CN116426391A - 一株出芽短梗霉Aureobasidium pullulans P1及其应用 - Google Patents
一株出芽短梗霉Aureobasidium pullulans P1及其应用 Download PDFInfo
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- CN116426391A CN116426391A CN202310309581.6A CN202310309581A CN116426391A CN 116426391 A CN116426391 A CN 116426391A CN 202310309581 A CN202310309581 A CN 202310309581A CN 116426391 A CN116426391 A CN 116426391A
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Abstract
本发明公开了一株出芽短梗霉AureobasidiumpullulansP1及其在稀有皂苷转化方面的应用,属于微生物及生物医药技术领域。菌株的保藏编号为:CGMCCNo.23227。本发明的出芽短梗霉AureobasidiumpullulansP1能够通过发酵产生β—糖苷酶转化人参皂苷Rb1制备稀有人参皂苷Rg3和CK,目标产物多样且明确,其他杂质干扰较小、产物易生成,转化特异性强、效率高,产品回收率超过60%,在大量发酵生产Rg3和CK的过程中存在明显的优势。
Description
技术领域
本发明涉及微生物及生物医药技术领域,更具体地说是涉及一株出芽短梗霉Aureobasidium pullulans P1及其在转化人参皂苷Rb1制备稀有人参皂苷中的应用。
背景技术
人参皂苷为三萜糖苷类化合物,是人参的主要药用活性成分,根据其在植物中的含量分为主要人参皂苷(Major ginsenoside,如Rb1、Rb2、Rc、Rd、Re和Rg1等占总皂苷的80%以上)和稀有人参皂苷(Rare ginsenoside,如Rg3、Rh1、Rh2、F1、F2、CK、C-O、CY和C-Mc)等。其中稀有人参皂苷是指在人参中含量极低或者不存在的皂苷,但其药理活性优于上述高含量人参皂苷,例如,稀有人参皂苷Rg3具有抗肿瘤、防治心脑血管疾病和冠心病、抑制癌细胞转移、保肝、保护神经、提高免疫力等多种药理活性;稀有人参皂苷CK具有很好的抗细胞突变、抑制肿瘤细胞转移、诱导肿瘤细胞凋亡、逆转肿瘤细胞耐药性、抗过敏和抗炎活性。
虽然人参皂苷Rg3和CK具有很高的药用价值,但由于它们在自然界中含量极其稀少,很难通过传统的方法从人参等植物中分离得到,这对稀有人参皂苷的生产造成很大限制。目前,制备稀有人参皂苷的方法主要是直接酸水解法和微生物/酶转化法。直接酸水解法通常选择性较差、产率低、不易纯化、同时容易造成环境污染。微生物/酶转化法则具备区域选择性、立体选择性和基团选择性高、靶向性好、得率高、副产物少、无污染和容易工业化生产等优点,能完成一些用化学方法难以进行的反应,获得化学法难以得到的稀有人参皂苷如Rg3和CK,被认为是生产稀有人参皂苷最具有潜力的方法。
以一种人参皂苷为底物转化生成特定的稀有人参皂苷,能提高资源利用率和降低人参皂苷原料的提取成本,但各种酶对底物的专一性不同,降解的糖苷键类型也各不相同。目前报道的能转化人参皂苷Rb1同时生成Rg3和CK的菌株很少,且产品回收率比较低、不能满足大规模生产Rg3和CK的实际应用。因此,寻找能发酵生产高效、特异性降解人参皂苷Rb1生产人参皂苷Rg3和CK的糖苷水解酶的菌株成为了研究关键。
发明内容
有鉴于此,本发明提供了一株出芽短梗霉Aureobasidium pullulans P1及其应用。
为了实现上述目的,本发明采用如下技术方案:
一株出芽短梗霉(Aureobasidium pullulan)P1,于2021年9月10日保藏于中国微生物菌种保藏管理委员会普通微生物中心,保藏编号为CGMCC No.23227,保藏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,分类命名为出芽短梗霉Aureobasidium pullulans。
本发明的另一目的是提供上述出芽短梗霉P1在转化人参皂苷Rb1制备稀有人参皂苷中的应用。
优选的,所述稀有人参皂苷为稀有人参皂苷Rg3和稀有人参皂苷CK。
优选的,所述转化人参皂苷Rb1制备稀有人参皂苷是通过出芽短梗霉P1发酵产生β-葡萄糖苷酶实现的。
优选的,所述人参皂苷Rb1转化为稀有人参皂苷的转化途径为Rb1→Rd→Rg3;人参皂苷Rb1转化为稀有人参皂苷CK的转化途径为Rb1→Rd→F2→CK。
经由上述的技术方案可知,与现有技术相比,本发明公开提供了一株出芽短梗霉Aureobasidium pullulans P1菌株,该菌株能够将主要人参皂苷Rb1转化至已商品化且附加值高的稀有人参皂苷Rg3和CK,目标产物多样且明确,其他杂质干扰较小、产物易生成,在大量发酵生产Rg3和CK的过程中存在明显的优势。此外,转化的特异性强,转化Rb1到稀有人参皂苷效率高,产品回收率超过60%。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍,显而易见地,下面描述中的附图仅仅是本发明的实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据提供的附图获得其他的附图。
图1为出芽短梗霉菌P1菌株菌落形态特征;
图2为出芽短梗霉菌株***发育树构建;
图3为出芽短梗霉菌Aureobasidium pullulans P1菌株的发酵转化前后发酵液中人参皂苷Rb1和转化产物的HPLC检测图;
图4为主要人参皂苷Rb1转化稀有人参皂苷的转化途径;
图5为出芽短梗霉Aureobasidium pullulans P1发酵制备β-糖苷酶,并将糖苷酶用于转化主要人参皂苷Rb1前后的人参皂苷HPLC检测图。
具体实施方式
下面对本发明实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本发明一部分实施例,而不是全部的实施例。基于本发明中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本发明保护的范围。
为了进一步理解本发明,下面结合实施例对本发明进行详细阐述,其中如无特殊说明,实施例中涉及的试剂均可以通过商业渠道采购,未提及的方法为常规实验方法,在此不再一一赘述。
实施例1:出芽短梗霉Aureobasidium pullulans P1的鉴定
1、菌株的分离与培养
该菌株来源于植物扣子七内生真菌,具体培养方法为将采自秦岭的野生扣子七的根组织切成1厘米大小,依次用75%乙醇表面消毒1min,2.5%的次氯酸钠表面消毒2min,75%乙醇表面消毒1min,最后用无菌水清洗3-5次后,放入事先灭菌备用的培养皿中,用无菌镊子将组织块***到含有100U/mL硫酸阿卡米星的PDA培养基平板上,然后于28℃恒温培养箱中倒置培养5~7d,直至长出菌丝体。
2.菌株的筛选与鉴定
将PDA平板上生长的菌丝体接种到含有七叶苷的R2A培养基上,于28℃恒温培养箱中倒置培养5~7d,七叶苷-R2A琼脂培养基的组成为:1g/L七叶苷,0.5g/L柠檬酸铁,15.2g/L R2A商品培养基。培养基121℃灭菌20min。
如果菌株能够产生β-糖苷酶将七叶苷水解,则在菌株生长的周围会产生红色的晕。经过以上检测后筛选得到一株能够产生β-糖苷酶的菌株,将其命名为P1菌株,其菌落特征见图1。
将该菌株送华大生物技术有限公司测序,该菌株的ITS序列如SEQ ID NO.1所示。测序结果在NCBI核酸数据库中进行Blast相似性分析,经Blast序列比对及进化树分析(见图2),确定其为出芽短梗霉菌。
实施例2:出芽短梗霉Aureobasidium pullulans P1对Rb1的发酵性能鉴定
将芽短梗霉Aureobasidium pullulans P1菌株培养在PDA培养基中,28℃培养5d,然后将菌块接种到液体培养液中,液体培养液的成分为:20g/L葡萄糖,10g/L酵母粉,0.5g/L NaNO3,1g/L KH2PO4,2g/LNa2HPO4,0.2g/L FeSO4,0.1g/L ZnSO4,0.2g/L CuSO4,1g/LCaCl2,0.5g/L MgSO4·7H2O。初始pH6.5,在培养温度28℃,转速180rpm的条件下培养72h。
用2%的甲醇水溶液溶解人参皂苷Rb1,配制成浓度为200mg/L的人参皂苷Rb1母液,过滤除菌之后,等体积加入到上述出芽短梗霉Aureobasidium pullulans P1发酵72h后的发酵液中继续培养6h;取上清液2mL加入2倍体积的正丁醇进行萃取,然后将上层的正丁醇相移出到新的培养皿,将培养皿放置到通风橱进行挥干至恒重,得干燥物。
用甲醇溶解干燥物,然后进行液相HPLC检测,色谱条件为:色谱柱采用YMC-PackODS-AQ,规格为250mm×4.6mm,5μm;流动相采用A相-B相,A相为水,B相为23wt%乙腈,梯度洗脱方式为:0~12min,采用77wt%~48wt%A;12~35min,采用48wt%~25wt%A;35~36min,采用25wt%A;36~42min,采用25wt%~77wt%A。梯度洗脱过程的体积流量1mL/min,柱温30℃,进样量为10μL。所有样品均在203nm处进行吸收检测。
对菌株发酵Rb1产物进行HPLC检测,将其与标准品系列人参皂苷的HPLC谱图和数据进行比对(见图3),在转化产物中,Rb1的含量降低,出现了人参皂苷Rd,F2,Rg3和CK,确定菌株能够将人参皂苷Rb1转化为稀有皂苷Rg3和CK,其转化的途径(见图4),分别为Rb1→Rd→Rg3和Rb1→Rd→F2→CK。
实施例3:出芽短梗霉Aureobasidium pullulans P1发酵制备的β-糖苷酶对Rb1的发酵性能鉴定
将芽短梗霉Aureobasidium pullulans P1菌株培养在PDA培养基中,28℃培养5d,然后将菌块接种到液体培养液中,液体培养液的成分为:20g/L葡萄糖,10g/L酵母粉,0.5g/L NaNO3,1g/L KH2PO4,2g/L Na2HPO4,0.2g/L FeSO4,0.1g/L ZnSO4,0.2g/L CuSO4,1g/LCaCl2,0.5g/L MgSO4·7H2O。初始pH6.5,在培养温度28℃,转速180rpm的条件下培养96h。
将上述培养结束后的发酵液8000rpm/mim离心去除菌体,上清中加入70%的硫酸铵进行盐析,4℃过夜,离心获得粗蛋白,将粗蛋白溶于水,透析袋进行脱盐,脱盐的酶液进行冻干获得酶样品;用pH 5.0的磷酸缓冲盐分别溶解酶样品和人参皂苷Rb1,配制成200mg/L的酶溶液和200mg/L的人参皂苷Rb1溶液,然后将酶溶液与人参皂苷Rb1溶液等体积混匀,置于温度为55℃的水浴锅中反应1h。反应结束后,用2倍体积的正丁醇对反应液进行萃取,将上层的正丁醇相移出到新的培养皿,然后将培养皿放置到通风橱进行挥干至恒重,得干燥物。
用甲醇溶解干燥物,然后进行液相HPLC检测,色谱条件为:色谱柱采用YMC-PackODS-AQ,规格为250mm×4.6mm,5μm;流动相采用A相-B相,A相为水,B相为23wt%乙腈,梯度洗脱方式为:0~12min,采用77wt%~48wt%A;12~35min,采用48wt%~25wt%A;35~36min,采用25wt%A;36~42min,采用25wt%~77wt%A。梯度洗脱过程的体积流量1mL/min,柱温30℃,进样量为10μL。所有样品均在203nm处进行吸收检测。
对菌株发酵Rb1产物进行HPLC检测,将其与标准品系列人参皂苷的HPLC谱图和数据进行比对(见图5),在转化产物中,Rb1完全被降解,仅检测出稀有人参皂苷Rg3和CK,通过峰面积计算获得稀有人参皂苷Rg3和CK的回收率超过60%。
本说明书中各个实施例采用递进的方式描述,每个实施例重点说明的都是与其他实施例的不同之处,各个实施例之间相同相似部分互相参见即可。对于实施例公开的装置而言,由于其与实施例公开的方法相对应,所以描述的比较简单,相关之处参见方法部分说明即可。
对所公开的实施例的上述说明,使本领域专业技术人员能够实现或使用本发明。对这些实施例的多种修改对本领域的专业技术人员来说将是显而易见的,本文中所定义的一般原理可以在不脱离本发明的精神或范围的情况下,在其它实施例中实现。因此,本发明将不会被限制于本文所示的这些实施例,而是要符合与本文所公开的原理和新颖特点相一致的最宽的范围。
Claims (6)
1.一株出芽短梗霉(Aureobasidiumpullulans)P1,其特征在于,保藏编号为:CGMCCNo.23227。
2.如权利要求1所述一株出芽短梗霉P1在转化人参皂苷Rb1制备稀有人参皂苷中的应用。
3.如权利要求2所述的应用,其特征在于,所述稀有人参皂苷为稀有人参皂苷Rg3和稀有人参皂苷CK。
4.如权利要求2所述的应用,其特征在于,通过出芽短梗霉P1发酵产生β-葡萄糖苷酶实现转化。
5.如权利要求2所述的应用,其特征在于,人参皂苷Rb1转化为稀有人参皂苷的转化途径为Rb1→Rd→Rg3。
6.如权利要求2所述的应用,其特征在于,人参皂苷Rb1转化为稀有人参皂苷CK的转化途径为Rb1→Rd→F2→CK。
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CN116836818B (zh) * | 2023-07-28 | 2024-03-22 | 陕西省微生物研究所 | 一株青霉菌f8816及其应用 |
CN117089465A (zh) * | 2023-08-22 | 2023-11-21 | 陕西省微生物研究所 | 一种疣梗曲霉及应用 |
CN117089465B (zh) * | 2023-08-22 | 2024-05-03 | 陕西省微生物研究所 | 一种疣梗曲霉及应用 |
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