Preparation method of uridine diphosphate glucuronic acid
阅读说明：本技术 一种尿苷二磷酸葡萄糖醛酸的制备方法 (Preparation method of uridine diphosphate glucuronic acid ) 是由 何丽丽 窦文芳 吕海超 于 2020-12-30 设计创作，主要内容包括：本发明涉及一种尿苷二磷酸葡萄糖醛酸的制备方法。本发明利用尿苷二磷酸葡萄糖脱氢酶氧化尿苷二磷酸葡萄糖生成尿苷二磷酸葡萄糖醛酸,同时加入乳酸脱氢酶进行双酶偶联反应,实现NAD～+的循环再生,减少NAD～+的添加,节约了成本,与此同时NAD～+/NADH的循环减少副产物NADH对产物尿苷二磷酸葡萄糖醛酸的反馈抑制作用,目标产物的生成量可达到1.01mg/mL。本发明对乳酸脱氢酶的核苷酸序列进行优化改造,使得目的蛋白能更好的在大肠杆菌中表达,有利于高效合成目标产物尿苷二磷酸葡萄糖醛酸。本发明采用体外反应,反应体系更加可控,相比胞内反应,体外反应可在限定条件下更利于反应的进行。(The invention relates to a preparation method of uridine diphosphate glucuronate. The invention utilizes uridine diphosphate glucose dehydrogenase to oxidize uridine diphosphate glucose to generate uridine diphosphate glucuronic acid, and simultaneously adds lactate dehydrogenase to carry out a double-enzyme coupling reaction to realize NAD + Cyclic regeneration of, reducing NAD + While at the same time NAD saves cost + The feedback inhibition effect of the byproduct NADH on the product uridine diphosphate glucuronic acid is reduced by the circulation of NADH, and the generation amount of the target product can reach 1.01 mg/mL. The invention optimizes and reforms the nucleotide sequence of the lactate dehydrogenase, leads the target protein to be better expressed in escherichia coli, and is beneficial to efficiently synthesizing the target productUridine diphosphate glucuronate. The invention adopts in vitro reaction, the reaction system is more controllable, and compared with intracellular reaction, the in vitro reaction can be more beneficial to the reaction under the limited condition.)
1. A method for preparing uridine diphosphate glucuronate, which is characterized by comprising the following steps: uridine diphosphate glucose and NAD+Under the coupling action of uridine diphosphate glucose dehydrogenase and lactate dehydrogenase, carrying out an in vitro catalytic reaction to obtain uridine diphosphate glucuronic acid.
2. The method according to claim 1, wherein the amino acid sequence of uridine diphosphate glucose dehydrogenase is represented by SEQ ID NO. 1.
3. The method according to claim 1, wherein the lactate dehydrogenase has an amino acid sequence represented by SEQ ID NO. 2.
4. The method according to claim 1, wherein the uridine diphosphate glucose dehydrogenase is added in an amount of not less than 200 mg/L.
5. The method according to claim 1, wherein the lactate dehydrogenase is added in an amount of not less than 80 mg/L.
6. The method of claim 1, wherein the system for catalyzing the reaction further comprises Tris buffer, NAD+Beta-mercaptoethanol and sodium pyruvate.
7. The method according to claim 6, wherein the Tris buffer has a pH of 7 to 11.
8. The method of claim 6, wherein the NAD is present in a predetermined amount+The concentration is 1.0 mM-3.0 mM.
9. The method according to claim 6, wherein the β -mercaptoethanol is 1 μ L/mL, and the concentration of sodium pyruvate is 50 mM-300 mM.
10. The preparation method according to claim 1, wherein the reaction temperature of the catalytic reaction is 15-35 ℃ and the reaction time is 6-18 h.
Technical Field
The invention relates to the technical field of enzyme catalysis, in particular to a preparation method of uridine diphosphate glucuronate.
Background
Nucleotide sugars are commonly used as high-energy forms activated by monosaccharides in nature, such as uridine diphosphate (UDP-sugar), thymidyldiphosphate (TDP-sugar), guanosine diphosphate (GTP-sugar), and the like. Particularly uridine diphosphate sugars are most widely used, UDP-glucose being the starting substrate for the formation of other UDP-monosaccharides, and UDP-glucuronic acid being the key substrate for the conversion of UDP-monosaccharides from six-carbon sugars to five-carbon sugars.
The derivative UDP-glucuronic acid obtained by dehydrogenation of UDP-glucose is an important glycosyl donor in cells and participates in a plurality of metabolic pathways of organisms. In microbial cells, UDP-glucuronic acid is a key synthetic substrate for capsular polysaccharides, and the synthesis of capsular polysaccharides directly affects the pathogenic properties of pathogenic microorganisms. In plant cells, UDP-glucuronic acid, as a precursor of nucleotides, is of great importance in the biosynthesis of pectin and hemicellulose polymers, and inseparable in the biosynthesis of cell walls. In animal cells, UDP-glucuronic acid also has an indispensable role in the synthesis of metabolites such as mucopolysaccharides, hyaluronic acid and heparin, and is also involved in glycosylation of a part of the metabolites.
Because the current synthesis difficulty is high and the price is high, the exploration of a method for efficiently synthesizing UDP-glucuronic acid becomes a research hotspot. The current UDP-glucuronic acid methods mainly include chemical methods and biological methods. The chemical method is often long in synthesis time, low in synthesis efficiency, expensive in cost, poor in stereoselectivity and not beneficial to industrial development, while the enzymatic reaction is mild in conditions, strong in selectivity, low in energy consumption and environment-friendly in process, which is incomparable with chemical synthesis. UGD of an escherichia coli source is subjected to recombinant expression and optimization in the morning, UGPase and UGD are added into the system, two-step coupling catalytic reaction is carried out to synthesize UDP-glucuronic acid, UGPase enzyme takes UTP as a substrate to catalytically synthesize UDP-glucose, UGD takes UDP-glucose as a substrate to catalytically synthesize UDP-glucuronic acid, and the substrate conversion rate is found to be not ideal. The Jixiaohu takes fresh cattle liver as a raw material, a stepped salting-out-thermal denaturation method is adopted to prepare a UGD crude product, and a commercially available rabbit-muscle lactic dehydrogenase one-pot enzyme method is utilized to synthesize UDP-glucuronic acid, although the UDP-glucuronic acid has better advantages in the aspects of yield and byproducts than the commercially available rabbit-muscle lactic dehydrogenase one-pot enzyme method, the commercially available rabbit-muscle lactic dehydrogenase is adopted, the raw material cost is higher, and the UGD crude enzyme solution needs to be extracted from the fresh cattle liver, so that the preparation process is more complex.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of uridine diphosphate glucuronate, UGD enzyme of streptococcus pyogenes source is used as a catalyst and coupled with LDH enzyme of pig source, and the dual-enzyme coupling catalyzes UDP-glucose to be oxidized into UDP-glucuronate, so that the method is a simple and efficient biological catalysis method.
The first object of the present invention is to provide a method for preparing uridine diphosphate glucuronate, which comprises the following steps: uridine diphosphate glucose and NAD+Under the coupling action of uridine diphosphate glucose dehydrogenase and lactate dehydrogenase, carrying out an in vitro catalytic reaction to obtain uridine diphosphate glucuronic acid.
Furthermore, the amino acid sequence of the uridine diphosphate glucose dehydrogenase is shown in SEQ ID NO. 1.
Furthermore, the amino acid sequence of the lactate dehydrogenase is shown as SEQ ID NO. 2.
The nucleotide sequence of the uridine diphosphate glucose dehydrogenase is shown as SEQ ID No.3, the nucleotide sequence of the lactate dehydrogenase is shown as the sequence shown as SEQ ID No.4, the sequence shown as SEQ ID No.5 is obtained through codon optimization, and codons which are low in prokaryotic bioavailability and cannot be expressed in the sequence are changed into codon sequences with higher escherichia coli utilization rate, so that the target protein can be better expressed in an escherichia coli system.
Furthermore, the addition amount of the uridine diphosphate glucose dehydrogenase is not less than 200 mg/L.
Furthermore, the addition amount of the lactate dehydrogenase is not less than 80 mg/L.
Further, a system for catalyzing the reaction also comprises Tris buffer solution and NAD+Beta-mercaptoethanol and sodium pyruvate.
Further, the pH value of the Tris buffer solution is 7-11.
Further, the NAD+The concentration is 1.0 mM-3.0 mM.
Furthermore, the beta-mercaptoethanol is 1 mu L/mL, and the concentration of the sodium pyruvate is 50 mM-300 mM.
Further, the reaction temperature of the catalytic reaction is 15-35 ℃, and the reaction time is 6-18 h.
By the scheme, the invention at least has the following advantages:
the invention utilizes uridine diphosphate glucose dehydrogenase to oxidize uridine diphosphate glucose to generate uridine diphosphate glucuronic acid, and simultaneously adds lactate dehydrogenase to carry out a double-enzyme coupling reaction to realize NAD+Cyclic regeneration of, reducing NAD+While at the same time NAD saves cost+The feedback inhibition effect of the byproduct NADH on the product uridine diphosphate glucuronic acid is reduced by the circulation of NADH, and the generation amount of the target product can reach 1.01 mg/mL. The invention optimizes and reforms the nucleotide sequence of lactate dehydrogenase, so that the target protein can be better expressed in escherichia coli, and the high-efficiency synthesis of the target product uridine diphosphate glucuronic acid is facilitated. The invention adopts in vitro reaction, the reaction system is more controllable, and compared with intracellular reaction, the in vitro reaction can be more beneficial to the reaction under the limited condition.
The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.
Drawings
FIG. 1 is a liquid phase map of uridine diphosphate glucose production using an immobilized enzyme;
FIG. 2 is a liquid phase diagram of UDP-glucose;
FIG. 3 is NAD+Liquid phase spectrum of (a);
FIG. 4 is a liquid phase diagram of NADH;
FIG. 5 is a liquid phase spectrum of a uridine diphosphate glucuronic acid standard;
FIG. 6 shows uridine diphosphate glucose and NAD+Liquid phase atlas of single enzyme reaction liquid;
FIG. 7 shows uridine diphosphate glucose and NAD+Liquid phase atlas of double enzyme coupling reaction liquid;
FIG. 8 is a mass spectrum of a uridine diphosphate glucuronate standard;
FIG. 9 is a mass spectrum of the reaction product uridine diphosphate glucuronate;
FIG. 10 is a nuclear magnetic hydrogen spectrum of the reaction product uridine diphosphate glucuronate;
FIG. 11 shows the catalysis of uridine diphosphate glucose and NAD without lactate dehydrogenase+Liquid phase atlas of the reaction solution;
FIG. 12 is a graph of the catalysis of uridine diphosphate glucose and NAD without beta-mercaptoethanol+Liquid phase atlas of reaction liquid.
Detailed Description
The following examples are given to further illustrate the embodiments of the present invention. The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Example 1: preparation of UDP-glucose
Weighing 50g of anion exchange resin in a 250mL beaker, adding 50mL of 10% isopropanol, soaking for 20min, carrying out suction filtration, leaching with 10% isopropanol, leaching with deionized water, carrying out suction filtration, leaching with 0.5M HCl, carrying out suction filtration, suspending 45mL of 0.5M HCl, soaking for 2h, carrying out suction filtration, leaching with deionized water, and stirring 45mL of CoCl2 aqueous solution overnight. The 50mM phosphate buffer pH7.5 was rinsed clean for use. Preparing 20mM Tris buffer solution, adjusting the pH value to 7.0, uniformly mixing the volume of the enzyme solution and the volume of the buffer solution according to a ratio of 1:4, putting 194mg of purified protein GlmU enzyme and 3mg of PPA enzyme into a triangular flask, respectively adding 21.5g and 0.15g of treated resin, putting the mixture into a shaking table, rotating at a speed of 120rpm, keeping the temperature at 25 ℃ and keeping the time for 1 h. Suction filtration, washing with 50mM phosphate buffer (pH7.5), and standing at 4 deg.C for further use.
2.16g of glucose and 3.05g of ATP were weighed and placed in a triangular flask, 25mL of 1M Tris buffer and 0.5mL of 1M MgCl2 solution were added in this order, mixed and dissolved, the pH was adjusted to 7.5, 20mL of 3.9g/L NaHK enzyme was added, and the reaction was carried out at 30 ℃ and 100rpm overnight.
2.1g of UTP, 42. mu.L of DTT, and 0.28mL of 1M MgCl2 were added to the reaction mixture in this order, and after mixing, the immobilized GlmU enzyme and PPA enzyme were added and reacted at 30 ℃ and 180rpm overnight. UDP-glucose was detected by passing 100. mu.L of the reaction mixture through a 0.22 μm pore size aqueous membrane and analyzing the reaction mixture by high performance liquid chromatography (see FIG. 1).
Example 2: single enzyme catalysis UDP-glucose oxidation to UDP-glucuronic acid
Respectively mixing 2.0mL of 10mM UDPG, 2.5M of 10mM NAD, 100 μ L of 1M Tris, 10 μ L of beta-mercaptoethanol, 20 μ L of 1M MgCl2 and 3.37mL of deionized water uniformly, then adding 2.5mL of 1.0mg/mL UGD enzyme solution, and reacting at the optimum temperature of 25 ℃ overnight; 100. mu.L of the reaction solution was passed through a 0.22 μm-pore aqueous filter, and high performance liquid chromatography was performed (as shown in FIG. 6), and UDP-glucose and NAD as reaction substrates were added+And performing high performance liquid chromatography (as shown in FIGS. 2, 3, 4 and 5) on the NADH and UDP-glucuronic acid products, wherein FIG. 6 shows that new substances NADH and UDP-glucuronic acid are generated, and the amount of UDP-glucuronic acid generated is about 0.51 mg/mL.
Example 3: double-enzyme coupling catalysis UDP-glucose oxidation to generate UDP-glucuronic acid
Respectively mixing 2.4mL of 10mM UDPG, 1.5mL of 10mM NAD, 100 μ L of 1M Tris, 10 μ L of beta-mercaptoethanol, 20 μ L of 1M MgCl2, 1mL of 1M sodium pyruvate and 1.97mL of deionized water uniformly, then sequentially adding 2.5mL of 1.0mg/mL UGD enzyme solution and 0.5mL of 2.7mg/mL LDH enzyme solution, and reacting at the optimum temperature of 25 ℃ overnight; when 100. mu.L of the reaction solution was passed through a 0.22 μm pore size aqueous membrane and subjected to HPLC analysis, UDP-glucuronic acid production was detected with a yield of about 1.01mg/mL, which was nearly doubled (see FIG. 7).
Respectively carrying out mass spectrum detection on a UDP-glucuronic acid standard product and a reaction product UDP-glucuronic acid, and carrying out LC-MS analysis by adopting WATERS MALDI SYNAPT MS, wherein as shown in figures 8 and 9, the detection result of a sample is consistent with that of the UDP-glucuronic acid standard product, and the molecular weight is 579.0 and the actual molecular weight is 580.0 because a negative ion source is adopted for detection.
To further determine the structure of UDP-glucuronic acid, the purified lyophilized product was deuteratedDissolving in water, and further preparing1And (4) detecting by using H NMR (nuclear magnetic resonance),1h NMR spectra were determined using a Bruker Avance iii 400MHZ nuclear magnetic resonance apparatus (TMS is an internal standard) and the results are shown in fig. 10, 1H NMR (400MHZ, Deuterium Oxide) δ 7.97(d, J ═ 8.2Hz,1H),6.00(s,1H),5.63(dd, J ═ 7.6,3.5Hz,1H),4.38(t, J ═ 4.2Hz,2H),4.30(p, J ═ 2.7Hz,1H),4.23(dd, J ═ 4.5,2.4Hz,1H),4.20(dd, J ═ 5.5,2.8Hz,1H),4.15(d, J ═ 10.2Hz,1H),3.79(t, J ═ 9.5Hz,1H),3.74(s,1H),3.58 (s, 3.8 Hz,1H), 3.47 (d, 3.47H, 1H).
Comparative example 1:
UDP-glucuronic acid was prepared as in example 3, except that lactate dehydrogenase was not added to the system. 100 μ L of the reaction mixture was passed through a 0.22 μm pore size aqueous membrane and analyzed by high performance liquid chromatography, and the results are shown in FIG. 11. The yield was about 0.52mg/mL in terms of the peak area of the product without adding lactate dehydrogenase. The yield of UDP-glucuronic acid is obviously improved by about 1 time after the lactate dehydrogenase is added, which shows that the lactate dehydrogenase can reduce the feedback inhibition effect of a high-energy product NADH on the product, thereby being more beneficial to the forward direction of the reaction.
Comparative example 2:
UDP-glucuronic acid was prepared as in example 3, with the only difference that no beta-mercaptoethanol was added to the system. 100 μ L of the reaction solution was passed through a 0.22 μm pore size aqueous membrane and subjected to high performance liquid chromatography, and the results are shown in FIG. 12. According to the conversion of the peak area of the product, the yield is about 0.22mg/mL under the condition of not adding beta-mercaptoethanol. The beta-mercaptoethanol antioxidant can play a certain role in protecting the substrate.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
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