CN117343844A - 一种青霉菌及其发酵产Atpenin A5的方法 - Google Patents
一种青霉菌及其发酵产Atpenin A5的方法 Download PDFInfo
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- OVULNOOPECCZRG-UHFFFAOYSA-N Atpenin A5 Natural products COC=1NC(O)=C(C(=O)C(C)CC(C)C(Cl)CCl)C(=O)C=1OC OVULNOOPECCZRG-UHFFFAOYSA-N 0.000 title claims abstract description 37
- OVULNOOPECCZRG-CIUDSAMLSA-N atpenin-a5 Chemical compound COC=1NC(=O)C(C(=O)[C@@H](C)C[C@H](C)[C@@H](Cl)CCl)=C(O)C=1OC OVULNOOPECCZRG-CIUDSAMLSA-N 0.000 title claims abstract description 37
- 241000228143 Penicillium Species 0.000 title claims abstract description 26
- 238000000855 fermentation Methods 0.000 title claims description 37
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- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 24
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 24
- 239000007788 liquid Substances 0.000 claims description 21
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/14—Fungi; Culture media therefor
- C12N1/145—Fungal isolates
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- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
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- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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Abstract
本发明公开了一株菌株青霉菌(Penicillium sp.)HDCC00055(CGMCC NO.23056)为一株全新的Atpenin A5产生菌,生产能力高,其发酵积累Atpenin A5的能力比现有技术中其他菌种有了大幅度的提高,Atpenin A5的效价可达418mg/L以上,便于工业化生产。
Description
技术领域
本发明涉及工业微生物发酵技术领域,具体涉及一种青霉菌及其发酵产AtpeninA5的方法。
背景技术
Atpenin A5是一种有效的复合物II(琥珀酸-泛醌氧化还原酶) 抑制剂,复合物II是线粒体电子传递链上重要功能复合物,已经成为农用杀菌剂方面的关键靶标,该类杀菌剂在植物病原真菌保护中发挥着重要的作用。基于复合物II的呼吸系统抑制剂被广泛用于世界范围内的真菌病害,该类抑制剂通过与复合物II的泛醌还原位点结合而抑制真菌的呼吸,作用方式独特,与其他类别的杀菌剂如苯并咪唑类、strobilurins类、氨基吡啶类均无交互抗性,因此成为改善杀菌剂抗性和提高病害控制的优秀候选品种。
1988年,由日本Kitasato公司从土壤中分离得到的一株菌株 Penicilliumsp.FO-125的代谢产物中分离获得。Atpenin A5是一种胞内脂溶性抗真菌类抗生素,在筛选脂代谢微生物抑制剂的过程中, Hidetoshi Kumagai等发现了从青霉菌FO-125的培养液中分离到一系列抗真菌抗生素atpenins,包含三种有效组分,A4,A5和B,他们都具有抗真菌活性,尤其是对Trichophyton sp.真菌的抑制能力最强,其中又以Atpenin A5的抗真菌活性最强。
Masaki Ohtaw等,Enantioselective total synthesis of atpenin A5,报道了Atpenin A5具有比较出众的活性,对其进行了全合成研究,也完成了合成产物的结构确证。但化学合成存在反应步骤长,收率低,对安全性要求高,且成本方面也没有明显优势。Satoshi Omura等, ATPENINS,NEW ANTIFUNGAL ANTIBIOTICS PRODUCED BY PENICILLIUMSP.,报道Atpenin A5的产量为1-3mg/L。
针对现有技术中发酵获得Atpenin A5存在发酵产量低,而化学合成制备工艺中存在的合成步骤长、收率低,且用到的试剂易燃易爆,较不安全等一系列的问题,因此,本发明寻求一种新的微生物,并可通过简单的发酵即可获得较高发酵产量的Atpenin A5,从而实现 Atpenin A5较低的生产成本。
发明内容
为了解决Atpenin A5现有制备方法不足的问题,本发明的目的之一在于提供一种青霉菌(Penicillium sp.)HDCC00055保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)(地址:北京市朝阳区北辰西路1号院3号,中国科学院微生物研究所),保藏编号为 CGMCC NO.23056,保藏日期为2021年07月26日,并登记在册,证明存活。
本发明的目的还在于提供了一种青霉菌(Penicillium sp.)HDCC00055在制备Atpenin A5或者含有Atpenin A5的药物组合物的应用。
本发明还提供了一种Atpenin A5的制备方法,该包括采用青霉菌 (Penicilliumsp.)HDCC00055在含有可同化的碳源和/或氮源的营养培养基里,进行有氧发酵。
在优选的实施方案中,所述可同化的碳源选自葡萄糖、甘油、山梨醇、蔗糖、麦芽糖、糊精、淀粉之一或者任意几种的组合,优选葡萄糖、甘油、山梨醇。
在优选的实施方案中,所述可同化的氮源选自黄豆饼粉、棉籽饼粉、玉米蛋白粉、玉米粉、小麦胚芽粉、酵母浸粉、蛋白胨、酵母膏、玉米浆之一或者任意几种的组合;优选为黄豆饼粉、棉籽饼粉。
在优选的实施方案中,所述营养培养基还包括无机盐,所述无机盐选为硫酸铵、磷酸二氢钾、硫酸镁、氯化钾、氯化钙之一或任意几种的组合。
在优选的实施方案中,所述营养培养基含有:
葡萄糖0~4%
山梨醇1~9%
淀粉0~3%
酵母浸粉0~3%
蛋白胨0~3%
硫酸铵0~1.5%
磷酸二氢钾0.1~0.5%
硫酸镁0.05~0.2%
氯化钾0.05~0.15%
氯化钙0.05~0.15%
在优选的实施方案中,所述有氧发酵的温度为23~30℃,优选25~27℃;培养基pH为5.0~7.0,优选为5.0~6.0;培养时间为96~144 小时,优选为96~120小时;通气量为0.5~1.2vvm,优选为0.8~1.0vvm。
在优选的实施方案中,所述青霉菌(Penicillium sp.)HDCC00055 是通过种子液接种至所述营养培养基中进行所述发酵培养的;
其中,所述种子液是将权利要求1所述的青霉菌(Penicillium sp.)HDCC00055在种子培养基里进行种子培养得到的。
在优选的实施方案中,所述的种子培养基含有:
葡萄糖2~6%
硫酸铵0.8~1.5%
磷酸二氢钾0.02~0.1%
硫酸镁0.02~0.1%
在优选的实施方案中,所述种子培养的条件为:种子培养的温度为23~30℃,优选25~27℃;培养基pH为5.0~7.0,优选为5.0~6.0;培养时间为24~72小时,优选为30~40小时。
在优选的实施方案中,所述的种子培养液,移种时的关键控制指标 pH为2.5~4.0。
本发明Atpenin A5通过以下条件进行HPLC检测:
本发明Atpenin A5效价检测所用的液相检测方法条件如下:
HPLC检测方法为:
色谱柱:C18 4.6*250mm 5um
柱温:35℃
进样量:5ul
流动相:A(水):B(0.1%甲酸乙腈溶液)=65:35
流速:1.0ml/min。
本发明所述青霉菌(Penicillium sp.)HDCC00055(CGMCC NO.23056)主要生物学特征为:菌落正圆形,边缘整齐,中心孢子丰满,表面呈墨绿色,边缘为白色气生菌丝、微凸,反面浅黄色,无渗出液,无可溶性色素。显微镜下观察,该菌种菌丝粗、网状、顶端产生多细胞的分生孢子梗,分生孢子呈现扫帚状,染色较好。
本发明所述菌株青霉菌(Penicillium sp.)HDCC00055(CGMCC NO.23056)为一株全新的Atpenin A5产生菌,生产能力高,其发酵积累Atpenin A5的能力比现有技术中其他菌种有了大幅度的提高, Atpenin A5的效价可达314mg/L以上,便于工业化生产。
附图说明
图1为菌株青霉菌(Penicillium sp.)HDCC00055(CGMCC NO.23056)菌落特征图。
图2为原始生产菌株青霉菌(Penicillium sp.)ATP-434通过发酵培养后,对菌体进行分离提取,经HPLC检测的Atpenin A5图谱。
具体实施方式
下述实施例中所用的实验方法如无特殊说明,均为常规方法。
下述实施例中采用的材料、试剂等如无特殊说明,皆为普通市售品,皆可于市场购得。
以下结合具体实施例,对本发明作进一步说明,应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
实施例1菌株来源
取我国安徽安庆菱湖景区的土壤样品,加入到50m L无菌水中,在250rpm振荡10min后得到土壤菌原液。分别取上述菌原液1ml,接种到含有50mg/L青霉素的PDB培养基中,于25℃、250rpm过夜富集培养,之后用无菌水分别依次稀释为10-2、10-3、10-4、10-5 四个不同浓度的菌液,将各浓度菌液取100μL涂布于含有50mg/L 青霉素的PDA平板,放置于25℃培养箱中培养4天。待菌落长出后,挑选合适浓度涂布的单菌落的平板,从平板上挑取圆形、青色孢子量丰满的单菌落,分别在PDA平板上划线分离纯化,纯化后的菌株制成PDA斜面菌苔。斜面菌苔分别制成孢子悬液,以滤纸片法接种到涂满白色念珠菌的PDA平板表面,置于25℃培养箱中培养5天,观察是否有青霉菌生长且抑菌圈明显。挑选抑菌圈大的青霉菌落,采用 8mm打孔器挖取菌落及底部琼脂培养基加入到10ml离心管中,加入无水乙醇1ml,用3颗玻璃珠振荡打散30min,离心,上清液过滤后采用HPLC进行检测,保留与Atpenin A5对照品有相同保留时间的菌株和样品,进一步采用LCMS进行分子量测定,最终获得了生产Atpenin A5的原始生产菌种(原始编号为ATP-434)。
实施例2Atpenin A5高产菌种筛选
以原始菌种(ATP-434)为出发菌株,甘油管0.1ml接种PDA斜面,置于25℃培养7天,用pH6.5的磷酸缓冲液将新鲜菌苔孢子洗下,加玻璃珠振荡打散,获得孢子菌悬液。孢子菌悬液与860μg/ml的 NTG母液等体积混合,置于25℃摇床上诱变处理30min。取诱变液于离心管中,14000rpm高速离心,弃上清后用0.1%硫代硫酸钠溶液重悬,如此反复离心洗涤3次后进行梯度稀释。取不同稀释梯度的稀释液涂布到PDA平板上,置于25℃培养3天,获得分离单菌落。分离单菌落一对一点种到新鲜PDA平板,置于25℃培养5天,得到纯化点种单菌落,用接种铲刮取少量单菌落孢子,接种到液体初筛发酵培养基中并搅散,置于25℃、250rpm摇床上振荡培养5天,获得初筛发酵液。取发酵液2ml,用2ml无水乙醇浸泡超声30min,离心过滤后进行HPLC检测。挑选出最高产菌株即青霉菌(Penicillium sp.)HDCC00055(CGMCCNO.23056)。
初筛发酵培养基为:40g/L葡萄糖,20g/L黄豆饼粉,硫酸铵0.7%, 1g/L氯化钠,pH6.0。
HPLC检测方法为:
色谱柱:C18 4.6*250mm 5um
柱温:35℃
进样量:5ul
流动相:A(水):B(0.1%甲酸乙腈溶液)=65:35
流速:1.0ml/min。
实施例3Atpenin A5高产菌种(CGMCC NO.23056)形态学检查、18S rDNA鉴定
取CGMCC NO.23056菌种甘油管,稀释涂布于PDA平板,置于 25℃培养3天,菌落正圆形,边缘整齐,中心孢子丰满,表面呈墨绿色,边缘为白色气生菌丝、微凸,反面浅黄色,无渗出液,无可溶性色素。显微镜下观察,该菌种菌丝粗、网状、顶端产生多细胞的分生孢子梗,分生孢子呈现扫帚状,染色较好。pH试验结果表明该菌种最适生长pH范围为4.0~6.0,温度试验结果表明最适生长温度范围为 23~28℃。
实施例4菌种鉴定
青霉菌(Penicillium sp.)HDCC00055(CGMCC NO.23056)的18S rDNA序列分析参照《分子克隆实验指南》书中的有关内容进行实验。收集菌体,然后用真菌DNA提取试剂盒抽提总DNA。
利用通用引物进行PCR扩增(美国BioRad公司,PTC200扩增仪),用0.9%的琼脂糖凝胶电泳鉴定PCR产物,纯化回收采用 AxyPrep凝胶回收试剂盒,纯化后的产物鉴定委托浙江工业大学生物工程研究所进行18S rDNA序列测定。
通用引物序列如下:NS1 GTAGTCATATGCTTGTCTC
NS6 GCATCACAGACCTGTTATTGCCTC
菌株(Penicillium sp.)HDCC00055所测得到的18S rDNA序列 (SEQ ID NO:1)经校对后与GenBank数据库中相关种、属的序列进行同源序列BLAST比较,最终确定该菌株为青霉菌(Penicillium sp.)。
该菌种18S rDNA序列(SEQ ID NO:1)和BLAST比对结果如表1 所示(表中只列出同源性较高的模式菌株):
表1菌株(Penicillium sp.)HDCC00055和典型模式菌株的同源性
实施例5Atpenin A5摇瓶发酵制备
取CGMCC NO.23056菌株甘油管,划线法接种PDA斜面,置于 25℃培养3-5天,获得斜面孢子,用接种铲刮取少量孢子,接种到液体种子培养基,置于25℃培养40h,获得摇瓶种子液,种子液pH达到2.5~4.0之间移种,发酵移种量为5%。移种完成后的摇瓶发酵放置到25℃,250rpm的摇床上振荡培养120h后结束,取发酵液1ml用无水乙醇浸泡后离心过滤,上清液采用HPLC检测。该批发酵液中 Atpenin A5的效价为396mg/L。
种子培养基组成为:葡萄糖2%,硫酸铵0.8%,磷酸二氢钾0.02%,硫酸镁0.1%,pH 6.0。
发酵培养基组成为:山梨醇9%,淀粉3%,酵母浸粉3%,硫酸铵1.5%,磷酸二氢钾0.5%,硫酸镁0.05%,氯化钾0.15%,氯化钙 0.05%,pH为6.0。
实施例6Atpenin A5摇瓶发酵制备
取CGMCC NO.23056菌株甘油管,划线法接种PDA斜面,置于25℃培养3-5天,获得斜面孢子,用接种铲刮取少量孢子,接种到液体种子培养基,置于27℃培养30h,获得摇瓶种子液,种子液pH达到2.5~4.0之间移种,发酵移种量为20%。移种完成后的摇瓶发酵放置到27℃,250rpm的摇床上振荡培养96h后结束,取发酵液1ml用无水乙醇浸泡后离心过滤,上清液采用HPLC检测。该批发酵液中 Atpenin A5的效价为412mg/L。
种子培养基组成为:葡萄糖6%,硫酸铵1.5%,磷酸二氢钾0.1%,硫酸镁0.02%,pH 5.0。
发酵培养基组成为:葡萄糖4%,山梨醇1%,蛋白胨3%,磷酸二氢钾0.1%,硫酸镁0.2%,氯化钾0.05%,氯化钙0.15%,pH为5.0。
实施例7Atpenin A5 50L罐发酵制备
取CGMCC NO.23056菌株甘油管,划线法接种PDA斜面,置于 25℃培养3-5天,获得斜面孢子,用接种铲刮取少量孢子,接种到液体种子培养基,置于27℃培养40h,获得一级摇瓶种子液。一级摇瓶种子液以0.05%的比例接种到装有10L液体培养基的种子罐中,设定温度27℃,罐压0.05Mpa,空气流量1vvm,初始搅拌100rpm,搅拌联动控制溶氧≥40%,培养周期30h,二级种子液pH达到2.5~4.0之间移种。二级种子液以10%的比例接种到装有30L液体发酵培养基的发酵罐中,设定温度26℃,罐压0.05Mpa,空气流量1vvm,初始搅拌100rpm,待溶氧下降到20%以下,开启搅拌联动,控制溶氧≥ 10%。发酵24h以后用碱水控制pH,使之维持在5.0~6.0之间。发酵 24h后每天取样检测效价,具体方法为发酵液1ml,用无水乙醇浸泡,离心过滤后进行HPLC检测。最终,发酵液中Atpenin A5的效价达到418mg/L。
种子培养基组成为:葡萄糖4%,硫酸铵1.0%,磷酸二氢钾0.1%,硫酸镁0.1%,pH6.0。
发酵培养基组成为:葡萄糖2%,山梨醇6%,玉米淀粉2%,酵母浸粉1%,蛋白胨2%,磷酸二氢钾0.5%,硫酸镁0.2%,氯化钾0.15%,氯化钙0.15%,pH为6.0。
序列表
<110> 浙江珲达生物科技有限公司
<120> 一种青霉菌及其发酵产Atpenin A5的方法
<130> P0102022060594
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 576
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atttccgtag gggaacctgc ggaaggatca ttaccgagtg agttccctct gacctcccac 60
ccgtgtttat tttaccttgt tgcttacgcg agcctgcctt cgggctgccg gggggcatct 120
gcccccgggt ccgcgctcgc cggagacacc tcgaactctg tctgaagatt gtagtctgag 180
acaaaatata aattatttaa aactatcaac aacggatctc ttggttccgg catcgatgaa 240
agacgcagcg aaatgcgata cgtaatgtga attgcagaat tcagtgaatc atcgagtctt 300
tgaacctaca ttgcgccctc tggtattccg gagcccatgc ctgtccgagc gtcattgctg 360
ccctcaagca cggcttgtgt gttgggctcc gtcctccttc tggggggacg ggcccgaaag 420
gcagcggcgg caccgcgtcc ggtcctcgag cgtatggggc tttgtcaccc gctctgtagg 480
actggccggc gcctgccgat caaccaaact tttttccagg ttgacctcgg atcaggtagg 540
gatacccgct gaacttaagc atatcaataa ttgccg 576
Claims (14)
1.一种青霉菌(Penicillium sp.)HDCC00055保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC),保藏编号为CGMCC NO.23056,保藏日期为2021年07月26日。
2.根据权利要求1所述的青霉菌(Penicillium sp.)HDCC00055在制备Atpenin A5或者含有Atpenin A5的药物组合物的应用。
3.一种含权利要求1所述的青霉菌(Penicillium sp.)HDCC00055的发酵液。
4.一种Atpenin A5的制备方法,其特征在于:包括采用如权利要求1所述的青霉菌(Penicillium sp.)HDCC00055进行发酵制备。
5.根据权利要求4所述的方法,其特征在于:所述的发酵过程包括在含有可同化的碳源和/或氮源的营养培养基里,进行有氧发酵。
6.根据权利要求5所述的方法,其特征在于:所述可同化的碳源选自葡萄糖、甘油、山梨醇、蔗糖、麦芽糖、糊精、淀粉之一或者任意几种的组合,优选葡萄糖、淀粉、山梨醇。
7.根据权利要求5所述的方法,其特征在于:所述可同化的氮源选自黄豆饼粉、棉籽饼粉、玉米蛋白粉、玉米粉、小麦胚芽粉、酵母浸粉、蛋白胨、酵母膏、玉米浆之一或者任意几种的组合;优选为黄豆饼粉、棉籽饼粉之一或者任意几种的组合。
8.根据权利要求5所述的方法,其特征在于:所述营养培养基还包括无机盐,所述无机盐为硫酸铵、磷酸二氢钾、硫酸镁、氯化钾、氯化钙中的一种或任意几种的组合。
9.根据权利要求5所述的方法,其特征在于:所述营养培养基含有:葡萄糖0~4%、山梨醇1~9%、淀粉0~3%、酵母浸粉0~3%、蛋白胨0~3%、硫酸铵0~1.5%、磷酸二氢钾0.1~0.5%、硫酸镁0.05~0.2%、氯化钾0.05~0.15%、氯化钙0.05~0.15%。
10.根据权利要求5所述的方法,其特征在于:所述有氧发酵的温度为23~30℃,优选25~27℃;培养基pH为5.0~7.0,优选为5.0~6.0;培养时间为96~144小时,优选为96~120小时;通气量为0.5~1.2vvm,优选为0.8~1.0vvm。
11.根据权利要求4-10任一项所述的方法,其特征在于:所述青霉菌(Penicilliumsp.)HDCC00055是通过种子液接种至所述营养培养基中进行所述发酵培养的;
其中,所述种子液是将权利要求1所述的青霉菌(Penicillium sp.)HDCC00055在种子培养基里进行种子培养得到的。
12.根据权利要求11所述的方法,其特征在于:所述的种子培养基含有葡萄糖2~6%、硫酸铵0.8~1.5%、磷酸二氢钾0.02~0.1%、硫酸镁0.02~0.1%。
13.如权利要求11所述的方法,其特征在于:所述种子培养的条件为:种子培养温度为23~30℃,优选25~27℃;培养基pH为5.0~7.0,优选为5.0~6.0;培养时间为24~72小时,优选为30~40小时。
14.根据权利要求13所述的方法,其特征在于:所述的种子培养液,移种时的关键控制指标pH为2.5~4.0。
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