WO2019104608A1 - 一种酶催化合成人参皂苷Rh2的方法 - Google Patents

一种酶催化合成人参皂苷Rh2的方法 Download PDF

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WO2019104608A1
WO2019104608A1 PCT/CN2017/113837 CN2017113837W WO2019104608A1 WO 2019104608 A1 WO2019104608 A1 WO 2019104608A1 CN 2017113837 W CN2017113837 W CN 2017113837W WO 2019104608 A1 WO2019104608 A1 WO 2019104608A1
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ginsenoside
positions
mutation
glucosidase
glutamic acid
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PCT/CN2017/113837
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French (fr)
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傅荣昭
刘立辉
张贵慰
江名
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邦泰生物工程(深圳)有限公司
江西邦泰绿色生物合成生态产业园发展有限公司
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Priority to PCT/CN2017/113837 priority Critical patent/WO2019104608A1/zh
Priority to CN201780002296.2A priority patent/CN108064309B/zh
Priority to CN201910291028.8A priority patent/CN109837261B/zh
Publication of WO2019104608A1 publication Critical patent/WO2019104608A1/zh

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    • C12P33/00—Preparation of steroids
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)

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  • the invention belongs to the field of synthesis of natural compounds, and in particular relates to a method for catalyzing the synthesis of ginsenoside Rh2 by an enzyme.
  • Ginseng has a long history and is a traditional Chinese medicine with excellent medicinal value.
  • the main active ingredient of ginseng is ginsenoside, including three types: Protopanaxadiol type ginsenoside (PPD), Protopanaxatriol type ginsenoside (PPT) and ginsenoside Ro.
  • Most of the saponins in ginseng are Rb1, Rb2, Rc, Rd, Re and Rg1 ginsenosides.
  • the rare ginsenoside Rh2 has very little content in nature.
  • Ginsenoside Rh2 is a newly discovered anti-tumor and anti-metastatic natural botanical ingredient. It is the drug of choice for radiotherapy and chemotherapy, and it is the first choice for anti-cancer health care products.
  • ginsenosides The chemical structure of ginsenosides is: Its type and structural characteristics are shown in Table 1.
  • Glu glucose
  • Araf arabinofuranose
  • Arap arabinose
  • Rha rhamnose
  • CN102352402 B discloses extracting an extract containing ginseng autosaponin and other water-soluble substances from ginseng, and reacting with the original ginseng glycol saponin to prepare Rh2 mixed saponin.
  • CN 101565694 A discloses the use of Panax notoginseng saponins to convert notoginsenosides to higher activity Rg3, CK, Rh2 secondary saponins and aglycones.
  • CN 101385519A discloses an enzyme solution produced by using a geranase-rich cereal bud, mixed with ginseng powder or ginseng total saponin or ginsenoside Rb1, and stirred at 40-50 ° C for 10-24 hours to produce F2, Rg3, Ginsenosides such as CK and Rh2.
  • the present invention mutates the wild type glucosidase to obtain a glucosidase mutant having greatly improved enzyme activity and greatly improved ginsenoside Rh2 conversion.
  • One of the objects of the present invention is to provide a glucosidase mutant.
  • Another object of the invention is to provide the use of a glucosidase mutant.
  • Another object of the present invention is to provide a method for producing ginsenoside Rh2 of a glucosidase mutant.
  • ginsenoside Rh2 An enzyme-catalyzed synthesis of ginsenoside Rh2, using ginsenoside Rg3 as a substrate, catalyzed by glucosidase to form ginsenoside Rh2; the glucosidase is derived from Terrabacter ginsenosidimutans; the temperature of the catalytic reaction is 25 ⁇ 50 ° C;
  • the concentration of ginsenoside Rg3 in the catalytic reaction system is 1% to 5% w/v, and the amount of glucosidase is 0.01 to 0.06 times the weight of the substrate ginsenoside Rg3, the balance being water or phosphate buffer solution, and for dissolving a cosolvent for ginsenoside Rg3;
  • the pH of the catalytic reaction system is 6.5 to 9.5.
  • amino acid sequence of the glucosidase is represented by SEQ ID NO 2, or the amino acid sequence represented by SEQ ID NO 2 is obtained by at least one of the following mutations:
  • the serine S and proline P at positions 109 and 114 were respectively mutated to alanine A and glycine G;
  • the tyrosine Y at position 469 was mutated to phenylalanine F.
  • the cosolvent of the ginsenoside Rg3 is at least one selected from the group consisting of methanol, ethanol, and 1% to 20% v/v DMSO.
  • the cosolvent of the ginsenoside Rg3 is 1% to 20% v/v DMSO.
  • the catalytic reaction time is 4 to 12 hours.
  • the phosphate buffer solution is a 0.08-0.12 M phosphate buffer solution having a pH of 7.2 to 7.8.
  • the ginsenoside Rg3 is first dissolved in the co-solvent, and then slowly added to the glucosidase, and stirred while being added. Slow addition prevents the Rg3 dissolved in the cosolvent from forming a viscous liquid, which causes the stirrer to fail to rotate.
  • this phenomenon is the lowest in DMSO, and the other two solvents are more likely to occur.
  • the glucosidase is added in the form of an enzyme powder or an enzyme solution.
  • the enzyme solution is obtained by ultrasonically breaking the cells with a phosphate buffer solution and centrifuging the supernatant.
  • a glucosidase mutant obtained from at least one of the following amino acid sequences of SEQ ID NO:
  • the serine S and proline P at positions 109 and 114 were respectively mutated to alanine A and glycine G;
  • the tyrosine Y at position 469 was mutated to phenylalanine F.
  • glucosidase mutant is obtained from the nucleotide sequence shown in SEQ ID NO: 1 by at least one of the following mutations:
  • the base sequences AGC and CCG of positions 325 to 327 and 340 to 342 were respectively mutated to GCC and GGT;
  • the base sequences GAG and GAT of positions 1234 to 1236 and 1246 to 1248 were respectively mutated to GAC and GAG;
  • the base sequence TAT at positions 1405 to 1407 was mutated to TTT.
  • ginsenoside Rg3 is used as a substrate.
  • the glucosidase derived from Terrabacter ginsenosidimutans and the mutant enzyme activity thereof have a conversion rate of more than 95.89% of the substrate Rg3 into Rh2, the conversion rate is greatly improved, the reaction selectivity is high, the by-products are few, and the Rh2 content is High, can be used to prepare high purity Rh2, which is beneficial to further industrial production of ginsenoside Rh2 by enzymatic method.
  • the process for producing ginsenoside Rh2 of the invention is simple, the reaction selectivity is high, and the by-products are few.
  • the glucosidase and the mutant thereof of the invention are easily obtained by Escherichia coli fermentation, and the production cost and product quality are superior to the chemical method, and are suitable for industrialization. produce.
  • the reaction conditions are mild and belong to an environmentally friendly production process, which is in line with the national green environmental protection industry standards.
  • the method of the present invention adopts a catalytic reaction temperature of 25 to 50 ° C, a pH of 6.5 to 9.5, a substrate Rg3 concentration of 1% to 5% w/v, and a glucosidase dosage of Rg3 of 0.01 to 0.06 times. It is more conducive to the catalytic activity of Terrabacter ginsenosidimutans glucosidase and its mutant enzyme activity on the substrate Rg3, and improves the conversion rate of Rg3 to Rh2, which is up to 95.89%, reduces the production of by-products and improves the obtained Rh2. The purity is more conducive to the industrialization of ginsenoside Rh2 by enzymatic production.
  • the amino acid sequence of ⁇ -glucosidase Rh2-015 derived from Terrabacter ginsenosidimutans (the gene sequence is represented by SEQ ID NO: 1, the protein sequence encoded as shown in SEQ ID NO: 2) is obtained by at least one of the following mutations.
  • Glucosidase mutant The amino acid sequence of ⁇ -glucosidase Rh2-015 derived from Terrabacter ginsenosidimutans (the gene sequence is represented by SEQ ID NO: 1, the protein sequence encoded as shown in SEQ ID NO: 2) is obtained by at least one of the following mutations.
  • Glucosidase mutant is obtained by at least one of the following mutations.
  • P135A alanine A
  • F168Y tyrosine Y
  • Y469F phenylalanine F
  • a single-double mutant library was prepared by site-directed mutagenesis of Rh2-015 by reverse PCR.
  • the ⁇ -glucosidase gene Rh2-015 derived from Terrabacter ginsenosidimutans utilizes primer A (SEQ ID NO: 3, respectively).
  • primer B SEQ ID NO: 4 was obtained by PCR amplification technique and then digested, and inserted into the Nde I and EcoR I sites of the expression vector pET22b (+) to obtain the recombinant plasmid pET22b-Rh2- 015.
  • reverse primers were designed at the position of mutation (mutation primer sequences are shown in Table 2), and the target fragment was amplified by the upstream and downstream mutation primers, and the corresponding mutation was introduced into the primer to recombine the plasmid pET22b.
  • -Rh2-015 was used as a template for reverse PCR, and the PCR product was treated with Dpn I digestion template (the commonly used plasmid DNA was extracted from E. coli host due to endogenous dam methylase, The adenine of the sequence has been methylated and thus can be cleaved by DpnI.
  • DNA synthesized by PCR or the like cannot be cleaved due to methylation, and can not be cleaved, eliminating false positives) and transformed into E. coli Rosetta (de3), after Amp After screening, the colonies were picked for sequencing. After the determination is correct, a recombinant bacterium having successful mutation is obtained, and a glucosidase mutant can be obtained by inducing expression of the successfully mutated recombinant strain.
  • the PCR reaction procedure is:
  • the ⁇ -glucosidase gene Rh2-015 derived from Terrabacter ginsenosidimutans utilizes primer A (SEQ ID NO: 3, respectively).
  • primer B SEQ ID NO: 4
  • the recombinant plasmid was transferred to E. coli Rosetta (de3).
  • the obtained recombinant Escherichia coli was inoculated into a small volume of LB medium (containing 100 ⁇ g/mL of Amp), and cultured at 30 to 37 ° C overnight, and then transferred to a 1 L volume of LB medium at a dose of 1 to 5% (containing 100 ⁇ g/mL of Amp), continue to culture OD 600 at 30-37 ° C to 0.6-1.0, add isopropyl- ⁇ -D-thiogalactoside (IPTG) at a final concentration of 0.1 mM to 1 mM, at 20-37 After induction of expression for 10-20 hours at °C, the cells were collected by centrifugation. The fermenting cells were suspended in 4 volumes of 50-100 mM phosphate buffer (pH 7.4) and ultrasonically disrupted for 20 min, and the supernatant was centrifuged to obtain an enzyme solution of Rh2-015.
  • IPTG isopropyl- ⁇ -D-thiogalactoside
  • ⁇ -glucosidase Rh2-015 enzyme solution reaction using pNP-Glc as a substrate, adding 1 mL of 150 mM pNP-Glc, 100 ⁇ L of diluted enzyme solution in a 3 mL reaction system, and reacting at pH 7.0 and 37 ° C for a certain period of time. The reaction was terminated by 0.5 M sodium carbonate for 5 min. The product p-nitrophenol was measured for an increase in absorbance at 405 nm.
  • One unit of enzyme activity is defined as: 1 ⁇ M of p-nitrophenol per minute is 1 U.
  • the ⁇ -glucosidase mutant enzyme activity assay method is the same as the wild type ⁇ -glucosidase, and the recombinant strain successfully mutated in Example 2 is induced to express, and the induced expression method is the same as the above (1), and the above (2) is utilized.
  • the same method was used to measure the enzyme activity, and the enzyme activity and stability of the wild type and the mutant (preserving more than 80% of the enzyme activity) were compared.
  • the liquid phase was analyzed using Kinetex 2.6 ⁇ m C18 100A as the analytical column, acetonitrile and water were fluid, the column temperature was room temperature, the detection wavelength was 203 nm, and the flow rate was 1.0 mL/min. After 10 hours of reaction, the yield of Rh2 was measured.
  • the reaction conditions of the wild type and the mutant ⁇ -glucosidase of the present invention are identical.

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Abstract

公开了一种酶催化合成人参皂苷Rh2的方法，该方法以人参皂苷Rg3为底物加入来源于 Terrabacter ginsenosidimutans的葡萄糖苷酶进行催化反应生成人参皂苷Rh2；催化反应的温度为25～50℃；pH值为6.5～9.5。还公开了该葡萄糖苷酶及其变体。

Description

一种酶催化合成人参皂苷Rh2的方法 技术领域
本发明属于天然化合物合成领域，具体涉及一种酶催化合成人参皂苷Rh2的方法。
背景技术
人参历史悠久，作为一种传统名贵中药，具有优良的药用价值。人参主要活性成分为人参皂苷，包括三种类型，分别为：原人参二醇类皂苷(Protopanaxadiol type ginsenoside，PPD)、原人参三醇类皂苷(Protopanaxatriol type ginsenoside，PPT)和人参皂苷Ro。人参中绝大部分皂苷是Rb1、Rb2、Rc、Rd、Re和Rg1人参皂苷。其中稀有人参皂苷Rh2在自然界含量极少。人参皂苷Rh2是一种新近发现的抗肿瘤、抗转移的天然植物成分，是配合放疗、化疗增效减毒的首选药物，作为新型抗癌强身保健品深受市场欢迎。
人参皂苷的化学结构通式为：
Figure PCTCN2017113837-appb-000001
其种类及结构特征如表1所示。
表1人参皂苷的种类及结构特征
Figure PCTCN2017113837-appb-000002
Figure PCTCN2017113837-appb-000003
注：Glu：葡萄糖；Araf：阿拉伯呋喃糖；Arap：阿拉伯吡喃糖；Rha：鼠李糖
现有技术中存在许多制备人参皂苷Rh2的方法，如CN102352402 B公开了从参根中提取含有人参自身皂苷酶和其它水溶性物质的提取物，与原人参二元醇类皂苷反应制备Rh2混合皂苷。CN 101565694 A公开了利用三七人参皂苷酶转化三七皂苷为活性更高的Rg3、CK、Rh2次生皂苷和苷元。CN 101385519A公开了利用一种皂苷酶活性高的谷物芽产生的酶液，与人参粉或人参总皂苷或人参皂苷Rb1混合，在40-50℃搅拌反应10-24小时，产生含F2、Rg3、CK、Rh2等人参皂苷。还有些方法需使用全细胞进行反应，不利于酶和底物的充分接触，从而会影响转化率，造成反应选择性低，Rh2产量低、纯度低；如CN 1105781C专利，利用β-葡萄糖苷酶水解人参皂苷得到少量的人参皂苷Rh2，转化率低，造成反应选择性低，Rh2产量低、纯度低。可见，现有常见的人参皂苷Rh2的制备方法，存在许多缺点，如选择性低，Rh2含量低不能制备高纯物，工艺粗糙，不适宜放大生产等。
发明内容
为了解决上述存在的问题，本发明对野生型葡萄糖苷酶进行突变，获得酶活性大大提高、人参皂苷Rh2转化率大大提高的葡萄糖苷酶突变体。
本发明的目的之一在于提供葡萄糖苷酶突变体。
本发明的另一目的在于提供葡萄糖苷酶突变体的应用。
本发明的另一目的在于提供葡萄糖苷酶突变体的人参皂苷Rh2的制备方法。
本发明所采取的技术方案是：
一种酶催化合成人参皂苷Rh2的方法，以人参皂苷Rg3为底物，在葡萄糖苷酶的催化下，反应生成人参皂苷Rh2；所述葡萄糖苷酶来源于Terrabacter ginsenosidimutans；催化反应的温度为25～50℃；
催化反应体系中人参皂苷Rg3的浓度为1％～5％w/v，葡萄糖苷酶用量为底物人参皂苷Rg3重量的0.01～0.06倍，余量为水或磷酸盐缓冲液，以及用于溶解人参皂苷Rg3的助溶剂；
所述催化反应体系的pH值为6.5～9.5。
进一步的，所述葡萄糖苷酶的氨基酸序列如SEQ ID NO 2所示，或者为SEQ ID NO 2所示氨基酸序列经以下至少一种突变情况所得：
将第71和72位的谷氨酸E、缬氨酸V分别突变为苏氨酸T、谷氨酸E；
将第92位的谷氨酰胺Q突变为组氨酸H；
将第109和114位的丝氨酸S、脯氨酸P分别突变为丙氨酸A、甘氨酸G；
将第135位的脯氨酸P突变为丙氨酸A；
将第168位的苯丙氨酸F突变为酪氨酸Y；
将第240位的谷氨酰胺Q突变为谷氨酸E；
将第344位的赖氨酸K突变为精氨酸R；
将第412和416位的谷氨酸E、天冬氨酸D分别突变为天冬氨酸D、谷氨酸E；
将第469位的酪氨酸Y突变为苯丙氨酸F。
进一步的，所述人参皂苷Rg3的助溶剂选自甲醇、乙醇、1％～20％v/v的DMSO中的至少一种。
更进一步的，所述人参皂苷Rg3的助溶剂为1％～20％v/v的DMSO。
进一步的，所述催化反应的时间为4～12h。
进一步的，所述磷酸盐缓冲液为pH 7.2～7.8的0.08～0.12M磷酸盐缓冲液。
进一步的，人参皂苷Rg3需先溶解于助溶剂中，再缓慢加入葡萄糖苷酶中，边加入边搅拌。缓慢加入可避免溶于助溶剂中的Rg3形成粘稠状的液体，从而导致搅拌子无法转动。在上述三种助溶剂中，以DMSO出现此类现象的程度最低，另两种溶剂更容易出现这种现象。
进一步的，所述葡萄糖苷酶以酶粉或酶液形式加入。
进一步的，所述酶液是将菌体用磷酸缓冲液超声波破胞、离心取上清得到。
葡萄糖苷酶突变体，由SEQ ID NO 2所示氨基酸序列经以下至少一种突变情况所得：
将第71和72位的谷氨酸E、缬氨酸V分别突变为苏氨酸T、谷氨酸E；
将第92位的谷氨酰胺Q突变为组氨酸H；
将第109和114位的丝氨酸S、脯氨酸P分别突变为丙氨酸A、甘氨酸G；
将第135位的脯氨酸P突变为丙氨酸A；
将第168位的苯丙氨酸F突变为酪氨酸Y；
将第240位的谷氨酰胺Q突变为谷氨酸E；
将第344位的赖氨酸K突变为精氨酸R；
将第412和416位的谷氨酸E、天冬氨酸D分别突变为天冬氨酸D、谷氨酸E；
将第469位的酪氨酸Y突变为苯丙氨酸F。
进一步的，葡萄糖苷酶突变体由SEQ ID NO 1所示核苷酸序列经以下至少一种突变情况所得：
将第211～216位的碱基序列GAGGTG突变为ACGGAG；
将第274～276位的碱基序列CAG突变为CAC；
将第325～327位和340～342位的碱基序列AGC、CCG分别突变为GCC、GGT；
将第403～405位的碱基序列CCG突变为GGC；
将第502～504位的碱基序列TTC突变为TAC；
将第718～720位的碱基序列CAA突变为GAA；
将第1030～1032位的碱基序列CGG突变为AAG；
将第1234～1236位和1246～1248位的碱基序列GAG、GAT分别突变为GAC、GAG；
将第1405～1407位的碱基序列TAT突变为TTT。
上述任一项所述的葡萄糖苷酶突变体在制备人参皂苷Rh2中的应用。
进一步的，上述应用中以人参皂苷Rg3为底物。
本发明的有益效果是：
(1)本发明来源于Terrabacter ginsenosidimutans的葡萄糖苷酶及其突变体酶活对底物Rg3转化成Rh2的转化率达95.89％以上，转化率大大提高，反应选择性高，副产物少，Rh2含量高，可用于制备高纯Rh2，有利于进一步通过酶法工业化生产人参皂苷Rh2。
(2)本发明生产人参皂苷Rh2的工艺简单，反应选择性高，副产物少，本发明葡萄糖苷酶及其突变体经大肠杆菌发酵易得，生产成本和产品质量优于化学法，适合工业化生产。反应条件温和，属于环境友好型生产工艺，符合国家推广的绿色环保工业标准。
(3)本发明方法中采用催化反应温度25～50℃、pH值6.5～9.5、底物Rg3浓度为1％～5％w/v、葡萄糖苷酶用量为Rg3重量0.01～0.06倍等技术特征，更有利于Terrabacter ginsenosidimutans葡萄糖苷酶及其突变体酶活对底物Rg3的催化活性，提高其将Rg3转化成Rh2的转化率，可达95.89％以上，降低了副产物的产生，提高所得Rh2的纯度，更有利于酶法生产人参皂苷Rh2的工业化。
具体实施方式
下面结合具体实施例对本发明作进一步的说明。
实施例1葡萄糖苷酶及其突变体
将来源于Terrabacter ginsenosidimutans的β-葡萄糖苷酶Rh2-015(基因序列如SEQ ID NO:1所示，编码的蛋白序列如SEQ ID NO:2所示)的氨基酸序列经以下至少一种突变获得葡 萄糖苷酶突变体：
将第71和72位的谷氨酸E、缬氨酸V分别突变为苏氨酸T、谷氨酸E(简称E71T+V72E)；
或/和将第92位的谷氨酰胺Q突变为组氨酸H(简称Q92H)；
或/和将第109和114位的丝氨酸S、脯氨酸P分别突变为丙氨酸A、甘氨酸G(简称S109A+P114G)；
或/和将第135位的脯氨酸P突变为丙氨酸A(简称P135A)；
或/和将第168位的苯丙氨酸F突变为酪氨酸Y(简称F168Y)；
或/和将第240位的谷氨酰胺Q突变为谷氨酸E(简称Q240E)；
或/和将第344位的赖氨酸K突变为精氨酸R(简称K344R)；
或/和将第412和416位的谷氨酸E、天冬氨酸D分别突变为天冬氨酸D、谷氨酸E(简称E412D+D416E)；
或/和将第469位的酪氨酸Y突变为苯丙氨酸F(简称Y469F)。
实施例2葡萄糖苷酶突变体的制备
通过反向PCR技术对Rh2-015进行定点突变，制备单双突变体库。
将来源于Terrabacter ginsenosidimutans的β-葡萄糖苷酶基因Rh2-015(基因序列如SEQ ID NO:1所示，编码的蛋白序列如SEQ ID NO:2所示)分别利用引物A(SEQ ID NO:3)和引物B(SEQ ID NO:4)通过PCR扩增技术获得PCR产物后经过酶切处理，同时插入到表达载体pET22b(+)的Nde I和EcoR I位点，得到重组质粒pET22b-Rh2-015。
按照实施例1所述的突变位置，在突变位置设计反向引物(突变引物序列如表2所示)，利用上下游突变引物扩增目的片段，并在引物上引入相应突变，以重组质粒pET22b-Rh2-015作为模板进行反向PCR，PCR产物经Dpn I酶消化模板处理后(一般使用的质粒DNA是从大肠杆菌宿主中抽提出来的，由于内源性的dam甲基化酶，该序列的腺嘌呤已被甲基化，因此可以被DpnⅠ切断。而用PCR等合成的DNA由于未被甲基化，因此不能被切断，消除假阳性)转化到大肠杆菌Rosetta(de3)，经过Amp的筛选后挑取菌落送测序。测定正确后，获得突变成功的重组菌，对突变成功的重组菌进行诱导表达，即可获得葡萄糖苷酶突变体。
上述反向PCR的体系为：
Figure PCTCN2017113837-appb-000004
Figure PCTCN2017113837-appb-000005
PCR反应程序为：
98℃、2min；98℃、10s，55-65℃、30s，30个循环；72℃、7min；72℃、10min。
表2突变位点的突变引物
Figure PCTCN2017113837-appb-000006
实施例3葡萄糖苷酶突变体酶活力的检测
(1)野生型β-葡萄糖苷酶重组大肠杆菌的诱导表达及其酶液的制备
将来源于Terrabacter ginsenosidimutans的β-葡萄糖苷酶基因Rh2-015(基因序列如SEQ ID NO:1所示，编码的蛋白序列如SEQ ID NO:2所示)分别利用引物A(SEQ ID NO:3)和引物B(SEQ ID NO:4)通过PCR扩增技术获得PCR产物后经过酶切处理，同时插入到表达载 体pET22b(+)的Nde I和EcoR I位点，得到表达重组质粒pET22b-Rh2-015。经测序验证后，将重组质粒转入大肠杆菌Rosetta(de3)。获得的重组大肠杆菌接种在小体积的LB培养基(含有100μg/mL的Amp)，30～37℃过夜培养后，以1～5％的接种量转接到1L体积的LB培养基中(含有100μg/mL的Amp)，在30～37℃继续培养OD600达到0.6～1.0加入终浓度为0.1mM～1mM的异丙基-β-D-硫代半乳糖苷(IPTG)，在20～37℃诱导表达10～20h后离心收集菌体。发酵菌体悬浮于4倍体积的50～100mM的磷酸缓冲液(pH 7.4)并超声波破胞20min，离心取上清得到Rh2-015的酶液。
(2)野生型β-葡萄糖苷酶酶活力测定方法
β-葡萄糖苷酶Rh2-015酶液反应以pNP-Glc为底物，在一个3mL的反应体系中加入1mL的150mM pNP-Glc，100μL的稀释酶液，在pH 7.0和37℃反应一定时间，0.5M碳酸钠5min终止反应。产物p-硝基酚在405nm处测定吸光值增加。1单位酶活定义为：每分钟产生1μM p-硝基酚为1U。
(3)β-葡萄糖苷酶突变体酶活力测定方法
β-葡萄糖苷酶突变体酶活力测定方法同野生型β-葡萄糖苷酶，将实施例2中突变成功的重组菌进行诱导表达，诱导表达方法同上述(1)，并利用与上述(2)相同的方法进行酶活力测定，将野生型和突变体的酶活力及其稳定性(保存80％以上的酶活力)进行比较。
比较结果如表3所示，从中可以看出，本发明突变后的葡萄糖苷酶活性大大提高，同时温度稳定范围和pH稳定范围不受影响，甚至稳定范围更广。
表3野生型和突变体β-葡萄糖苷酶的酶活力及稳定性
Figure PCTCN2017113837-appb-000007
实施例4葡萄糖苷酶突变体和野生型催化人参皂苷Rg3合成人参皂苷Rh2
方法：不同反应器中分别加入经诱导表达的野生型、本发明不同突变型的β-葡萄糖苷酶的酶液，各组分别缓慢加入等量的经助溶剂甲醇溶解的4g人参皂苷Rg3(边加入边搅拌)和纯水，使各组反应体系中Rg3的终浓度4％w/v，葡萄糖苷酶的终浓度为0.04％w/v。反应在温度40℃、300rpm和pH 8的条件下进行反应。反应过程中每隔一定时间取反应液用流动相稀释50～100倍，微孔过滤后进样进行液相分析，检测反应情况。液相检测使用Kinetex 2.6μm C18 100A为分析柱，乙腈和水为流动性，柱温为室温，检测波长为203nm，流速为1.0mL/min。反应10小时后，检测Rh2的产量。野生型和本发明突变型β-葡萄糖苷酶的反应条件完全相同。
结果：检测结果如表4所示，葡萄糖苷酶及其突变体酶活对底物Rg3转化生成Rh2的转化率达95.89％以上，其中Q240E生成Rh2转化率达99.68％。
表4野生型和突变体β-葡萄糖苷酶生成Rh2的效果
Figure PCTCN2017113837-appb-000008
实施例5葡萄糖苷酶突变体催化人参皂苷Rg3合成人参皂苷Rh2
方法：向反应釜中加入16ml 0.1M磷酸盐缓冲液(pH7.5)同时搅拌，之后加入葡萄糖苷酶突变体(Q240E)0.04g，搅拌，另外，称量4g底物Rg3投入烧杯中，加入20ml DMSO，400rpm磁力搅拌。待底物溶解后，缓慢加入反应釜中(边加入边搅拌)，加纯水至100mL，使反应中的底物与酶充分混合，50℃水浴，氢氧化钠调节pH值为7-8。反应6小时后，检测Rh2的产量。
结果：实验结果显示，反应6小时后经纯化，得Rh2产量3.16g，可见突变型葡萄糖苷 酶对底物的转化率为99.68％。
实施例6葡萄糖苷酶突变体催化人参皂苷Rg3合成人参皂苷Rh2
方法：在反应器中加入突变型β-葡萄糖苷酶(P135A)和0.1M磷酸盐缓冲液(pH7.5)，再缓慢加入经助溶剂甲醇溶解的人参皂苷Rg3(边加入边搅拌)，使Rg3的终浓度4％w/v，葡萄糖苷酶的浓度为0.12％w/v。反应在温度25℃、400rpm和pH 9.5的条件下进行反应。反应12小时后，检测Rh2的产量，突变型葡萄糖苷酶对底物的转化率为99.05％。
实施例7葡萄糖苷酶突变体催化人参皂苷Rg3合成人参皂苷Rh2
方法：在反应器中加入突变型β-葡萄糖苷酶(E412D+D416E)和0.12M磷酸盐缓冲液(pH7.8)，再缓慢加入经助溶剂乙醇溶解的人参皂苷Rg3(边加入边搅拌)，使Rg3的终浓度1％w/v，葡萄糖苷酶的浓度为0.06％w/v。反应在温度50℃、200rpm和pH 6.5的条件下进行反应。反应4小时后，检测Rh2的产量，突变型葡萄糖苷酶对底物的转化率为99.37％。
上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。

Claims (10)

  1. 一种酶催化合成人参皂苷Rh2的方法，其特征在于：以人参皂苷Rg3为底物，在葡萄糖苷酶的催化下，反应生成人参皂苷Rh2；所述葡萄糖苷酶来源于Terrabacter ginsenosidimutans；催化反应的温度为25～50℃；
    催化反应体系中人参皂苷Rg3的浓度为1％～5％w/v，葡萄糖苷酶用量为底物人参皂苷Rg3重量的0.01～0.06倍，余量为水或磷酸盐缓冲液，以及用于溶解人参皂苷Rg3的助溶剂；
    所述催化反应体系的pH值为6.5～9.5。
  2. 根据权利要求1所述的方法，其特征在于，所述葡萄糖苷酶的氨基酸序列如SEQ ID NO 2所示，或者为SEQ ID NO 2所示氨基酸序列经以下至少一种突变情况所得：
    将第71和72位的谷氨酸E、缬氨酸V分别突变为苏氨酸T、谷氨酸E；
    将第92位的谷氨酰胺Q突变为组氨酸H；
    将第109和114位的丝氨酸S、脯氨酸P分别突变为丙氨酸A、甘氨酸G；
    将第135位的脯氨酸P突变为丙氨酸A；
    将第168位的苯丙氨酸F突变为酪氨酸Y；
    将第240位的谷氨酰胺Q突变为谷氨酸E；
    将第344位的赖氨酸K突变为精氨酸R；
    将第412和416位的谷氨酸E、天冬氨酸D分别突变为天冬氨酸D、谷氨酸E；
    将第469位的酪氨酸Y突变为苯丙氨酸F。
  3. 根据权利要求1所述的方法，其特征在于，所述人参皂苷Rg3的助溶剂选自甲醇、乙醇、1％～20％v/v的DMSO中的至少一种。
  4. 根据权利要求1所述的方法，其特征在于，所述催化反应的时间为4～12h。
  5. 根据权利要求1所述的方法，其特征在于，所述磷酸盐缓冲液为pH 7.2～7.8的0.08～0.12M磷酸盐缓冲液。
  6. 根据权利要求1所述的方法，其特征在于，所述人参皂苷Rg3需先溶解于所述助溶剂中，再缓慢加入所述葡萄糖苷酶中，边加入边搅拌。
  7. 葡萄糖苷酶突变体，其特征在于，由SEQ ID NO 2所示氨基酸序列经以下至少一种突变情况所得：
    将第71和72位的谷氨酸E、缬氨酸V分别突变为苏氨酸T、谷氨酸E；
    将第92位的谷氨酰胺Q突变为组氨酸H；
    将第109和114位的丝氨酸S、脯氨酸P分别突变为丙氨酸A、甘氨酸G；
    将第135位的脯氨酸P突变为丙氨酸A；
    将第168位的苯丙氨酸F突变为酪氨酸Y；
    将第240位的谷氨酰胺Q突变为谷氨酸E；
    将第344位的赖氨酸K突变为精氨酸R；
    将第412和416位的谷氨酸E、天冬氨酸D分别突变为天冬氨酸D、谷氨酸E；
    将第469位的酪氨酸Y突变为苯丙氨酸F。
  8. 根据权利要求7所述的葡萄糖苷酶突变体，其特征在于，由SEQ ID NO 1所示核苷酸序列经以下至少一种突变情况所得：
    将第211～216位的碱基序列GAGGTG突变为ACGGAG；
    将第274～276位的碱基序列CAG突变为CAC；
    将第325～327位和340～342位的碱基序列AGC、CCG分别突变为GCC、GGT；
    将第403～405位的碱基序列CCG突变为GGC；
    将第502～504位的碱基序列TTC突变为TAC；
    将第718～720位的碱基序列CAA突变为GAA；
    将第1030～1032位的碱基序列CGG突变为AAG；
    将第1234～1236位和1246～1248位的碱基序列GAG、GAT分别突变为GAC、GAG；
    将第1405～1407位的碱基序列TAT突变为TTT。
  9. 权利要求7～8任一项所述的葡萄糖苷酶突变体在制备人参皂苷Rh2中的应用。
  10. 根据权利要求9所述的应用，其特征在于，以人参皂苷Rg3为底物。
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