原薯蓣皂苷糖苷酶-1型的特性及其基因的克隆与表达
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摘要
薯蓣属植物中的主要活性成分是甾体皂苷,但天然的甾体皂苷常常以低活性的多糖基形式存在,不易被人体吸收。为制备高活性的、易吸收的低糖基甾体皂苷,本文采用微生物酶法转化穿龙薯蓣中的甾体皂苷,并研究了其特异性的甾体皂苷糖苷酶的分离纯化、酶性质以及基因的克隆和表达。
本研究首先采用乙醇浸提法对穿龙薯蓣植物中总皂苷进行提取,并对提取物进行脱脂、脱糖处理。从4kg穿龙薯蓣植物干燥根中共提取得到了250g的总皂苷,得率为6.3%。通过硅胶柱层析法和制备色谱法,从总皂苷中分离纯化得到了两种主要甾体皂苷,结构经鉴定分别为：26-0-p-D-吡喃葡萄糖基25(R)-22-羟基-呋甾-/△5(6)-烯-3β,26-二羟基-3-O-α-L-吡喃鼠李糖基(1→2)-[α-L-吡喃鼠李糖基(1→4)]-β-D-吡喃葡萄糖苷(原薯蓣皂苷),薯蓣皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-[α-L-吡喃鼠李糖基(1→4)]-β-D-吡喃葡萄糖苷(薯蓣皂苷)。
研究了甾体皂苷酶的生产菌,筛选获得了转化穿龙薯蓣总皂苷效果较好的米曲霉(DLFCC-38),其粗酶液几乎完全转化穿龙薯蓣总皂苷,主要生成薯蓣次皂苷A、纤细皂苷,以及一定量的薯蓣皂苷元。以薯蓣次皂苷A的产量为目标,对微生物发酵产酶的条件进行了优化,最佳条件为：最佳诱导物为穿龙薯蓣提取物,最佳诱导物浓度为2%(v/v),在30℃条件下发酵72h。其次,对酶转化的反应条件进行了优化,最佳酶反应条件为：反应温度为50℃,反应体系pH5.0,反应时间9h,反应体系中底物最终浓度20mg/ml。在此基础之上,米曲霉(DLFCC-38)大量发酵制备粗酶液,批量定向酶解制备低糖基甾体皂苷。总皂苷160g经酶转化后共制备得到产物粗品117g,产物经硅胶柱层析法分离纯化,得到3种单体皂苷成分,分别为：60.3g的薯蓣次皂苷A,得率为51.5%；纤细皂苷11.54g,得率为9.86%；3.01g的薯蓣皂苷元,得率为2.57%。
采用分子筛层析法和离子交换层析法,对米曲霉(DLFCC-38)诱导产粗酶液进行分离纯化。经三次柱法层析,分离得到了原薯蓣皂苷糖苷酶的纯酶。经SDS-PAGE法测定,该酶的分子量约为55kDa。酶反应的最适温度和pH分别为50℃和5.0,酶活力在60℃以下,pH为3.0-8.0范围内相对稳定。K+、Na+、Mg2+、Ca2+四种金属离子对酶活力几乎没有影响；Zn2+对酶活力具有一定的抑制作用；而低浓度的Cu2+和Fe3+对酶活力都有明显的抑制作用。
纯化得到的原薯蓣甾体皂苷糖苷酶,能水解原薯蓣皂苷上的26-O-β-D-葡萄糖基生成薯蓣皂苷,然后进一步水解薯蓣皂苷的3-O-α-L-(1→4)-鼠李糖基生成薯蓣次皂苷A。该酶水解原薯蓣皂苷葡萄糖基的Km=4.59mM,Vmax=3.86mM/h,水解薯蓣皂苷鼠李糖基的Km=42.6mM,Vmax=0.15mM/h。此外,该酶还能水解对硝基苯基-α-D-半乳糖苷(pNP-α-D-Ga1)、对硝基苯基-β-D-葡萄糖苷(pNP-β-D-Glc)和对硝基苯基-β-D-半乳糖苷(pNP-β-D-Gal)。该酶呈现出新的水解特性,不同于国际酶学委员会公布的184种糖苷酶(一种糖苷酶只能水解一种糖苷键),也不同于其他已报道过的甾体皂苷糖苷酶,是一种新酶,将其命名为原薯蓣皂苷糖苷酶-1型(protodioscin-Glycosidase-1, PGase-1)。
采用RT-PCR技术以及RACE技术,对原薯蓣皂苷酶-1型的基因全长序列进行调取。该酶基因全长为1725bp,包含一个1497bp的开放阅读框(ORF),编码498个氨基酸,N端前20个氨基酸序列为信号肽序列。通过亚克隆技术,将原薯蓣皂苷糖苷酶-1型基因重组至真核表达载体pPIC9K,并电转化至巴斯德毕赤酵母GS115。重组酵母菌诱导表达的酶液具有原薯蓣皂苷糖苷酶-1型的酶活力,且最佳诱导培养时间为144h、甲醇的最终浓度为0.5%。经SDS-PAGE分析,诱导表达酶的分子量约为55kDa,与野生型原薯蓣皂苷糖苷酶-1型的分子量55kDa相近。这说明原薯蓣皂苷糖苷酶-1型基因在毕赤酵母中成功的进行了异源表达。经序列相似性比较,发现原薯蓣皂苷糖苷酶-1型的基因与α-淀粉酶的基因序列有很高的相似性。基于两者序列的相似性,原薯蓣皂苷糖苷酶-1型应分在糖苷水解酶家族GH13(Glycoside Hydrolase Family13)中；但是两者具有完全不同的水解特性。因此,酶蛋白的基因序列不能完全代表酶的特性。
The steroidal saponins with long sugar chains are physiologically active ingredients in medicinal plant of Dioscorea L., but these natural steroidal saponins generally containing long sugar chains have low activity and low absorption in human bodies. In this paper, the enzyme from microorganism was used for converting the steroidal saponins in Dioscorea nipponica Makino to more active and easy-absorbing steroidal saponins with low sugar chains. In addition, a novel steroidal saponin-glycosidase was purified, characterized, cloned and expressed in Pichia pastoris GS115.
Firstly, the total steroidal saponins of250g were obtained from4kg dried roots of D. nipponica Makino after by ethanol extraction, desugaration and decoloration. The extraction yield was about6.3%. Tow main steroidal saponins were separated by silica gel chromatography, and the structure of these tow steroidal saponins was identified as protodioscin, i. e.,26-O-β-D-glucopyranosyl-25(R)-22-hydroxyl-5-ene-furostane-3β,26-diol-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyrano side, and dioscin dioscin, i.e., diosgenin-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside, respectively.
Aspergillus oryzae DLFCC-38was screened as the enzyme producing microorganism, the enzyme from this microorganism could almost convert the total steroidal saponins of Dioscorea nipponica Makino to major progenin Ⅲ and gracillin, and small amount of diosgenin. The optimal culture conditions for enzyme production by A. oryzae DLFCC-38were investigated. For enzyme production, the strain was cultured for72h at30℃, in the medium containing2%(v/v) extract of D. nipponica as the enzyme inducer. The crude enzyme converted total steroidal saponins into major progenin III with a high yield when the reaction was carried out for9h, at50℃and pH5.0, with the20mg/ml of substrate. In the scale-up preparation of progenin Ⅲ,117g of crude product was obtained from160g of substrate.117g of the crude product was purified with silica gel column to obtain60.3g progenin with the yield of51.5%,11.54g gracillin with the yield of9.86%, and3.01g diosgenin with the yield of2.57%.
After3-steps, a protodioscin-glycosidase was purified from the A. oryzae DLFCC-38culture by the methods of molecular sieve chromatography and ion exchange chromatography. The molecular mass of this enzyme was determined to be about55kDa based on SDS-polyacrylamide gel electrophoresis. The optimum temperature and pH of the enzyme was5.0and50℃, respectively, and the enzyme was stable at pH3.0-8.0, below60 ℃. The activity of this enzyme was not obviously affected by K+, Na+, Mg2+and Ca2+ions, but slightly inhibited by Zn2+and strictly inhibited Cu2+and Fe3+ions.
The purified protodioscin-glycosidase was able to hydrolyze the terminal26-O-β-D-glucopyranoside of protodioscin to produce dioscin, and then further hydrolyze the terminal3-O-(1→4)-α-L-rhamnopyranoside of dioscin to form progenin Ⅲ. The Km and Vmax for protodioscin were4.59mM and3.86mM/h, respectively. The Km and Vmax for dioscin were42.6mM and0.15mM/h, respectively. In addition, this enzyme also could hydrolyze the a-D-galactopyranoside, β-D-glucopyranoside and β-D-galactopyranoside of p-nitrophenyl-glycosides. These new properties of the protodioscin-glycosidase are significantly different from those of previously described steroidal saponin-glycosidases and the glycosidases currently described in Enzyme Nomenclature by the NC-IUBMB where typically one enzyme hydrolyzes one type of glycoside. It therefore represents the advent of a novel enzyme, and the new enzyme was named protodioscin-glycosidase-1(PGase-1).
The complete gene sequence of protodioscin-glycosidase-1was amplified by the methods of RT-PCR and RACE, and the sequence length is1725bp. The full-length cDNA contains a1497-bp open reading frame (ORF) which encoding498amino acid residues, and20amino acids were cleaved from the N terminus of the mature form and seemed to be a signal sequence. The protodioscin-glycosidase-1gene cloned from A. oryzae was subcloned into the vector pPIC9K and then transformed into Pichia pastoris GS115. The optimal culture condition of recombinant P. pastoris GS115was identified as:the culture time was144h and the final methnol content was0.5%. The recombinant protodioscin-glycosidase-1from recombinant P. pastoris GS115also showed the activity hydrolyzing glycosides of steroidal saponins and the molecular mass of recombinant protodioscin-glycosidase-1was determined to be about55kDa based on SDS-polyacrylamide gel electrophoresis, which were both similar to that of the wild-type protodioscin-glycosidase-1from A. oryzae. The results show that gene has been successfully expressed in P. pastoris GS115. The protodioscin-glycosidase-1gene is highly similar to a-amylase (EC3.2.1.1), and the protodioscin-glycosidase-1should be classified as glycoside hydrolase family13by the method of gene sequence-based classification. But the enzyme properties of this enzyme are different from those of a-amylase in this family. Therefore, gene sequence of the enzyme can not completely represent enzyme properties.
本研究首先采用乙醇浸提法对穿龙薯蓣植物中总皂苷进行提取,并对提取物进行脱脂、脱糖处理。从4kg穿龙薯蓣植物干燥根中共提取得到了250g的总皂苷,得率为6.3%。通过硅胶柱层析法和制备色谱法,从总皂苷中分离纯化得到了两种主要甾体皂苷,结构经鉴定分别为：26-0-p-D-吡喃葡萄糖基25(R)-22-羟基-呋甾-/△5(6)-烯-3β,26-二羟基-3-O-α-L-吡喃鼠李糖基(1→2)-[α-L-吡喃鼠李糖基(1→4)]-β-D-吡喃葡萄糖苷(原薯蓣皂苷),薯蓣皂苷元-3-O-α-L-吡喃鼠李糖基(1→2)-[α-L-吡喃鼠李糖基(1→4)]-β-D-吡喃葡萄糖苷(薯蓣皂苷)。
研究了甾体皂苷酶的生产菌,筛选获得了转化穿龙薯蓣总皂苷效果较好的米曲霉(DLFCC-38),其粗酶液几乎完全转化穿龙薯蓣总皂苷,主要生成薯蓣次皂苷A、纤细皂苷,以及一定量的薯蓣皂苷元。以薯蓣次皂苷A的产量为目标,对微生物发酵产酶的条件进行了优化,最佳条件为：最佳诱导物为穿龙薯蓣提取物,最佳诱导物浓度为2%(v/v),在30℃条件下发酵72h。其次,对酶转化的反应条件进行了优化,最佳酶反应条件为：反应温度为50℃,反应体系pH5.0,反应时间9h,反应体系中底物最终浓度20mg/ml。在此基础之上,米曲霉(DLFCC-38)大量发酵制备粗酶液,批量定向酶解制备低糖基甾体皂苷。总皂苷160g经酶转化后共制备得到产物粗品117g,产物经硅胶柱层析法分离纯化,得到3种单体皂苷成分,分别为：60.3g的薯蓣次皂苷A,得率为51.5%；纤细皂苷11.54g,得率为9.86%；3.01g的薯蓣皂苷元,得率为2.57%。
采用分子筛层析法和离子交换层析法,对米曲霉(DLFCC-38)诱导产粗酶液进行分离纯化。经三次柱法层析,分离得到了原薯蓣皂苷糖苷酶的纯酶。经SDS-PAGE法测定,该酶的分子量约为55kDa。酶反应的最适温度和pH分别为50℃和5.0,酶活力在60℃以下,pH为3.0-8.0范围内相对稳定。K+、Na+、Mg2+、Ca2+四种金属离子对酶活力几乎没有影响；Zn2+对酶活力具有一定的抑制作用；而低浓度的Cu2+和Fe3+对酶活力都有明显的抑制作用。
纯化得到的原薯蓣甾体皂苷糖苷酶,能水解原薯蓣皂苷上的26-O-β-D-葡萄糖基生成薯蓣皂苷,然后进一步水解薯蓣皂苷的3-O-α-L-(1→4)-鼠李糖基生成薯蓣次皂苷A。该酶水解原薯蓣皂苷葡萄糖基的Km=4.59mM,Vmax=3.86mM/h,水解薯蓣皂苷鼠李糖基的Km=42.6mM,Vmax=0.15mM/h。此外,该酶还能水解对硝基苯基-α-D-半乳糖苷(pNP-α-D-Ga1)、对硝基苯基-β-D-葡萄糖苷(pNP-β-D-Glc)和对硝基苯基-β-D-半乳糖苷(pNP-β-D-Gal)。该酶呈现出新的水解特性,不同于国际酶学委员会公布的184种糖苷酶(一种糖苷酶只能水解一种糖苷键),也不同于其他已报道过的甾体皂苷糖苷酶,是一种新酶,将其命名为原薯蓣皂苷糖苷酶-1型(protodioscin-Glycosidase-1, PGase-1)。
采用RT-PCR技术以及RACE技术,对原薯蓣皂苷酶-1型的基因全长序列进行调取。该酶基因全长为1725bp,包含一个1497bp的开放阅读框(ORF),编码498个氨基酸,N端前20个氨基酸序列为信号肽序列。通过亚克隆技术,将原薯蓣皂苷糖苷酶-1型基因重组至真核表达载体pPIC9K,并电转化至巴斯德毕赤酵母GS115。重组酵母菌诱导表达的酶液具有原薯蓣皂苷糖苷酶-1型的酶活力,且最佳诱导培养时间为144h、甲醇的最终浓度为0.5%。经SDS-PAGE分析,诱导表达酶的分子量约为55kDa,与野生型原薯蓣皂苷糖苷酶-1型的分子量55kDa相近。这说明原薯蓣皂苷糖苷酶-1型基因在毕赤酵母中成功的进行了异源表达。经序列相似性比较,发现原薯蓣皂苷糖苷酶-1型的基因与α-淀粉酶的基因序列有很高的相似性。基于两者序列的相似性,原薯蓣皂苷糖苷酶-1型应分在糖苷水解酶家族GH13(Glycoside Hydrolase Family13)中；但是两者具有完全不同的水解特性。因此,酶蛋白的基因序列不能完全代表酶的特性。
The steroidal saponins with long sugar chains are physiologically active ingredients in medicinal plant of Dioscorea L., but these natural steroidal saponins generally containing long sugar chains have low activity and low absorption in human bodies. In this paper, the enzyme from microorganism was used for converting the steroidal saponins in Dioscorea nipponica Makino to more active and easy-absorbing steroidal saponins with low sugar chains. In addition, a novel steroidal saponin-glycosidase was purified, characterized, cloned and expressed in Pichia pastoris GS115.
Firstly, the total steroidal saponins of250g were obtained from4kg dried roots of D. nipponica Makino after by ethanol extraction, desugaration and decoloration. The extraction yield was about6.3%. Tow main steroidal saponins were separated by silica gel chromatography, and the structure of these tow steroidal saponins was identified as protodioscin, i. e.,26-O-β-D-glucopyranosyl-25(R)-22-hydroxyl-5-ene-furostane-3β,26-diol-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyrano side, and dioscin dioscin, i.e., diosgenin-3-O-α-L-rhamnopyranosyl-(1→2)-[α-L-rhamnopyranosyl-(1→4)]-β-D-glucopyranoside, respectively.
Aspergillus oryzae DLFCC-38was screened as the enzyme producing microorganism, the enzyme from this microorganism could almost convert the total steroidal saponins of Dioscorea nipponica Makino to major progenin Ⅲ and gracillin, and small amount of diosgenin. The optimal culture conditions for enzyme production by A. oryzae DLFCC-38were investigated. For enzyme production, the strain was cultured for72h at30℃, in the medium containing2%(v/v) extract of D. nipponica as the enzyme inducer. The crude enzyme converted total steroidal saponins into major progenin III with a high yield when the reaction was carried out for9h, at50℃and pH5.0, with the20mg/ml of substrate. In the scale-up preparation of progenin Ⅲ,117g of crude product was obtained from160g of substrate.117g of the crude product was purified with silica gel column to obtain60.3g progenin with the yield of51.5%,11.54g gracillin with the yield of9.86%, and3.01g diosgenin with the yield of2.57%.
After3-steps, a protodioscin-glycosidase was purified from the A. oryzae DLFCC-38culture by the methods of molecular sieve chromatography and ion exchange chromatography. The molecular mass of this enzyme was determined to be about55kDa based on SDS-polyacrylamide gel electrophoresis. The optimum temperature and pH of the enzyme was5.0and50℃, respectively, and the enzyme was stable at pH3.0-8.0, below60 ℃. The activity of this enzyme was not obviously affected by K+, Na+, Mg2+and Ca2+ions, but slightly inhibited by Zn2+and strictly inhibited Cu2+and Fe3+ions.
The purified protodioscin-glycosidase was able to hydrolyze the terminal26-O-β-D-glucopyranoside of protodioscin to produce dioscin, and then further hydrolyze the terminal3-O-(1→4)-α-L-rhamnopyranoside of dioscin to form progenin Ⅲ. The Km and Vmax for protodioscin were4.59mM and3.86mM/h, respectively. The Km and Vmax for dioscin were42.6mM and0.15mM/h, respectively. In addition, this enzyme also could hydrolyze the a-D-galactopyranoside, β-D-glucopyranoside and β-D-galactopyranoside of p-nitrophenyl-glycosides. These new properties of the protodioscin-glycosidase are significantly different from those of previously described steroidal saponin-glycosidases and the glycosidases currently described in Enzyme Nomenclature by the NC-IUBMB where typically one enzyme hydrolyzes one type of glycoside. It therefore represents the advent of a novel enzyme, and the new enzyme was named protodioscin-glycosidase-1(PGase-1).
The complete gene sequence of protodioscin-glycosidase-1was amplified by the methods of RT-PCR and RACE, and the sequence length is1725bp. The full-length cDNA contains a1497-bp open reading frame (ORF) which encoding498amino acid residues, and20amino acids were cleaved from the N terminus of the mature form and seemed to be a signal sequence. The protodioscin-glycosidase-1gene cloned from A. oryzae was subcloned into the vector pPIC9K and then transformed into Pichia pastoris GS115. The optimal culture condition of recombinant P. pastoris GS115was identified as:the culture time was144h and the final methnol content was0.5%. The recombinant protodioscin-glycosidase-1from recombinant P. pastoris GS115also showed the activity hydrolyzing glycosides of steroidal saponins and the molecular mass of recombinant protodioscin-glycosidase-1was determined to be about55kDa based on SDS-polyacrylamide gel electrophoresis, which were both similar to that of the wild-type protodioscin-glycosidase-1from A. oryzae. The results show that gene has been successfully expressed in P. pastoris GS115. The protodioscin-glycosidase-1gene is highly similar to a-amylase (EC3.2.1.1), and the protodioscin-glycosidase-1should be classified as glycoside hydrolase family13by the method of gene sequence-based classification. But the enzyme properties of this enzyme are different from those of a-amylase in this family. Therefore, gene sequence of the enzyme can not completely represent enzyme properties.
引文
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