Method for synthesizing fructose 1, 6-diphosphate by enzyme method

Abstract

The invention relates to the field of enzyme catalysis production of fructose 1, 6-diphosphate, in particular to a method for synthesizing fructose 1, 6-diphosphate by an enzyme method. According to the method, glucose is used as a substrate, ATP and acetyl phosphoric acid are used as phosphorus source donors, a multienzyme system is used for reaction, and 1, 6-fructose diphosphate is synthesized through catalysis. The method specifically comprises the following steps: adding glucose, magnesium chloride, ATP and acetyl phosphoric acid into a reaction container, and uniformly mixing; then adjusting the pH value to 6.0-8.0, adding glucokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase and acetokinase, and carrying out stirring reaction at 25-40 DEG C for 1-3 hours, so as to synthesize the 1, 6-fructose diphosphate. The synthesis method is simple to operate, short in production period, easy to separate and purify, low in production cost and gt in conversion efficiency; therefore, the method has an important industrial application prospect.

Description

Technical Field

[0001] The invention relates to the field of enzyme-catalyzed production of fructose-1,6-diphosphate, and in particular to a method for synthesizing fructose-1,6-diphosphate by enzymatic method. Background Art

[0002] Fructose 1,6-diphosphate is an intermediate product of intracellular energy metabolism and plays a very important role in energy metabolism. It can improve myocardial ischemia and can be used as an auxiliary treatment for angina pectoris, acute myocardial infarction, heart failure and arrhythmia. It can also promote liver metabolism and improve liver function. It can be used in the symptomatic treatment of hepatitis and reduce liver cell damage. In addition, it can regulate blood sugar, promote insulin secretion and improve the symptoms of diabetes.

[0003] At present, the synthesis method of fructose 1,6-diphosphate is mainly prepared by microbial conversion, using the enzyme system in the microorganism to react glucose or sucrose with inorganic phosphate to generate fructose 1,6-diphosphate, mainly including yeast, thermophilic Bacillus and filamentous fungi. In the production process, it is necessary to add chemical reagents or change the permeability of the cell membrane through mechanical cell wall breaking, which is cumbersome to operate and pollutes the environment. At the same time, there are also problems such as low product concentration, low conversion rate and difficult separation. Therefore, it is necessary to develop a new method for enzymatic synthesis of fructose 1,6-diphosphate that is green, efficient and low in production cost. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for synthesizing fructose 1,6-diphosphate by enzymatic method in view of the deficiencies of the prior art. The specific reaction synthesis pathway of the method is as follows: Figure 1 shown.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A method for synthesizing fructose 1,6-diphosphate by an enzymatic method, using glucose as a substrate, ATP and acetyl phosphate as phosphorus source donors, magnesium chloride as a magnesium ion donor, utilizing glucokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase to carry out a catalytic reaction, and synthesizing fructose 1,6-diphosphate;

[0007] The glucose kinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase are obtained by heterologous expression in Escherichia coli, respectively.

[0008] The glucokinase is derived from Saccharomyces cerevisiae, and its amino acid sequence is shown in SEQ ID NO.1, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0009] The glucose-6-phosphate isomerase is derived from Saccharomyces cerevisiae, and its amino acid sequence is shown in SEQ ID NO.3, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.4.

[0010] The 6-phosphofructokinase 1 is derived from Saccharomyces cerevisiae, and its amino acid sequence is shown in SEQ ID NO.5, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.6.

[0011] The acetate kinase is derived from Citrobacter freundii, and its amino acid sequence is shown in SEQ ID NO.7, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.8.

[0012] The gene sequences of the above-mentioned glucose kinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase were all commissioned to General Biotechnology (Anhui) Co., Ltd. for full gene synthesis. To facilitate subsequent cloning, six bases of CATATG were added to the 5′ end of the gene fragment to form an NdeI restriction site, and six bases of CTCGAG were added to the 3′ end to form an XhoI restriction site.

[0013] The glucose kinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase are obtained by heterologous expression in Escherichia coli, specifically, the nucleotide sequences of the glucose kinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase genes of the present invention are respectively connected to various commercially available empty vectors to obtain recombinant expression vectors, which are then transformed into Escherichia coli.

[0014] Specifically, the commercially available empty vector can be any conventional plasmid vector in the art, as long as the recombinant expression vector can replicate normally in the corresponding expression host and express the glucose kinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase.

[0015] In some embodiments of the present invention, the commercially available empty vector is a pET28a vector for the host Escherichia coli BL21 (DE3).

[0016] The catalytic reaction has a reaction system of: 100-300 mM glucose, 1-5 mM ATP, 10-100 mM magnesium chloride, 200-700 mM acetyl phosphate, 1-5 g / L glucose kinase, 2-10 g / L glucose-6-phosphate isomerase, 1-5 g / L 6-phosphofructokinase 1 and 0.1-1 g / L acetate kinase.

[0017] Preferably, the reaction system of the catalytic reaction is: 100-200 mM glucose, 1-2 mM ATP, 60-100 mM magnesium chloride, 300-500 mM acetyl phosphate, 1-2.5 g / L glucose kinase, 5-6 g / L glucose-6-phosphate isomerase, 1-2.5 g / L 6-phosphofructokinase 1 and 0.1-0.5 g / L acetate kinase.

[0018] More preferably, the reaction system of the catalytic reaction is: 200 mM glucose, 2 mM ATP, 100 mM magnesium chloride, 460 mM acetyl phosphate, 2.5 g / L glucose kinase, 5 g / L glucose-6-phosphate isomerase, 2.5 g / L 6-phosphofructokinase 1 and 0.5 g / L acetate kinase.

[0019] The catalytic reaction is carried out under the following conditions: 25-40°C, pH 6.0-8.0 for 1-3 hours.

[0020] Specifically, glucose, magnesium chloride, ATP and acetyl phosphate are uniformly mixed in a reaction container, the pH is adjusted to 6.0-8.0, and then glucose kinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase are added at 25-40° C. to carry out a catalytic reaction to synthesize fructose 1,6-diphosphate.

[0021] Beneficial effects: The present invention provides a method for synthesizing fructose 1,6-diphosphate by enzymatic method, which introduces the ATP energy cycle system, uses acetyl phosphate as a phosphate and energy donor, uses acetate kinase to regenerate ATP, and only needs to add a very small amount of ATP, which greatly reduces the cost. In addition, the substrates glucose and acetyl phosphate are converted into fructose 1,6-diphosphate by enzymatic method, and the conversion rate is >99.5%, which has the advantages of simple steps, short production cycle, easy separation and purification, and low production cost. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The present invention will be further described in detail below with reference to the accompanying drawings, and the above and / or other advantages of the present invention will become more clear.

[0023] Figure 1 It is the reaction pathway for the enzymatic synthesis of fructose 1,6-diphosphate. DETAILED DESCRIPTION

[0024] The present invention will be further described in detail below in conjunction with specific implementations, and the above and / or other advantages of the present invention will become more clear.

[0025] The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials described are commercially available unless otherwise specified.

[0026] Example 1: Construction of catalytic enzyme strains

[0027] The genes of glucose kinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase were all entrusted to General Biotechnology (Anhui) Co., Ltd. for full gene synthesis. To facilitate subsequent cloning, six bases of CATATG were added to the 5′ end of the gene fragment to form an NdeI restriction site, and six bases of CTCGAG were added to the 3′ end to form an XhoI restriction site.

[0028] Among them, the amino acid sequence of the glucose kinase is shown in SEQ ID NO.1, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.2; the amino acid sequence of the glucose-6-phosphate isomerase is shown in SEQ ID NO.3, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.4; the amino acid sequence of the 6-phosphofructokinase 1 is shown in SEQ ID NO.5, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.6; the amino acid sequence of the acetate kinase is shown in SEQ ID NO.7, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.8.

[0029] The synthesized glucokinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase genes and pET28a vectors were double-digested with NdeI and XhoI (purchased from TaKaRa), and the fragments were recovered using a DNA gel kit (purchased from TaKaRa). The gene fragments were connected to the linearized pET28a vector, and the connection system was as follows: 2 μL DNA fragment purification product, 3 μL linearized pET28a vector, 5 μL Solution I was reacted at 16 ° C for 2 h to obtain a recombinant expression vector, and then the recombinant expression vector was transformed into Escherichia coli BL21 (DE3) competent cells, and cultured at 37 ° C for 12 hours on LB solid plates (containing kanamycin at a final concentration of 50 mg / L) to obtain clones. Three clones were picked and cultured in LB medium (containing kanamycin at a final concentration of 50 mg / L) at 37 ° C and 200 rpm for 12 hours and sent for sequencing to obtain recombinant Escherichia coli with correct sequencing.

[0030] Example 2: Expression and fragmentation of catalytic enzymes

[0031] The recombinant E. coli colonies with correct sequencing were inoculated into 20 mL LB medium (containing kanamycin at a final concentration of 50 mg / L) and cultured at 37°C, 200 rpm for 12 hours. Then, the inoculum was transferred to 1 L TB medium (containing kanamycin at a final concentration of 50 mg / L) at a 1% v / v inoculum and cultured at 37°C, 200 rpm until OD 600 When the pH value was 0.6-0.8, IPTG was added at a final concentration of 0.2 mM, and the induction culture was continued at 28°C and 150 rpm for 12-16 h. The culture solution was centrifuged at 8000 rpm for 10 min, and the cells of each recombinant Escherichia coli were collected and stored in a -80°C refrigerator.

[0032] Weigh 2g of bacteria into 50mL centrifuge tubes, wash twice with 0.1mol / L PBS (pH7.5) solution, collect bacteria by centrifugation, add 20mL of 0.1mol / L PBS (pH7.5) solution to resuspend to obtain 100g / L bacterial solution, and use ultrasonic disruptor to disrupt cells in ice water bath. The instrument parameters are set to power 200W, ultrasonic 3s pause 7s, about 40min until the bacterial solution is clear. After the end, centrifuge at 8000rpm for 10min at 4℃, and the supernatant in the centrifuge tube is the crude enzyme solution. Finally, crude enzyme solutions of glucose kinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase are obtained.

[0033] Example 3: Synthesis of fructose 1,6-diphosphate

[0034] The catalytic reaction system for enzymatic synthesis of fructose 1,6-diphosphate includes: 100-300 mM glucose, 1-5 mM ATP, 10-100 mM magnesium chloride, 200-700 mM acetyl phosphate, 1-5 g / L glucose kinase, 2-10 g / L glucose-6-phosphate isomerase, 1-5 g / L 6-phosphofructokinase 1 and 0.1-1 g / L acetate kinase; the reaction conditions are: 25-40°C, pH 6.0-8.0 for 1-3 hours.

[0035] The addition amount of each component in the reaction system was optimized, and the optimization results are shown in Table 1. From the results in the table, it can be seen that the catalytic effect of reaction group 2 is the best, followed by reaction group 1. The specific catalytic reaction system of reaction group 2 is that in a 50mL reaction system, 200mM glucose, 2mM ATP, 100mM magnesium chloride, and 460mM acetyl phosphate are added respectively, and after adjusting the pH to 7.5, crude enzyme solution with a final concentration of 2.5g / L glucose kinase, 5g / L glucose-6-phosphate isomerase, 2.5g / L 6-phosphofructokinase 1 and 0.5g / L acetate kinase is added at 37°C, and 1,6-diphosphate fructose is obtained after reacting for 2h. The concentration of the product 1,6-diphosphate fructose is measured to be 67.8g / L, and the conversion rate is 99.7%.

[0036] The determination method of fructose 1,6-diphosphate is as follows: high performance liquid chromatography is used, using a differential refractometer and AMINEX HPX-87H, a 300mm×7.8mm chromatographic column, a 5mM dilute sulfuric acid aqueous solution as the mobile phase, a flow rate of 0.6mL / min, a column temperature of 60°C, and a detection cell temperature of 35°C.

[0037] Table 1 Optimization of synthesis of fructose 1,6-diphosphate

[0038]

[0039] The present invention provides a method and idea for synthesizing fructose 1,6-diphosphate by enzymatic method. There are many methods and approaches to realize the technical solution. The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the protection scope of the present invention. All components not specified in this embodiment can be realized by existing technologies.

Claims

1. A method for enzymatically synthesizing fructose 1,6-diphosphate, characterized in that: With glucose as substrate, ATP and acetyl phosphate as phosphorus donors, magnesium chloride as magnesium ion donor, glucose kinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase are used to carry out catalytic reaction to synthesize fructose 1,6-diphosphate; The glucose kinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase are obtained by heterologous expression in Escherichia coli, respectively.

2. The method according to claim 1, characterized in that The amino acid sequence of the glucose kinase is shown in SEQ ID NO.1, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

3. The method according to claim 1, characterized in that The amino acid sequence of the glucose-6-phosphate isomerase is shown in SEQ ID NO.3, and the nucleotide sequence of the corresponding encoding gene is shown in SEQ ID NO.

4.

4. The method according to claim 1, characterized in that: The amino acid sequence of the 6-phosphofructokinase 1 is shown in SEQ ID NO.5, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.

6.

5. The method according to claim 1, characterized in that The amino acid sequence of the acetate kinase is shown in SEQ ID NO.7, and the corresponding nucleotide sequence of the encoding gene is shown in SEQ ID NO.

8.

6. The method according to claim 1, characterized in that The catalytic reaction has a reaction system comprising: 100-300 mM glucose, 1-5 mM ATP, 10-100 mM magnesium chloride, 200-700 mM acetyl phosphate, 1-5 g / L glucose kinase, 2-10 g / L glucose-6-phosphate isomerase, 1-5 g / L 6-phosphofructokinase 1 and 0.1-1 g / L acetate kinase.

7. The method according to claim 6, characterized in that The catalytic reaction has a reaction system comprising: 100-200 mM glucose, 1-2 mM ATP, 60-100 mM magnesium chloride, 300-500 mM acetyl phosphate, 1-2.5 g / L glucose kinase, 5-6 g / L glucose-6-phosphate isomerase, 1-2.5 g / L 6-phosphofructokinase 1 and 0.1-0.5 g / L acetate kinase.

8. The method according to claim 7, characterized in that The catalytic reaction has a reaction system of: 200 mM glucose, 2 mM ATP, 100 mM magnesium chloride, 460 mM acetyl phosphate, 2.5 g / L glucose kinase, 5 g / L glucose-6-phosphate isomerase, 2.5 g / L 6-phosphofructokinase 1 and 0.5 g / L acetate kinase.

9. The method according to claim 1, characterized in that: The catalytic reaction has the following reaction conditions: 25-40° C., pH 6.0-8.0, and reaction time of 1-3 hours.

10. The method according to any one of claims 6 to 9, characterized in that: Glucose, magnesium chloride, ATP and acetyl phosphate are uniformly mixed in a reaction container, and after adjusting the pH to 6.0-8.0, glucose kinase, glucose-6-phosphate isomerase, 6-phosphofructokinase 1 and acetate kinase are added at 25-40° C. to carry out catalytic reaction to synthesize fructose 1,6-diphosphate.

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