CN115161295B - 一种能将黄酮氧苷转化为黄酮碳苷的酶组合物及其应用 - Google Patents
一种能将黄酮氧苷转化为黄酮碳苷的酶组合物及其应用 Download PDFInfo
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Abstract
本发明涉及一种能将黄酮氧苷类化合物转化为黄酮碳苷类化合物的酶组合物及其应用。该组合物由蛋白DgpA、DgpB和DgpC组成,可将黄酮氧苷类化合物诸如大豆苷和染料木苷转化为黄酮碳苷类化合物诸如葛根素和染料木素‑8‑C‑葡萄糖苷。本发明提供的利用酶组合物生产黄酮碳苷类化合物的方法具有步骤简便、成本低、经济和环保等特点,可用于医药、食品、饲料、化工原料制造和研发等目的。
Description
技术领域
本发明涉及一种能将黄酮氧苷类化合物转化为黄酮碳苷类化合物的酶组合物及其应用。
背景技术
黄酮碳苷类化合物是广泛存在于植物中的一类化合物,其糖基以C-C键直接与黄酮母核相连(吴新安等.解放军药学学报,2005,21(2):135-138)。该类化合物的获得主要通过从植物中分离纯化,或者化学法合成。从植物中分离纯化黄酮碳苷类化合物牡荆素需要提取、分离及层析等多步流程(谢运昌,蒋小华.CN201610283820.5);染料木素-8-C-葡萄糖苷则主要采用化学法合成,步骤繁多(Jesus AR,et al.J Med Chem,2014,57(22):9463-9472)。来源于人粪便的梭菌属菌株PUE所含的基因dgpA、dgpB和dgpC所表达的蛋白质DgpA、DgpB和DgpC具有糖基转移酶活性,在三种酶的共同作用下,可将葛根素脱糖基生成大豆苷元(Nakamura K,et al.Biol Pharm Bull,2019,42(3):417-423;Nakamura K,et al.ApplEnviron Microbiol,2020,86(14):e00607-20)。DgpA、DgpB和DgpC酶组合物也对氧苷类化合物具有脱糖基作用。
发明内容
本发明提供了一种能将黄酮氧苷类化合物转化为黄酮碳苷类化合物的酶组合物,该组合物可用于黄酮碳苷类化合物的制备及结构鉴定。将dgpA、dgpB和dgpC三个基因在大肠杆菌中异源表达,经纯化后得到蛋白质DgpA、DgpB和DgpC。以大豆苷为底物,在三种蛋白酶的共同催化下,可生成葛根素和大豆苷元两种物质;以染料木苷为底物,在三种蛋白酶的共同催化下,可生成染料木素-8-C-葡萄糖苷和染料木素两种物质。将以上两种混合物经分子筛柱层析分离纯化可分别得到染料木素-8-C-葡萄糖苷和葛根素。经液质联用检测鉴定,两种纯化的物质分别为染料木素-8-C-葡萄糖苷和葛根素。本发明提供的利用酶组合物生产黄酮碳苷类化合物的方法具有步骤简便、成本低、经济和环保等特点,可用于医药、食品、饲料、化工原料制造和研发等目的。
本发明涉及的dgpA、dgpB和dgpC三个基因公开于GenBank数据库,具体信息如下:
dgpA(GenBank:BBG22493.1)
dgpB(GenBank:BBG22494.1)
dgpC(GenBank:BBG22495.1)
附图说明
图1是蛋白质DgpA、DgpB和DgpC转化染料木苷的高效液相色谱图
如图1所示,其中a为对照品染料木苷和染料木素的色谱图,1为染料木苷的色谱峰,2为染料木素的色谱峰;b为转化前检测到的染料木苷的色谱图;c转化后检测到的染料木素-8-C-葡萄糖苷和染料木素的色谱图,3为染料木素-8-C-葡萄糖苷的色谱峰。
图2是分子筛柱层析纯化前的染料木苷反应物高效液相色谱图
如图2所示,分子筛柱层析纯化前,反应体系中主要有两种物质染料木素-8-C-葡萄糖苷和染料木素,1为染料木素-8-C-葡萄糖苷的色谱峰,2为染料木素的色谱峰。
图3是分子筛柱层析纯化后的染料木苷反应物高效液相色谱图
如图3所示,分子筛柱层析纯化后,反应体系中主要有一种物质染料木素-8-C-葡萄糖苷,1为染料木素-8-C-葡萄糖苷的色谱峰。
图4是液质联用法鉴定纯化后的染料木素-8-C-葡萄糖苷谱图
如图4所示,根据质谱鉴定该物质为染料木素-8-C-葡萄糖苷。
图5是蛋白质DgpA、DgpB和DgpC转化大豆苷的高效液相色谱图
如图5所示,其中a为对照品大豆苷和大豆苷元的色谱图,1为大豆苷的色谱峰,2为大豆苷元的色谱峰;b为转化前检测到的大豆苷的色谱图;c转化后检测到的葛根素和大豆苷元的色谱图,3为葛根素的色谱峰。
图6是分子筛柱层析纯化前的大豆苷反应物高效液相色谱图
如图6所示,分子筛柱层析纯化前,反应体系中主要有两种物质葛根素和大豆苷元,1为葛根素的色谱峰,2为大豆苷元的色谱峰。
图7是分子筛柱层析纯化后的大豆苷反应物高效液相色谱图
如图7所示,分子筛柱层析纯化后,反应体系中主要有一种物质葛根素,1为葛根素的色谱峰。
图8是液质联用法鉴定纯化后的葛根素谱图
如图8所示,根据质谱鉴定该物质为葛根素。
具体实施方式
下面结合具体实施例,对本发明作进一步说明,但本发明并不局限于以下实施例。
实施例1
本发明DgpA、DgpB和DgpC基因的获得及三种蛋白质表达纯化
A:三个基因dgpA(GenBank:BBG22493.1),dgpB(GenBank:BBG22494.1)和dgpC(GenBank:BBG22495.1)于NCBI数据库获取。合成DgpA、DgpB和DgpC,构建三个基因的pET-28a重组表达质粒。提取质粒后进行验证,验证正确后,通过热激法将重组质粒导入E.coliBL21(DE3)中,用含卡那霉素的平板筛选阳性克隆进行菌落PCR验证。采用pET-28a通用引物T7启动子(序列)和T7终止子(序列)作为菌落PCR的检测引物,挑取阳性转化子接种到含有卡那霉素的LB液体培养基中。在37℃、180rpm培养至OD600值约为0.4~0.5后,加入IPTG(异丙基-β-D-硫代半乳糖苷)诱导培养20h以上。培养液经4000rpm离心15min后收集菌体,并用结合缓冲液(50mM Tris(三羟基甲基氨基甲烷),0.5M NaCl(氯化钠),pH 8.0)重悬菌体。将细胞破碎,12000×g离心30min,取上清液进行蛋白纯化。
B:将上清液通过蠕动泵泵入Ni-IDA层析柱,使蛋白质吸附在层析柱上,弃去废液。然后用洗脱缓冲液(1M咪唑,50mM Tris,0.5M NaCl,pH 8.0)将NI-IDA层析柱上吸附的蛋白梯度洗脱,收集洗脱液进行SDS-PAGE电泳,确定蛋白含量及分子量大小。将蛋白含量较高的洗脱液用半透膜与透析缓冲液(50mM Tris,0.1M NaCl,pH 8.5)透析12h。将透析完的洗脱液用阴离子交换柱纯化,梯度洗脱后收集洗脱液取样,通过蛋白质洗脱时的UV吸收曲线和SDS-PAGE电泳确定蛋白质纯度及分子量大小。再将洗脱液浓缩后泵入HiloadTM 16/600SeperdexTM 200pg凝胶柱,利用蛋白质分子量大小不同进一步分离纯化,观察紫外吸收曲线确定蛋白质性质,最后用超滤管浓缩得到均一的蛋白质样品,即为纯化的蛋白质DgpA、DgpB和DgpC。
实施例2
DgpA、DgpB和DgpC对染料木苷的生物转化
A.分别取1mg纯化的蛋白质DgpA、DgpB和DgpC加入到含有染料木苷对照品、氯化锰以及NAD+的磷酸盐缓冲液中,对照组不加蛋白质,空白组以不含染料木苷对照品的磷酸盐缓冲液代替。37℃下恒温静置反应24h。
B.分别取转化后样品200μL,置于1.5mL离心管中,加入甲醇600μL,混匀。在4℃,14800rpm离心15min除去蛋白。各取上清液500μL,用于高效液相分析。由图5可知,在24h内酶组合物能够将染料木苷降解生成苷元染料木素及染料木素-8-C-葡萄糖苷。
C.将B中所得反应产物混合物染料木素及染料木素-8-C-葡萄糖苷采用SephdexLH20分子筛层析进行分离。将分离得到的染料木素-8-C-葡萄糖苷通过高效液相检测其纯度,收集纯度较高的组分,采用液质联用的方法对染料木素-8-C-葡萄糖苷进行鉴定。
实施例3
DgpA、DgpB和DgpC对大豆苷的生物转化
A.分别取1mg纯化的蛋白质DgpA、DgpB和DgpC加入到含有大豆苷对照品、氯化锰以及NAD+的磷酸盐缓冲液中,对照组不加蛋白质,空白组以不含大豆苷对照品的磷酸盐缓冲液代替。37℃下恒温静置培养24h。
B.分别取转化后样品200μL,置于1.5mL EP管中,加入甲醇600μL,混匀。在4℃,14800rpm离心15min除去蛋白。各取上清液500μL,用于高效液相分析。由图1可知,在24h内酶组合物能够将大豆苷降解生成大豆苷元及葛根素。
C.将B中所得反应产物混合物大豆苷元及葛根素采用Sephadex LH20分子筛层析进行分离。将分离得到的葛根素通过高效液相检测其纯度,收集纯度较高的组分,采用液质联用的方法对葛根素进行鉴定。
Claims (1)
1.一种能将黄酮氧苷类化合物转化为黄酮碳苷类化合物的酶组合物的应用,所述酶组合物由DgpA、DgpB、DgpC组成,其特征在于:
所述应用为将大豆苷转化为葛根素、大豆苷元,或将染料木苷转化为染料木素-8-C-葡萄糖苷、染料木素;所述应用包括如下步骤:
A.分别取1mg纯化的蛋白质DgpA、DgpB和DgpC加入到含有大豆苷对照品、氯化锰以及NAD+的磷酸盐缓冲液中,对照组不加蛋白质,空白组以不含大豆苷对照品的磷酸盐缓冲液代替,37℃下恒温静置培养24h;
B.分别取转化后样品200μL,置于1.5mLEP管中,加入甲醇600μL,混匀;在4℃,14800rpm离心15min除去蛋白;各取上清液500μL,用于高效液相分析;
C.将B中所得反应产物混合物大豆苷元及葛根素采用Sephadex LH20分子筛层析进行分离;将分离得到的葛根素通过高效液相检测其纯度,收集纯度较高的组分,采用液质联用的方法对葛根素进行鉴定;
或者,
A.分别取1mg纯化的蛋白质DgpA、DgpB和DgpC加入到含有染料木苷对照品、氯化锰以及NAD+的磷酸盐缓冲液中,对照组不加蛋白质,空白组以不含染料木苷对照品的磷酸盐缓冲液代替,37℃下恒温静置反应24h;
B.分别取转化后样品200μL,置于1.5mL离心管中,加入甲醇600μL,混匀,在4℃,14800rpm离心15min除去蛋白;各取上清液500μL,用于高效液相分析;
C.将B中所得反应产物混合物染料木素及染料木素-8-C-葡萄糖苷采用Sephdex LH20分子筛层析进行分离;将分离得到的染料木素-8-C-葡萄糖苷通过高效液相检测其纯度,收集纯度较高的组分,采用液质联用的方法对染料木素-8-C-葡萄糖苷进行鉴定。
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