Method for synthesizing NADPH (nicotinamide adenine dinucleotide phosphate) by biological enzyme method

Abstract

The invention discloses a method for synthesizing NADPH (Nicotinamide Adenine Dinucleotide Phosphate) by a biological enzyme method, which comprises the following steps: adding a substrate NAD < + >, formic acid, formate dehydrogenase, NADH kinase and a buffer solution into a reaction kettle, catalyzing in vitro or in cells to generate NADPH, centrifugally separating the product, and taking the supernatant to obtain an NADH product solution. The target product can be obtained only through two steps, the yield is high, product separation is simple, cost is low, and the method is suitable for industrial production.

Description

Technical Field

[0001] The invention relates to the technical field of biosynthesis, and in particular to a method for synthesizing NADPH using a bioenzymatic method. Background Art

[0002] Biocatalysis uses enzymes as catalysts, which have the characteristics of high efficiency and product specificity, and is widely used in the synthesis of chiral drugs and their intermediates. However, most enzyme reduction processes require an equimolar amount of reduced coenzyme nicotinamide adenine dinucleotide (NADH) or its phosphorylated form (NADPH) as a hydrogen source, and the coenzyme is expensive. Currently, the synthesis of NADPH is mainly carried out by biological or chemical methods.

[0003] The mainstream synthesis method of NADPH reported in relevant research is mainly biological method, which mainly studies the regeneration system of NADPH and related technologies and processes in the process of metabolic synthesis reaction in cells. The production process of NADPH is rarely involved. The conventional synthesis route with the most industrial application and research is the route that uses NAD+ as the starting material, first converts it into NADP+, and then converts it into NADPH. On the one hand, the cost of NAD+ is relatively high, generally reaching several thousand yuan / kg. On the other hand, the yield of this route is not very ideal, generally only about 20%.

[0004] The chemical synthesis of NADPH mainly replaces the catalytic production with inorganic catalysts, and is still designed according to the idea of ​​coenzyme recycling regeneration. For example, patent CN117816244A has developed a supported metal nanoparticle catalyst that can convert NADP+ into NADPH.

[0005] At present, whether it is a biological preparation method or a chemical preparation method, the overall idea is still limited to the strengthening and improvement of the intracellular NADPH concentration, the strengthening of the recycling and regeneration of NADPH, and the intracellular production and intracellular utilization of NADPH. Since the metabolic network in the cell is undergoing thousands of chemical reactions all the time, the content or concentration of NADPH is always in a dynamic balance, which seriously limits the extraction efficiency of NADPH. The yield obtained is often extremely low, and the subsequent separation and purification is also difficult, and there is a high loss in the preparation process. In patent CN107557412A, NAD+ is used as the starting material, NADP+ is generated under the catalysis of NAD kinase, and then glucose is added to generate NADPH under the catalysis of glucose dehydrogenase. The conversion rate of NADPH is about 70%, and the purity is about 70%. When using this route to produce NADPH, glucose needs to be added as a reaction substrate during the period, which increases the difficulty of purifying the reaction product in the later stage.

[0006] The idea of ​​the chemical preparation method is simply to replace the inorganic catalyst and apply it to the mutual conversion between NADP+ and NADPH. It cannot be expanded to the single production of NADPH. Due to the high price of NADP+, this method cannot produce cost-effective NADPH products with commercial value.

[0007] Based on the current NADPH preparation methods, there is an urgent need to develop a cost-effective NADPH synthesis route that has cheap and readily available starting materials, simple operation, the shortest synthesis route, and is suitable for industrial production. Summary of the invention

[0008] The object of the present invention is to provide a method for synthesizing NADPH by bioenzymatic method, which can obtain the target product in only two steps, has high yield, simple product separation, low cost and is suitable for industrial production.

[0009] The technical solution adopted by the present invention to solve its technical problem is: A method for synthesizing NADPH in vitro by bioenzymatic method, wherein substrate NAD+, formic acid, formate dehydrogenase, NADH kinase and buffer are added into a reactor to catalyze the generation of NADPH in vitro or in cells, the product is separated by centrifugation, and the supernatant is taken to obtain a NADH product solution; the formic acid is added in the form of ammonium formate; The catalytic process is: Substrate NAD+ and formate are catalyzed by formate dehydrogenase to produce CO2 and NADH; NADH is catalyzed to generate NADPH with energy provided by NADH kinase and ATP.

[0010] In the reaction system, the amount of NAD+ is 1-100 g / L, the amount of ammonium formate is 30-60 g / L, the amount of formate dehydrogenase is 10-500 mg / L, and the amount of NADH kinase is 10-500 mg / L.

[0011] The pH of the reaction system is 6.5-7.8; the reaction temperature is 20-40° C.; and the reaction time is 10-20 hours.

[0012] Formate dehydrogenase and NADH kinase were measured based on the amount of wet bacteria, immobilized enzyme or immobilized cells.

[0013] The buffer is phosphate buffer or Tris-HCl buffer.

[0014] The ATP used is generated by the ATP cycle system, which includes ADP kinase, ATP for cycle start and sodium pyrophosphate, wherein the concentration of ADP kinase is 0.1-10 mg / L, the initial concentration of ATP for cycle start is 50-1000 mg / L, the initial concentration of sodium pyrophosphate is 50-1000 mg / L, and sodium pyrophosphate is added every 1.5-2 hours. The ATP cycle system is used to provide ATP during in vitro synthesis.

[0015] The ATP used is provided by the cells. When cells are used for the production of NADPH, the ATP is provided by the cells themselves.

[0016] Formate dehydrogenase and NADH kinase are both expressed and produced by an engineering strain of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Pichia pastoris.

[0017] The international system classification number of the formate dehydrogenase is EC 1.17.1.9; the international system classification number of NADH kinase is EC 2.7.1.86.

[0018] As a preferred embodiment, the formate dehydrogenase is obtained by enzyme modification of formate dehydrogenase EC 1.17.1.9, specifically, mutating the lysine K at position 121 of formate dehydrogenase EC 1.17.1.9 to serine S, mutating the leucine L at position 284 to phenylalanine F, and mutating the phenylalanine A at position 353 to cysteine ​​C; The NADH kinase is obtained by enzyme modification of NADH kinase EC 2.7.1.86, specifically, the 173rd amino acid of NADH kinase EC2.7.1.86 is mutated from leucine L to cysteine ​​C, the 235th amino acid is mutated from serine S to aspartic acid D, and the 268th amino acid is mutated from alanine A to phenylalanine F.

[0019] This patent uses a mutated enzyme, which has higher enzyme activity than the initial unmutated enzyme and a higher conversion rate for the catalytic reaction. The tolerance of the enzyme has been improved after enzyme evolution, making the reaction more stable.

[0020] The beneficial effects of the present invention are as follows: the NAD+ starting substrate used in the NADPH synthesis route of the present invention is cheap, the price is about 1,000 yuan / kg, and it has good economic applicability. The entire synthesis route only requires two steps to obtain the target product, the yield is high, and it is suitable for industrial production and application. The substrate of the present invention is replaced with formic acid, and the product is carbon dioxide. Compared with the chemical synthesis method and the biological extraction method, there are fewer impurities, and there is no need to add other substrates such as glucose, so the later separation and purification becomes extremely convenient and more environmentally friendly.

[0021] The in vitro enzymatic synthesis route of the present invention avoids the consumption of the target product by microbial cells and improves the overall yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is the synthetic route diagram of NADPH of the present invention; Figure 2 It is the SDS-PAGE electrophoresis spectrum of the biological enzyme used in the present invention, wherein 1 is NADH kinase; 2 is formate dehydrogenase; Figure 3 It is an agarose gel electrophoresis diagram of the biological enzyme gene sequence used in the present invention, wherein 1 is NADH kinase; 2 is formate dehydrogenase; Figure 4 This is the HPLC detection spectrum of the product of Example 2; Figure 5 This is the HPLC detection spectrum of the product of Example 3; Figure 6 This is the HPLC detection spectrum of the product of Example 4; Figure 7 This is the HPLC detection spectrum of the product of Example 5; Figure 8 This is the HPLC detection spectrum of the product of Example 6; Fig. 9 This is the HPLC detection spectrum of the product of Example 7; Fig.10 This is the HPLC detection spectrum of the product of Comparative Example 1; Fig.11 It is the HPLC detection spectrum of the product of Comparative Example 2. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described in detail below through specific embodiments.

[0024] In the present invention, unless otherwise specified, the raw materials and equipment used can be purchased from the market or are commonly used in the art. The methods in the following embodiments, unless otherwise specified, are all conventional methods in the art.

[0025] The NADPH synthesis route designed by the present invention is shown in the attached Figure 1 The relevant enzyme proteins are obtained by microbial fermentation. In the present invention, each enzyme expressed by the plasmid is connected to a His tag at its N-terminus for protein purification.

[0026] The relevant genes used in the present invention can be obtained by the PCR method well known in the biological field. The agarose nucleic acid electrophoresis diagram of the relevant key enzyme genes is shown in the attached Figure 3 .

[0027] The enzyme proteins used in the present invention are all obtained by microbial fermentation. The SDS-PAGE protein electrophoresis diagram of the relevant key enzymes is shown in the attached Figure 2 .

[0028] In the present invention, the formula of the Escherichia coli seed culture medium is: 12 g / L yeast extract, 24 g / L peptone, 4 mL / L glycerol, 16.43 g / L dipotassium hydrogen phosphate, and 2.31 g / L potassium dihydrogen phosphate.

[0029] In the present invention, all proteins are attached with His tags for protein purification operations. The tags, usage of the tags and protein purification methods are general methods well known in the art.

[0030] In the present invention, the LB medium formula used is applied to the culture of Escherichia coli and Bacillus subtilis. The LB liquid medium formula is: tryptone 10g / L, yeast extract 5g / L, NaCl 5g / L; the LB solid medium formula is: tryptone 10g / L, yeast extract 5g / L, NaCl 5g / L, agar 15g / L.

[0031] In the present invention, the fermentation medium used has the formula of: peptone: 10 g / L, yeast powder: 15 g / L, K2HPO4: 2 g / L. The fermentation medium is applied to the fermentation production of enzyme protein by Escherichia coli and Bacillus subtilis.

[0032] In the present invention, the YPD medium formula used is applied to the normal culture and fermentation culture of brewer's yeast. The YPD solid medium formula is: 10g / L yeast extract, 20g / L peptone, 20g / L glucose, 20g / L agar; the YPD liquid medium formula is: 10g / L yeast extract, 20g / L peptone, 20g / L glucose.

[0033] In the present invention, the MD medium formula used is applied to the normal culture of Pichia pastoris. MD plate (MD solid medium) formula: 13.4g / L YNB (yeast nitrogen source base, containing ammonium sulfate but not amino acids), 4×10-4g / L biotin, 20g / L glucose, 15g / L agar.

[0034] In the present invention, the point mutation method used to change a certain amino acid in the enzyme protein is a universal method in this field.

[0035] In the present invention, the biosynthesis of NADPH uses relatively cheap NAD+ as a starting material, and the target product NADPH can be obtained through two steps of enzyme catalysis.

[0036] In the present invention, the enzyme protein expression host microorganism used can be selected from commonly used host bacteria in the biological field such as Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae, and Pichia pastoris. Among them, Escherichia coliBL21 (DE3) is recommended for Escherichia coli, and other Escherichia coli types used for protein expression can also be used, Bacillus subtilis can use Bacillus subtilis 168, Bacillus subtilis WB600 and other Bacillus subtilis types used for protein expression, Saccharomyces cerevisiae can use Saccharomyces cerevisiae CEN.PK2-1C, Saccharomyces cerevisiaeS288C and other Saccharomyces cerevisiae types that can be used for protein expression, and Pichia pastoris can use Pichia pastorisGS115 and other Pichia pastoris types that can be used for protein expression.

[0037] In the present invention, the plasmid corresponding to the host bacteria can be selected without special requirements, such as pET28 plasmid for Escherichia coli, pHT43 plasmid for Bacillus subtilis, pRS425 plasmid for Saccharomyces cerevisiae, and pPIC9K plasmid for Pichia pastoris. There is no special requirement for the promoter, and the promoter on the plasmid can be directly used.

[0038] In the present invention, the signal peptide sequence used is a common amylase signal peptide: MFAKRFKTSLLPLFAGFLLLFYLVLAGPAAA (SEQ ID NO: 5).

[0039] The following is a detailed description of the NADPH biosynthesis route of the present invention in conjunction with specific examples.

[0040] Example 1 The present invention provides a method for preparing formate dehydrogenase and NADH kinase, which specifically comprises the following steps: 1. Prepare formate dehydrogenase and NADH kinase production strains.

[0041] The gene fragments of formate dehydrogenase and NADH kinase were sent for synthesis (Shanghai Biotech). The amino acid sequence of formate dehydrogenase is shown in SEQ ID NO: 1, and the gene sequence is shown in SEQ ID NO: 3; the amino acid sequence of NADH kinase is shown in SEQ ID NO: 2, and the gene sequence is shown in SEQ ID NO: 4; the protein electrophoresis results of the two enzymes are shown in Figure 2 As shown, the results of nucleic acid electrophoresis are as follows Figure 3As shown. The commercial plasmid pET28a was used to insert the target genes into pET28 for expression. Two prokaryotic expression plasmids, pET28-formate dehydrogenase and pET28-NADH kinase, were obtained. After sequencing was correct, they were transformed into Escherichia coli BL21 strains, and high-expression strains were selected as formate dehydrogenase and NADH kinase production strains.

[0042] 2. Preparation of free formate dehydrogenase and NADH kinase by fermentation tank fermentation The preserved production strain tube was thawed at room temperature, and a loop was gently taken with an inoculation loop to streak on a seed culture plate (containing 5 mg / L kanamycin sulfate, 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, 15 g / L agar), and activated by overnight culture at 37°C. Then a single colony was picked and placed in two bottles containing 5 mL of liquid seed culture solution (containing 5 mg / L kanamycin sulfate, 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl), and cultured overnight at 37°C. Subsequently, 1% inoculation was inoculated into 500 mL seed culture medium for shake flask culture, with 200 mL of liquid in each bottle, and the flask was cultured at 220 rpm at 37°C for 12 hours as secondary seeds.

[0043] Prepare 3.5L fermentation medium, sterilize at 121℃ for 20min, cool and set aside, add the fermentation medium to a 5L fermentation tank, transfer the prepared secondary seeds to it, and culture at 37℃ with stirring at 300rpm.

[0044] Take samples from the fermentation tank, dilute and measure OD600. When OD600 reaches 20-25, add IPTG to a final concentration of 0.1 mM to induce expression.

[0045] When the OD value of the sample in the fermentation tank no longer continues to rise, the fermentation is completed (about 12 to 20 hours). The fermentation tank is opened and filtered to collect the bacteria. The bacteria are weighed to obtain BL21 (DE3) -pET28a - formate dehydrogenase bacteria and BL21 (DE3) -pET28a -NADH kinase bacteria, record them and store them at -20 ° C for use.

[0046] Weigh 100 mg of wet bacteria using a balance, add 500 mL of distilled water and stir evenly, then crush with a high-pressure homogenizer, filter to obtain crude enzyme solutions of formate dehydrogenase and NADH kinase, and store at -20°C for use.

[0047] 3. Preparation of immobilized formate dehydrogenase and NADH kinase by embedding method The enzyme obtained above was embedded in a sodium alginate solution to obtain an immobilized enzyme. The specific operation process is as follows: 50 mL of crude enzyme solution was mixed with 50 mL of 20 g / L sodium alginate at 25°C and stirred for 20 min, then added dropwise to a 1 wt% sterile CaCl2 solution, fixed at room temperature for 1 h under stirring conditions, filtered, washed, and dried to obtain two spherical immobilized enzymes of formate dehydrogenase and NADH kinase, and the immobilized enzymes were stored in a Tris-HCl buffer (pH = 7.1).

[0048] Example 2 This example illustrates the steps and effects of expressing the enzyme in bacterial cells and carrying out whole-cell catalysis in a catalytic system buffer to produce the target product NADPH. The bacterial cells and substrate are added together into the buffer to carry out enzyme catalysis to produce the target product NADPH.

[0049] 10 mg of each of BL21(DE3)-pET28a-formate dehydrogenase wet bacteria and BL21(DE3)-pET28a-NADH kinase wet bacteria obtained in Example 1 were added to 1 L of Tris-HCl buffer at pH 6.8.

[0050] Add 1 g of NAD+ and 30 g of ammonium formate to the Tris-HCl buffer containing wet bacteria, the reaction temperature is 20°C, and the reaction time is 10 hours. After the catalytic reaction is completed, boil at 100°C for 10 minutes to terminate the reaction, and centrifuge to obtain the supernatant, which is the NADH product solution.

[0051] 5) The product was determined using HPLC.

[0052] The final NADPH yield was 52%, and the HPLC detection spectrum is attached. Figure 4 .

[0053] Example 3 This example illustrates the steps and effects of expressing the enzyme in bacterial cells, obtaining the enzyme protein, immobilizing the enzyme, and performing a series of catalytic reactions in a buffer system to prepare the target product NADPH.

[0054] 1) 200 mg of each of the immobilized formate dehydrogenase and NADH kinase enzymes obtained in Example 1 were added to 1 L of Tris-HCl buffer at pH 7.5.

[0055] 2) In the 1L reaction system containing formate dehydrogenase and NADH kinase immobilized enzyme, 50g of NAD+ and 45g of ammonium formate were added, and the ATP used was generated by the ATP cycle system, which included ADP kinase, ATP for cycle start and sodium pyrophosphate, wherein the ADP kinase addition concentration was 0.1mg / L, the ATP addition concentration was 50mg / L, the sodium pyrophosphate addition concentration was 50mg / L, and the sodium pyrophosphate was added every 1.5 hours, the reaction temperature was set to 35°C, and the reaction time was 15 hours. Then the reaction was terminated by boiling at 100°C for 10min, and the supernatant was centrifuged to obtain the NADH product solution.

[0056] 3) The product was determined using HPLC.

[0057] The final NADPH yield was 55%, and the HPLC detection spectrum is attached. Figure 5 .

[0058] Example 4 This example illustrates the steps and effects of expressing the enzyme in bacterial cells and reacting in series to produce the target product NADPH in the cells.

[0059] 1) The difference between this example and Example 1 is that the plasmid is replaced with pCOLADuet. Since pCOLADuet has a dual expression frame, formate dehydrogenase and NADH kinase genes can be inserted into it at the same time. The remaining steps are carried out according to Example 1. Culture at 37°C and 300rpm, add 0.1mM IPTG for induction, and add 70g of NAD+ to 1L of culture medium, and the amount of ammonium formate added is 60g. Keep pH 7.0 and temperature 30°C, continue fermentation for 20 hours, and then centrifuge at 12000rpm for 10min. The supernatant is taken as the NADH product solution.

[0060] 2) The product was determined using HPLC.

[0061] The final NADPH yield was 57%. See the attached HPLC detection spectrum Figure 6 .

[0062] Example 5 This example illustrates the steps and effects of expressing the enzyme in E. coli cells without enzyme immobilization and directly using the enzyme to catalyze the reaction in a buffer system to prepare the target product NADPH.

[0063] 1) Take 50 mL of the crude formate dehydrogenase enzyme solution and the crude NADH kinase enzyme solution obtained in Example 1 and add them into 1 L of Tris-HCl buffer at pH 7.0.

[0064] 2) In the above 1L reaction system, add 90g of NAD+ and 55g of ammonium formate. The ATP cycle system includes an ADP kinase concentration of 10mg / L, an initial concentration of ATP added to start the cycle of 1000mg / L, an initial concentration of sodium pyrophosphate added of 1000mg / L, and sodium pyrophosphate added every 2 hours. Set the reaction temperature to 30°C and the reaction time to 10 hours. Then boil at 100°C for 10 minutes to terminate the reaction, and centrifuge to obtain the supernatant, which is the NADH product solution.

[0065] 3) The product was determined using HPLC.

[0066] The final NADPH yield was 61%, and the HPLC detection spectrum is attached. Figure 7 .

[0067] Example 6 This example illustrates the steps and effects of expressing the enzyme in bacterial cells, immobilizing the cells and performing a series of catalytic reactions in a buffer system to prepare the target product NADPH.

[0068] 1) The difference between this embodiment and embodiment 1 is that the obtained wet bacterial cells are embedded with sodium alginate solution to obtain immobilized cells. The specific operation process is as follows: 50 mL of bacterial liquid (bacterial concentration 1 mg / mL) resuspended with Tris-HCl was mixed with 50 mL of 20 g / L sodium alginate and stirred at 25°C for 20 min, then added dropwise to a 1 wt% sterile CaCl2 solution and fixed at room temperature for 1 h under stirring. The mixture was filtered, washed, and dried to obtain two spherical immobilized cells, BL21-formate dehydrogenase and BL21-NADH kinase, which were then stored in Tris-HCl buffer (pH = 7.1).

[0069] 2) In a 1L reaction system, add 10g NAD+ and 60g ammonium formate, 500mg BL21-formate dehydrogenase immobilized cells, and 500mg BL21-NADH kinase immobilized cells. The buffer system is Tris-HCl buffer at pH 7.6, the reaction temperature is 28°C, and the reaction time is 12 hours. Then boil at 100°C for 10 minutes to terminate the reaction, and centrifuge to obtain the supernatant, which is the NADH product solution.

[0070] 3) The product was determined using HPLC.

[0071] The final NADPH yield was 63%, and the HPLC detection spectrum is attached. Figure 8 .

[0072] Example 7 The difference between this example and example 1 is that formate dehydrogenase and NADH kinase are subjected to exocytotic expression treatment (signal peptide addition), and crude enzyme solution can be directly obtained after fermentation, that is, the enzyme protein is expressed outside the cell. The crude enzyme solution is fixed to obtain the immobilized enzyme.

[0073] To 1 L of Tris-HCl buffer at pH 7.3, 300 mg of each of immobilized formate dehydrogenase and NADH kinase were added.

[0074] In the above 1L reaction system: add 100g of NAD+, 55g of ammonium formate, and the ATP cycle system includes: ADP kinase concentration of 50mg / L, initial concentration of ATP added for cycle start-up of 600mg / L, initial concentration of sodium pyrophosphate added of 600mg / L, and sodium pyrophosphate added every 2 hours. Set the reaction temperature to 30℃ and the reaction time to 20 hours. Then boil at 100℃ for 10min to terminate the reaction, centrifuge and take the supernatant, which is the NADH product solution. The final NADPH yield is 65%, and the HPLC detection spectrum is as follows Fig. 9 shown.

[0075] Example 8 The difference between this example and example 7 is that a point mutation is introduced into the NADH kinase gene sequence to change the amino acid at the key position to obtain a crude enzyme solution. The amino acid at position 173 of the NADH kinase in SEQ ID NO.2 is mutated from leucine (L) to cysteine ​​(C). The final NADPH yield is 81%.

[0076] Example 9 The difference between this example and Example 7 is that point mutations were introduced into the NADH kinase gene sequence to change the amino acids at key positions to obtain a crude enzyme solution. The amino acid at position 173 of the NADH kinase in SEQ ID NO.2 was mutated from leucine (L) to cysteine ​​(C), and the amino acid at position 235 was mutated from serine (S) to aspartic acid (D). The final NADPH yield was 84%.

[0077] Example 10 The difference between this example and Example 7 is that point mutations are introduced into the NADH kinase gene sequence to change the amino acids at key positions to obtain a crude enzyme solution. The amino acid at position 173 of the NADH kinase in SEQ ID NO.2 is mutated from leucine (L) to cysteine ​​(C), the amino acid at position 235 is mutated from serine (S) to aspartic acid (D), and the amino acid at position 268 is mutated from alanine (A) to phenylalanine (F). The final NADPH yield is 86%.

[0078] Embodiment 11 The difference between this example and example 10 is that, on the basis of introducing amino acid mutation into NADH kinase, point mutation is introduced into the gene sequence of formate dehydrogenase to change the amino acid at the key position and obtain crude enzyme solution. Lysine (K) at position 121 of formate dehydrogenase in SEQ ID NO.1 is mutated into serine (S). The final NADPH yield is 88%.

[0079] Example 12 The difference between this example and Example 10 is that, on the basis of introducing amino acid mutation into NADH kinase, point mutation is introduced into the gene sequence of formate dehydrogenase to change the amino acid at the key position and obtain crude enzyme solution. Lysine (K) at position 121 of formate dehydrogenase in SEQ ID NO.1 is mutated into serine (S), and leucine (L) at position 284 is mutated into phenylalanine (F). The final NADPH yield is 87%.

[0080] Example 13 The difference between this example and example 10 is that, on the basis of introducing amino acid mutation into NADH kinase, point mutation is introduced into the formate dehydrogenase gene sequence to change the amino acid at the key position and obtain the crude enzyme solution. The lysine (K) at position 121 of the formate dehydrogenase in sequence 1 is mutated into serine (S), the leucine (L) at position 284 is mutated into phenylalanine (F), and the phenylalanine (A) at position 353 is mutated into cysteine ​​(C). The final NADPH yield is 94%.

[0081] Table 1. Changes in product yield after enzyme modification Example No. Enzymes for introducing point mutations Yield (%) 2 No mutation 52 3 No mutation 55 4 No mutation 57 5 No mutation 61 6 No mutation 63 7 No mutation 65 8 NADH kinase 81 9 NADH kinase 84 10 NADH kinase 86 11 Formate dehydrogenase + NADH kinase 88 12 Formate dehydrogenase + NADH kinase 87 13 Formate dehydrogenase + NADH kinase 94 As can be seen from Table 1, the effect of the enzyme modification is that the product yield is significantly improved compared to Example 7 before mutation.

[0082] Comparative Example 1 This comparative example illustrates the comparative effect of NADPH catalytic synthesis in the absence of formate dehydrogenase and the complete NADPH synthesis route provided by the present invention.

[0083] The difference between this example and Example 3 is that no immobilized formate dehydrogenase is added, and the rest is the same as Example 3.

[0084] The final NADPH yield was 0%, and the HPLC detection spectrum is attached. Fig.10 .

[0085] Comparative Example 2 This comparative example illustrates the comparative effect of catalyzing the synthesis of NADPH by directly adding ATP in the absence of an ATP cycle (the ATP in the embodiment is continuously supplied by the ATP cycle, while the amount of ATP in Comparative Example 2 is a single addition that cannot be regenerated) and the NADPH synthesis route provided by the present invention.

[0086] The difference between this example and Example 3 is that 50 mg of ATP is directly added to the 1 L system, and the rest is the same as Example 3.

[0087] The final NADPH yield was 30%, and the HPLC detection spectrum was as follows: Fig.11 shown.

[0088] Comparative Example 3 This comparative example illustrates the comparative effect of catalytic synthesis of NADPH in the absence of ammonium formate and the complete synthesis route of NADPH given in the present invention.

[0089] The difference between this example and Example 3 is that no ammonium formate enzyme is added, and the rest is the same as Example 3.

[0090] The final NADPH yield was 0%.

[0091] MAMRQAAKATIRACSSSSSSGYFARRQFNASSGKKDSKKIVGVFYKANEYATKNPNFLGCVEN ALGIRDWLESQGHQYIVTDDKEGPDCELEKHIPDLHVLISTPFHPAYVTAERIKKAKNLKLL LTAGIGSDHIDLQAAAAAGLTVAEVTGSNVVSVAEDELMRILILMRNFVPGYNQVVKGEWN VAGIAYRAYDLEGKTIGTVGAGRIGKLLLQRLKPFGCNLLYHDRLQMAPELEKETGAKFVE DLNEMLPKCDVIVINMPLTEKTRGMFNKELIGKLKKGVLIVNNARGAIMERQAVVDAVESG HIGGYSGDVWDPQPAPKDHPWRYMPNQAMTPHTSGTTIDAQLRYAAGTKDMLERYFKGEDFPTENYIVKDGELAPQYR (SEQ ID NO: 1).

[0092] ATGGCGATGCGTCAGGCGGCGAAAGCGACCATCCGTGCGTGCTCTTCTTCTTCTTCTTCT GGTTACTTCGCGCGTCGTCAGTTCAACGCGTCTTCTGGTGACTCTAAAAAAATCGTTGG TGTTTTCTACAAAGCGAACGAATACGCGACCAAAAACCCGAACTTCCTGGGTTGCGTTG AAAACGCGCTGGGTATCCGTGACTGGCTGGAATCTCAGGGTCACCAGTACATCGTTACC GACGACAAAGAAGGTCCGGACTGCGAACTGGAAAAACACATCCCGGACCTGCACGTTC TGATCTCTACCCCGTTCCACCCGGCGTACGTTACCGCGGAACGTATCAAAAAAGCGAAA AACCTGAAACTGCTGCTGACCGCGGGTATCGGTTCTGACCACATCGACCTGCAGGCGGC GGCGGCGGCGGGTCTGACCGTTGCGGAAGTTACCGGTTCTAACGTTGTTTCTGTTGCGG AAGACGAACTGATGCGTATCCTGATCCTGATGCGTAACTTCGTTCCGGGTTACAACCAGG TTGTTAAAGGTGAATGGAACGTTGCGGGTATCGCGTACCGTGCGTACGACCTGGAAGGT AAAACCATCGGTACCGTTGGTGCGGGTCGTATCGGTAAACTGCTGCTGCAGCGTCTGAA ACCGTTCGGTTGCAACCTGCTGTACCACGACCGTCTGCAGATGGCGCCGGAACTGGAA AAAGAAACCGGTGCGAAATTCGTTGAAGACCTGAACGAAATGCTGCCGAAATGCGACG TTATCGTTATCAACATGCCGCTGACCGAAAAAACCCGTGGTATGTTCAACAAAGAACTG ATCGGTAAACTGAAAAAAGGTGTTCTGATCGTTAACAACGCGCGTGGTGCGATCATGGA ACGTCAGGCGGTTGTTGACGCGGTTGAATCTGGTCACATCGGTGGTTACTCTGGTGACG TTTGGGACCCGCAGCCGGCGCCGAAAGACCACCCGTGGCGTTACATGCCGAACCAGGC GATGACCCCGCACACCTCTGGTACCACCATCGACGCGCAGCTGCGTTACGCGGCGGGTA CCAAAGACATGCTGGAACGTTACTTCAAAGGTGAAGACTTCCCGACCGAAAACTACATCGTTAAAGACGGTGAACTGGCGCCGCAGTACCGTTAA(SEQ ID NO:3)。

[0093] MFVRVKLNKPVKWYRFYSTLDSHSLKLQSGSKFVKIKPVNNLRSSSSADFVSPPNSKLQSLI WQNPLQNVYITKKPWTPSTREAMVEFITHLHESYPEVNVIVQPDVAEEISQDFKSPLENDPN RPHILYTGPEQDIVNRTDLLVTLGGDGTILHGVSMFGNTQVPPVLAFALGTLGFLSPFDFKE HKKVFQEVISSRAKCLHRTRLECHLKKKDSNSSIVTHAMNDIFLHRGNSPHLTNLDIFIDGEF LTRTTADGVALATPTGSTAYSLSAGGSIVSPLVPAILMTPICPRSLSFRPLILPHSSHIRIKIGSKL NQKPVNSVVKLSVDGIPQQDLDVGDEIYVINEVGTIYIDGTQLPTTRKTENDFNNSKKPKRSGIYCVAKTENDWIRGINELLGFNSSFRLTKRQTDND(SEQ ID NO:2)。

[0094] ATGTTCGTTCGTGTTAAACTGAACAAACCGGTTAAATGGTACCGTTTCTACTCTACCCTG GACTCTCACTCTCTGAAACTGCAGTCTGGTTCTAAATTCGTTAAAATCAAACCGGTTAAC AACCTGCGTTCTTCTTCTTCTGCGGACTTCGTTTCTCCGCCGAACTCTAAACTGCAGTCT CTGATCTGGCAGAACCCGCTGCAGAACGTTTACATCACCAAAAAACCGTGGACCCCGTC TACCCGTGAAGCGATGGTTGAATTCATCACCCACCTGCACGAATCTTACCCGGAAGTTAA CGTTATCGTTCAGCCGGACGTTGCGGAAGAAATCTCTCAGGACTTCAAATCTCCGCTGG AAAACGACCCGAACCGTCCGCACATCCTGTACACCGGTCCGGAACAGGACATCGTTAA CCGTACCGACCTGCTGGTTACCCTGGGTGGTGACGGTACCATCCTGCACGGTGTTTCTAT GTTCGGTAACACCCAGGTTCCGCCGGTTCTGGCGTTCGCGCTGGGTACCCTGGGTTTCC TGTCTCCGTTCGACTTCAAAGAACACAAAAAAGTTTTCCAGGAAGTTATCTCTTCTCGT GCGAAATGCCTGCACCGTACCCGTCTGGAATGCCACCTGAAAAAAAAAGACTCTAACTC TTCTATCGTTACCCACGCGATGAACGACATCTTCCTGCACCGTGGTAACTCTCCGCACCT GACCAACCTGGACATCTTCATCGACGGTGAATTCCTGACCCGTACCACCGCGGACGGTG TTGCGCTGGCGACCCCGACCGGTTCTACCGCGTACTCTCTGTCTGCGGGTGGTTCTATCG TTTCTCCGCTGGTTCCGGCGATCCTGATGACCCCGATCTGCCCGCGTTCTCTGTCTTTCC GTCCGCTGATCCTGCCGCACTCTTCTCACATCCGTATCAAAATCGGTTCTAAACTGAACC AGAAACCGGTTAACTCTGTTGTTAAACTGTCTGTTGACGGTATCCCGCAGCAGGACCTG GACGTTGGTGACGAAATCTACGTTATCAACGAAGTTGGTACCATCTACATCGACGGTACC CAGCTTGCCGACCACCCGTAAAACCGAAAACGACTTCAACAACTCTAAAAAACCGAAAC GTTCTGGTATCTACTGCGTTGCGAAAACCGAAAACGACTGGATCCGTGGTATCAACGAA CTGCTGGGTTTCAACTCTTCTTTCCGTCTGACCAAACGTCAGACCGACAACGACTAA (SEQ ID NO: 4).

[0095] The above-described embodiment is only a preferred solution of the present invention and does not limit the present invention in any form. There are other variations and modifications without exceeding the technical solution described in the claims.

Claims

1. A method for synthesizing NADPH by bioenzymatic method, characterized in that, The substrate NAD+, formic acid, formate dehydrogenase, NADH kinase and buffer are added into a reactor to catalyze the generation of NADPH in vitro or in cells, the product is separated by centrifugation, and the supernatant is taken to obtain an NADH product solution; the formic acid is added in the form of ammonium formate; The catalytic process is: Substrate NAD+ and formate are catalyzed by formate dehydrogenase to produce CO2 and NADH; NADH is catalyzed to generate NADPH with energy provided by NADH kinase and ATP.

2. The method according to claim 1, characterized in that In the reaction system, the amount of NAD+ is 1-100 g / L, the amount of ammonium formate is 30-60 g / L, the amount of formate dehydrogenase is 10-500 mg / L, and the amount of NADH kinase is 10-500 mg / L.

3. The method according to claim 2, characterized in that The pH of the reaction system is 6.5 to 7.8; the reaction temperature is 20 to 40° C.; and the reaction time is 10 to 20 hours.

4. The method according to claim 2, characterized in that: Formate dehydrogenase and NADH kinase were measured based on the amount of wet bacteria, immobilized enzyme or immobilized cells.

5. The method according to claim 1 or 2, characterized in that: The buffer is phosphate buffer or Tris-HCl buffer.

6. The method according to claim 1 or 2, characterized in that: The ATP used is generated by the ATP circulation system, which includes ADP kinase, ATP for cycle start and sodium pyrophosphate, wherein the concentration of ADP kinase is 0.1-10 mg / L, the initial addition concentration of ATP for cycle start is 50-1000 mg / L, the initial addition concentration of sodium pyrophosphate is 50-1000 mg / L, and sodium pyrophosphate is supplemented every 1.5-2 hours.

7. The method according to claim 1 or 2, characterized in that: The ATP used is provided by the cells.

8. The method according to claim 1 or 2, characterized in that: Formate dehydrogenase and NADH kinase are both expressed and produced by an engineering strain of Escherichia coli, Bacillus subtilis, Saccharomyces cerevisiae and Pichia pastoris.

9. The method according to claim 1 or 2, characterized in that: The international system classification number of the formate dehydrogenase is EC 1.17.1.9; the international system classification number of NADH kinase is EC 2.7.1.

86.

10. The method according to claim 9, characterized in that The formate dehydrogenase is obtained by enzyme modification of formate dehydrogenase EC1.17.1.9, specifically, mutating the 121st lysine K to serine S, the 284th leucine L to phenylalanine F, and the 353rd phenylalanine A to cysteine ​​C of formate dehydrogenase EC 1.17.1.9; The NADH kinase is NADH kinase EC 2.7.1.86 is obtained after enzyme modification, specifically: the 173rd amino acid of NADH kinase EC2.7.1.86 is mutated from leucine L to cysteine ​​C, the 235th amino acid is mutated from serine S to aspartic acid D, and the 268th amino acid is mutated from alanine A to phenylalanine F.

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