伞枝犁头霉Absidia corymbifera AS2生物转化富集黄芪甲苷研究
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摘要
黄芪甲苷(ASI)是中药黄芪中一种具有多种药理活性的皂苷类化合物,为黄芪制剂质量鉴定的指标成分。黄芪甲苷生物活性突出,中药一类新药“黄芪甲苷葡萄糖注射液”作为治疗缺血性心肌炎的药物正在进行Ⅲ期临床研究。但由于黄芪甲苷结构复杂,无法进行化学全合成,因此其来源主要依靠从黄芪植物中提取。而其在黄芪中含量又非常低,需要大量植物资源和有机溶剂,使大规模制备成为瓶颈。
微生物转化反应具有专一性强、污染少和转化率高的优点,广泛用于天然产物的结构改造。本研究在国家重大科技专项(2009ZX09301-011)的资助下,借鉴前人的研究经验,采用微生物转化富集技术,以黄芪总皂苷作为底物,通过发酵转化提高黄芪甲苷含量。主要内容是从筛选菌种开始,建立了发酵转化和从发酵液中分离提取黄芪甲苷的新方法,并成功分离获得新的关键酶—黄芪皂苷去乙酰化酶。此外,通过对可转化为黄芪甲苷的前体化合物的鉴定和转化研究,假设了一条全新的微生物去乙酰化富集黄芪甲苷的途径。具体研究结果如下：
1、本研究首先从不同来源的霉菌中筛选到能够高效转化黄芪总皂苷生成黄芪甲苷的菌种—Absidia corymbifera AS2。以黄芪总皂苷为底物,在投料浓度为5g/L的情况下,转化60h,转化率最高达到90.3%,黄芪甲苷的含量是原料中的3倍。产物黄芪甲苷主要集中在发酵上清液中,经D101大孔吸附树脂分离纯化后,总回收率最高达到了62%,产品色谱纯度达到95%以上。该发酵及纯化工艺黄芪甲苷收率高,成本低,操作简便,避免使用有机溶剂,有利于其工业化生产。
2、本研究对黄芪总皂苷中可能转化为黄芪甲苷的前体化合物进行了分析。采用常规硅胶柱和反相硅胶柱分离的方法,并以A. corymbifera AS2微生物转化为指导,进行前体化合物的分离和确认,最终共分离到4个可转化为黄芪甲苷的皂苷类化合物。经结构鉴定4个前体化合物分别是astragaloside Ⅰ (AS-Ⅰ), isoastragaloside Ⅰ (isoAS-Ⅰ), astragaloside Ⅱ (AS-Ⅱ)和isoastragaloside Ⅱ (isoAS-Ⅱ)。通过ELSD检测与DAD扫描相结合的方法对四个前体化合物进行纯度鉴定,并采用ELSD吸收图谱的归一化法对四个标准品的纯度进行标定,结果分别为AS-Ⅰ(97%)、isoAS-Ⅰ(97.4%).AS-Ⅱ(98%)和isoAS-Ⅱ(98.7%).建立了黄芪甲苷和4个前体化合物HPLC分析方法和标准曲线,通过对发酵转化以及酶转化过程的实时分析,进行代谢途径的研究。
3、从化学结构分析来看,AS-Ⅰ(2'3'-di-OAc)和isoAS-Ⅰ(2'4'-di-OAc)比黄芪甲苷多两个乙酰基；AS-Ⅱ(2'-OAc)和isoAS-Ⅱ(3'-OAc)比黄芪甲苷多一个乙酰基。这些结构均可完全被A.corymbifera AS2转化为黄芪甲苷,提示该菌株可能存在去乙酰化酶。为了进一步研究微生物转化黄芪总皂苷生成黄芪甲苷的机制,本研究继续对该微生物中可能存在的去乙酰化酶进行分离纯化。首先采用硫酸铵分步沉淀法浓缩粗酶液中的目标蛋白,再进行Q-sepharose HP离子交换层析,在0.1mol/L氯化钠洗脱组份中发现了较高的酶活性。该组份经phenyl-sepharose6FF疏水层析,收集在0.7mol/L硫酸铵洗脱的活性组份继续进行CHT陶瓷型羟基磷灰石柱层析,在24.5mmol/L磷酸钾洗脱组份中发现有较高的活性。最后采用1mL Resource Q预装柱层析获得电泳纯的活性蛋白。酶活力回收率和蛋白回收率分别为0.32%和0.075‰。
4、进一步对分离获得的黄芪皂苷去乙酰化酶进行酶学性质研究。以p-NPA为底物在pH7.0和30℃条件下的动力学常数：米氏常数Km值为3.76mmol/L;最大反应速率Vmax为1.64mmol/min/mg.以p-NPA为底物的最适反应pH为8.0,在pH7.0-9.5范围内稳定；最适反应温度为35-45℃；在小于45℃温度范围内相对稳定。MS检测分子量为36067Da。采用SPITC修饰的PSD-MALDI质谱进行序列分析,显示该酶可能为之前没有报道过的新酶。
5、最后对黄芪皂苷去乙酰化酶的酶解途径进行研究。结果显示4个前体化合物的转化途径分别是：AS-Ⅱ→ASI；isoAS-Ⅱ→AS-Ⅱ→ASI；AS-Ⅰ→(AS-Ⅱ、 isoAS-Ⅱ)→ASI:isoAS-Ⅰ→AS-Ⅱ→ASI.
Astragaloside IV (ASI) is believed to be the main active constituent that is normally used as a marker for quality control of Radix Astragali. ASI has been proved to have special biological activities. A new medicine, ASI glucose injection, for treatment of ischemic myocarditis is on the third period clinical research. Now ASI is mainly obtained from plant extraction for lacking of total synthesis method. Due to the low content of ASI in natural plants, the isolation of ASI from natural products is extremely difficult and cost.
Microbial transformation which has high specificity and environmental compatibility is increasingly being used as a useful tool in the structure modification of natural compounds. In this study, a new method by microbial transformation to enrich ASI was reported. A new deacetylase was purified from A. corymbifera AS2by combination of different traditional protein purification chromatographics. Four precursors were identified and a new hydrolyzing pathway of these four precursors by the purified deacetylase was conjectured. The dissertation is thankful to the financial support of Chinese National Science and Technology Major Project (2009ZX09301-011).
1, Absidia corymbifera AS2, which has the highest ability of biotransforming astragalosides to ASI was screened from various fungi. Total astragalosides as substrate with the concentration of5g/L, transformed for60h, the mol degree of conversion arrived at90.3%in5L flask. The yield of ASI was improved by about3folds and the product of ASI was mainly found in the supernatant. A simple, stable and effective method for isolation and purification of ASI from fermentation medium with D101resin was established. After purification, the chromatographic purity and recovery of ASI arrived at95%and62%, respectively.
2.. The precursors of astragalosides which can be transformed to ASI by microorganisms were investigated. By the methods of silica gel and reverse-phase silica gel chromatography and microbial transformation tracking, Astragaloside I (AS-Ⅰ), Isoastragaloside Ⅰ (isoAS-I), Astragalside Ⅱ (AS-Ⅱ) and Isoastragaloside Ⅱ (isoAS-Ⅱ) were finally separated and identified. These four precursors were analyzed by HPLC-ELSD-DAD to assure the quantity level. A convenient HPLC method for studying the metabolic pathways was established and used to study the metabolic pathways of ASI, AS-Ⅰ, isoAS-Ⅰ, AS-Ⅱ and isoAS-Ⅱ.
3、Compared with the chemical structure of ASI, there are two more acetyls on3-xylose residue of AS-Ⅰ (2'3'-di-OAc) and isoAS-Ⅰ (2'4'-di-OAc), and one more acetyls on3-xylose residue of isoAS-II (3'-OAc) and AS-Ⅱ (2'-OAc). It points that A. corymbifera AS2has deacetylases which can hydrolyze acetyls residues of some astragalosides to ASI. The purification of deacetylases from A. corymbifera AS2was continued to be studied. Finally, one deacetylase was purified from A. corymbifera AS2by combination of different traditional protein purification chromatographics: ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, CHT ceramic hydroxyapatite and Resource Q prepacked column. The activity recovery and the protein recovery of the purified enzyme were0.32%and0.075%o, respectively.
4、The properties of the purified enzyme were also studied. The molecular weight (MW) of enzyme subunits was38KDa by SDS-PAGE and36067Da by MS. The purified deacetylase had optimal activities at pH8.0and was stable within pH7.0-9.5. The enzyme had optimal temperature of35-45and was stable lower than45℃. The kinetic parameters, Km and Fmax values, of the purified enzyme against p-NPA were determined by typical Lineweaver-Burk double reciprocal plots under pH7.0and30℃. The Km values was3.76mmol/L and the Vmax values was17.64mmol/min/mg. The amino acid sequences of deacetylase were further analyzed with the PSD-MALDIMS method sulfonated by SPITC.
5、The hydrolyzing pathway of the four precursors by the purified deacetylase was further studied. The result showed that the hydrolyzing pathways were AS-Ⅱ→ASI; isoAS-Ⅱ→-AS-Ⅱ→ASI; AS-Ⅰ→(AS-Ⅱ、isoAS-Ⅱ)-ASI; isoAS-Ⅰ-AS-Ⅱ-ASI。
微生物转化反应具有专一性强、污染少和转化率高的优点,广泛用于天然产物的结构改造。本研究在国家重大科技专项(2009ZX09301-011)的资助下,借鉴前人的研究经验,采用微生物转化富集技术,以黄芪总皂苷作为底物,通过发酵转化提高黄芪甲苷含量。主要内容是从筛选菌种开始,建立了发酵转化和从发酵液中分离提取黄芪甲苷的新方法,并成功分离获得新的关键酶—黄芪皂苷去乙酰化酶。此外,通过对可转化为黄芪甲苷的前体化合物的鉴定和转化研究,假设了一条全新的微生物去乙酰化富集黄芪甲苷的途径。具体研究结果如下：
1、本研究首先从不同来源的霉菌中筛选到能够高效转化黄芪总皂苷生成黄芪甲苷的菌种—Absidia corymbifera AS2。以黄芪总皂苷为底物,在投料浓度为5g/L的情况下,转化60h,转化率最高达到90.3%,黄芪甲苷的含量是原料中的3倍。产物黄芪甲苷主要集中在发酵上清液中,经D101大孔吸附树脂分离纯化后,总回收率最高达到了62%,产品色谱纯度达到95%以上。该发酵及纯化工艺黄芪甲苷收率高,成本低,操作简便,避免使用有机溶剂,有利于其工业化生产。
2、本研究对黄芪总皂苷中可能转化为黄芪甲苷的前体化合物进行了分析。采用常规硅胶柱和反相硅胶柱分离的方法,并以A. corymbifera AS2微生物转化为指导,进行前体化合物的分离和确认,最终共分离到4个可转化为黄芪甲苷的皂苷类化合物。经结构鉴定4个前体化合物分别是astragaloside Ⅰ (AS-Ⅰ), isoastragaloside Ⅰ (isoAS-Ⅰ), astragaloside Ⅱ (AS-Ⅱ)和isoastragaloside Ⅱ (isoAS-Ⅱ)。通过ELSD检测与DAD扫描相结合的方法对四个前体化合物进行纯度鉴定,并采用ELSD吸收图谱的归一化法对四个标准品的纯度进行标定,结果分别为AS-Ⅰ(97%)、isoAS-Ⅰ(97.4%).AS-Ⅱ(98%)和isoAS-Ⅱ(98.7%).建立了黄芪甲苷和4个前体化合物HPLC分析方法和标准曲线,通过对发酵转化以及酶转化过程的实时分析,进行代谢途径的研究。
3、从化学结构分析来看,AS-Ⅰ(2'3'-di-OAc)和isoAS-Ⅰ(2'4'-di-OAc)比黄芪甲苷多两个乙酰基；AS-Ⅱ(2'-OAc)和isoAS-Ⅱ(3'-OAc)比黄芪甲苷多一个乙酰基。这些结构均可完全被A.corymbifera AS2转化为黄芪甲苷,提示该菌株可能存在去乙酰化酶。为了进一步研究微生物转化黄芪总皂苷生成黄芪甲苷的机制,本研究继续对该微生物中可能存在的去乙酰化酶进行分离纯化。首先采用硫酸铵分步沉淀法浓缩粗酶液中的目标蛋白,再进行Q-sepharose HP离子交换层析,在0.1mol/L氯化钠洗脱组份中发现了较高的酶活性。该组份经phenyl-sepharose6FF疏水层析,收集在0.7mol/L硫酸铵洗脱的活性组份继续进行CHT陶瓷型羟基磷灰石柱层析,在24.5mmol/L磷酸钾洗脱组份中发现有较高的活性。最后采用1mL Resource Q预装柱层析获得电泳纯的活性蛋白。酶活力回收率和蛋白回收率分别为0.32%和0.075‰。
4、进一步对分离获得的黄芪皂苷去乙酰化酶进行酶学性质研究。以p-NPA为底物在pH7.0和30℃条件下的动力学常数：米氏常数Km值为3.76mmol/L;最大反应速率Vmax为1.64mmol/min/mg.以p-NPA为底物的最适反应pH为8.0,在pH7.0-9.5范围内稳定；最适反应温度为35-45℃；在小于45℃温度范围内相对稳定。MS检测分子量为36067Da。采用SPITC修饰的PSD-MALDI质谱进行序列分析,显示该酶可能为之前没有报道过的新酶。
5、最后对黄芪皂苷去乙酰化酶的酶解途径进行研究。结果显示4个前体化合物的转化途径分别是：AS-Ⅱ→ASI；isoAS-Ⅱ→AS-Ⅱ→ASI；AS-Ⅰ→(AS-Ⅱ、 isoAS-Ⅱ)→ASI:isoAS-Ⅰ→AS-Ⅱ→ASI.
Astragaloside IV (ASI) is believed to be the main active constituent that is normally used as a marker for quality control of Radix Astragali. ASI has been proved to have special biological activities. A new medicine, ASI glucose injection, for treatment of ischemic myocarditis is on the third period clinical research. Now ASI is mainly obtained from plant extraction for lacking of total synthesis method. Due to the low content of ASI in natural plants, the isolation of ASI from natural products is extremely difficult and cost.
Microbial transformation which has high specificity and environmental compatibility is increasingly being used as a useful tool in the structure modification of natural compounds. In this study, a new method by microbial transformation to enrich ASI was reported. A new deacetylase was purified from A. corymbifera AS2by combination of different traditional protein purification chromatographics. Four precursors were identified and a new hydrolyzing pathway of these four precursors by the purified deacetylase was conjectured. The dissertation is thankful to the financial support of Chinese National Science and Technology Major Project (2009ZX09301-011).
1, Absidia corymbifera AS2, which has the highest ability of biotransforming astragalosides to ASI was screened from various fungi. Total astragalosides as substrate with the concentration of5g/L, transformed for60h, the mol degree of conversion arrived at90.3%in5L flask. The yield of ASI was improved by about3folds and the product of ASI was mainly found in the supernatant. A simple, stable and effective method for isolation and purification of ASI from fermentation medium with D101resin was established. After purification, the chromatographic purity and recovery of ASI arrived at95%and62%, respectively.
2.. The precursors of astragalosides which can be transformed to ASI by microorganisms were investigated. By the methods of silica gel and reverse-phase silica gel chromatography and microbial transformation tracking, Astragaloside I (AS-Ⅰ), Isoastragaloside Ⅰ (isoAS-I), Astragalside Ⅱ (AS-Ⅱ) and Isoastragaloside Ⅱ (isoAS-Ⅱ) were finally separated and identified. These four precursors were analyzed by HPLC-ELSD-DAD to assure the quantity level. A convenient HPLC method for studying the metabolic pathways was established and used to study the metabolic pathways of ASI, AS-Ⅰ, isoAS-Ⅰ, AS-Ⅱ and isoAS-Ⅱ.
3、Compared with the chemical structure of ASI, there are two more acetyls on3-xylose residue of AS-Ⅰ (2'3'-di-OAc) and isoAS-Ⅰ (2'4'-di-OAc), and one more acetyls on3-xylose residue of isoAS-II (3'-OAc) and AS-Ⅱ (2'-OAc). It points that A. corymbifera AS2has deacetylases which can hydrolyze acetyls residues of some astragalosides to ASI. The purification of deacetylases from A. corymbifera AS2was continued to be studied. Finally, one deacetylase was purified from A. corymbifera AS2by combination of different traditional protein purification chromatographics: ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography, CHT ceramic hydroxyapatite and Resource Q prepacked column. The activity recovery and the protein recovery of the purified enzyme were0.32%and0.075%o, respectively.
4、The properties of the purified enzyme were also studied. The molecular weight (MW) of enzyme subunits was38KDa by SDS-PAGE and36067Da by MS. The purified deacetylase had optimal activities at pH8.0and was stable within pH7.0-9.5. The enzyme had optimal temperature of35-45and was stable lower than45℃. The kinetic parameters, Km and Fmax values, of the purified enzyme against p-NPA were determined by typical Lineweaver-Burk double reciprocal plots under pH7.0and30℃. The Km values was3.76mmol/L and the Vmax values was17.64mmol/min/mg. The amino acid sequences of deacetylase were further analyzed with the PSD-MALDIMS method sulfonated by SPITC.
5、The hydrolyzing pathway of the four precursors by the purified deacetylase was further studied. The result showed that the hydrolyzing pathways were AS-Ⅱ→ASI; isoAS-Ⅱ→-AS-Ⅱ→ASI; AS-Ⅰ→(AS-Ⅱ、isoAS-Ⅱ)-ASI; isoAS-Ⅰ-AS-Ⅱ-ASI。
引文
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