WO2018072213A1 - 一种酶法制备瑞鲍迪甙n的方法 - Google Patents

一种酶法制备瑞鲍迪甙n的方法 Download PDF

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WO2018072213A1
WO2018072213A1 PCT/CN2016/102948 CN2016102948W WO2018072213A1 WO 2018072213 A1 WO2018072213 A1 WO 2018072213A1 CN 2016102948 W CN2016102948 W CN 2016102948W WO 2018072213 A1 WO2018072213 A1 WO 2018072213A1
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ugt
sequence
udp
amino acid
rebaudioside
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PCT/CN2016/102948
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English (en)
French (fr)
Inventor
陶军华
李国庆
王文霞
郑雷雷
朱春磊
梁晓亮
陈车翘
李托马斯
芬纳蒂⋅麦克
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苏州汉酶生物技术有限公司
百事可乐公司
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Priority to AU2016427130A priority Critical patent/AU2016427130B2/en
Application filed by 苏州汉酶生物技术有限公司, 百事可乐公司 filed Critical 苏州汉酶生物技术有限公司
Priority to EP16919469.3A priority patent/EP3530745A4/en
Priority to CA3041152A priority patent/CA3041152A1/en
Priority to BR112019008048A priority patent/BR112019008048A2/pt
Priority to RU2019112909A priority patent/RU2737118C2/ru
Priority to CN202310412340.4A priority patent/CN116515929A/zh
Priority to MX2019004628A priority patent/MX2019004628A/es
Priority to PCT/CN2016/102948 priority patent/WO2018072213A1/zh
Priority to CN201680089986.1A priority patent/CN109890973B/zh
Priority to JP2019520995A priority patent/JP6972123B2/ja
Priority to US16/343,335 priority patent/US11352653B2/en
Publication of WO2018072213A1 publication Critical patent/WO2018072213A1/zh
Priority to US17/663,631 priority patent/US11976313B2/en

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L27/00—Spices; Flavouring agents or condiments; Artificial sweetening agents; Table salts; Dietetic salt substitutes; Preparation or treatment thereof
    • A23L27/30—Artificial sweetening agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1048—Glycosyltransferases (2.4)
    • C12N9/1051—Hexosyltransferases (2.4.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/04—Polysaccharides, i.e. compounds containing more than five saccharide radicals attached to each other by glycosidic bonds
    • C12P19/08—Dextran
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/44—Preparation of O-glycosides, e.g. glucosides
    • C12P19/56—Preparation of O-glycosides, e.g. glucosides having an oxygen atom of the saccharide radical directly bound to a condensed ring system having three or more carbocyclic rings, e.g. daunomycin, adriamycin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y204/00—Glycosyltransferases (2.4)
    • C12Y204/01—Hexosyltransferases (2.4.1)
    • C12Y204/01017—Glucuronosyltransferase (2.4.1.17)

Definitions

  • the invention relates to a preparation method of rebaudioside N, in particular to a biological preparation method of rebaudioside N.
  • Sweeteners are a class of food additives that are widely used in the production of foods such as beverages and confectionery. They can be added during the production of foods or appropriately diluted as a substitute for sucrose during home baking. Sweeteners include natural sweeteners and artificial sweeteners, the former including sucrose, high fructose corn syrup, honey, and the like, the latter including aspartame, saccharin, and the like.
  • Stevia is a natural sweetener extracted from plant stevia and is currently widely used in foods and beverages. The extract of Stevia has a variety of steviosides including rebaudioside, and the different batch components of naturally extracted stevioside vary greatly, requiring subsequent purification.
  • the ratio of rebaudioside in stevia leaves is less than 1.5%. It is extremely difficult to obtain high-purity Rebaudioside N by traditional extraction methods, which limits the in-depth study of Rebaudio N and hinders Its commercial application.
  • the technical problem to be solved by the present invention is to overcome the deficiencies of the prior art and provide a method for preparing rebaudioside N by enzymatic method, which can produce high purity rebaudioside at a low cost and in a short cycle. N product.
  • the present invention adopts the following technical solutions:
  • the glycosyl donor comprises one or two of a glucose-based donor, a rhamnosyl donor, UDP-glucose or UDP consisting of sucrose, sucrose synthase and UDP.
  • a rhamnose donor is UDP-rhamnose.
  • a UDP-glucose regeneration system composed of sucrose, sucrose synthase, and UDP is preferred, and UDP glucose is expensive, and the cost can be greatly reduced by the UDP-glucose regeneration system.
  • the UDP-glycosyltransferase ie uridine diphosphate glucosyltransferase, abbreviated UGT, is known
  • UGT uridine diphosphate glucosyltransferase
  • It includes one or two of UGT-A from Stevia rebaudiana and UGT-B from rice (Oryza sativa).
  • the UDP-glycosyltransferase comprises UGT-A from Stevia and UGT-B from rice; the UDP-glycosyltransferase is added to the reaction system in two steps, and the first step is to add UGT- B, the second step is to join UGT-A.
  • the amino acid sequence of UGT-A has at least 60% identity with sequence 2 shown in the sequence listing; and/or the amino acid sequence of UGT-B has at least 60% with sequence 4 shown in the sequence listing. Consistency.
  • the amino acid sequence of the UGT-A is at least 70% identical to the sequence 2 shown in the sequence listing; and/or the amino acid sequence of the UGT-B and the sequence 4 shown in the sequence listing. Has at least 70% consistency.
  • amino acid sequence of the UGT-A has at least 80% identity with the sequence 2 shown in the sequence listing; and/or, the amino acid sequence of the UGT-B has the sequence 4 shown in the sequence listing. At least 80% consistency.
  • amino acid sequence of the UGT-A has at least 90% identity with the sequence 2 shown in the sequence listing; and/or the amino acid sequence of the UGT-B and the sequence 4 shown in the sequence listing Has at least 90% consistency.
  • amino acid sequence of UGT-A is identical to sequence 2 shown in the sequence listing; and/or the amino acid sequence of UGT-B is identical to sequence 4 shown in the sequence listing. .
  • the UDP-glycosyltransferase is UGT-A from Stevia, the amino acid sequence of the UGT-A having at least 60% identity to the sequence 2 shown in the Sequence Listing.
  • amino acid sequence of UGT-A is at least 70% identical to sequence 2 shown in the Sequence Listing.
  • amino acid sequence of UGT-A has at least 80% identity to sequence 2 shown in the sequence listing.
  • amino acid sequence of UGT-A has at least 90% identity to sequence 2 shown in the sequence listing.
  • amino acid sequence of UGT-A is identical to sequence 2 shown in the Sequence Listing.
  • the reaction can be carried out in an aqueous phase system at a temperature of 4 to 50 ° C and a pH of 5.0 to 9.0.
  • the reaction is carried out in an aqueous phase system at a temperature of 35-45 ° C and a pH of 7.5-8.5.
  • reaction is carried out in a phosphate buffer solution.
  • the reaction system contains a recombinant cell of UDP-glycosyltransferase and a cell permeable agent.
  • the cell penetrating agent is toluene, and the volume ratio of toluene in the reaction system is 1-3%.
  • Rebaudio N product that meets the requirements for use can be obtained by purification treatment.
  • a specific purification method is post-treatment including separation of the resin, according to which the rebaudioside having a purity of up to 95% can be obtained. N product.
  • the recombinant cell is a microbial cell. More preferably, the microorganism is Escherichia coli, Saccharomyces cerevisiae or Pichia pastoris.
  • the first step reaction substrate is rebaudioside A
  • the UDP-glycosyltransferase is UGT-B from rice
  • the amino acid sequence of UGT-B from rice has at least 80 % consistency
  • the second step reaction substrate is a reaction solution containing the first step reaction product Rebaudioside J
  • the UDP-glycosyltransferase is UGT-A from Stevia
  • the amino acid sequence of UGT-A from Stevia has at least 80% consistency.
  • the substrate is Rebaudioside J
  • the UDP-glycosyltransferase is UGT-A from Stevia
  • the amino acid sequence of UGT-A from Stevia has at least 80% consistency.
  • the present invention has the following advantages compared with the prior art:
  • the method for preparing rebaudioside N by the enzymatic method provided by the invention has important application value. Since the growth rate of microorganisms is much faster than that of plants, the method can greatly reduce the production cost, shorten the production cycle, and greatly improve the competitiveness of the product. In addition, the stevia content in plants is low, and there are many different structures of stevioside, it is difficult to extract a relatively pure product, compared with the technique of extracting rebaudioside N from stevia leaves, the invention is The enzymatic synthesis method can provide higher purity products, and will certainly promote the research and application of the new stevioside rebaudioside N.
  • the invention mainly provides two routes for synthesizing Rebaudio N:
  • the UGT-A or UGT-B used in the present invention may be present in the form of an enzyme lyophilized powder or present in recombinant cells.
  • UGT-A or UGT-B can be obtained as follows:
  • Recombinant Escherichia coli (or other microbial) expression strains of UGT-A or UGT-B were obtained by molecular cloning technology and genetic engineering technology, and then recombinant Escherichia coli was fermented to prepare UGT-A or UGT.
  • -B recombinant cells, or lyophilized powder of UGT-A or UGT-B prepared from the above recombinant cells.
  • the recombinant plasmid was transformed into Escherichia coli BL21 (DE3), and the expression of the target protein was induced by IPTG to obtain a recombinant Escherichia coli expression strain of UGT-A or UGT-B.
  • the recombinant Escherichia coli expression strain containing UGT-A or UGT-B was inoculated into 4 ml of liquid LB medium at a ratio of 1%, shake culture (200 rpm) overnight at 37 ° C, and the overnight culture was inoculated at 1%. Transfer to 50 ml of liquid LB medium, shake culture (200 rpm) at 37 ° C until the OD600 value reached 0.6-0.8, and incubate at a final concentration of 0.4 mM IPTG at 20 ° C overnight.
  • the cells were collected by centrifugation (8,000 rpm, 10 min), and the cells were resuspended in 5 ml of 2 mmol/L phosphate buffer (pH 7.0) to obtain the recombinant cells, and the cells were further ultrasonically disrupted in an ice bath, and the disrupted solution was centrifuged ( 8,000 rpm, 10 min), the supernatant was collected and lyophilized for 24 h to obtain the lyophilized powder.
  • phosphate buffer pH 7.0
  • Example 1 Preparation of recombinant E. coli cells containing UGT-A
  • the UGT strain was inoculated into 4 ml of liquid LB medium at a ratio of 1%, shake culture (200 rpm) overnight at 37 ° C, and the overnight culture was transferred to 50 ml of liquid LB medium at a 1% inoculum and shake cultured at 37 ° C (200 rpm).
  • the OD 600 value was 0.6-0.8, and the final concentration of 0.4 mM IPTG was added to shake culture at 20 ° C overnight.
  • the cells were collected by centrifugation (8,000 rpm, 10 min), and the cells were resuspended in 5 ml of 2 mmol/L phosphate buffer (pH 7.0) to obtain recombinant cells containing UGT-A for catalysis.
  • the recombinant cells of UGT-A prepared in Example 1 were ultrasonically disrupted in an ice bath, and the disrupted solution was centrifuged (8,000 rpm, 10 min), and the supernatant was collected and lyophilized for 24 hours to obtain a lyophilized powder of UGT-A.
  • the UGT strain was inoculated into 4 ml of liquid LB medium at a ratio of 1%, shake culture (200 rpm) overnight at 37 ° C, and the overnight culture was transferred to 50 ml of liquid LB medium at a 1% inoculum and shake cultured at 37 ° C (200 rpm).
  • the OD 600 value was 0.6-0.8, and the final concentration of 0.4 mM IPTG was added to shake culture at 20 ° C overnight.
  • the cells were collected by centrifugation (8,000 rpm, 10 min), and the cells were resuspended in 5 ml of 2 mmol/L phosphate buffer (pH 7.0) to obtain recombinant cells containing UGT-B for catalysis.
  • the recombinant cells of UGT-B prepared in Example 3 were ultrasonically disrupted in an ice bath, and the disrupted solution was centrifuged (8,000 rpm, 10 min), and the supernatant was collected and lyophilized for 24 hours to obtain a lyophilized powder of UGT-B.
  • the UGT-A lyophilized powder prepared according to the method of Example 2 was used to catalyze the synthesis of Rebaudioside N.
  • sucrose, a sucrose synthase derived from Arabidopsis thaliana (hereinafter referred to as AtSUS1), and a UDP-glucose regeneration system composed of UDP were used as the glucosyl donor.
  • the conversion rate of rebaudioside J is over 90%. After purification by silica gel resin, crystallization and the like, it was purified to obtain Rebaudioside N 0.61 g, and the purity was more than 95%.
  • the UGT-A lyophilized powder prepared according to the method of Example 2 and the UGT-B lyophilized powder prepared according to the method of Example 4 were used for catalytic synthesis of Rebaudioside N.
  • the first step reaction 1L 0.05mol/L phosphate buffer (pH 8.0), 2g UDP rhamnose, 1g rebaudioside A, UGT-B lyophilized powder 10g are uniformly added to the reaction system and then placed in 40 The reaction was stirred for 24 h at 300 rpm in a water bath.
  • the second step reaction after the first step, the reaction solution is boiled for 10 min, the pH is adjusted to 8.0, 0.5 g UDP, 5 g of sucrose, 10 g of UGT-A lyophilized powder, and 3 g of AtSUS1 lyophilized powder are uniformly mixed and placed. The reaction was stirred at 300 rpm for 24 h in a 40 ° C water bath.
  • Example 7 Synthesis of Rebaudi under the catalysis of recombinant cells containing UDP-glycosyltransferase using Rebaudioside J as substrate ⁇ N
  • Example 8 Synthesis of Rebaudi under the catalysis of recombinant cells containing UDP-glycosyltransferase using Rebaudioside A as substrate ⁇ N
  • First step reaction 1L 0.05mol/L phosphate buffer (pH 8.0), 2g UDP rhamnose, 1g rebaudioside A, UTG-B whole cell 40g were mixed and evenly placed in the reaction system and then placed at 40 °C. The reaction was stirred for 24 h at 300 rpm in a water bath.
  • the second step reaction After the first step, the reaction solution is boiled for 10 minutes, the pH is adjusted to 8.0, 0.5 g UDP, 5 g of sucrose, 40 g of UGT-A whole cells, and 10 g of AtSUS1 whole cells are uniformly mixed and placed at 40 ° C. The reaction was stirred for 24 h at 300 rpm in a water bath.

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Abstract

提供一种酶法制备瑞鲍迪甙N的方法，该方法以瑞鲍迪甙A或瑞鲍迪甙J为底物，使所述底物在糖基供体存在下，在UDP-糖基转移酶和/或含有UDP-糖基转移酶的重组细胞的催化下反应生成瑞鲍迪甙N。

Description

一种酶法制备瑞鲍迪甙N的方法 技术领域
本发明涉及一种瑞鲍迪甙N的制备方法，特别涉及一种瑞鲍迪甙N的生物制备方法。
背景技术
甜味剂是一类广泛应用于饮料及糖果等食品生产的食品添加剂，其既可以在食品的生产过程中添加，也可以在家庭烘焙时经过适当稀释作为蔗糖的替代品使用。甜味剂包括天然甜味剂和人工甜味剂，前者包括蔗糖、高果糖玉米糖浆、蜜糖等，后者包括阿斯巴甜、糖精等。甜菊糖是一类从植物甜菊中提取出来的天然甜味剂，目前已被广泛用在食品及饮料中。甜菊的提取物中具有包含瑞鲍迪甙在内的多种甜菊糖，天然提取的甜菊糖不同的批次成分差异较大，需要后续的提纯。
瑞鲍迪甙N在甜菊叶中甜菊糖的比例不超过1.5％，用传统提取的方法得到高纯度的瑞鲍迪甙N极其困难，限制了对瑞鲍迪甙N的深入研究，也阻碍了其商业化的应用。
发明内容
本发明所要解决的技术问题是克服现有技术的不足，提供一种酶法制备瑞鲍迪甙N的方法，该方法可以较低的成本、较短的周期生产出高纯度的瑞鲍迪甙N产品。
为解决以上技术问题，本发明采取如下技术方案：
一种酶法制备瑞鲍迪甙N的方法，以瑞鲍迪甙A为底物，在糖基供体存在下，在含有UDP-糖基转移酶的重组细胞和/或其制备的UDP-糖基转移酶的催化下，反应生成瑞鲍迪甙N。
一种酶法制备瑞鲍迪甙N的方法，以瑞鲍迪甙J为底物，在糖基供体存在下，在含有UDP-糖基转移酶的重组细胞和/或其制备的UDP-糖基转移酶的催化下，反应生成瑞鲍迪甙N。
优选地，所述糖基供体包括葡萄糖基供体、鼠李糖基供体中一种或二种，所述葡萄糖基供体为UDP-葡萄糖或由蔗糖、蔗糖合成酶及UDP组成的UDP-葡萄糖再生体系(2007，FEBS Letters，581，2562-2566)，所述鼠李糖基供体为UDP-鼠李糖。其中，优选由蔗糖、蔗糖合成酶和UDP组成的UDP-葡萄糖再生体系，UDP葡萄糖价格较高，采用由UDP-葡萄糖再生体系可以大幅度降低成本。
优选地，所述UDP-糖基转移酶(即尿苷二磷酸葡萄糖基转移酶，简称UGT，是已知的) 包括来自甜菊(Stevia rebaudiana)的UGT-A、来自水稻(Oryza sativa)的UGT-B中的一种或二种。
优选地，所述UDP-糖基转移酶包括来自甜菊的UGT-A和来自水稻的UGT-B；所述UDP-糖基转移酶分两步加入到反应体系中，第一步先加入UGT-B，第二步再加入UGT-A。所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少60％的一致性；和/或，所述UGT-B的氨基酸序列与序列表中所示的序列4具有至少60％的一致性。
更优选地，所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少70％的一致性；和/或，所述UGT-B的氨基酸序列与序列表中所示的序列4具有至少70％的一致性。
进一步地，所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少80％的一致性；和/或，所述UGT-B的氨基酸序列与序列表中所示的序列4具有至少80％的一致性。
更进一步地，所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少90％的一致性；和/或，所述UGT-B的氨基酸序列与序列表中所示的序列4具有至少90％的一致性。
在某些具体实例中，所述UGT-A的氨基酸序列与序列表中所示的序列2完全一致；和/或，所述UGT-B的氨基酸序列与序列表中所示的序列4完全一致。
优选地，所述UDP-糖基转移酶为来自甜菊的UGT-A，所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少60％的一致性。
更优选地，所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少70％的一致性。
进一步地，所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少80％的一致性。
更进一步地，所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少90％的一致性。
在某些具体实例中，所述UGT-A的氨基酸序列与序列表中所示的序列2完全一致。
根据本发明，可以使所述反应在温度4-50℃以及pH 5.0-9.0的水相体系中进行。优选地，反应在35-45℃温度以及pH7.5-8.5的水相体系中进行。
更优选地，反应在磷酸缓冲溶液中进行。
更优选地，反应体系含有UDP-糖基转移酶的重组细胞和细胞通透剂。进一步地，所述细胞通透剂为甲苯，甲苯在反应体系中的体积比浓度为1-3％。
更优选地，将反应所用全部原料加入到反应釜中，混合均匀后置于设定温度下，搅拌反应。反应完毕后，通过提纯处理即可获得达到使用要求的瑞鲍迪甙N产品。一个具体的提纯方方法是经包括树脂分离在内的后处理，按照该提纯方法，可获得纯度高达95％的瑞鲍迪甙 N产品。
优选地，所述重组细胞为微生物细胞。更优选地，所述微生物为大肠埃希氏杆菌、酿酒酵母或毕赤酵母。
根据本发明的一个具体方面：第一步反应底物为瑞鲍迪甙A，UDP-糖基转移酶为来自水稻的UGT-B，来自水稻的UGT-B的氨基酸序列与序列4具有至少80％的一致性。第二步反应底物为含第一步反应产物瑞鲍迪甙J的反应液，UDP-糖基转移酶为来自甜菊的UGT-A，来自甜菊的UGT-A的氨基酸序列与序列2具有至少80％的一致性。
根据本发明的又一具体方面：底物为瑞鲍迪甙J，UDP-糖基转移酶为来自甜菊的UGT-A，该来自甜菊的UGT-A的氨基酸序列与序列2具有至少80％的一致性。
由于以上技术方案的实施，本发明与已有技术相比具有如下优势：
本发明提供的酶法制备瑞鲍迪甙N的方法具有重要的应用价值。由于微生物生长速度远远快于植物，采取所述方法可以较大幅度的降低生产成本，缩短生产周期，极大的提高产品的竞争力。此外，植物中的甜菊糖含量低，且有较多不同结构的甜菊糖，很难提取较纯的产品，与已有从甜菊叶中提取瑞鲍迪甙N的技术相比，采用本发明的酶法合成方法能够提供纯度更高的产品，必将推动对新型甜菊糖苷瑞鲍迪甙N的研究和应用。
具体实施方式
瑞鲍迪甙J、瑞鲍迪甙N两者的结构式分别参见式I和II。
Figure PCTCN2016102948-appb-000001
本发明主要提供两条合成瑞鲍迪甙N的路线：
路线一：
Figure PCTCN2016102948-appb-000002
路线二：
Figure PCTCN2016102948-appb-000003
本发明所用的UGT-A或UGT-B可以酶冻干粉形式存在或存在于重组细胞中。
UGT-A或UGT-B的获得方法如下：
利用分子克隆技术、基因工程技术获得UGT-A或UGT-B的重组大肠埃希氏杆菌(或其它微生物菌)表达菌株，然后将重组大肠埃希氏杆菌发酵，制备得到含有UGT-A或UGT-B的重组细胞，或由上述重组细胞制备得到UGT-A或UGT-B的冻干粉。
本发明所述的分子克隆技术和基因工程技术均是已知的。分子克隆技术可参见《分子克隆实验指南》第三版(J.沙姆布鲁克著，2005)。
采用基因工程技术构建本发明重组菌株的表达步骤如下：
(1)(根据序列表中所示的序列1及序列2，或根据序列3及序列4)基因合成所需的基因片段，连入pUC57载体，两端分别加上NdeI和BamHI酶切位点；
(2)通过双酶切、连接，将各基因片段插入表达载体pET30a相应的酶切位点中，使各基因置于T7启动子的控制之下；
(3)将重组质粒转化进入大肠埃希氏杆菌BL21(DE3)中，利用IPTG诱导目的蛋白表达，得到UGT-A或UGT-B的重组大肠埃希氏杆菌表达菌株。
利用含有UGT-A或UGT-B的重组大肠埃希氏杆菌表达菌株制备含有UGT-A或UGT-B的重组细胞、UGT-A或UGT-B的冻干粉的步骤如下：
以1％比例将含有UGT-A或UGT-B的重组大肠埃希氏杆菌表达菌株接种到4ml液体LB培养基中，，37℃振荡培养(200rpm)过夜，取过夜培养物以1％接种量转接于50ml液体LB培养基，37℃振荡培养(200rpm)至OD600值达到0.6-0.8，加入终浓度0.4mM IPTG于20℃振荡培养过夜。诱导结束后离心收集细胞(8,000rpm，10min)，用5ml 2mmol/L磷酸缓冲液(pH7.0)重悬细胞，获得所述重组细胞，进一步于冰浴中超声波破碎细胞，将破碎液离心(8,000rpm，10min)，收集上清液冻干24h，获得所述冻干粉。
下面结合具体的实施例对本发明作更为详细的描述。
实施例1：制备含UGT-A的重组大肠杆菌细胞
根据序列表中所示的序列1及序列2，基因合成UGT-A基因片段，两端分别加上NdeI和BamHI酶切位点，连入pUC57载体(苏州金唯智生物技术有限公司生产)。将UGT基因片段用限制性内切酶NdeI和BamHI酶切，回收纯化片段，加入T4连接酶将片段连入pET30a对应酶切位点，转化BL21(DE3)菌株。
以1％比例将UGT菌种接种到4ml液体LB培养基，37℃振荡培养(200rpm)过夜，取过夜培养物以1％接种量转接于50ml液体LB培养基，37℃振荡培养(200rpm)至OD600值达到0.6-0.8，加入终浓度0.4mM IPTG于20℃振荡培养过夜。诱导结束后离心收集细胞(8,000rpm，10min)，用5ml 2mmol/L磷酸缓冲液(pH7.0)重悬细胞，获得含UGT-A的重组细胞用于催化。
实施例2：制备UGT-A冻干粉
将实施例1中制得的UGT-A的重组细胞于冰浴中超声波破碎细胞，将破碎液离心(8,000rpm，10min)，收集上清液冻干24h，获得UGT-A的冻干粉。
实施例3：制备含UGT-B的重组大肠杆菌细胞
根据序列3及序列4，基因合成UGT-B基因片段，两端分别加上NdeI和BamHI酶切位点，连入pUC57载体(苏州金唯智生物技术有限公司生产)。将UGT基因片段用限制性内切酶NdeI和BamHI酶切，回收纯化片段，加入T4连接酶将片段连入pET30a对应酶切位点，转化BL21(DE3)菌株。
以1％比例将UGT菌种接种到4ml液体LB培养基，37℃振荡培养(200rpm)过夜，取过夜培养物以1％接种量转接于50ml液体LB培养基，37℃振荡培养(200rpm)至OD600值达到0.6-0.8，加入终浓度0.4mM IPTG于20℃振荡培养过夜。诱导结束后离心收集细胞(8,000rpm，10min)，用5ml 2mmol/L磷酸缓冲液(pH7.0)重悬细胞，获得含UGT-B的重组细胞用于催化。
实施例4：制备UGT-B冻干粉
将实施例3中制得的UGT-B的重组细胞于冰浴中超声波破碎细胞，将破碎液离心(8,000rpm，10min)，收集上清液冻干24h，获得UGT-B的冻干粉。
实施例5：以瑞鲍迪甙J为底物在UDP-糖基转移酶的催化下合成瑞鲍迪甙N(路线一)
在本实施例中，按照实施例2方法制备的UGT-A冻干粉被用于催化合成瑞鲍迪甙N。在本实施例中，使用蔗糖、来自拟南芥(Arabidopsis thaliana)的蔗糖合成酶(以下简称AtSUS1)以及UDP组成的UDP-葡糖糖再生体系作为葡糖糖基供体。
在反应体系中依次加入1L 0.05mol/L磷酸缓冲液(pH8.0)，0.5g UDP，1g瑞鲍迪甙J，蔗糖5g，UGT-A冻干粉10g，AtSUS1冻干粉0.5g混合均匀后置于40℃水浴，300rpm搅拌反应24h。反应结束后，取500μl反应液加入等体积无水甲醇混匀，8,000rpm离心10min 取上清液过滤膜后用高效液相色谱检测(色谱条件：色谱柱：Agilent eclipse SB-C18 4.6×150mm；检测波长：210nm；流动相：0.1％甲酸水溶液∶乙腈＝65％∶35％；流速：1.0mL/min；柱温：30℃)。瑞鲍迪甙J的转化率为90％以上。经硅胶树脂分离、结晶等后处理纯化后得到瑞鲍迪甙N 0.61g，纯度大于95％。
实施例6：以瑞鲍迪甙A为底物存UDP-糖基转移酶的催化下合成瑞鲍迪甙N(路线二)
在本实施例中，按照实施例2方法制备的UGT-A冻干粉和按照实施例4方法制备的UGT-B冻干粉被用于催化合成瑞鲍迪甙N。
第一步反应：在反应体系中依次加入1L 0.05mol/L磷酸缓冲液(pH 8.0)，2g UDP鼠李糖，1g瑞鲍迪甙A，UGT-B冻干粉10g混合均匀后置于40℃水浴，300rpm搅拌反应24h。第二步反应：第一步反应结束后将反应液煮沸10min，将pH值调至8.0，加入0.5g UDP，蔗糖5g，UGT-A冻干粉10g，AtSUS1冻干粉3g混合均匀后置于40℃水浴，300rpm搅拌反应24h。反应结束后，取500μl反应液加入等体积无水甲醇混匀，8,000rpm离心10min取上清液过滤膜后用高效液相色谱检测(色谱条件：色谱柱：Agilent eclipse SB-C18 4.6×150mm；检测波长：210nm；流动相：0.1％甲酸水溶液∶乙腈＝65％∶35％；流速：1.0mL/min；柱温：30℃)。瑞鲍迪甙A的转化率为90％以上。经硅胶树脂分离、结晶等后处理纯化后得到瑞鲍迪甙N 0.58g，纯度大于95％。
实施例7：以瑞鲍迪甙J为底物在含有UDP-糖基转移酶的重组细胞的催化下合成瑞鲍迪 甙N
在本实施例中，按照实施例1方法制备的含UGT-A的重组细胞用于催化合成瑞鲍迪甙N。
在反应体系中依次加入1L 0.05mol/L磷酸缓冲液(pH8.0)，0.5g UDP，1g瑞鲍迪甙J，蔗糖5g，UGT-A全细胞40g，AtSUS1全细胞10g混合均匀后置于40℃水浴，300rpm搅拌反应24h。反应结束后，取500μl反应液加入等体积无水甲醇混匀，8,000rpm离心10min取上清液过滤膜后用高效液相色谱检测(色谱条件：色谱柱：Agilent eclipse SB-C18 4.6×150mm；检测波长：210nm；流动相：0.1％甲酸水溶液∶乙腈＝65％∶35％；流速：1.0mL/min；柱温：30℃)。瑞鲍迪甙J的转化率为90％以上。经硅胶树脂分离、结晶等后处理纯化后得到瑞鲍迪甙N 0.54g，纯度大于95％。
实施例8：以瑞鲍迪甙A为底物在含有UDP-糖基转移酶的重组细胞的催化下合成瑞鲍迪 甙N
第一步反应：在反应体系中依次加入1L 0.05mol/L磷酸缓冲液(pH 8.0)，2g UDP鼠李糖，1g瑞鲍迪甙A，UGT-B全细胞40g混合均匀后置于40℃水浴，300rpm搅拌反应24h。第二步反应：第一步反应结束后将反应液煮沸10min，将pH值调至8.0，加入0.5g UDP，蔗糖5g，UGT-A全细胞40g，AtSUS1全细胞10g混合均匀后置于40℃水浴，300rpm搅拌反应24h。反应结束后，取500μl反应液加入等体积无水甲醇混匀，8,000rpm离心10min取上清液过滤膜后用高效液相色谱检测(色谱条件：色谱柱：Agilent eclipse SB-C18 4.6×150mm；检测波长：210nm；流动相：0.1％甲酸水溶液∶乙腈＝65％∶35％；流速：1.0mL/min；柱温：30℃)。瑞鲍迪甙A的转化率为90％以上。经硅胶树脂分离、结晶等后处理纯化后得到瑞鲍迪甙N 0.53g，纯度大于95％。
上述实施例只为说明本发明的技术构思及特点，其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施，并不能以此限制本发明的保护范围，凡根据本发明精神实质所作的等效变化或修饰，都应涵盖在本发明的保护范围之内。

Claims (20)

  1. 一种酶法制备瑞鲍迪甙N的方法，其特征在于：以瑞鲍迪甙A为底物，在糖基供体存在下，在含有UDP-糖基转移酶的重组细胞和/或其制备的UDP-糖基转移酶的催化下，反应生成瑞鲍迪甙N。
  2. 一种酶法制备瑞鲍迪甙N的方法，其特征在于：以瑞鲍迪甙J为底物，在糖基供体存在下，在含有UDP-糖基转移酶的重组细胞和/或其制备的UDP-糖基转移酶的催化下，反应生成瑞鲍迪甙N。
  3. 根据权利要求1或2所述的方法，其特征在于：所述糖基供体包括葡萄糖基供体、鼠李糖基供体中一种或二种，所述葡萄糖基供体为UDP-葡萄糖或由蔗糖、蔗糖合成酶及UDP组成的UDP-葡萄糖再生体系，所述鼠李糖基供体为UDP-鼠李糖。
  4. 根据权利要求1或2所述的方法，其特征在于：所述UDP-糖基转移酶包括来自甜菊的UGT-A、来自水稻的UGT-B中的一种或二种。
  5. 根据权利要求1所述的方法，其特征在于：所述UDP-糖基转移酶包括来自甜菊的UGT-A和来自水稻的UGT-B；所述UDP-糖基转移酶分两步加入到反应体系中，第一步先加入UGT-B，第二步再加入UGT-A。
  6. 根据权利要求5所述的方法，其特征在于：所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少60％的一致性；和/或，所述UGT-B的氨基酸序列与序列表中所示的序列4具有至少60％的一致性。
  7. 根据权利要求6所述的方法，其特征在于：所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少70％的一致性；和/或，所述UGT-B的氨基酸序列与序列表中所示的序列4具有至少70％的一致性。
  8. 根据权利要求7所述的方法，其特征在于：所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少80％的一致性；和/或，所述UGT-B的氨基酸序列与序列表中所示的序列4具有至少80％的一致性。
  9. 根据权利要求8所述的方法，其特征在于：所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少90％的一致性；和/或，所述UGT-B的氨基酸序列与序列表中所示的序列4具有至少90％的一致性。
  10. 根据权利要求2所述的方法，其特征在于：所述UDP-糖基转移酶为来自甜菊的 UGT-A，所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少60％的一致性。
  11. 根据权利要求10所述的方法，其特征在于：所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少70％的一致性。
  12. 根据权利要求11所述的方法，其特征在于：所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少80％的一致性。
  13. 根据权利要求12所述的方法，其特征在于：所述UGT-A的氨基酸序列与序列表中所示的序列2具有至少90％的一致性。
  14. 根据权利要求1或2所述的方法，其特征在于：反应在35-45℃温度以及pH7.5-8.5的水相体系中进行。
  15. 根据权利要求14所述的方法，其特征在于：反应在磷酸缓冲溶液中进行。
  16. 根据权利要求14所述的方法，其特征在于：反应体系含有UDP-糖基转移酶的重组细胞和细胞通透剂。
  17. 根据权利要求16所述的方法，其特征在于：所述细胞通透剂为甲苯，甲苯在反应体系中的体积比浓度为1-3％。
  18. 根据权利要求14所述的方法，其特征在于：将反应所用全部原料加入到反应釜中，混合均匀后置于设定温度下，搅拌反应。
  19. 根据权利要求1或2所述的方法，其特征在于：所述重组细胞为微生物细胞。
  20. 根据权利要求19所述的方法，其特征在于：所述微生物为大肠埃希氏杆菌、酿酒酵母或毕赤酵母。
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