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WO2025043715A1 - 利用生物酶法高效制备莱鲍迪苷m的方法 - Google Patents

利用生物酶法高效制备莱鲍迪苷m的方法 Download PDF

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WO2025043715A1
WO2025043715A1 PCT/CN2023/116557 CN2023116557W WO2025043715A1 WO 2025043715 A1 WO2025043715 A1 WO 2025043715A1 CN 2023116557 W CN2023116557 W CN 2023116557W WO 2025043715 A1 WO2025043715 A1 WO 2025043715A1
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seq
salt
rebaudioside
mixed solution
enzyme
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PCT/CN2023/116557
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English (en)
French (fr)
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宋云飞
王筱
谢永富
刘彦雪
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桂林莱茵生物科技股份有限公司
嘉兴欣贝莱生物科技有限公司
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Publication of WO2025043715A1 publication Critical patent/WO2025043715A1/zh

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    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/18—Preparation of compounds containing saccharide radicals produced by the action of a glycosyl transferase, e.g. alpha-, beta- or gamma-cyclodextrins
    • 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

Definitions

  • the invention belongs to the technical field of biocatalytic conversion, and specifically relates to a method for efficiently preparing rebaudioside M by utilizing a bioenzyme method.
  • Stevioside is a natural sweetener obtained from the leaves of Stevia (S. rebaudiana) by plant extraction. Stevia originates from the original forests of Paraguay and Brazil in South America. For hundreds of years, locals have been grinding Stevia leaves and using them as sweeteners. This sweetness mainly comes from the mixture of various steviol glycosides contained in the leaves. Stevioside is a glycoside mixture formed by some diterpenoid glycoside compounds. Its chemical structure is a condensation of sugar ligands and steviol aglycones.
  • Rebaudioside M which is present in very small amounts (0.3%) in stevia leaves, has a fast, pure sweetness and very low calories. It is most similar to sucrose in terms of sensory properties and is of great research significance in the food and pharmaceutical industries. It is expected to replace sucrose, a traditional sugar source, as a natural sweetener for widespread use and is known as the next generation of sweeteners.
  • rebaudioside A The current commercial product, rebaudioside A, is not as sweet as rebaudioside M.
  • rebaudioside M which has the best taste in stevia, is seriously lacking in stevia and has a very low natural content. It is impossible to directly extract and purify it from stevia leaves for commercialization, as is the case with high-content stevioside and rebaudioside A. This limits the large-scale production and supply of rebaudioside M.
  • the production of rebaudioside M can be increased by genetically modifying stevia or producing it by microbial methods, but both methods require a full understanding of the biosynthetic pathway of rebaudioside M, especially the glycosylation process.
  • the biosynthetic pathway of steviol glycosides has been basically clarified, and a series of glycosyltransferases (UGTs) using UDP-glucose (UDPG) as a glycosyl donor have been identified.
  • UDP-glucose UDP-glucose
  • Analysis of the biosynthetic pathway of rebaudioside M shows that glycosylation in the C19-carboxyl direction of rebaudioside A, i.e., the formation of a ⁇ (1-2) glycosidic bond at this position, is an essential step in the production of rebaudioside M and determines the natural content of rebaudioside M.
  • glycosylation of steviol glycosides depends on four glycosyltransferases (UGTs) located in the cytoplasm of Stevia (S. rebaudiana). They are all annotated as members of the GT1 family in the Carbohydrate Active Enzyme (CAZy) database (www.cazy.org) and use UDP-glucose as a glycosyl donor.
  • UGTs glycosyltransferases
  • UGT85C2 and UGT74G1 respectively connect the first glucose group at the C13 hydroxyl and C19 carboxyl positions of steviol aglycones with ⁇ -1 glycosidic bonds, followed by UGT91D2 adding a glycosyl group at the C2 position of the first glucose group through a ⁇ (1-2) glycosidic bond, and finally UGT76G1 adding a glycosyl group at the C3 position of the first glucose group.
  • Zhang et al. found that stevia itself lacks the ability to form a ⁇ (1-2) glycosidic bond in the C19-carboxyl direction of steviol glycosides, which is the direct reason for the low natural content of steviol glycoside DM.
  • rebaudioside M through glycosylation reaction using rebaudioside A as a substrate usually requires two-step conversion.
  • rebaudioside A is glycosylated to produce rebaudioside D, and then further glycosylated to produce rebaudioside M.
  • the disadvantage of the two-step catalytic method for producing rebaudioside M is that it is easy to cause the accumulation of the intermediate product rebaudioside D, which is not conducive to subsequent separation and purification.
  • the two-step method for catalyzing rebaudioside A to produce rebaudioside M usually requires multiple enzymes, and the steps are relatively cumbersome.
  • UDP-glycosyl donor-dependent glycosyltransferases require the use of expensive and unstable UDP-glycosyl donors, such as UDP-glucose (UDPG).
  • UDP-glycosyl donor such as UDP-glucose (UDPG).
  • UDPG is too expensive and its use as a glycosyl donor will incur huge costs and is not suitable for large-scale industrial production, which seriously limits the industrial application of UGTs.
  • Patent CN110734944B applied for by Tianjin University - a one-step method for synthesizing rebaudioside M.
  • the invention uses rebaudioside A as a substrate, adds uridine diphosphate glucose, magnesium sulfate or magnesium chloride, and methanol, and utilizes recombinant engineered bacteria UGT1 and UGT2 that can secrete and express glycosyltransferases to catalyze the reaction to obtain rebaudioside M.
  • This method adds a large amount of metal ions and organic solvents, which is not conducive to subsequent separation and purification and food-grade production.
  • expensive uridine diphosphate glucose is added to the reaction, and the production cost is too high.
  • Patent CN109750071A applied by Nanjing University of Technology -
  • a method for biocatalytic synthesis of rebaudioside M the application is to use tomato-derived UDP-glycosyltransferase and potato-derived sucrose synthase, stevioside as raw material to synthesize rebaudioside E through glycosylation reaction; then, stevia-derived UDP-glycosyltransferase and potato-derived sucrose synthase are used to further synthesize rebaudioside M through glycosylation reaction with rebaudioside E as raw material.
  • This method requires the generation of rebaudioside E first, and then further generates rebaudioside M.
  • This method requires two or more steps of catalytic reaction to obtain the final product, the reaction speed is slow, the catalytic efficiency is low, and there are many by-products, which is not conducive to separation and purification.
  • the plant extraction method has complicated steps, low yield, high cost, and difficulty in separation and purification.
  • the catalytic reaction system contains a large amount of metal ions and organic solvents, which is not conducive to subsequent separation and purification and food-grade production.
  • the sugar donor usually required is expensive uridine diphosphate glucose, which has high production cost and is not suitable for large-scale industrial production.
  • the purpose of the present invention is to provide a method for efficiently preparing rebaudioside M using a bioenzymatic method.
  • the method provided by the present invention is to obtain glycosyltransferase based on a gene mining method, then construct an engineering bacterium for protein expression, and in vitro catalyze rebaudioside A as a substrate to perform a glycosylation reaction to synthesize rebaudioside M in one step.
  • the synthesis method has high catalytic efficiency and short reaction time, and the product is single and easy to separate, and has great industrial application prospects.
  • the glycosyltransferase provided by the present invention can also be coupled with sucrose synthase for catalysis, using sucrose as a glycosyl donor, and rebaudioside A is catalyzed to generate rebaudioside M in one step.
  • the method for efficiently preparing rebaudioside M by using a bioenzymatic method provided by the present invention can use rebaudioside A as a substrate to catalyze the generation of rebaudioside M in one step, thereby realizing efficient biosynthesis of rebaudioside M.
  • the method can avoid the generation of the intermediate product rebaudioside D, facilitate subsequent separation and purification, and simplify the process flow of the catalytic reaction.
  • the method uses a bioenzymatic method to efficiently synthesize and prepare a high-sweetness, low-calorie natural sweetener-rebaudioside M.
  • the present invention provides a method for efficiently preparing rebaudioside M by using a biological enzyme method, comprising the following steps:
  • Rebaudioside A uridine diphosphate glucose, glycosyltransferase and salt are added into a buffer solution, mixed evenly to obtain a mixed solution, and an enzyme-catalyzed reaction is carried out under stirring to obtain the rebaudioside M.
  • the concentration of rebaudioside A is 1-100 g/L;
  • the concentration of rebaudioside A is 1 g/L.
  • the concentration of UDP-glucose is 0.5-20 mM
  • the concentration of UDP-glucose is 1 mM.
  • the salt is one or more of calcium salt, cobalt salt, iron salt, magnesium salt, manganese salt, ammonium salt, nickel salt and zinc salt;
  • the salt is one or more of calcium salt, magnesium salt, manganese salt and iron salt;
  • the salt is a magnesium salt.
  • the concentration of salt is 0.1-10 mM
  • the concentration of salt is 1 mM.
  • the pH of the buffer solution is 5.0-9.0;
  • the pH of the buffer is 6.0-8.0;
  • the pH of the buffer is 8.0;
  • stirring rate under stirring is 50-220 rpm
  • the stirring rate in the stirring state is 200 rpm.
  • the temperature of the enzyme catalyzed reaction is 20-45°C
  • the time of the enzyme catalyzed reaction is 1-5h.
  • the temperature of the enzyme catalyzed reaction is 30-37°C.
  • the temperature of the enzyme catalyzed reaction is 37°C.
  • the concentration of the glycosyltransferase is 1-20 g/L;
  • the concentration of the glycosyltransferase is 5 g/L.
  • sequence of the glycosyltransferase is one or more of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.
  • amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO.1.
  • the glycosyltransferase used has a high efficiency in catalyzing the glycosylation reaction.
  • UDPG uridine diphosphate glucose
  • the enzyme can well catalyze the glycosylation of rebaudioside A to produce the high-value sweetener rebaudioside M in one step without the need to add any other enzymes.
  • the optimum pH of the glycosylation reaction of the glycosyltransferase used is 8.0, and the optimal reaction temperature is 37°C. It is a metal ion-dependent enzyme, and Mg 2+ , Ca 2+ , and Mn 2+ have a significant promoting effect on the catalytic efficiency of the enzyme, among which Mg 2+ has the strongest promoting effect.
  • the optimal reaction conditions of the enzyme are: the final concentrations of various substances in the reaction system are respectively 1 g/L of rebaudioside A, 1 mM of UDPG, 5 mM of MgCl2, the reaction temperature is 37°C, the rotation speed is 220 rpm, and the reaction time is 1-2 h.
  • the enzyme has a high catalytic efficiency in catalyzing the glycosylation of rebaudioside A to generate rebaudioside M, and the conversion rate can reach more than 95%.
  • Another method for efficiently preparing rebaudioside M by using a biological enzyme method provided by the present invention comprises the following steps:
  • Rebaudioside A sucrose, UDP (5-uridine diphosphate disodium salt), glycosyltransferase, sucrose synthase and salt are added to a buffer solution, mixed evenly to obtain a mixed solution, and an enzyme-catalyzed reaction is carried out under stirring to obtain the rebaudioside M.
  • the concentration of rebaudioside A is 1-100 g/L;
  • the concentration of rebaudioside A is 10 g/L.
  • the concentration of sucrose is 1-500 mM
  • the concentration of sucrose is 100 mM.
  • the concentration of UDP (5-uridine diphosphate disodium salt) is 0.1-20 mM;
  • the concentration of UDP is 2 mM.
  • the salt is one or more of calcium salt, cobalt salt, iron salt, magnesium salt, manganese salt, ammonium salt, nickel salt and zinc salt;
  • the salt is one or more of calcium salt, magnesium salt, manganese salt and iron salt;
  • the concentration of salt is 0.1-10 mM
  • the concentration of salt is 0.1-5 mM.
  • the salt concentration is 1 mM.
  • the pH of the buffer solution is 4.0-9.0;
  • the pH of the buffer is 6.0-8.0;
  • the pH of the buffer is 8.0;
  • stirring rate under stirring is 50-220 rpm
  • the stirring rate in the stirring state is 200 rpm.
  • the temperature of the enzyme catalyzed reaction is 20-45°C
  • the time of the enzyme catalyzed reaction is 1-5h.
  • the concentration of the glycosyltransferase is 5 g/L.
  • sequence of the glycosyltransferase is one or more of the amino acid sequences shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12.
  • amino acid sequence of the glycosyltransferase is as shown in SEQ ID NO.1.
  • the concentration of the sucrose synthase is 1-10 g/L;
  • the concentration of sucrose synthase is 2 g/L.
  • amino acid sequence of the sucrose synthase is one or more of SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO.18, SEQ ID NO.19 and SEQ ID NO.20.
  • amino acid sequence of the sucrose synthase is as shown in SEQ ID NO.13.
  • the second method for efficiently preparing rebaudioside M by using a biological enzyme method provided by the present invention is to use sucrose as a glycosyl donor.
  • the glycosyltransferase can be coupled with sucrose synthase for catalytic coupling, so that rebaudioside A can generate a high-value sweetener, rebaudioside M, in one step without generating other intermediates or by-products.
  • the optimum pH of the dual enzyme coupled catalytic reaction is 8.0
  • the optimal reaction temperature is 37°C
  • Mg2 + has a significant promoting effect on the catalytic efficiency of the coupled catalytic reaction.
  • the reaction time of the dual enzyme coupled catalysis is short, and rebaudioside A is completely converted into rebaudioside M within 2 hours.
  • This method utilizes the coupled catalytic action of glycosyltransferase and sucrose synthase, uses sucrose as the glycosyl donor to replace expensive UDPG, and efficiently catalyzes RA to generate RM in one step, thereby reducing production costs.
  • glycosyltransferases with high catalytic efficiency are used, which can use rebaudioside A as a substrate to catalyze the generation of rebaudioside M in one step, thereby realizing efficient biosynthesis of rebaudioside M.
  • These two methods can reduce impurities such as metal ions and by-products in the system, reduce the cost of separation and purification, and realize green biosynthesis of rebaudioside M.
  • sucrose is used as the glycosyl donor, which greatly reduces the production cost.
  • the present invention has the following advantages and beneficial effects:
  • the enzyme used can efficiently catalyze rebaudioside A to generate rebaudioside M in one step under the optimal reaction conditions. No other enzymes need to be added during the catalytic process.
  • the enzyme can specifically catalyze the glycosylation reaction to generate rebaudioside M without intermediates or other by-products.
  • the reaction time is short and the catalytic efficiency is high.
  • the yield of the product rebaudioside M can reach more than 95%, which is conducive to the separation and purification of the product.
  • rebaudioside A can be catalyzed to generate rebaudioside M in one step under the optimal reaction conditions through dual enzyme coupling catalysis, without the generation of by-products, with good specificity, which is conducive to the subsequent separation and purification of the product.
  • Sucrose is used as the glycosyl donor, the production cost is low, and it is conducive to the large-scale biosynthesis of rebaudioside M.
  • the final reaction system contains only trace metal ions (Mg 2+ ), no intermediate products or by-products, and the impurity content is extremely low and easy to separate, which is conducive to subsequent separation and purification in industrial-scale production.
  • FIG1 is a graph showing the catalytic efficiency of glycosyltransferases at different pH values
  • FIG2 is a graph showing the catalytic efficiency of glycosyltransferases at different temperatures
  • FIG3 is a graph showing the effect of different metal ions on the catalytic efficiency of glycosyltransferase
  • FIG4 is a liquid phase detection diagram of the synthesis of rebaudioside M from rebaudioside A catalyzed by using UDPG as a glycosyl donor;
  • Figure 5 is a liquid phase detection diagram of the synthesis of rebaudioside M from rebaudioside A using sucrose as the glycosyl donor.
  • the glycosyltransferase (UGTs) gene was used as the probe sequence, and several homologous sequences were obtained through homology search. Multiple sequence alignment was performed using mega software (MEGA-X). Then, an evolutionary tree was constructed with the sequences after extracting the conserved sites. The evolutionary tree can intuitively display the evolutionary relationship of each homologous sequence. Several corresponding enzyme protein gene sequences that were close to the probe sequence species were selected as potential candidate genes. Then, molecular substrate docking was performed after Rosetta homology modeling, and free energy, binding energy, etc. were calculated to further screen the candidate gene sequences for subsequent functional verification.
  • MTTs glycosyltransferase
  • the selected glycosyltransferase gene was sequence and codon optimized, and the selected glycosyltransferase gene fragment was synthesized (six his tags were added to the C-terminus of the gene fragment during the synthesis process to facilitate the subsequent separation and purification of the recombinant enzyme using a Ni2 + affinity chromatography column), and then the recombinant gene fragment was ligated into the E. coli expression vector pET28a using restriction endonucleases BamH I and EcoR I to obtain a recombinant plasmid carrying the target gene.
  • the heat shock transformation method was used to construct recombinant E. coli.
  • Several synthesized recombinant plasmids were transferred into E. coli DH5 ⁇ (for PCR amplification of recombinant plasmids) and E. coli BL21 (DE3) (for heterologous expression of recombinase protein) competent cells to obtain the corresponding recombinant E. coli containing the target gene.
  • Activation culture Take 5 ⁇ L of glycerol bacteria and inoculate it into 5 mL of LB liquid culture medium containing Kan resistance (final concentration of 100 ⁇ g/mL), and place it in a biochemical incubator at a temperature of 37°C and a rotation speed of 220 r/min for overnight culture (culture time is 16 hours).
  • Expanded culture Use a pipette to transfer 8 mL of activated bacterial solution into a 1-L conical flask containing 800 mL of 2 ⁇ YT liquid culture medium (containing Kan resistance). Culture the culture in a biochemical incubator at 37°C and 220 r/min for a certain period of time, and monitor the OD value at the same time.
  • a univariate factor exploration experiment of the enzyme catalytic reaction conditions was carried out on the glycosyltransferase (as shown in SEQ ID NO.1) to identify its enzyme properties.
  • the optimum pH of the enzyme is 8.0, and when the pH is equal to 6.0, the catalytic efficiency can maintain 65%, indicating that the enzyme has a certain potential for large-scale biocatalytic production of rebaudioside M.
  • the optimum pH and catalytic efficiency of the remaining amino acid sequences shown in SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11 and SEQ ID NO.12 are similar to those of the amino acid sequence shown in SEQ ID NO.1, as shown in Figure 1.
  • Cu 2+ , Zn 2+ and Ni 2+ have a significant inhibitory effect on the catalytic efficiency of the enzyme, among which Cu 2+ ions have the strongest inhibitory effect on the enzyme.
  • the enzyme loses its activity and the yield of rebaudioside M is 0.
  • the catalytic efficiency of the enzyme is only half of that of the blank control group.
  • the enzyme is a metal-dependent enzyme, among which Mg 2+ has the strongest promoting effect on the enzyme, and Cu 2+ ions have the strongest inhibitory effect on the enzyme, which can directly destroy the enzyme protein structure and thus make it inactive.
  • High performance liquid chromatography was used to detect the content of substrate and product in the system after enzyme reaction.
  • Chromatographic column model InertsilTM ODS-3 reverse phase C18 column; column temperature: 40°C; detector: PDA (ultraviolet) detector; detection wavelength: 218nm; mobile phase A is acetonitrile containing 0.1% formic acid; mobile phase C is ultrapure water containing 0.1% formic acid, % is volume percentage; liquid phase gradient elution program is: 0min (25% A, 75% C), 4min (40% A, 60% C), 15min (42% A, 58% C), 16min (100% A, 0% C), 21min (5% A, 95% C), 30min (25% A, 75% C) flow rate: 0.6mL/min; injection volume: 10 ⁇ L.
  • Example 6 Enzymatic synthesis of rebaudioside M from rebaudioside A using UDPG (uridine diphosphate glucose) as a glycosyl donor
  • sucrose, 10g/L rebaudioside A glycosyltransferase crude enzyme solution ( ⁇ 5mg/mL total protein), sucrose synthase crude enzyme solution ( ⁇ 2mg/mL total protein), 2mM uridine diphosphate disodium salt, 5mM magnesium chloride to the reaction system, and add PBS (50mM) buffer with a pH of 8.0 to the final reaction volume.
  • React at 37°C, 200rpm for 3h take a sample and place it in a 100°C water bath for 10min, centrifuge at 12000rpm for 1min at room temperature, take the supernatant and filter it with a 0.45 ⁇ m sterile filter membrane into a liquid phase vial, and analyze it by HPLC liquid phase detection.
  • High performance liquid chromatography was used to detect the content of substrate and product in the system after enzyme reaction.
  • Chromatographic column model InertsilTM ODS-3 reverse phase C18 column; column temperature: 40°C; detector: PDA (ultraviolet) detector; detection wavelength: 218nm; mobile phase A is acetonitrile containing 0.1% formic acid; mobile phase C is ultrapure water containing 0.1% formic acid, % is volume percentage; liquid phase gradient elution program is: 0min (25% A, 75% C), 4min (40% A, 60% C), 15min (42% A, 58% C), 16min (100% A, 0% C), 21min (5% A, 95% C), 30min (25% A, 75% C) flow rate: 0.6mL/min; injection volume: 10 ⁇ L.

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Abstract

本发明公开了利用生物酶法高效制备莱鲍迪苷M的方法。该方法包括：将莱鲍迪苷A、尿苷二磷酸葡萄糖、糖基转移酶、盐加入缓冲液中，混匀，得到混合液，在搅拌状态下进行酶催化反应，得到所述莱鲍迪苷M。所述糖基转移酶的氨基酸序列为如SEQ ID NO.1-12所示的中的一种以上。该糖基转移酶还可以与蔗糖合成酶进行偶联催化，以蔗糖作为糖基供体，将莱鲍迪苷A一步催化生成莱鲍迪苷M。

Description

利用生物酶法高效制备莱鲍迪苷M的方法 技术领域
本发明属于生物催化转化技术领域，具体涉及一种利用生物酶法高效制备莱鲍迪苷M的方法。
背景技术
人们发现以甜菊糖苷为代表的一类绿色、健康的天然甜味剂可以广泛的作为代糖使用。甜菊糖苷是从甜叶菊(S.rebaudiana)的叶片中通过植物提取法得到的天然甜味剂。甜叶菊产地源自于南美巴拉圭和巴西的原始森林，几百年以来，当地人一直将甜叶菊的叶子研磨后作为甜味剂使用，这种甜味主要是源于叶子中含有的多种甜菊醇醇糖苷混合物。甜菊糖苷是由一些二萜类糖苷化合物形成的糖苷混合物，它的化学结构是糖配体与甜菊醇苷元连接缩合，甜菊糖苷中的糖苷键大部分无法消化，因此释放的能量很少，可以成为非常有吸引力的糖替代品。甜叶菊叶片中含量最丰富的是甜菊苷(Stevioside)和莱鲍迪苷A(RebaudiosideA)，其甜度是蔗糖的200倍，是最早开始商业化使用的甜味剂。然而，甜菊苷和莱鲍迪苷A会作用于味蕾中的苦味受体hTAS2R4和hTAS2R14，导致其具有明显的后苦味，口感不好，而这降低了人们对二者的接受度，影响了其应用范围及市场接受度。甜叶菊叶片中含量非常少(0.3％)的莱鲍迪苷M具有快速、纯正的甜味且热量极低，在感官特性上与蔗糖最为相似，在食品、医药行业非常具有研究意义，有望代替蔗糖这一传统糖源作为天然甜味剂来广泛使用，被誉为下一代甜味剂。
目前商业化的产品莱鲍迪苷A甜味味质不如莱鲍迪苷M，然而甜叶菊中口感最佳的莱鲍迪苷M在甜叶菊中严重缺乏，天然含量非常低，不可能像含量高的甜菊苷和莱鲍迪苷A一样，直接从甜叶菊叶片进行商品化的提炼和纯化，这限制了莱鲍迪苷M的大批量规模化生产和供给。通过转基因甜叶菊或者微生物法来生产莱鲍迪苷M可以提高其产量，但以上两种方法均需要充分了解莱鲍迪苷M的生物合成途径，特别是糖基化过程。
目前甜菊糖苷的生物合成途径已经基本明晰，并鉴定出了一系列利用尿苷二磷酸葡萄糖(UDP-glucose，UDPG)作为糖基供体的糖基转移酶(UGTs)。分析莱鲍迪苷M生物合成途径可以发现，位于莱鲍迪苷A的C19-羧基方向的糖基化，即在该位置形成β(1-2)糖苷键，是生成莱鲍迪苷M的必需步骤，决定了莱鲍迪苷M的天然含量大小。甜菊糖苷的糖基化依赖于位于甜叶菊(S.rebaudiana)细胞质中的4个糖基转移酶(UGTs)。它们在碳水化合物活性酶(CAZy)数据库(www.cazy.org)中均被标注为GT1家族成员，并利用尿苷二磷酸葡萄糖(UDP-glucose)作为糖基供体。糖基转移酶UGT85C2和UGT74G1分别在甜菊醇苷元的C13羟基和C19羧基位置上以β-1糖苷键连接第一个葡萄糖基，接着UGT91D2通过β(1-2)糖苷键在第一个葡萄糖基的C2添加一个糖基，最后UGT76G1在第一个葡萄糖基的C3位添加一个糖基。张等发现甜叶菊自身缺乏在甜菊糖苷C19-羧基方向形成β(1-2)糖苷键的能力，是造成甜菊糖苷DM天然含量低的直接原因。
目前以莱鲍迪苷A作为底物经糖基化反应生成莱鲍迪苷M，通常需要经过两步转化，首先莱鲍迪苷A糖基化生成莱鲍迪苷D，然后再进一步糖基化生成莱鲍迪苷M，两步催化法生成莱鲍迪苷M的缺点是容易造成中间产物莱鲍迪苷D的累计，不利于后续分离提纯，并且两步法催化莱鲍迪苷A生成莱鲍迪苷M通常需要多种酶，步骤较为繁琐。
通常UDP-糖基供体依赖的糖基转移酶(UGTs)催化的糖基化反应需要使用价格昂贵且稳定性差的UDP-糖基供体，如UDP-葡萄糖(UDPG)等，UDPG过于昂贵，作为糖基供体使用会产生巨额的成本，不适用于大规模的工业化生产，这严重限制了UGTs的工业化应用。
天津大学申请的专利CN110734944B-一步法合成莱鲍迪苷M的方法，该发明以莱鲍迪苷A为底物，加入尿苷二磷酸葡萄糖，硫酸镁或氯化镁，甲醇，利用能够分泌表达糖基转移酶的重组工程菌UGT1和UGT2催化反应，得到莱鲍迪苷M，该方法添加了较多的金属离子和有机溶剂，不利于后续的的分离纯化和食品级生产，且反应中添加了昂贵的尿苷二磷酸葡萄糖，生产成本过高。南京工业大学申请的专利CN109750071A-一种生物催化合成莱鲍迪苷M的方法，该申请是利用番茄来源UDP-糖基转移酶和马铃薯来源蔗糖合成酶，以甜菊甙为原料糖基化反应合成莱鲍迪苷E；随后，利用甜叶菊来源UDP-糖基转移酶和马铃薯来源蔗糖合成酶，以莱鲍迪苷E为原料进一步糖基化反应合成莱鲍迪苷M，该方法需要先生成莱鲍迪苷E，再进一步生成莱鲍迪苷M。该方法需要经过两步或多步催化反应才能得到最终产物，反应速度慢，催化效率低，副产物较多，不利于分离纯化。
因此目前现有生产莱鲍迪苷M的技术中，存在如下的缺点：
1)植物提取法步骤繁琐，产量低，成本高，分离提纯困难。
2)植物提取法需要大量的甜叶菊，浪费耕地面积。
3)化学合成法产生大量有害气体和废液，不利于环境保护。
4)催化反应体系中含有较多的金属离子和有机溶剂，不利于后续的的分离纯化和食品级生产。
5)需要经过两步或多步催化反应才能得到最终产物，反应速度慢，催化效率低，副产物较多，不利于分离纯化。
6)通常所需糖基供体为昂贵的尿苷二磷酸葡萄糖，生产成本高，不适合大规模的工业化生产。
7)需要添加多种酶制剂进行催化反应，特异性差，产率较低。
所以亟需一种高效低成本生产莱鲍迪苷M的方法。
发明内容
为了克服现有技术存在的不足，本发明的目的是提供利用生物酶法高效制备莱鲍迪苷M的方法。
本发明提供的方法是基于基因挖掘的方法得到糖基转移酶，然后构建工程菌进行蛋白表达，体外催化以莱鲍迪苷A作为底物进行糖基化反应一步合成莱鲍迪苷M。该合成方法催化效率高反应时间短，产物单一易分离，有较大的工业应用前景。并且本发明提供的糖基转移酶还可与蔗糖合成酶进行偶联催化，以蔗糖作为糖基供体，将莱鲍迪苷A一步催化生成莱鲍迪苷M。
本发明提供的利用生物酶法高效制备莱鲍迪苷M的方法通过使用催化效率较高的糖基转移酶，能够以莱鲍迪苷A为底物，一步催化生成莱鲍迪苷M，实现莱鲍迪苷M的高效生物合成。该方法可以避免中间产物莱鲍迪苷D的生成，便于后续的分离提纯，精简催化反应的工艺流程。该方法用生物酶法高效合成制备高甜度低热量天然甜味剂-莱鲍迪苷M。
本发明的目的至少通过如下技术方案之一实现。
本发明提供的一种利用生物酶法高效制备莱鲍迪苷M的方法，包括如下步骤：
将莱鲍迪苷A、尿苷二磷酸葡萄糖、糖基转移酶、盐加入缓冲液中，混合均匀，得到混合液，在搅拌状态下进行酶催化反应，得到所述莱鲍迪苷M。
进一步地，在所述混合液中，莱鲍迪苷A的浓度为1-100g/L；
优选地，在所述混合液中，莱鲍迪苷A的浓度为1g/L。
进一步地，在所述混合液中，尿苷二磷酸葡萄糖的浓度为0.5-20mM；
优选地，在所述混合液中，尿苷二磷酸葡萄糖的浓度为1mM。
进一步地，所述盐为钙盐、钴盐、铁盐、镁盐、锰盐、铵盐、镍盐、锌盐中的一种以上；
优选地，所述盐为钙盐、镁盐、锰盐、铁盐中的一种以上；
进一步优选地，所述盐为镁盐。
进一步地，在所述混合液中，盐的浓度为0.1-10mM；
优选地，在所述混合液中，盐的浓度为1mM。
进一步地，所述缓冲液的pH为5.0-9.0；
优选地，所述缓冲液的pH为6.0-8.0；
进一步优选地，所述缓冲液的pH为8.0；
进一步地，所述在搅拌状态下的搅拌速率为50-220rpm；
优选地，所述在搅拌状态下的搅拌速率为200rpm。
进一步地，所述酶催化反应的温度为20-45℃，所述酶催化反应的时间为1-5h。
优选地，所述酶催化反应的温度为30-37℃。
进一步优选地，所述酶催化反应的温度为37℃。
进一步地，在所述混合液中，所述糖基转移酶的浓度为1-20g/L；
优选地，在所述混合液中，所述糖基转移酶的浓度为5g/L。
进一步地，所述糖基转移酶的序列为如SEQ ID NO.1所示、如SEQ ID NO.2所示、如SEQ ID NO.3所示、如SEQ ID NO.4所示、如SEQ ID NO.5所示、如SEQ ID NO.6所示、如SEQ ID NO.7所示、如SEQ ID NO.8所示、如SEQ ID NO.9所示、如SEQ ID NO.10所示、如SEQ ID NO.11所示及如SEQ ID NO.12所示的氨基酸序列中的一种以上。
优选地，所述糖基转移酶的氨基酸序列如SEQ ID NO.1所示。
本发明提供的方法中，使用的糖基转移酶催化糖基化反应效率较高，以尿苷二磷酸葡萄糖(UDPG)作为糖基供体，该酶可以很好地催化莱鲍迪苷A糖基化一步生成高价值甜味剂莱鲍迪苷M，且不需要添加其他任何酶。
本发明提供的方法中，使用的糖基转移酶的糖基化反应的最适pH为8.0，最佳反应温度为37℃，是一种金属离子依赖型酶，Mg2+、Ca2+、Mn2+对该酶的催化效率有明显的促进提升作用，其中Mg2+的促进作用最强。
本发明提供的方法中，该酶的最佳反应条件为：反应体系各种物质的终浓度分别是莱鲍迪苷A为1g/L，UDPG为1mM，MgCl2为5mM，反应温度37℃，转速220rpm，反应时间1-2h，在最佳反应条件下，该酶催化莱鲍迪苷A糖基化生成莱鲍迪苷M的催化效率较高，转化率可达95％以上。
本发明提供的另一种利用生物酶法高效制备莱鲍迪苷M的方法，包括如下步骤：
将莱鲍迪苷A、蔗糖、UDP(5-尿苷二磷酸二钠盐)、糖基转移酶、蔗糖合成酶、盐加入缓冲液中，混合均匀，得到混合液，在搅拌状态下进行酶催化反应，得到所述莱鲍迪苷M。
进一步地，在所述混合液中，莱鲍迪苷A的浓度为1-100g/L；
优选地，在所述混合液中，莱鲍迪苷A的浓度为10g/L。
进一步地，在所述混合液中，蔗糖的浓度为1-500mM；
优选地，在所述混合液中，蔗糖的浓度为100mM。
进一步地，在所述混合液中，UDP(5-尿苷二磷酸二钠盐)的浓度为0.1-20mM；
优选地，在所述混合液中，UDP(5-尿苷二磷酸二钠盐)的浓度为2mM。
进一步地，所述盐为钙盐、钴盐、铁盐、镁盐、锰盐、铵盐、镍盐、锌盐中的一种以上；
优选地，所述盐为钙盐、镁盐、锰盐、铁盐中的一种以上；
进一步地，在所述混合液中，盐的浓度为0.1-10mM；
优选地，在所述混合液中，盐的浓度为0.1-5mM。
进一步优选地，在所述混合液中，盐的浓度为1mM。
进一步地，所述缓冲液的pH为4.0-9.0；
优选地，所述缓冲液的pH为6.0-8.0；
进一步优选地，所述缓冲液的pH为8.0；
进一步地，所述在搅拌状态下的搅拌速率为50-220rpm；
优选地，所述在搅拌状态下的搅拌速率为200rpm。
进一步地，所述酶催化反应的温度为20-45℃，所述酶催化反应的时间为1-5h。
优选地，所述酶催化反应的温度为30-37℃。
进一步优选地，所述酶催化反应的温度为37℃。
进一步地，在所述混合液中，所述糖基转移酶的浓度为1-20g/L；
优选地，在所述混合液中，所述糖基转移酶的浓度为5g/L。
进一步地，所述糖基转移酶的序列为如SEQ ID NO.1所示、如SEQ ID NO.2所示、如SEQ ID NO.3所示、如SEQ ID NO.4所示、如SEQ ID NO.5所示、如SEQ ID NO.6所示、如SEQ ID NO.7所示、如SEQ ID NO.8所示、如SEQ ID NO.9所示、如SEQ ID NO.10所示、如SEQ ID NO.11所示及如SEQ ID NO.12所示的氨基酸序列中的一种以上。
优选地，所述糖基转移酶的氨基酸序列如SEQ ID NO.1所示。
进一步地，在所述混合液中，所述蔗糖合成酶的浓度为1-10g/L；
优选地，在所述混合液中，所述蔗糖合成酶的浓度为2g/L。
进一步地，所述蔗糖合成酶的氨基酸序列为如SEQ ID NO.13所示、如SEQ ID NO.14所示、如SEQ ID NO.15所示、如SEQ ID NO.16所示、如SEQ ID NO.17所示、如SEQ ID NO.18所示、如SEQ ID NO.19所示及如SEQ ID NO.20所示中的一种以上。
优选地，所述蔗糖合成酶的氨基酸序列如SEQ ID NO.13所示。
本发明提供的第二种利用生物酶法高效制备莱鲍迪苷M的方法，是以蔗糖作为糖基供体，在UDP存在的条件下，该糖基转移酶可以与蔗糖合成酶进行偶联催化，使莱鲍迪苷A一步生成高价值甜味剂莱鲍迪苷M，无其他中间产物或副产物生成。该方法中，双酶偶联催化反应的最适pH为8.0，最佳反应温度为37℃，Mg2+对该偶联催化反应的催化效率有明显的促进提升作用。该方法中，双酶偶联催化的反应时间短，2h内莱鲍迪苷A即被全部转化生成莱鲍迪苷M。
该方法利用糖基转移酶和蔗糖合成酶的偶联催化作用，以蔗糖为糖基供体取代昂贵的UDPG，高效催化RA一步生成RM，从而降低生产成本。
本发明提供的两个方法中，均使用了催化效率较高的糖基转移酶，能够以莱鲍迪苷A为底物，一步催化生成莱鲍迪苷M，实现莱鲍迪苷M的高效生物合成。这两个方法可以减少体系中的金属离子、副产物等杂质，降低分离纯化的成本，以实现莱鲍迪苷M的绿色生物合成。
本发明提供的第二个方法中，是以廉价的蔗糖作为糖基供体，大幅度降低生产成本。
与现有技术相比，本发明具有如下优点和有益效果：
(1)本发明实施例提供的方法中，使用的酶在最佳反应条件下可以高效催化莱鲍迪苷A一步生成莱鲍迪苷M，催化过程中不需要添加其他酶，能够专一性的催化糖基化反应生成莱鲍迪苷M，无中间产物或其他副产物，且反应时间短，催化效率高，产物莱鲍迪苷M的得率可达95％以上，有利于产物的分离提纯；
(2)本发明实施例提供的方法中，通过双酶偶联催化可以在最佳反应条件下将莱鲍迪苷A一步催化生成莱鲍迪苷M，无副产物产生，专一性较好，有利于后续的产物分离提纯，以蔗糖作为糖基供体，生产成本低，有利于莱鲍迪苷M大规模生物合成；
(3)本发明实施例提供的方法中，反应终体系中只含有微量金属离子(Mg2+)，无中间产物或副产物，杂质含量极低且易分离，有利于工业化规模生产的后续分离纯化。
附图说明
图1为不同pH下糖基转移酶的催化效率结果图；
图2为不同温度下糖基转移酶的催化效率结果图；
图3为不同金属离子对糖基转移酶催化效率的影响结果图；
图4为以UDPG为糖基供体催化莱鲍迪苷A合成莱鲍迪苷M的液相检测图；
图5为以蔗糖为糖基供体催化莱鲍迪苷A合成莱鲍迪苷M的液相检测图。
具体实施方式
以下结合实例对本发明的具体实施作进一步说明，但本发明的实施和保护不限于此。需指出的是，以下若有未特别详细说明之过程，均是本领域技术人员可参照现有技术实现或理解的。所用试剂或仪器未注明生产厂商者，视为可以通过市售购买得到的常规产品。
实施例1：基因挖掘
首先以糖基转移酶(UGTs)基因为探针序列，通过同源性搜索得到若干同源序列，用mega软件(MEGA-X)进行多重序列比对，然后以提取保守位点后的序列构建进化树，进化树可直观显示各同源性序列的进化关系，选取距离探针序列物种较近的若干对应酶蛋白基因序列作为潜在的候选基因，接着通过Rosetta同源建模后进行分子底物对接，计算自由能、结合能等，进一步筛选得到候选基因序列，进行后续的功能验证。
实施例2：重组质粒构建
将上述筛选出的糖基转移酶基因进行序列及密码子优化，合成上述筛选出的糖基转移酶基因片段(在合成过程中添加6个his标签于基因片段的C末端，便于后续利用Ni2+亲和层析柱进行重组酶的分离纯化)，再利用限制性内切酶BamH I和EcoR I将该重组基因片段连接到E.coli表达载体pET28a中，得到带有目的基因的重组质粒。
采用热激转化法来构建重组大肠杆菌。将合成的若干重组质粒分别转入E.coli DH5α(用于重组质粒的PCR扩增)和E.coli BL21(DE3)(用于重组酶的异源表达蛋白)感受态细胞，得到相应的含有目的基因的重组大肠杆菌。
实施例3：重组酶的表达
(1)活化培养：取甘油菌5μL接种至装有5mL含Kan抗性(终浓度为100μg/mL)的LB液体培养基中，放到温度为37℃，转速为220r/min的生化培养箱中过夜培养(培养时间为16小时)。
(2)扩大化培养：用移液枪吸取活化后的8mL菌液转入800m L 2×YT液体培养基(含Kan抗性)的1L锥形瓶中，同样温度为37℃，转速为220r/min的生化培养箱中培养一定的时间，同时对OD值进行监测。
(3)诱导表达：当菌液的OD值为0.6-0.8时，停止37摄氏度培养，在锥形瓶中加入400μL的IPTG(0.5mM)诱导剂，温度改变为16℃，转速不变，继续在恒温振荡培养箱中低温培养16-20h，诱导重组酶过量表达。
(4)离心收菌：将步骤(3)低温诱导表达之后的重组酶菌液，用落地式离心机进行离心，温度设置为4℃，转速设置为6000rpm，时间设置为10min，离心完成后，倒掉上清液，留下菌体沉淀。用50mM、pH＝8.0的PBS缓冲液重悬洗涤至40ml，在4℃，8000rpm条件下离心10min，弃去上清液，收集菌体沉淀。
(5)破菌：以上洗涤之后的菌体沉淀加入50mL的50mM、pH＝8.0的PBS缓冲液，进行重悬，然后用高压低温破碎仪高压下破碎细胞。8000rpm离心30min，取上清即为粗酶液。
(6)粗酶液蛋白浓度测量：用Pierce BCA蛋白定量试剂盒在多功能酶标仪中测量粗酶液中总蛋白的含量。
实施例4：糖基转移酶的单变量因素探究
对糖基转移酶(如SEQ ID NO.1所示)进行了酶催化反应条件的单变量因素探究实验，对其进行酶性质鉴定。
1)为研究反应体系pH对糖基转移酶催化效率的影响，在32.5℃条件下，采用乙酸盐缓冲液(50mM，pH缓冲范围4.0-5.5)、磷酸盐缓冲液(50mM，pH缓冲6.0-8.0)以及三羟甲基氨基甲烷盐酸盐缓冲液(50mM，pH缓冲值7.5-9.0)三种缓冲体系，然后配制成3种对应的反应体系(可参照表1所示)，检测在pH 4.0-9.0范围内的酶活力变化。
如图1所示，该酶的最适pH为8.0，而且当pH等于6.0的时候，可以保持65％的催化效率，表明该酶具有一定的莱鲍迪苷M生物催化规模生产的潜力，其余如SEQ ID NO.2所示、如SEQ ID NO.3所示、如SEQ ID NO.4所示、如SEQ ID NO.5所示、如SEQ ID NO.6所示、如SEQ ID NO.7所示、如SEQ ID NO.8所示、如SEQ ID NO.9所示、如SEQ ID NO.10所示、如SEQ ID NO.11所示及如SEQ ID NO.12所示的氨基酸序列的最适pH及催化效率与如SEQ ID NO.1所示的氨基酸序列相似，可参照图1所示。
2)为研究温度对糖基转移酶催化效率的影响，在pH 8.0条件下，分别检测在16℃、22℃、30℃、32.5℃、37℃、42℃、65℃条件下该酶的催化效率，该糖基转移酶在温度大于40℃时催化效率大幅度下降，在30℃～37℃内表现出较高的催化活性(如图2所示)。
如图2所示，37℃时莱鲍迪苷M的得率最高，因此，该酶(所有糖基转移酶的趋势都是一致的)在37℃温度条件下催 化效率最高。其余如SEQ ID NO.2所示、如SEQ ID NO.3所示、如SEQ ID NO.4所示、如SEQ ID NO.5所示、如SEQ ID NO.6所示、如SEQ ID NO.7所示、如SEQ ID NO.8所示、如SEQ ID NO.9所示、如SEQ ID NO.10所示、如SEQ ID NO.11所示及如SEQ ID NO.12所示的氨基酸序列的最适温度及催化活性与如SEQ ID NO.1所示的氨基酸序列相似，可参照图2所示。
3)本实验探究了多种不同的金属离子及铵根离子对糖基转移酶催化效率的影响。由图3可知，Mg2+、Ca2+、Mn2+及Fe3+对该酶的催化效率有明显的促进提升作用，其中Mg2+的促进作用最强，同等条件下莱鲍迪苷M的得率可达61％，比空白对照组提高了约6倍，Ca2+和Mn2+存在的条件下莱鲍迪苷M的得率约为56％，值得一提的是，NH4+对该酶也具有较强的促进提升作用，莱鲍迪苷M得率为55％。而Cu2+、Zn2+和Ni2+则对该酶的催化效率有明显抑制作用，其中Cu2+离子对该酶的抑制作用最强，铜离子存在的情况下，该酶失去活性，莱鲍迪苷M得率为0，Zn2+存在时该酶的催化效率仅为空白对照组的一半。
以上结果说明该酶是一种金属依赖型酶，其中Mg2+对该酶的促进提升作用最强，Cu2+离子对该酶的抑制作用最强，可直接破坏酶蛋白结构进而使其失去活性。
不同的金属离子及铵根离子对如SEQ ID NO.2所示、如SEQ ID NO.3所示、如SEQ ID NO.4所示、如SEQ ID NO.5所示、如SEQ ID NO.6所示、如SEQ ID NO.7所示、如SEQ ID NO.8所示、如SEQ ID NO.9所示、如SEQ ID NO.10所示、如SEQ ID NO.11所示及如SEQ ID NO.12所示的氨基酸序列的催化效率的影响与如SEQ ID NO.1所示的氨基酸序列相似，可参照图3所示。
实施例5：以蔗糖为糖基供体双酶法催化莱鲍迪苷A合成莱鲍迪苷M
1)催化反应体系
添加1mM的UDPG(尿苷二磷酸葡萄糖)、糖基转移酶粗酶液(～3mg/mL总蛋白)、1g/L莱鲍迪苷A、5mM氯化镁于反应体系中，补加pH为8.0的PBS(50mM)缓冲液至反应终体积。37℃，200rpm反应2h，取样置于100℃水浴加热10min，12000rpm室温离心1min，取上清液用0.45μm无菌滤膜过滤至液相小瓶中，用HPLC液相检测分析。
2)HPLC检测方法
采用高效液相色谱法(HPLC)检测酶反应后体系中的底物及产物含量。色谱柱型号：InertsilTM ODS-3反相C18柱；柱温：40℃；检测器：PDA(紫外)检测器；检测波长：218nm；流动相A为含0.1％甲酸的乙腈；流动相C为含0.1％甲酸的超纯水，％为体积百分比；液相梯度洗脱程序为：0min(25％A，75％C)，4min(40％A，60％C)，15min(42％A，58％C)，16min(100％A，0％C)，21min(5％A，95％C)，30min(25％A，75％C)流速：0.6mL/min；进样量：10μL。
3)结果
如图4所示，根据液相检测结果进行分析计算，反应2h后，莱鲍迪苷A几乎全部转化为莱鲍迪苷M，转化率高达98％，无副产物生成。
实施例6：以UDPG(尿苷二磷酸葡萄糖)为糖基供体酶法催化莱鲍迪苷A合成莱鲍迪苷M
1)催化反应体系
添加34g/L的蔗糖、10g/L莱鲍迪苷A、糖基转移酶粗酶液(～5mg/mL总蛋白)、蔗糖合成酶粗酶液(～2mg/mL总蛋白)、2mM尿苷二磷酸二钠盐、5mM氯化镁于反应体系中，补加pH为8.0的PBS(50mM)缓冲液至反应终体积。37℃，200rpm反应3h，取样置于100℃水浴加热10min，12000rpm室温离心1min，取上清液用0.45μm无菌滤膜过滤至液相小瓶中，用HPLC液相检测分析。
2)HPLC检测方法
采用高效液相色谱法(HPLC)检测酶反应后体系中的底物及产物含量。色谱柱型号：InertsilTM ODS-3反相C18柱；柱温：40℃；检测器：PDA(紫外)检测器；检测波长：218nm；流动相A为含0.1％甲酸的乙腈；流动相C为含0.1％甲酸的超纯水，％为体积百分比；液相梯度洗脱程序为：0min(25％A，75％C)，4min(40％A，60％C)，15min(42％A，58％C)，16min(100％A，0％C)，21min(5％A，95％C)，30min(25％A，75％C)流速：0.6mL/min；进样量：10μL。
3)结果
如图5所示，根据液相检测结果进行分析计算，以蔗糖为糖基供体双酶法催化莱鲍迪苷A反应3h后，莱鲍迪苷A几乎全部转化为莱鲍迪苷M，转化率高达95％，无副产物生成。
以上实施例仅为本发明较优的实施方式，仅用于解释本发明，而非限制本发明，本领域技术人员在未脱离本发明精神实质下所作的改变、替换、修饰等均应属于本发明的保护范围。

Claims (10)

  1. 一种利用生物酶法高效制备莱鲍迪苷M的方法，其特征在于，包括如下步骤：
    将莱鲍迪苷A、尿苷二磷酸葡萄糖、糖基转移酶、盐加入缓冲液中，混合均匀，得到混合液，在搅拌状态下进行酶催化反应，得到所述莱鲍迪苷M。
  2. 根据权利要求1所述的利用生物酶法高效制备莱鲍迪苷M的方法，其特征在于，在所述混合液中，莱鲍迪苷A的浓度为1g/L-100g/L；在所述混合液中，尿苷二磷酸葡萄糖的浓度为0.5mM-20mM；所述盐为钙盐、钴盐、铁盐、镁盐、锰盐、铵盐、镍盐、锌盐中的一种以上；在所述混合液中，盐的浓度为1-10mM。
  3. 根据权利要求2所述的利用生物酶法高效制备莱鲍迪苷M的方法，其特征在于，所述盐为钙盐、镁盐、锰盐、铁盐中的一种及以上；在所述混合液中，盐的浓度为0.1-5mM。
  4. 根据权利要要求1所述的利用生物酶法高效制备莱鲍迪苷M的方法，其特征在于，在所述混合液中，所述缓冲液的pH为5.0-9.0；所述在搅拌状态下的搅拌速率为50-220rpm；所述酶催化反应的温度为20-45℃，所述酶催化反应的时间为1-5h。
  5. 根据权利要求1所述的利用生物酶法高效制备莱鲍迪苷M的方法，其特征在于，在所述混合液中，所述糖基转移酶的浓度为0.1-20g/L；所述糖基转移酶的序列为如SEQ ID NO.1所示、如SEQ ID NO.2所示、如SEQ ID NO.3所示、如SEQ ID NO.4所示、如SEQ ID NO.5所示、如SEQ ID NO.6所示、如SEQ ID NO.7所示、如SEQ ID NO.8所示、如SEQ ID NO.9所示、如SEQ ID NO.10所示、如SEQ ID NO.11所示及如SEQ ID NO.12所示的氨基酸序列中的一种以上。
  6. 一种利用生物酶法高效制备莱鲍迪苷M的方法，其特征在于，包括如下步骤：
    将莱鲍迪苷A、蔗糖、5-尿苷二磷酸二钠盐、糖基转移酶、蔗糖合成酶、盐加入缓冲液中，混合均匀，得到混合液，在搅拌状态下进行酶催化反应，得到所述莱鲍迪苷M。
  7. 根据权利要求6所述的利用生物酶法高效制备莱鲍迪苷M的方法，其特征在于，在所述混合液中，莱鲍迪苷A的浓度为1-100g/L；在所述混合液中，蔗糖的浓度为1-500mM；在所述混合液中，5-尿苷二磷酸二钠盐的浓度为0.1-20mM；所述盐为钙盐、钴盐、铁盐、镁盐、锰盐、铵盐、镍盐、锌盐中的一种以上；在所述混合液中，盐的浓度为0.1-10mM；所述缓冲液的pH为5.0-9.0；所述在搅拌状态下的搅拌速率为50-220rpm；所述酶催化反应的温度为20-45℃，所述酶催化反应的时间为1-10h。
  8. 根据权利要求7所述的利用生物酶法高效制备莱鲍迪苷M的方法，其特征在于，所述盐为钙盐、镁盐、锰盐、铁盐中的一种以上；在所述混合液中，盐的浓度为0.1-5mM；所述缓冲液的pH为6.0-8.0；所述酶催化反应的温度为30-37℃。
  9. 根据权利要求6所述的利用生物酶法高效制备莱鲍迪苷M的方法，其特征在于，在所述混合液中，所述糖基转移酶的浓度为0.1-20g/L；所述糖基转移酶的序列为如SEQ ID NO.1所示、如SEQ ID NO.2所示、如SEQ ID NO.3所示、如SEQ ID NO.4所示、如SEQ ID NO.5所示、如SEQ ID NO.6所示、如SEQ ID NO.7所示、如SEQ ID NO.8所示、如SEQ ID NO.9所示、如SEQ ID NO.10所示、如SEQ ID NO.11所示及如SEQ ID NO.12所示的氨基酸序列中的一种以上。
  10. 根据权利要求6所述的利用生物酶法高效制备莱鲍迪苷M的方法，其特征在于，在所述混合液中，所述蔗糖合成酶的浓度为0.1-10g/L；所述蔗糖合成酶的序列为如SEQ ID NO.13所示、如SEQ ID NO.14所示、如SEQ ID NO.15所示、如SEQ ID NO.16所示、如SEQ ID NO.17所示、如SEQ ID NO.18所示、如SEQ ID NO.19所示及如SEQ ID NO.20所示的氨基酸序列中的一种以上。
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CN114921434A (zh) * 2022-05-27 2022-08-19 中化健康产业发展有限公司 催化Reb A生产Reb M的重组糖基转移酶

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