CN113699204A - 一株海洋假单胞菌产出高含量环二肽类化合物方法 - Google Patents
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Abstract
一株海洋假单胞菌产出高含量环二肽类化合物方法,其包括如下步骤,1)将一株海洋假单胞菌即F‑Q127菌种从固体斜面培养基中取适量接种到内装100mL液体培养基的三角瓶中,在28℃、180r·min-1的条件下摇床培养48h,作为种子培养液;2)将该种子培养液按体积分数7%的接种量接种于含150mL液体培养基的三角瓶中,共接种15L,在28℃180r·min-1的条件下摇床培养5d,再对培养液进行萃取,得到菌种发酵液的氯仿萃取物和菌丝体的氯仿萃取物;经薄层检查,菌种发酵液的氯仿萃取物和菌丝体的氯仿萃取物主要斑点相似,将两部分的氯仿萃取物合并得氯仿萃取物;3)对氯仿萃取物采用流式细胞术测定化合物的细胞周期抑制活性,得到环二肽类化合物。
Description
技术领域
本发明涉及环二肽类化合物制备,具体为一株海洋假单胞菌产出高含量环二肽类化合物方法。
背景技术
蛋白质可由酸、碱或酶水解,在水解过程中蛋白质逐步降解成为蛋白胨、多肽、三肽和二肽等越来越小的蛋白质碎片,直到最后成为氨基酸混合物。
在体内二肽通常能被二肽酶所水解而成为两个游离氨基酸。在体内肠黏膜细胞上也存在着吸收二肽或三肽的耗能主动运转体系,故有二肽吸收入细胞先于游离氨基酸之说。
二肽是一种化合物,是天然氨基酸即阿尔法氨基酸(氨基连在连有羧基的碳上)中的氨基于羧基脱水缩合而成的。其中只含一个肽键即-CO-NH-。可水解。
环二肽本质---氨基酸肽键形成的化合物活性肽,与动物营养、荷尔蒙、酵素抑制、调节免疫、抗菌、抗病毒、抗氧化作用直接。
现在环二肽类化合物类化合物制备不便,产量难以提升,同时制备方法繁琐。
发明内容
针对上述情况,为克服现有技术的缺陷,本发明提供一株海洋假单胞菌产出高含量环二肽类化合物方法,有效的解决了现在环二肽类化合物类化合物制备不便,产量难以提升,同时制备方法繁琐的问题。
为实现上述目的,本发明提供如下技术方案:本发明包括如下步骤,1)将一株海洋假单胞菌即F-Q127菌种从固体斜面培养基中取适量接种到内装100mL液体培养基的三角瓶中,在28℃、180r·min-1的条件下摇床培养48h,作为种子培养液;
2)将该种子培养液按体积分数7%的接种量接种于含150mL液体培养基的三角瓶中,共接种15L,在28℃180r·min-1的条件下摇床培养5d,再对培养液进行萃取,得到菌种发酵液的氯仿萃取物和菌丝体的氯仿萃取物;经薄层检查,菌种发酵液的氯仿萃取物和菌丝体的氯仿萃取物主要斑点相似,将两部分的氯仿萃取物合并得氯仿萃取物;
3)对氯仿萃取物采用流式细胞术测定化合物的细胞周期抑制活性,得到环二肽类化合物。
有益效果:通过本发明的制备方法,由一株海洋假单胞菌可产出高含量的环二肽类化合物,分别鉴定为环(丙-亮)[cyclo(Ala-Leu),1]、环(丙-异亮)[cyclo(Ala-Ile),2]、环(丙-缬)[cyclo(Ala-Val),3]、环(苯丙-亮)[cyclo(Phe-Leu),4]、环(丙-脯)[cyclo(Ala-Pro),5]和环(苯丙-缬)[cyclo(Phe-Val),通过不同诱导时机目的蛋白的表达实验方法得到:海洋假单胞菌F-Q127生长5h时加入终浓度0.3mmol/L的诱导剂IPTG诱导4h,为目的蛋白表达的最优条件。
具体实施方式
下面对本发明的具体实施方式做进一步详细说明。
实施例,本发明提供一株海洋假单胞菌产出高含量环二肽类化合物方法,包括如下步骤,1)将一株海洋假单胞菌即F-Q127菌种从固体斜面培养基中取适量接种到内装100mL液体培养基的三角瓶中,在28℃、180r·min-1的条件下摇床培养48h,作为种子培养液;
2)将该种子培养液按体积分数7%的接种量接种于含150mL液体培养基的三角瓶中,共接种15L,在28℃180r·min-1的条件下摇床培养5d,再对培养液进行萃取,得到菌种发酵液的氯仿萃取物和菌丝体的氯仿萃取物;经薄层检查,菌种发酵液的氯仿萃取物和菌丝体的氯仿萃取物主要斑点相似,将两部分的氯仿萃取物合并得氯仿萃取物。
3)对氯仿萃取物采用流式细胞术测定化合物的细胞周期抑制活性,得到环二肽类化合物,即从氯仿提取物中分离得到6个环二肽类化合物,分别鉴定为环(丙-亮)[cyclo(Ala-Leu),1]、环(丙-异亮)[cyclo(Ala-Ile),2]、环(丙-缬)[cyclo(Ala-Val),3]、环(苯丙-亮)[cyclo(Phe-Leu),4]、环(丙-脯)[cyclo(Ala-Pro),5]和环(苯丙-缬)[cyclo(Phe-Val)。
对一株海洋假单胞菌培养的代谢物进行鉴定:得到化合物1:白色无定型粉末,正离子TOF-MS在m/z185处给出伪分子离子峰[M+H]+峰,与分子式C9H16O2N2相符,红外光谱在3193cm-1(m)和1689cm-1(s)处有吸收峰,示有酰亚胺基团,1H-NMR(600MHz,CD3OD)谱给出δ:3.99(1H,qd,J=6.2、0.8Hz,H-6)、3.93(1H,ddd,J=8.5、4.8、0.8Hz,H-3)、1.84(1H,m,H-8)、1.72(1H,m,H-7)、1.63(1H,m,H-7)、1.44(3H,d,J=7.0Hz,H-11)、0.97(3H,d,J=6.6Hz,H-9)和0.95(3H,d,J=6.6Hz,H-10)质子信号,鉴定为环(丙-亮)化合物;
还可采用如下方法制备:取活化的种子菌,以1:100的接种量分别接种于10只5ml含氨苄青霉素LB液体培养基的试管中,37℃、200r/min摇床培养,每隔1h取出一只试管,紫外分光光度计测定OD590值,同时以同样的方法培养E.coliTB1和pMAL-c2X-TB1菌株做对照,即可得到环二肽类化合物。
实验例
海洋假单胞菌F-Q127目的蛋白表达条件的优化生长的实验方法,海洋假单胞菌F-Q127在不同的培养基中生长速率不同,将鉴定正确的转化菌分别接种于TB、GS选择培养基中(均含有100ug/ml氨苄青霉素,0.2%(w/v)葡萄糖),通过紫外分光光度计测定该菌在相同的时间内,在不同的培养基中的生长情况(以OD表示),绘制生长曲线,不同诱导时机目的蛋白的表达,根据结果,分别取活化的种子菌以1:100的接种量接种于5只5ml含氨苄青霉素LB液体培养基的试管中,37℃、200r/min摇床培养,分别在工程菌延迟期(0.5h)、对数期(2h、3h、4h、5h)加入诱导剂IPTG(终浓度为0.3mmol/L),继续震荡4h,取1ml菌液,SDS-PAGE电泳;
结果显示:海洋假单胞菌F-Q127生长5h时加入终浓度0.3mmol/L的诱导剂IPTG诱导4h,为目的蛋白表达的最优条件。
有益效果:通过本发明的制备方法,由一株海洋假单胞菌可产出高含量的环二肽类化合物,分别鉴定为环(丙-亮)[cyclo(Ala-Leu),1]、环(丙-异亮)[cyclo(Ala-Ile),2]、环(丙-缬)[cyclo(Ala-Val),3]、环(苯丙-亮)[cyclo(Phe-Leu),4]、环(丙-脯)[cyclo(Ala-Pro),5]和环(苯丙-缬)[cyclo(Phe-Val),通过不同诱导时机目的蛋白的表达实验方法得到:海洋假单胞菌F-Q127生长5h时加入终浓度0.3mmol/L的诱导剂IPTG诱导4h,为目的蛋白表达的最优条件。
最后应说明的是:以上所述仅为本发明的优选实施例而已,并不用于限制本发明,尽管参照前述实施例对本发明进行了详细的说明,对于本领域的技术人员来说,其依然可以对前述各实施例所记载的技术方案进行修改,或者对其中部分技术特征进行等同替换。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
Claims (1)
1.一株海洋假单胞菌产出高含量环二肽类化合物方法,其特征在于:包括如下步骤,1)将一株海洋假单胞菌即F-Q127菌种从固体斜面培养基中取适量接种到内装100mL液体培养基的三角瓶中,在28℃、180r·min-1的条件下摇床培养48h,作为种子培养液;
2)将该种子培养液按体积分数7%的接种量接种于含150mL液体培养基的三角瓶中,共接种15L,在28℃180r·min-1的条件下摇床培养5d,再对培养液进行萃取,得到菌种发酵液的氯仿萃取物和菌丝体的氯仿萃取物;经薄层检查,菌种发酵液的氯仿萃取物和菌丝体的氯仿萃取物主要斑点相似,将两部分的氯仿萃取物合并得氯仿萃取物;
3)对氯仿萃取物采用流式细胞术测定化合物的细胞周期抑制活性,得到环二肽类化合物,即从氯仿提取物中分离得到6个环二肽类化合物,分别鉴定为环(丙-亮)[cyclo(Ala-Leu),1]、环(丙-异亮)[cyclo(Ala-Ile),2]、环(丙-缬)[cyclo(Ala-Val),3]、环(苯丙-亮)[cyclo(Phe-Leu),4]、环(丙-脯)[cyclo(Ala-Pro),5]和环(苯丙-缬)[cyclo(Phe-Val)。
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