薯蓣皂素的酶促糖基化以及相关化合物的生物合成研究
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摘要
薯蓣皂素是一种重要的甾体皂甙化合物,它的葡萄糖苷(延龄草苷)是许多药用植物的活性成份,但含量相当少,不利于工业化生产。因此,薯蓣皂素人工糖基化是解决该问题的一条出路。化学法虽然可以有效地合成延龄草苷,但是繁琐的糖基保护和去保护过程是不可避免的,而且在该过程中会使用大量的有机溶剂,对环境压力很大；薯蓣皂素的酶促糖基化可能成为其产业化生产的一条捷径,原因在于：糖基转移酶催化的转糖基反应条件温和、糖基化位点唯一、可以避免糖基的保护和去保护等。但是糖基转移酶的糖基供体必须使用昂贵而且不稳定的核苷酸葡萄糖,这必然会增加酶法催化薯蓣皂素糖基化的成本。因此,为了降低酶催化合成延龄草苷的成本,本论文以廉价的麦芽糊精、UTP、磷酸盐和薯蓣皂素为底物,在一锅体系中,多酶组合催化合成了延龄草苷。同时本文以氨基化的磁性纳米颗粒为载体,将相关的生物催化剂制备成固定化酶,催化合成了尿苷二磷酸葡萄糖和葡萄糖-1-磷酸,它们是上述一锅合成体系中重要的中间体,也是酶催化转糖基过程中具有高附加值的高能糖基供体。同时本文还通过重组的半乳糖激酶催化合成了半乳糖-1-磷酸,为后续的薯蓣皂素糖基随机化改造做好铺垫。本论文的主要内容如下：
1.用于薯蓣皂素糖基化的生物催化剂制备
从E. coli K12中分别克隆了malpase, ugpase, ppase, udpgdh基因,从土豆芽中克隆了sgtl.1基因。这些基因分别在E.coli BL21(DE3)和E. coli DH5a中成功地表达了有活性的可溶蛋白。同时优化了重组蛋白的表达条件,并制成冻干酶制剂。
2.多酶一锅催化合成延龄草苷
在体系中加入麦芽糊精磷酸化酶(E1)、尿苷二磷酸葡萄糖焦磷酸化酶(E2)、无机焦磷酸酶(E3)、薯蓣皂素糖基转移酶(E4)以及麦芽糊精、UTP、磷酸盐、薯蓣皂素等底物,通过考察助溶剂添加效果、优化反应条件以及利用正交试验优化底物浓度的配比,最终确定了该体系的最优反应条件：6.58 U E1/1,125 U E2/1,7 U E3/1,0.75 U E4/1,5%(m/v)麦芽糊精,10 mM UTP,0.1 mM薯蓣皂素,5 mM MgCl2,100 mM (pH 9)磷酸盐缓冲液在40℃下连续反应48 h。在该条件下,延龄草苷的产率为89%,浓度为15.8 mg/l。
3.三种固定化酶组合催化合成尿苷二磷酸葡萄糖
利用化学共沉淀法制备的Fe3O4,经过氨基硅烷化和戊二醛活化后,分别和麦芽糊精磷酸化酶、尿苷二磷酸葡萄糖焦磷酸化酶、无机焦磷酸酶共价结合,制备成固定化酶。通过分别比较游离酶和固定化的理化性质,证明了磁性纳米颗粒适合在多酶催化合成尿苷二磷酸葡萄糖中使用。通过多步实验,确定多酶合成尿苷二磷酸葡萄糖的最佳反应条件如下：在体系中加入5%(m/v)麦芽糊精,2mMUTP,10mM MgCl2,100mM(pH 7.5)磷酸钠缓冲液,18 U E1/1,100 U E2/1,25 U E3/1,30℃恒温摇床上200 rpm连续反应12 h。然后在200ml体系中,利用优化后的反应条件,连续5批次酶促合成尿苷二磷酸葡萄糖。反应液经过SAX柱离子交换和ODS柱脱盐纯化后,得到0.63 g产品,分离得率为53.3%(以UTP为基准)。
4.用固定化的麦芽糊精磷酸化酶催化合成a-D-葡萄糖-1-磷酸
确定固定化的麦芽糊精磷酸化酶催化合成葡萄糖-1-磷酸的最佳反应条件为：5%(m/v)麦芽糊精,100 mM(pH7.5)磷酸钠缓冲液,24 U/I麦芽糊精磷酸化酶,在30℃恒温摇床上200 rpm连续反应。在该条件下连续8批,每批200 ml连续合成α-D-葡萄糖-1-磷酸。经过多步沉淀纯化,得到0.44 g产品,分离得率为70.5%。
5.利用半乳糖激酶催化制备α-D-半乳糖-1-磷酸
从E. coli K12中克隆了galk基因,在E. coli BL21(DE3)中进行了高效表达,并制备了半乳糖激酶制剂。实验确定半乳糖激酶的酶促反应最优条件为：在30℃下,22 mM ATP,20 mM半乳糖,3.5mM MgCl2,100mM(pH7.5)磷酸盐缓冲液,36 U/ml半乳糖激酶,连续反应8 h。100 ml体系中,在最优反应条件下合成了α-D-半乳糖-1-磷酸。反应液通过多步沉淀纯化,得到0.54克产品,产率为88.2%。
Trillin is a glucoside of diosgenin which is an important kind of steroidal saponins. It is the active ingredient in medicinal plants of dioscoreaceae. Although it can be isolated from plants, this is extremely difficult and uneconomic, especially in large amounts due to the the amount of trillin in plant is rather limited. Thus, glycosylations in vitro are important for the synthesis of steroidal saponins for the pharmaceutical industry. Chemical synthesis would provide a feasible route to obtain homogeneous saponins, however, the protection and the deprotection of hydroxy groups are unavoidable and it requires organic solvents. So the more economic and cleaner route should be enzymatic synthesis. In this work, a glucosyltransferase cloned from potato bud was used to glucosylate steroidal sapogenins and steroidal alkaloids, in which UDP-glucose was provided as an indispensable sugar donor. But UDP-glucose is extremely expensive and unstable as a kind of energy-rich compound. So avoiding the use of UDP-glucose can greatly increase predominance for enzymatic synthesis of trillin. A multiple-enzymes system for enzymatic synthesis of trillin is artificially designed, the glycosylation reactions in vitro, in which the low-cost maltodextrin, UTP, phosphate were used as substrates. UDP-glucose and glucose-1-phosphate are not only vital intermediates in the one-pot bioreaction system as mentioned above, but also high value-added energy-rich sugar donors in the reaction catalyzed by enzymes. This dissertation describes the biosynthesis of UDP-glucose and glucose-1-phosphate using the enzymes covalently immobilized on amino-functionalized magnetic nanoparticles. In addition, galactose-1-phosphate was also synthesized using a recombinant galactokinase cloned from E. coli in this work for the purpose of diosgenin glycorandomazation in the future.
The genes of malpase, ugpase,ppase, udpgdh from E. coli K12 and the gene of sgtl.1 from the bud of potato have been cloned. They were individually expressed with activity in E. coli BL21 (DE3) or E. coli DH5a. The expression condition for production of these recombinant proteins were optimized. And the recombinant enzymes were prepared and stored in the state of lyophilized powder.
The one-pot system for biocatalytic synthesis of diosgenyl-β-d-glucopyranoside contained maltodextrin phosphorylase (E1, MalPase), uridine diphosphate glucose pyrophosphorylase (E2, GIPTTase), inorganic pyrophosphatase (E3, PPase), solanidine glucosyltransferase (E4, SGT1.1) and maltodextrin, UTP, phosphate, diosgenin. To increase the yield of trillin, some factors such as cosolvents, reaction conditions and substrate concentration were optimized. Under the optimum conditions:the reaction mixture containing 6.58 U El/1,125 U E2/1,7 U E3/1,0.75 U E4/1,5 mM MgCl2,100 mM phosphate buffer (pH 9), maltodextrin (5%, m/v), 10 mM UTP and 0.1 mM diosgenin was incubated in a thermomixer at 200 rpm and 40℃for 48 h, giving trillin in 28% yield (ca.15.8 mg/l).
The amino-functionalized Fe3O4 nanoparticles prepared by chemical coprecipitation are an ideal material for immobilizing various enzymes which are needed for synthesis of UDP-glucose. The Fe3O4 nanoparticles activated by glutaraldehyde was found to be able to tightly bind with maltodextrin phosphorylase (E1), uridine diphosphate glucose pyrophosphorylase (E2) and inorganic pyrophosphatase (E3) respectively. The conditions for one-pot biocatalysis were optimized:18 U E1/l,100 U E2/l,25 U E3/l; maltodextrin,5% (m/v); UTP,2 mM; and MgCl2,10 mM; in 100 mM sodium phosphate buffer, pH 7.5; at 30℃for 10 h. Under the optimum conditions, the immobilized multiple-enzyme biocatalyst could be easily recovered and reused for five times in repeated syntheses of UDP-Glc. After purified by SAX column and ODS column orderly, approximately 630 mg of crystallized UDP-Glc was obtained from 1 litre of reaction mixture, with a moderate yield of 53.3% (based on UTP conversion).
The optimal reaction conditions for glucose-1-phosphate synthesis by immobilized MalPase was:MalPase,24 U/l; maltodextrin,5%(m/v); in 100 mM sodium phosphate buffer, pH 7.5; and reacting at 30℃for 8 h. The immobilized MalPase could be easily recovered and reused for eight times in repeated synthesis of glucose-1-phosphate. After simple purification, approximately 440 mg of a crude product was afforded, with a moderate isolation yield of 70.5%.
The genes of galk from E. coli K12 had been cloned and overexpressed in E. coli BL21(DE3). The reaction conditions for galactose-1-phosphate synthesis by galactokinase (GalK) was optimized as follows:the reaction mixture was composed of 36 U GalK/ml,22 mM ATP,20 mM galactose and 3.5 mM MgCl2 in 100 mM sodium phosphate buffer (pH 7.5), and incubated at 30℃for 8 h. Under the optimal conditions,0.54 g crude product was isolated from a 100-ml system, with a yield of 88.2%.
1.用于薯蓣皂素糖基化的生物催化剂制备
从E. coli K12中分别克隆了malpase, ugpase, ppase, udpgdh基因,从土豆芽中克隆了sgtl.1基因。这些基因分别在E.coli BL21(DE3)和E. coli DH5a中成功地表达了有活性的可溶蛋白。同时优化了重组蛋白的表达条件,并制成冻干酶制剂。
2.多酶一锅催化合成延龄草苷
在体系中加入麦芽糊精磷酸化酶(E1)、尿苷二磷酸葡萄糖焦磷酸化酶(E2)、无机焦磷酸酶(E3)、薯蓣皂素糖基转移酶(E4)以及麦芽糊精、UTP、磷酸盐、薯蓣皂素等底物,通过考察助溶剂添加效果、优化反应条件以及利用正交试验优化底物浓度的配比,最终确定了该体系的最优反应条件：6.58 U E1/1,125 U E2/1,7 U E3/1,0.75 U E4/1,5%(m/v)麦芽糊精,10 mM UTP,0.1 mM薯蓣皂素,5 mM MgCl2,100 mM (pH 9)磷酸盐缓冲液在40℃下连续反应48 h。在该条件下,延龄草苷的产率为89%,浓度为15.8 mg/l。
3.三种固定化酶组合催化合成尿苷二磷酸葡萄糖
利用化学共沉淀法制备的Fe3O4,经过氨基硅烷化和戊二醛活化后,分别和麦芽糊精磷酸化酶、尿苷二磷酸葡萄糖焦磷酸化酶、无机焦磷酸酶共价结合,制备成固定化酶。通过分别比较游离酶和固定化的理化性质,证明了磁性纳米颗粒适合在多酶催化合成尿苷二磷酸葡萄糖中使用。通过多步实验,确定多酶合成尿苷二磷酸葡萄糖的最佳反应条件如下：在体系中加入5%(m/v)麦芽糊精,2mMUTP,10mM MgCl2,100mM(pH 7.5)磷酸钠缓冲液,18 U E1/1,100 U E2/1,25 U E3/1,30℃恒温摇床上200 rpm连续反应12 h。然后在200ml体系中,利用优化后的反应条件,连续5批次酶促合成尿苷二磷酸葡萄糖。反应液经过SAX柱离子交换和ODS柱脱盐纯化后,得到0.63 g产品,分离得率为53.3%(以UTP为基准)。
4.用固定化的麦芽糊精磷酸化酶催化合成a-D-葡萄糖-1-磷酸
确定固定化的麦芽糊精磷酸化酶催化合成葡萄糖-1-磷酸的最佳反应条件为：5%(m/v)麦芽糊精,100 mM(pH7.5)磷酸钠缓冲液,24 U/I麦芽糊精磷酸化酶,在30℃恒温摇床上200 rpm连续反应。在该条件下连续8批,每批200 ml连续合成α-D-葡萄糖-1-磷酸。经过多步沉淀纯化,得到0.44 g产品,分离得率为70.5%。
5.利用半乳糖激酶催化制备α-D-半乳糖-1-磷酸
从E. coli K12中克隆了galk基因,在E. coli BL21(DE3)中进行了高效表达,并制备了半乳糖激酶制剂。实验确定半乳糖激酶的酶促反应最优条件为：在30℃下,22 mM ATP,20 mM半乳糖,3.5mM MgCl2,100mM(pH7.5)磷酸盐缓冲液,36 U/ml半乳糖激酶,连续反应8 h。100 ml体系中,在最优反应条件下合成了α-D-半乳糖-1-磷酸。反应液通过多步沉淀纯化,得到0.54克产品,产率为88.2%。
Trillin is a glucoside of diosgenin which is an important kind of steroidal saponins. It is the active ingredient in medicinal plants of dioscoreaceae. Although it can be isolated from plants, this is extremely difficult and uneconomic, especially in large amounts due to the the amount of trillin in plant is rather limited. Thus, glycosylations in vitro are important for the synthesis of steroidal saponins for the pharmaceutical industry. Chemical synthesis would provide a feasible route to obtain homogeneous saponins, however, the protection and the deprotection of hydroxy groups are unavoidable and it requires organic solvents. So the more economic and cleaner route should be enzymatic synthesis. In this work, a glucosyltransferase cloned from potato bud was used to glucosylate steroidal sapogenins and steroidal alkaloids, in which UDP-glucose was provided as an indispensable sugar donor. But UDP-glucose is extremely expensive and unstable as a kind of energy-rich compound. So avoiding the use of UDP-glucose can greatly increase predominance for enzymatic synthesis of trillin. A multiple-enzymes system for enzymatic synthesis of trillin is artificially designed, the glycosylation reactions in vitro, in which the low-cost maltodextrin, UTP, phosphate were used as substrates. UDP-glucose and glucose-1-phosphate are not only vital intermediates in the one-pot bioreaction system as mentioned above, but also high value-added energy-rich sugar donors in the reaction catalyzed by enzymes. This dissertation describes the biosynthesis of UDP-glucose and glucose-1-phosphate using the enzymes covalently immobilized on amino-functionalized magnetic nanoparticles. In addition, galactose-1-phosphate was also synthesized using a recombinant galactokinase cloned from E. coli in this work for the purpose of diosgenin glycorandomazation in the future.
The genes of malpase, ugpase,ppase, udpgdh from E. coli K12 and the gene of sgtl.1 from the bud of potato have been cloned. They were individually expressed with activity in E. coli BL21 (DE3) or E. coli DH5a. The expression condition for production of these recombinant proteins were optimized. And the recombinant enzymes were prepared and stored in the state of lyophilized powder.
The one-pot system for biocatalytic synthesis of diosgenyl-β-d-glucopyranoside contained maltodextrin phosphorylase (E1, MalPase), uridine diphosphate glucose pyrophosphorylase (E2, GIPTTase), inorganic pyrophosphatase (E3, PPase), solanidine glucosyltransferase (E4, SGT1.1) and maltodextrin, UTP, phosphate, diosgenin. To increase the yield of trillin, some factors such as cosolvents, reaction conditions and substrate concentration were optimized. Under the optimum conditions:the reaction mixture containing 6.58 U El/1,125 U E2/1,7 U E3/1,0.75 U E4/1,5 mM MgCl2,100 mM phosphate buffer (pH 9), maltodextrin (5%, m/v), 10 mM UTP and 0.1 mM diosgenin was incubated in a thermomixer at 200 rpm and 40℃for 48 h, giving trillin in 28% yield (ca.15.8 mg/l).
The amino-functionalized Fe3O4 nanoparticles prepared by chemical coprecipitation are an ideal material for immobilizing various enzymes which are needed for synthesis of UDP-glucose. The Fe3O4 nanoparticles activated by glutaraldehyde was found to be able to tightly bind with maltodextrin phosphorylase (E1), uridine diphosphate glucose pyrophosphorylase (E2) and inorganic pyrophosphatase (E3) respectively. The conditions for one-pot biocatalysis were optimized:18 U E1/l,100 U E2/l,25 U E3/l; maltodextrin,5% (m/v); UTP,2 mM; and MgCl2,10 mM; in 100 mM sodium phosphate buffer, pH 7.5; at 30℃for 10 h. Under the optimum conditions, the immobilized multiple-enzyme biocatalyst could be easily recovered and reused for five times in repeated syntheses of UDP-Glc. After purified by SAX column and ODS column orderly, approximately 630 mg of crystallized UDP-Glc was obtained from 1 litre of reaction mixture, with a moderate yield of 53.3% (based on UTP conversion).
The optimal reaction conditions for glucose-1-phosphate synthesis by immobilized MalPase was:MalPase,24 U/l; maltodextrin,5%(m/v); in 100 mM sodium phosphate buffer, pH 7.5; and reacting at 30℃for 8 h. The immobilized MalPase could be easily recovered and reused for eight times in repeated synthesis of glucose-1-phosphate. After simple purification, approximately 440 mg of a crude product was afforded, with a moderate isolation yield of 70.5%.
The genes of galk from E. coli K12 had been cloned and overexpressed in E. coli BL21(DE3). The reaction conditions for galactose-1-phosphate synthesis by galactokinase (GalK) was optimized as follows:the reaction mixture was composed of 36 U GalK/ml,22 mM ATP,20 mM galactose and 3.5 mM MgCl2 in 100 mM sodium phosphate buffer (pH 7.5), and incubated at 30℃for 8 h. Under the optimal conditions,0.54 g crude product was isolated from a 100-ml system, with a yield of 88.2%.
引文
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