CN117247842A - 一种转化人参皂苷Rb1的间座壳属真菌X-Z-5及其应用 - Google Patents
一种转化人参皂苷Rb1的间座壳属真菌X-Z-5及其应用 Download PDFInfo
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- GZYPWOGIYAIIPV-JBDTYSNRSA-N ginsenoside Rb1 Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H](O)[C@@H]1O)O)O[C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O GZYPWOGIYAIIPV-JBDTYSNRSA-N 0.000 title claims abstract description 49
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Abstract
本发明公开了一种转化人参皂苷Rb1的间座壳属真菌X‑Z‑5及其应用,该间座壳属真菌X‑Z‑5保藏于中国普通微生物菌种保藏管理中心,保藏地址是北京市朝阳区北辰西路1号院3号,保藏日期为2022年04月06日,保藏编号为CGMCC NO:40154。本发明的三七内生菌Diaporthe sp.X‑Z‑5将人参皂苷Rb1转化为Rd、F2、CK,转化率高达95.7%,产物Rd、F2、CK的摩尔比例约为8:2:1,转化途径为:Rb1→Rd→F2→CK。三七内生菌Diaporthesp.X‑Z‑5转化人参皂苷Rb1所需的转化率高、绿色无污染,可生产特定稀有人参皂苷,具有广阔应用前景。
Description
技术领域
本发明涉及一种间座壳属真菌,具体涉及一种转化人参皂苷Rb1的间座壳属真菌X-Z-5及其应用。
背景技术
三七,别名“田七”、“金不换”、“血参”等,来源于五加科人参属植物三七(Panaxnotoginseng(Burk.)F.H.Chen)的干燥茎或根,性温,味甘、微苦,归肝、肾、胃经,具有散瘀止血、消肿止痛的功效,是著名的中药材。
现代医学研究表明,皂苷类是三七主要的药理活性物质,到目前为止,已经从三七中分离、提取、鉴定出了近120多种皂苷类化合物,其中人参皂苷Rb1是主要的人参皂苷,在三七及人参总皂苷中的含量在20%左右(Acta Pharmaceutica Sinica B,2021,11:1813-1834)。药物代谢动力学研究表明人参皂苷Rb1等因达玛烷型骨架、侧链糖基数量多及糖基分子灵活度高,导致膜通透性差,易被胆道及泌尿系统排出,肠道生物利用率低,但去糖基化人参皂苷如Rd、Gyp17、F2等具有抗肿瘤、抗糖尿病、抗过敏活性,以及神经保护和心脏保护等药理活性而被广泛研究(Medicinal Research Reviews,2017,37:1140-1185)。
通过高温高压、酸碱水解等物理化学法可以将人参皂苷Rb1侧链糖基断裂得到高活性稀有人参皂苷,操作过程简单。但物理化学法在实际应用中存在众多缺点,如高温高压存在安全隐患,反应条件不易调控,副产物多,稀有人参皂苷得率低及过度使用酸碱试剂易对环境造成污染等。
相较于物理化学法而言,生物转化法因反应条件温和,产物单一,环境友好等特性而广受研究者青睐。其中,微生物转化法是利用微生物细胞生长发育过程中产生的代谢产物如酶等将底物转化为目标产物的过程。自然界微生物丰富,种类繁多,特别是真菌资源。真菌蕴含丰富的酶系,包括糖苷水解酶、蛋白酶、脂肪酶等,广泛应用于食品、饲料和生物技术等相关工业。目前用于人参皂苷生物转化的真菌研究多集中于曲霉属、青霉属等。
发明内容
本发明的目的是提供一种转化人参皂苷Rb1的间座壳属真菌X-Z-5及其应用,该间座壳属真菌X-Z-5能够将人参皂苷Rb1转化为Rd、F2、CK,转化率高达95.7%。
为了达到上述目的,本发明提供了一种转化人参皂苷Rb1为其他稀有人参皂苷的间座壳属真菌X-Z-5(Diaporthe sp.),该间座壳属真菌X-Z-5保藏于中国普通微生物菌种保藏管理中心,保藏地址是北京市朝阳区北辰西路1号院3号,保藏日期为2022年04月06日,保藏编号为CGMCC NO:40154。
本发明的另一目的是提供所述的间座壳属真菌X-Z-5或其代谢酶在转化人参皂苷Rb1为其他稀有人参皂苷中的应用。
优选地,所述其他稀有人参皂苷包含:稀有人参皂苷Rd、F2、CK。
本发明的另一目的是提供所述的间座壳属真菌X-Z-5或其代谢酶在医药、保健品或食品行业中的应用。
本发明的另一目的是提供一种转化人参皂苷Rb1为其他稀有人参皂苷的方法,该方法包含:采用所述的间座壳属真菌X-Z-5的代谢酶将人参皂苷Rb1侧链糖基水解,生成其他稀有人参皂苷。
优选地,所述其他稀有人参皂苷包含:稀有人参皂苷Rd、F2、CK。
优选地,所述间座壳属真菌X-Z-5于醋酸-醋酸钠缓冲液中配制成菌体悬液,用于转化人参皂苷Rb1。
优选地,所述醋酸-醋酸钠缓冲液的pH=5。
优选地,将人参皂苷Rb1溶于醋酸-醋酸钠缓冲液中并用滤膜过滤,制成无菌人参皂苷溶液,再与所述菌体悬液反应。
优选地,所述水解在28℃、160rpm条件下进行。
本发明的转化人参皂苷Rb1的间座壳属真菌X-Z-5及其应用,具有以下优点:
本发明从三七样品中分离出了一株新型的高效人参皂苷转化真菌菌株,通过对其进行菌种鉴定和转化特性的研究,发现该菌株是座壳属真菌(Diaporthe sp.),该菌株选择性转化人参皂苷Rb1,转化途径:Rb1→Rd→F2→CK,经检索未见间座壳属真菌(Diaporthesp.)转化人参皂苷的报道。
本发明的三七内生菌Diaporthe sp.X-Z-5将人参皂苷Rb1转化为Rd、F2、CK,转化率高达95.7%,产物Rd、F2、CK的摩尔比例约为8:2:1,转化途径为:Rb1→Rd→F2→CK。三七内生菌Diaporthe sp.X-Z-5转化人参皂苷Rb1所需的转化率高、绿色无污染,可生产特定稀有人参皂苷,具有广阔应用前景。
与现有技术相比,本发明的间座壳属真菌Diaporthe sp.X-Z-5转化人参皂苷Rb1转化工艺简单,易操作,转化率高,利于特定稀有人参皂苷的生产。
本发明的间座壳属真菌X-Z-5能转化人参皂苷Rb1为药理活性更高的稀有人参皂苷Rd、F2、CK,此类皂苷在医药、保健品或食品行业中具有较高的应用价值。
附图说明
图1为本发明Diaporthe sp.X-Z-5的菌落形态图。
图2为本发明Diaporthe sp.X-Z-5在光学显微镜下(400X)的形态图。
图3为本发明Diaporthe sp.X-Z-5在光学显微镜下(1000X)的形态图。
图4为本发明Diaporthe sp.X-Z-5基与18S rDNA序列系统发育树。
图5为本发明Diaporthe sp.X-Z-5转化人参皂苷Rb1 TLC分析;C为对照组;S为人参皂苷Rb1、Rd、F2、CK标准品;1~8分别为X-Z-5水解人参皂苷Rb11~8d分析。
图6为本发明Diaporthe sp.X-Z-5转化人参皂苷Rb1 HPLC分析;a为人参皂苷Rb1、Rd、F2、CK标准品HPLC分析;b为对照组HPLC分析;c为X-Z-5水解人参皂苷Rb18d结果分析。
图7为本发明Diaporthe sp.X-Z-5水解人参皂苷Rb1水解途径。
具体实施方式
下面将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。
本发明实施例中所用到的部分实验材料和试剂:
新鲜三七根采自云南文山;人参皂苷Rb1、人参皂苷Rd、人参皂苷F2、人参皂苷CK均购自上海源叶生物科技有限公司(纯度>98%),其他都为国产试剂(均可从普通生化试剂公司购买得到)。
实施例1菌株的筛选与鉴定
本实施方式中Diaporthe sp.X-Z-5,该菌株来自新鲜的三七根,按照以下方法进行分离培养:
1、筛选培养基和PDA培养基配制
筛选培养基配制:筛选培养基的成分为蛋白胨0.1g、NaCl 0.1g、琼脂2g、三七粉0.5g和ddH2O 100mL;在121℃,30min灭菌,4℃保存备用。
PDA培养基配制:20g马铃薯去皮,煮沸,过滤,取滤液;加入葡萄糖2g、琼脂2g和ddH2O 100mL;在121℃,15min灭菌,4℃保存备用。
2、三七内生菌的筛选
取新鲜三七根,自来水冲洗干净,取无菌滤纸吸干表面水分,放入超净工作台,经75%乙醇浸泡1min,无菌水冲洗5次,放入1%NaClO处理1min,无菌水冲洗5次。
用无菌手术刀将三七根切成直径约1cm小方块组织,无菌镊子将三七组织接入筛选培养基,常温倒置培养4~5d。
待组织块边缘长出菌丝后,反复转接纯化直至菌落形态一致,得到纯培养菌株,将其接种至斜面培养基,4℃冰箱保存备用。
以三七粉作为筛选培养基的唯一碳源,选取在筛选培养基上生长快,长势优的真菌菌株X-Z-5作为进一步研究对象。
3、菌丝形态
采用插片培养法观察真菌菌丝,具体方法如下:将上述菌株X-Z-5接种于新鲜PDA培养基内,用干净的镊子,将无菌的盖玻片以45°角度斜插入培养基中,倒置培养4~5d,待菌丝铺展开来并长过插片的位置后,取出盖玻片置于显微镜下观察菌丝形态。
菌株X-Z-5的主要形态特征为:在PDA固体培养基上,该菌株菌落初期为白色,菌丝短绒毛状,呈放射状(参见图1),一般5d后,菌落长满整个培养基,并逐渐变成深灰色;菌丝发达呈交织的分支状(参见图2),分生孢子呈椭圆形(参见图3)。
4、分子鉴定
用无菌接种环挑取纯培养菌株X-Z-5的菌落接入PDB培养基振荡培养3~4d,CTAB法提取真菌基因组,以真菌通用引物ITS1(SEQ ID NO.1)和ITS4(SEQ ID NO.2)为引物进行PCR扩增,产物送上海生工生物工程技术服务有限公司测序,将所测定的核酸序列在NCBI数据库中进行BLAST同源性比对,并应用软件MEGAX以邻接法(Neighbor-joining)构建系统发育树,确定目标菌株的分类地位。
ITS1(SEQ ID NO.1)序列如下:
5'-TCCGTAGGTGAACCTGCGG-3';
ITS4(SEQ ID NO.2)序列如下:
5'-TCCTCCGCTTATTGATATGC-3'。
菌株X-Z-5所获得的18s rDNA序列如序列表中的SEQ ID NO.3所示,基于ITS序列的邻接法对构建的系统发育树进行分析,结果表明菌株X-Z-5与Diaporthe eres(KP903572.1,MZ078592.1)和Diaporthe heterophyllae(MG600222.1)等亲缘关系最近,且与Diaporthe sp.菌株聚于同一分支(参见图4),结合目前数据及形态学分析,该菌株被鉴定属于真菌子囊菌门(Ascomycota)粪壳纲(Sordariomycetes)间座壳目(Diaporthales)间座壳科(Diaporthaceae)间座壳属(Diaporthe)。因此,菌株X-Z-5为间座壳属真菌Diaporthe sp.,将其定名为Diaporthe sp.X-Z-5,以下简称X-Z-5。
该间座壳属真菌X-Z-5(即Z5)保藏于中国普通微生物菌种保藏管理中心,保藏地址是北京市朝阳区北辰西路1号院3号,保藏日期为2022年04月06日,保藏编号为CGMCC NO:40154。
实施例2Diaporthe sp.X-Z-5真菌定向转化人参皂苷Rb1
1、X-Z-5菌丝体转化缓冲液的配制
(1)将斜面菌株X-Z-5接种于PDA固体培养基上培养5~6d;
(2)配制醋酸-醋酸钠缓冲液(50mM,pH=5),灭菌;
(3)刮取步骤(1)固体培养基上生长的X-Z-5菌体,加入步骤(2)配制的无菌缓冲液制成菌体悬液。
2、人参皂苷Rb1转化
将人参皂苷Rb1溶于上述配制的醋酸-醋酸钠缓冲液,并用滤膜过滤制成无菌人参皂苷溶液,上述菌体悬液混匀(反应体系中加入适量无菌玻璃珠),转化液中人参皂苷Rb1终浓度为0.5mg/mL,于28℃、160rpm条件下培养0~8d,以同等条件下处理但未加菌株悬液的样品作为对照。
3、人参皂苷转化产物鉴定
(1)薄层色谱(TLC)检测
分别取不同时间段(0~8d)的转化液,通过离心去除菌丝体,得到上清液,加入等体积饱和正丁醇水溶液萃取,超声15min,静置30min,取上清正丁醇层,此过程重复3次,旋转蒸发除去样品中的正丁醇,5mL甲醇复溶。
如图5所示,为本发明实施例2中Diaporthe sp.X-Z-5真菌定向转化人参皂苷Rb1的TLC结果,结果显示:与对照组相比较,添加了X-Z-5的转化体系中,在第1d时,人参皂苷Rb1部分侧链糖基被水解,生成人参皂苷Rd,第2d时,少量人参皂苷F2出现,第5d时,少量人参皂苷CK出现,随着时间的推移,人参皂苷Rd、F2、CK含量逐渐增加,而人参皂苷Rb1含量逐渐降低,直至第8d,人参皂苷Rb1几乎完全转化为其他稀有人参皂苷。
(2)高效液相色谱(HPLC)检测
将人参皂苷Rb1转化产物经色谱级甲醇稀释后,用高效液相色谱仪进行分析检测,具体条件如下:
仪器:ThermoFisherU-300;色谱柱:Welchrom C18,5μm,4.6mm×250mm;检测波长203nm,进样量10μL,柱温30℃,流速1.0mL/min。
人参皂苷分析,流动相条件,具体如下:
流动相A为乙腈,流动相B为水相;
0min,18%流动相A,82%流动相B;
0-40min,21%流动相A,79%流动相B;
40-42min,26%流动相A,74%流动相B;
42-46min,32%流动相A,68%流动相B;
46-66min,33.8%流动相A,66.2%流动相B;
66-71min,38%流动相A,62%流动相B;
71-82min,49.1%流动相A,50.9%流动相B;
82-83min,50.6%流动相A,49.4%流动相B;
83-88min,59.6%流动相A,40.4%流动相B;
88-97min,65%流动相A,35%流动相B;
97-109min,85%流动相A,15%流动相B;
109-115min,18%流动相A,82%流动相B。
如图6所示,为本发明实施例2中Diaporthe sp.X-Z-5真菌定向转化人参皂苷Rb1的HPLC图谱,结果表明人参皂苷Rb1、Rd、F2、CK的保留时间分别为58.050min、72.497min、81.230min、94.517min,转化结果与TLC一致,当转化时间增至8d时,人参皂苷Rb1几乎完全转化为人参皂苷Rd、F2、CK,转化率达到95.7%,产物Rd、F2、CK的摩尔比例约为8:2:1,结合图5,人参皂苷Rb1的转化途径为:Rb1→Rd→F2→CK(图7)。
综上所述,本发明从新鲜三七根中分离纯化得到一株真菌,经形态学及分子鉴定,该菌株属于间座壳属真菌Diaporthe sp.X-Z-5,进一步检验发现,该菌株具有人参皂苷Rb1水解能力,可水解人参皂苷Rb1生成稀有人参皂苷Rd、F2、CK,人参皂苷Rb1转化率高达95.7%。
尽管本发明的内容已经通过上述优选实施例作了详细介绍,但应当认识到上述的描述不应被认为是对本发明的限制。在本领域技术人员阅读了上述内容后,对于本发明的多种修改和替代都将是显而易见的。因此,本发明的保护范围应由所附的权利要求来限定。
Claims (10)
1.一种转化人参皂苷Rb1为其他稀有人参皂苷的间座壳属真菌X-Z-5(Diaporthesp.),其特征在于,该间座壳属真菌X-Z-5保藏于中国普通微生物菌种保藏管理中心,保藏地址是北京市朝阳区北辰西路1号院3号,保藏日期为2022年04月06日,保藏编号为CGMCCNO:40154。
2.如权利要求1所述的间座壳属真菌X-Z-5或其代谢酶在转化人参皂苷Rb1为其他稀有人参皂苷中的应用。
3.根据权利要求2所述的应用,其特征在于,所述其他稀有人参皂苷包含:稀有人参皂苷Rd、F2、CK。
4.如权利要求1所述的间座壳属真菌X-Z-5或其代谢酶在医药、保健品或食品行业中的应用。
5.一种转化人参皂苷Rb1为其他稀有人参皂苷的方法,其特征在于,该方法包含:
采用如权利要求1所述的间座壳属真菌X-Z-5的代谢酶将人参皂苷Rb1侧链糖基水解,生成其他稀有人参皂苷。
6.根据权利要求5所述的方法,其特征在于,所述其他稀有人参皂苷包含:稀有人参皂苷Rd、F2、CK。
7.根据权利要求5所述的方法,其特征在于,所述间座壳属真菌X-Z-5于醋酸-醋酸钠缓冲液中配制成菌体悬液,用于转化人参皂苷Rb1。
8.根据权利要求7所述的方法,其特征在于,所述醋酸-醋酸钠缓冲液的pH=5。
9.根据权利要求7所述的方法,其特征在于,将人参皂苷Rb1溶于醋酸-醋酸钠缓冲液中并用滤膜过滤,制成无菌人参皂苷溶液,再与所述菌体悬液反应。
10.根据权利要求7所述的方法,其特征在于,所述水解在28℃、160rpm条件下进行。
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