糖苷酶转化人参皂苷的研究
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摘要
人参皂苷是人参的主要活性成分之一，目前已从人参中分离纯化出约四十种人参皂苷。研究表明，不同的皂苷活性不同，一些稀有人参皂苷，具有显著的药理学活性，如人参皂苷C-K，具有抗肿瘤、抗炎症、抗衰老等活性。而这些稀有皂苷在天然人参中含量甚微，只能通过人工方法制备。因此，制备稀有人参皂苷成为目前研究的热点。由于稀有皂苷与某些高含量人参皂苷相比，其皂苷母环结构相同，只是糖基数目不同。因此，理论上可以通过水解大量皂苷的糖基来制备稀有皂苷。
水解人参皂苷的糖基的主要方法有酸水解、碱水解、加热、糖苷酶催化等。加热或酸、碱水解等化学法条件剧烈，易产生副产物。糖苷酶介导的生物转化法由于条件温和、专一性高，是潜在的转化方法，也是目前研究的热点之一。通过生物转化制备糖苷化合物或寡糖等，具有重要的理论和应用意义。因此，寻找活性高、专一性好的糖苷酶，用以转化大量皂苷从而制备稀有皂苷，是本课题的研究重点之一。该论文的主要研究成果如下：
1.研究了12种商品糖类水解酶对人参二醇型皂苷混合物和单体皂苷Rb1的水解。其中1种酶不转化人参二醇型皂苷，其余11种酶均能不同程度地转化人参二醇型皂苷混合物和单体皂苷Rb1，但水解终产物与速率不同。8种酶水解人参皂苷Rb1产生唯一产物Rd，水解路径为：Rb1→Rd；1种酶水解Rb1至F2，水解路径为：Rb1→Rd→F2；2种酶水解Rb1至终产物Compound K（C-K），水解路径分别为Rb1→Rd→F2→C-K；Rb1→Rd/GXVII→F2→C-K。此外，测试了这11种酶转化Rb1的效率。
2.从人参种植土壤中筛选到一株真菌sp.68，该菌能高效转化人参二醇型皂苷混合物，产生一系列稀有人参皂苷产物，其终产物为人参皂苷C-K。该菌株用分子生物学方法鉴定为草酸青霉。对草酸青霉转化人参二醇型皂苷的能力进行了研究。首先，草酸青霉发酵液经过滤、离心后的上清经DEAE-纤维素柱层析和30-90%硫酸铵沉淀后，得到草酸青霉胞外酶液。用该酶液转化人参二醇型皂苷Rb1、Rb2、Rc，得到如下转化路径：Rb1→Rd→F2→C-K；Rb2→CO→CY→C-K；Rc→Mb→Mc→C-K。对各转化产物用TLC、HPLC和~(13)C-NMR进行了鉴定。此外，对草酸青霉胞外酶液转化人参皂苷制备稀有皂苷C-K的条件进行了优化，得到如下优化结果：底物，Rb1；反应pH，3.5；反应温度，55℃；底物浓度，0.5mg/ml。
3.对草酸青霉产生的发酵液进行分离纯化，经过一系列色谱层析分离纯化后，得到三个纯的胞外糖苷酶GH1、GH3-1和GH3-2。对这三种酶的酶学性质进行研究。底物专一性结果表明：以人参皂苷作为底物时，GH1只水解人参皂苷Rb1至唯一产物Rd，而不水解Rb2和Rc。GH3-1和GH3-2对人参皂苷Rb1、Rb2和Rc均能水解，转化路径为：Rb1→Rd→F2→C-K；Rb2→CO→CY→C-K；Rc→Mb→Mc→C-K。此外，对这三种酶的分子量、等电点、最适pH及pH稳定性、最适温度及温度稳定性等进行了研究。这三种酶虽然由同一种菌分泌到胞外，但行使不同的功能。这些糖苷酶对于人参皂苷的转化尤其是一系列中间代谢产物的制备具有重要的应用意义。
4.从革兰氏阳性菌——粪肥杆菌中克隆、表达并纯化了七个糖苷水解酶。其中，cfi-01、cfi-08、cfi-11和cfi-13属于糖苷水解酶家族3；cfi-02属于糖苷水解酶家族31；cfi-04属于糖苷水解酶家族42；cfi-10属于糖苷水解酶家族1。对各糖苷水解酶的底物专一性进行了系统研究。在家族3的糖苷水解酶中，cfi-01是双功能酶，同时具有β-D-木糖苷酶/-L-呋喃阿拉伯糖苷酶的活性；cfi-08和cfi-11是β-D-葡萄糖苷酶；cfi-13只水解pNP-人工糖苷，而不水解二糖、寡糖，因此属于芳香基-β-D-糖苷酶。家族31的cfi-02是-木糖苷酶。家族42的cfi-04是β-D-半乳糖苷酶，同时也表现出微弱的β-D-果糖苷酶活性。家族1的cfi-10活性广泛，同时具有β-D-半乳糖苷酶、β-D-葡萄糖苷酶、β-D-果糖苷酶和β-D-木糖苷酶活性。此外，对克隆表达得到的7个糖苷水解酶转化人参皂苷的能力进行了研究。结果表明，cfi-08和cfi-10可水解人参皂苷Rb1，产生唯一产物Rd。其它5种糖苷水解酶不能转化人参皂苷。对草酸青霉中纯化得到的三个糖苷水解酶和粪肥杆菌中克隆表达得到的七个糖苷水解酶在底物专一性方面进行了系统的比较。这些糖苷水解酶可被应用于糖苷化合物的转化。
Ginsenosides are the principle components responsible for the pharmaceuticalactivities of ginseng. So far, about forty kinds of different ginsenosides have been isolatedfrom different parts of ginseng. Some minor ginsenosides exhibited various biological andpharmacological activities, including anti-tumor, immune-modulatory, anti-inflammatoryand anti-aging effects. These minor ginsenosides, however, exsited in very low content oreven did not naturally present in ginseng. The minor ginsenosides, which had sameginsengenin as the high-content ginsenosides, could therefore be prepared by hydrolyzingthe sugar moieties in high-content ginsenosides.
Methods including heating, acid hydrolysis, microbial and enzymatic transformationwere used in the hydrolysis of high-content ginsenosides to prepare minor ginsenosides.Among these methods, microbial and enzymatic transformations are more potential due totheir high specificity, low side reaction and mild conditions. Biotransformation ofglycosides to get useful compounds was one hot spotpoint in glycoside application.Therefore, in this study, we focus on looking for suitable strains or enzymes which couldbe used in ginsenoside biotransformation. The main results are as follows:
1. In this study, twelve kinds of commercial enzymes were tested for their ability totransform protopanaxadiol-type ginsenosides mixture and single ginsenoside Rb1. Amongthem, only one enzyme had no transformation activity against the substrates, the othereleven enzymes could transform protopanaxadiol-type ginsenosides mixture andginsenoside Rb1with different pathways and efficiencies. Among the eleven enzymes,eight kinds of enzymes transformed ginsenoside Rb1to Rd, the transformation pathwaywas Rb1→Rd; one enzyme transformed Rb1to ginsenoside F2, the pathway wasRb1→Rd→F2; two enzymes transformed Rb1to final product C-K, the transformationpathways were Rb1→Rd→F2→C-K and Rb1→Rd/GXVII→F2→C-K, respectively.
2. A fungus, sp.68, which was found to be able to transform protopanaxadiol-typeginsenosides into bioactive C-K with high activity and specificity, was isolated from theginseng-cultivating soil. The strain was identified to be Penicillium oxalicum by18S rDNAand ITS sequencing. Then the crude enzyme was prepared by DEAE-cellulosechromatography and sulfate ammonium precipitation from the fermentation broth of P.oxalicum. The crude enzyme was used in ginsenoside transformation. Ginsenoside Rb1,Rb2and Rc were transformed to the final product C-K by the crude enzyme via differentpathways. The pathways are as following: Rb1→Rd→F2→C-K；Rb2→CO→CY→C-K； Rc→Mb→Mc→C-K. The conditions for C-K production by crude enzyme of P. oxalicumwere optimized, as well. Following optimization conditions were obtained: substrate,ginsenoside Rb1; pH,3.5; temperature,55oC; concentration of Rb1,0.5mg/ml.
3. Three extracellular glycosidases GH1, GH3-1and GH3-2were purified from thefermentation broth of P. oxalicum. The substrate specificity results showed that GH1couldhydrolyze Rb1to sole product Rd, without hydrolyzing Rb2, Rc and Rd. GH3-1andGH3-2exhibited similar transformation pathways against Rb1, Rb2and Rc, the pathwaysare: Rb1→Rd→F2→C-K, Rb2→CO→CY→C-K, Rc→Mb→Mc→C-K. The enzymeswere characterized as well. These enzymes would be useful in ginsenosides transformation.
4. Seven glycoside hydrolases were cloned, expressed and purified fromCellulomonas fimi. Among them, cfi-01, cfi-08, cfi-11and cfi-13belonged to glycosidehydrolase family3while cfi-02, cfi-04and cfi-10belonged to family31,42and1,respectively. The substrate specificities of these recombinant enzymes were symtemicallystudied. Among the glycoside hydrolase family3enzymes, cfi-01was bifunctional enzymewith both β-D-xylosidase and-L-arabinofuranosidase activities. The enzymes cfi-08andcfi-11were β-D-glucosidases. Cfi-13hydrolyzed p-nitrophenyl substrates, withouthydrolyzing disaccharides or oligosaccharides, therefore, cfi-13was aryl-β-D-glucosidase.The enzyme cfi-02, which belonged to glycoside hydrolase family31, was-xylosidase.Cfi-10exhibited β-D-galactosidase, β-D-glucosidase, β-D-fucosidase and β-D-xylosidaseactivities. Also, the transformation ability of these seven recombinant glycosidases againstginsenosides were studied. The results showed that cfi-08and cfi-10transformedginsenoside Rb1to give the sole product Rd. Other glycosidases exhibited no activityagainst tested ginsenosides. The substrate specificities of the glycoside hydrolases from P.oxalicum and C. fimi were symtemically studied and compared. These enzymes would beuseful for hydrolysis of glycoside compounds or oligosaccharides.
水解人参皂苷的糖基的主要方法有酸水解、碱水解、加热、糖苷酶催化等。加热或酸、碱水解等化学法条件剧烈，易产生副产物。糖苷酶介导的生物转化法由于条件温和、专一性高，是潜在的转化方法，也是目前研究的热点之一。通过生物转化制备糖苷化合物或寡糖等，具有重要的理论和应用意义。因此，寻找活性高、专一性好的糖苷酶，用以转化大量皂苷从而制备稀有皂苷，是本课题的研究重点之一。该论文的主要研究成果如下：
1.研究了12种商品糖类水解酶对人参二醇型皂苷混合物和单体皂苷Rb1的水解。其中1种酶不转化人参二醇型皂苷，其余11种酶均能不同程度地转化人参二醇型皂苷混合物和单体皂苷Rb1，但水解终产物与速率不同。8种酶水解人参皂苷Rb1产生唯一产物Rd，水解路径为：Rb1→Rd；1种酶水解Rb1至F2，水解路径为：Rb1→Rd→F2；2种酶水解Rb1至终产物Compound K（C-K），水解路径分别为Rb1→Rd→F2→C-K；Rb1→Rd/GXVII→F2→C-K。此外，测试了这11种酶转化Rb1的效率。
2.从人参种植土壤中筛选到一株真菌sp.68，该菌能高效转化人参二醇型皂苷混合物，产生一系列稀有人参皂苷产物，其终产物为人参皂苷C-K。该菌株用分子生物学方法鉴定为草酸青霉。对草酸青霉转化人参二醇型皂苷的能力进行了研究。首先，草酸青霉发酵液经过滤、离心后的上清经DEAE-纤维素柱层析和30-90%硫酸铵沉淀后，得到草酸青霉胞外酶液。用该酶液转化人参二醇型皂苷Rb1、Rb2、Rc，得到如下转化路径：Rb1→Rd→F2→C-K；Rb2→CO→CY→C-K；Rc→Mb→Mc→C-K。对各转化产物用TLC、HPLC和~(13)C-NMR进行了鉴定。此外，对草酸青霉胞外酶液转化人参皂苷制备稀有皂苷C-K的条件进行了优化，得到如下优化结果：底物，Rb1；反应pH，3.5；反应温度，55℃；底物浓度，0.5mg/ml。
3.对草酸青霉产生的发酵液进行分离纯化，经过一系列色谱层析分离纯化后，得到三个纯的胞外糖苷酶GH1、GH3-1和GH3-2。对这三种酶的酶学性质进行研究。底物专一性结果表明：以人参皂苷作为底物时，GH1只水解人参皂苷Rb1至唯一产物Rd，而不水解Rb2和Rc。GH3-1和GH3-2对人参皂苷Rb1、Rb2和Rc均能水解，转化路径为：Rb1→Rd→F2→C-K；Rb2→CO→CY→C-K；Rc→Mb→Mc→C-K。此外，对这三种酶的分子量、等电点、最适pH及pH稳定性、最适温度及温度稳定性等进行了研究。这三种酶虽然由同一种菌分泌到胞外，但行使不同的功能。这些糖苷酶对于人参皂苷的转化尤其是一系列中间代谢产物的制备具有重要的应用意义。
4.从革兰氏阳性菌——粪肥杆菌中克隆、表达并纯化了七个糖苷水解酶。其中，cfi-01、cfi-08、cfi-11和cfi-13属于糖苷水解酶家族3；cfi-02属于糖苷水解酶家族31；cfi-04属于糖苷水解酶家族42；cfi-10属于糖苷水解酶家族1。对各糖苷水解酶的底物专一性进行了系统研究。在家族3的糖苷水解酶中，cfi-01是双功能酶，同时具有β-D-木糖苷酶/-L-呋喃阿拉伯糖苷酶的活性；cfi-08和cfi-11是β-D-葡萄糖苷酶；cfi-13只水解pNP-人工糖苷，而不水解二糖、寡糖，因此属于芳香基-β-D-糖苷酶。家族31的cfi-02是-木糖苷酶。家族42的cfi-04是β-D-半乳糖苷酶，同时也表现出微弱的β-D-果糖苷酶活性。家族1的cfi-10活性广泛，同时具有β-D-半乳糖苷酶、β-D-葡萄糖苷酶、β-D-果糖苷酶和β-D-木糖苷酶活性。此外，对克隆表达得到的7个糖苷水解酶转化人参皂苷的能力进行了研究。结果表明，cfi-08和cfi-10可水解人参皂苷Rb1，产生唯一产物Rd。其它5种糖苷水解酶不能转化人参皂苷。对草酸青霉中纯化得到的三个糖苷水解酶和粪肥杆菌中克隆表达得到的七个糖苷水解酶在底物专一性方面进行了系统的比较。这些糖苷水解酶可被应用于糖苷化合物的转化。
Ginsenosides are the principle components responsible for the pharmaceuticalactivities of ginseng. So far, about forty kinds of different ginsenosides have been isolatedfrom different parts of ginseng. Some minor ginsenosides exhibited various biological andpharmacological activities, including anti-tumor, immune-modulatory, anti-inflammatoryand anti-aging effects. These minor ginsenosides, however, exsited in very low content oreven did not naturally present in ginseng. The minor ginsenosides, which had sameginsengenin as the high-content ginsenosides, could therefore be prepared by hydrolyzingthe sugar moieties in high-content ginsenosides.
Methods including heating, acid hydrolysis, microbial and enzymatic transformationwere used in the hydrolysis of high-content ginsenosides to prepare minor ginsenosides.Among these methods, microbial and enzymatic transformations are more potential due totheir high specificity, low side reaction and mild conditions. Biotransformation ofglycosides to get useful compounds was one hot spotpoint in glycoside application.Therefore, in this study, we focus on looking for suitable strains or enzymes which couldbe used in ginsenoside biotransformation. The main results are as follows:
1. In this study, twelve kinds of commercial enzymes were tested for their ability totransform protopanaxadiol-type ginsenosides mixture and single ginsenoside Rb1. Amongthem, only one enzyme had no transformation activity against the substrates, the othereleven enzymes could transform protopanaxadiol-type ginsenosides mixture andginsenoside Rb1with different pathways and efficiencies. Among the eleven enzymes,eight kinds of enzymes transformed ginsenoside Rb1to Rd, the transformation pathwaywas Rb1→Rd; one enzyme transformed Rb1to ginsenoside F2, the pathway wasRb1→Rd→F2; two enzymes transformed Rb1to final product C-K, the transformationpathways were Rb1→Rd→F2→C-K and Rb1→Rd/GXVII→F2→C-K, respectively.
2. A fungus, sp.68, which was found to be able to transform protopanaxadiol-typeginsenosides into bioactive C-K with high activity and specificity, was isolated from theginseng-cultivating soil. The strain was identified to be Penicillium oxalicum by18S rDNAand ITS sequencing. Then the crude enzyme was prepared by DEAE-cellulosechromatography and sulfate ammonium precipitation from the fermentation broth of P.oxalicum. The crude enzyme was used in ginsenoside transformation. Ginsenoside Rb1,Rb2and Rc were transformed to the final product C-K by the crude enzyme via differentpathways. The pathways are as following: Rb1→Rd→F2→C-K；Rb2→CO→CY→C-K； Rc→Mb→Mc→C-K. The conditions for C-K production by crude enzyme of P. oxalicumwere optimized, as well. Following optimization conditions were obtained: substrate,ginsenoside Rb1; pH,3.5; temperature,55oC; concentration of Rb1,0.5mg/ml.
3. Three extracellular glycosidases GH1, GH3-1and GH3-2were purified from thefermentation broth of P. oxalicum. The substrate specificity results showed that GH1couldhydrolyze Rb1to sole product Rd, without hydrolyzing Rb2, Rc and Rd. GH3-1andGH3-2exhibited similar transformation pathways against Rb1, Rb2and Rc, the pathwaysare: Rb1→Rd→F2→C-K, Rb2→CO→CY→C-K, Rc→Mb→Mc→C-K. The enzymeswere characterized as well. These enzymes would be useful in ginsenosides transformation.
4. Seven glycoside hydrolases were cloned, expressed and purified fromCellulomonas fimi. Among them, cfi-01, cfi-08, cfi-11and cfi-13belonged to glycosidehydrolase family3while cfi-02, cfi-04and cfi-10belonged to family31,42and1,respectively. The substrate specificities of these recombinant enzymes were symtemicallystudied. Among the glycoside hydrolase family3enzymes, cfi-01was bifunctional enzymewith both β-D-xylosidase and-L-arabinofuranosidase activities. The enzymes cfi-08andcfi-11were β-D-glucosidases. Cfi-13hydrolyzed p-nitrophenyl substrates, withouthydrolyzing disaccharides or oligosaccharides, therefore, cfi-13was aryl-β-D-glucosidase.The enzyme cfi-02, which belonged to glycoside hydrolase family31, was-xylosidase.Cfi-10exhibited β-D-galactosidase, β-D-glucosidase, β-D-fucosidase and β-D-xylosidaseactivities. Also, the transformation ability of these seven recombinant glycosidases againstginsenosides were studied. The results showed that cfi-08and cfi-10transformedginsenoside Rb1to give the sole product Rd. Other glycosidases exhibited no activityagainst tested ginsenosides. The substrate specificities of the glycoside hydrolases from P.oxalicum and C. fimi were symtemically studied and compared. These enzymes would beuseful for hydrolysis of glycoside compounds or oligosaccharides.
引文
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