菌、金黄色葡萄球菌、枯草杆菌以10-8个/ml菌落密度倒MH平板;白色念珠菌、黑曲霉以10-8个/ml菌落密度倒沙氏平板。分别取上述纯化得到的FW-Z2与FW-Z5溶解于甲醇溶液,取8μl待测样品于6mm直径的圆形滤纸片上,等滤纸片上甲醇会发完全后,将载有样品的滤纸片贴于含有上述浓度的测试菌(大肠杆菌、金黄色葡萄球菌、枯草杆菌、白色念珠菌、黑曲霉)平板上,同时,以甲醇溶液作为阴性对照,28℃恒温培养24-48小时。观察记录抑菌圈直径大小,抑菌圈直径越大则说明该菌株的抗真菌活性越强。
实验结果表明,涉及的两个化合物FW-Z2和FW-Z5都表现出了抗真菌活性,抑菌圈直径9-12mm。因此,FW-Z2与FW-Z5可作为抗真菌活性先导化合物。
综上,从该大环内酯化合物的体外抑制真菌活性试验表明,上述化合物FW-Z2和FW-Z5具有抑制真菌活性,为研究开发新的抑制真菌药物提供了先导化合物,对开发利用中国的海洋药物资源具有重要价值。
显然,上述实施例仅仅是为清楚地说明所作的举例,而并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (8)
1.一种大环内酯类化合物FW-Z,其特征在于,所述大环内酯类化合物FW-Z具有如下式(Ⅰ)所示的结构:
其中,R1为=O时,R2为-CH3;R1为-OH时,R2为CH2CH3。
2.根据权利要求1所述大环内酯类化合物FW-Z,其特征在于,所述大环内酯类化合物FW-Z为化合物FW-Z2;其中,R1为=O,R2为-CH3。
3.根据权利要求1所述大环内酯类化合物FW-Z,其特征在于,所述大环内酯类化合物FW-Z为化合物FW-Z5;其中,R1为-OH,R2为-CH2CH3。
4.一种海洋小单孢菌株,其分类命名为放线菌小单孢菌属Micromonosporasp.FIMYZ51,于2021年12月9日保藏于中国科学院微生物研究所,保藏编号为CGMCCNo.24067。
5.权利要求4所述海洋小单孢菌株用于发酵制备权利要求1-3任一项所述大环内酯类化合物FW-Z的用途。
6.一种发酵制备权利要求1-3任一项所述大环内酯类化合物FW-Z的方法,其特征在于,包括如下步骤:
(1)取斜面保存的所述海洋小单孢菌Micromonospora sp.FIMYZ51接入液体种子培养基中进行恒温培养,收集种子液,备用;
所述液体种子培养基的组分包括:可溶性淀粉1-2wt%、葡萄糖0.1-1wt%、蛋白胨0.1-1wt%、酵母提取物0.1-1wt%、MgSO4·7H2O 0.01-0.1wt%、NaCl0.01-0.1wt%、(NH4)2SO40.01-0.1wt%、CaCO3 0.05-0.2wt%,pH 6.0-8.5;
所述培养步骤的温度为25-35℃,培养时间1-3天;
(2)将所述种子液转接至发酵培养基中进行恒温培养,即得含有所需大环内酯类化合物FW-Z的发酵液;
所述发酵培养基的组分包括:可溶性淀粉3-5wt%、葡萄糖0.1-1wt%、黄豆饼粉2-3wt%、酵母粉0.1-1wt%、MgSO4·7H2O 0.01-0.1wt%、K2HPO40.01-0.1wt%、CaCO3 0.05-0.2wt%,pH 6.0-8.5;
所述培养步骤的温度为25-35℃,培养时间3-6天。
7.根据权利要求6所述发酵制备所述大环内酯类化合物FW-Z的方法,其特征在于,所述方法还包括对所述大环内酯类化合物FW-Z进行提取及纯化的步骤,具体包括:
提取:将收集的发酵液和菌丝体进行固液分离,其中,所述发酵液用大孔树脂HP20进行吸附,吸附后经乙醇解吸回收并浓缩后得到粗提物A;所述菌丝体经醇溶剂浸提,收集提取液减压浓缩得到粗提物B;
纯化:将上述粗提物A和粗提物B进行合并,采用正相硅胶柱层析以体积比10:0-0:10的石油醚:乙酸乙酯溶剂梯度洗脱,薄层层析检测,合并浓缩10:1-1:1洗脱液;反向C18柱层析色谱法以体积比50%-100%的甲醇:水溶剂梯度洗脱分离,合并收集甲醇:水的体积比为70%-90%区段含大环内酯化合物FW-Z的洗脱液;经过制备型C18反相高压液相色谱以甲醇水60-100%梯度洗脱,得到具有抗真菌活性的大环内酯化合物FW-Z2与FW-Z5纯品。
8.权利要求1-3任一项所述大环内酯类化合物FW-Z用于制备医用、兽用或农用非治疗目的黑曲霉抑制剂的用途。
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