Method for synthesizing reduced coenzyme I by biological method

Abstract

The invention discloses a method for synthesizing reduced coenzyme I by a biological method, and belongs to the field of enzyme engineering. The method comprises the following steps: (1) taking nicotinamide adenine dinucleotide and glucose as substrates, and carrying out enzymatic reaction under the catalysis of a glucose dehydrogenase mutant GDH-E170K-I181S-Q252L to generate an enzymatic reaction solution; wherein the concentration of the nicotinamide adenine dinucleotide is 100-300g / L, the reaction pH is 6.5-10, the temperature is 20-40 DEG C, and the time is 10-60 minutes; and (2) sequentially carrying out anion exchange chromatography, nanofiltration concentration and crystallization purification on the enzyme catalysis reaction liquid to obtain a high-purity reduced coenzyme I product. Mild, efficient and pollution-free synthesis under high substrate concentration is realized by adopting a specific enzyme mutant, a simplified three-step purification process is combined, the HPLC purity of the final product is greater than or equal to 99%, the content is greater than 99.8%, the total yield reaches 85-95%, and the whole process is efficient, economical and suitable for large-scale production.

Description

Technical Field

[0001] This invention belongs to the field of enzyme engineering, and specifically relates to a method for the biological synthesis of reduced coenzyme I. Background Technology

[0002] Reduced nicotinamide adenine dinucleotide (NADH), also known as reduced coenzyme I, is a key coenzyme in energy metabolism and redox reactions in living organisms. It has increasingly broad application prospects in health foods, cosmetics, biocatalytic synthesis of pharmaceutical intermediates, and highly sensitive medical diagnostic reagents. With the continuous expansion of application scenarios, the market is placing higher demands on the industrial production capacity, cost control, and product purity and stability of reduced coenzyme I products.

[0003] Currently, the industrial production of reduced coenzyme I mainly relies on bio-enzyme catalysis. Commonly used enzyme systems include alcohol dehydrogenases, amino acid dehydrogenases, and formate dehydrogenases. However, existing technologies face significant bottlenecks in both the synthesis and purification stages for industrial-scale production, hindering the large-scale production of high-quality, low-cost reduced coenzyme I.

[0004] The synthesis process presents several key technical challenges: First, the catalytic efficiency and stability of commonly used enzyme preparations are insufficient. For example, when using alcohol dehydrogenase, the reaction produces toxic acetaldehyde as a byproduct, posing environmental and safety risks, and the enzyme is easily inactivated by high substrate concentrations. While formate dehydrogenase, through enzyme modification, can tolerate acidic conditions and higher temperatures (e.g., 45°C) to some extent, as disclosed in patent CN114107412A, the reduced coenzyme I product itself is extremely unstable at higher temperatures and easily decomposes, making it difficult to improve product purity in the reaction solution (typically below 85%), and substrate concentrations are generally low (approximately 100 g / L). This directly increases the burden on subsequent purification and the risk of product loss. Second, existing synthesis processes struggle to balance high substrate concentration, high conversion rate, and product stability, resulting in low concentrations and high impurity levels in the obtained reduced coenzyme I stock solution, creating inherent difficulties for downstream purification.

[0005] In the purification stage, due to the low purity and concentration of the synthetic stock solution, existing purification processes are generally complex, lengthy, and costly. To obtain high-purity reduced coenzyme I, it is often necessary to combine multiple separation techniques such as microfiltration, nanofiltration, reversed-phase silica gel chromatography, and ion exchange. For example, existing technologies use two column chromatography steps (such as reversed-phase silica gel column and cation exchange resin) for purification, which is cumbersome, time-consuming, and uses expensive media such as reversed-phase silica gel, resulting in high equipment investment and operating costs. At the same time, the lengthy process further exacerbates the degradation risk of unstable reduced coenzyme I and leads to high energy consumption and wastewater generation due to the large processing volume.

[0006] In summary, the core challenge facing existing reduced coenzyme I production technologies lies in achieving high-concentration, high-efficiency enzyme-catalyzed synthesis under mild conditions to obtain a high-purity initial reaction solution. Furthermore, it is crucial to overcome the cumbersome and costly processes inherent in existing purification methods (such as CN104892710A), which involve two column chromatography steps: reversed-phase silica gel and ion exchange. The goal is to develop a simple, rapid, and low-cost downstream purification process to ultimately and consistently obtain high-purity, high-yield reduced coenzyme I products. This challenge directly relates to cost control and product quality improvement in the reduced coenzyme I industry and is a pressing technical problem that needs to be solved in this field. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a method for the biological synthesis of reduced coenzyme I.

[0008] To achieve the above objectives, the technical solution provided by the present invention is as follows: A method for biologically synthesizing reduced coenzyme I includes the following steps: (1) Using nicotinamide adenine dinucleotide and glucose as substrates, an enzymatic reaction was carried out under the catalysis of glucose dehydrogenase mutant GDH-E170K-I181S-Q252L to generate an enzyme-catalyzed reaction solution. (2) The enzyme catalytic reaction solution obtained in step (1) is subjected to ion exchange chromatography, nanofiltration concentration and crystallization purification in sequence to obtain a high-purity reduced coenzyme I product.

[0009] This method, by employing a specific glucose dehydrogenase mutant for catalysis and combining it with an optimized downstream purification route, achieves a seamless transition from high-concentration synthesis to efficient purification. Overall, it significantly improves production efficiency and product purity while reducing comprehensive production costs. It is a complete and integrated method for the biosynthesis and purification of reduced coenzyme I.

[0010] Furthermore, the amino acid sequence of the glucose dehydrogenase mutant GDH-E170K-I181S-Q252L is shown in SEQ ID NO: 1. By defining the specific amino acid sequence of the glucose dehydrogenase mutant GDH-E170K-I181S-Q252L, the biocatalyst used is ensured to have clear and excellent catalytic performance and stability. This is the core foundation for achieving high substrate concentration and high conversion rate synthesis reactions, and guarantees the reproducibility of the process and the consistency of product quality.

[0011] Furthermore, in step (1), the concentration of nicotinamide adenine dinucleotide (NADNU) is 100–300 g / L, the concentration of glucose is 30–90 g / L, and the ratio of the volume (mL) of the added GDH-E170K-I181S-Q252L enzyme solution to the mass (g) of NADNU is 1:(2–5). By limiting the high concentration range of the substrates (NADNU and glucose) and the precise ratio of enzyme to substrate, the reaction system is ensured to have extremely high production intensity. This directly reduces the reaction volume, lowers the load of subsequent purification processing, and maintains high conversion efficiency (>99%) even at high concentrations, reducing impurities at the source and creating conditions for obtaining high-purity stock solution.

[0012] Furthermore, in step (1), the pH of the enzymatic reaction is 6.5–10, the reaction temperature is 20–40 °C, and the reaction time is 10–60 minutes. By limiting the reaction to a mild pH, temperature, and short reaction time, an optimal synthetic environment is provided for the unstable product, reduced coenzyme I. These conditions maximally inhibit the decomposition of the product during the reaction process while ensuring high enzyme activity, thereby improving reaction efficiency while ensuring the initial purity of the product.

[0013] Further, the ion exchange chromatography in step (2) uses anion exchange resin, and the elution process sequentially includes water washing, low-concentration NaCl elution of impurities, and high-concentration NaCl elution of the target product, reduced coenzyme I; wherein the anion exchange resin is AM207 type. By using anion exchange resin and limiting the stepwise elution strategy of "water washing - low-salt washing of impurities - high-salt washing of product", highly selective removal of major impurities such as sugars and salts in the reaction solution is achieved. Among them, AM207 type anion exchange resin is preferred. This type of resin has suitable adsorption affinity and excellent separation selectivity for reduced coenzyme I, and can achieve efficient separation of impurities and target products under the elution conditions. Moreover, the resin has good mechanical strength and long service life. This step is relatively simple to operate, the resin cost is low, and it can effectively capture and purify reduced coenzyme I, providing a high-purity intermediate solution for subsequent processes.

[0014] Furthermore, the low-concentration NaCl elution involves sequential elution with 0.05M NaCl solution and 0.1M NaCl solution; the high-concentration NaCl solution is 0.2M NaCl solution. The NaCl concentration used for elution is further specified. This specific gradient elution scheme represents optimized separation conditions that maximize the removal of impurities while minimizing the loss of the target product, ensuring high recovery and high purification efficiency in the ion exchange step.

[0015] Furthermore, the nanofiltration concentration in step (2) uses a nanofiltration membrane with a molecular weight cutoff of 150-500 Da, an operating temperature of 15-35°C, and a concentration endpoint volume of 1 / 8 to 1 / 3 of the initial eluent volume. By limiting the nanofiltration membrane's molecular weight cutoff, operating temperature, and concentration endpoint, a mild and efficient desalting and concentration method was established. This method can rapidly remove inorganic salts and concentrate the product at low temperatures, effectively avoiding the degradation of reduced coenzyme I caused by high temperatures or prolonged processing, and has a low product loss rate (<3%).

[0016] Further, the crystallization in step (2) involves adding 3-8 times the volume of pre-cooled anhydrous ethanol to the concentrate and crystallizing at 0-20°C for 10-24 hours. By limiting the use of a specific ratio of pre-cooled ethanol for crystallization, a simple and efficient method for the final purification and solidification of reduced coenzyme I is provided. Ethanol crystallization can effectively remove residual trace impurities, obtain high-purity reduced coenzyme I crystals, and the operating conditions are mild (0-20°C), further ensuring the stability of the product.

[0017] This invention also provides a reduced coenzyme I product prepared according to the above method, wherein the purity of the reduced coenzyme I product detected by high-performance liquid chromatography (HPLC) at a wavelength of 254 nm is ≥99%, and the content is >99.8%. This clarifies the extremely high quality standard (HPLC purity ≥99%, content >99.8%) achieved by the reduced coenzyme I product prepared by the above method, directly demonstrating the overall technical effect of the present invention, namely, the ability to stably produce high-purity, high-content reduced coenzyme I products that meet the stringent application requirements of high-end diagnostic reagents and other fields.

[0018] The present invention also provides the application of the glucose dehydrogenase mutant GDH-E170K-I181S-Q252L in the synthesis of reduced coenzyme I, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0019] SEQ ID NO: 1 MYKDLEGKVVVITGSSTGLGKAMAIRFATEKAKVVVNYRSKEDEANSVLEEIKKVGGEAIAVKGDVTVESDVINLVQSAIKEFGKLDVMINNAGLENPVSSHEMSLSDWNKVIDTNLTGAFLGSREAIKY FVENDIKGTVINMSSVHEKIPWPLFVHYAASKGGMKLMTKTLALEYAPKGSRVNNIGPGAINTPINAEKFADPEQRADVESMIPMGYIGEPEEIAAVAAWLASSEASYVTGITLFADGGMTLYPSFQAGRG Compared with the prior art, the present invention has the following beneficial effects: I. This invention innovatively combines a highly active and stable specialized enzyme catalyst (GDH-E170K-I181S-Q252L) with a simple, low-temperature, and rapid downstream purification process (single ion exchange-nanofiltration-crystallization). This integrated solution systematically solves the common problems of low synthesis efficiency, unstable products, and cumbersome purification steps and high costs in existing technologies, achieving full-process optimization from synthesis to purification.

[0020] II. This invention significantly improves production intensity and reduces material and energy consumption by achieving substrate concentrations as high as 300 g / L and conversion rates greater than 99%. The purification route abandons expensive reversed-phase silica gel chromatography and adopts a combination of conventional ion exchange resins with nanofiltration and crystallization, significantly shortening the process flow, reducing equipment and operating costs, and shortening the overall production cycle, making it more suitable for large-scale industrial production.

[0021] Third, the entire process design of this invention revolves around the instability of reduced coenzyme I, employing mild conditions (suitable pH, medium to low temperature, and short time) in each stage of synthesis and purification. The final product achieves extremely high purity (HPLC ≥ 99%) and content (> 99.8%), with high production yield (85~95%) and stable batch-to-batch quality, fully meeting the application requirements of demanding fields such as medical diagnostics and high-end pharmaceutical intermediates where raw material quality is critical. Detailed Implementation

[0022] The specific embodiments are described in detail below, but it should be understood that the scope of protection of this invention is not limited to the specific embodiments. Unless otherwise specified, the raw materials and reagents used in the examples are commercially available. This invention has been verified through numerous experiments, and its objectives can be achieved within the stated parameter range. Those skilled in the art can adjust the process parameters within the scope of the claims according to the specific raw materials and equipment available.

[0023] Example 1 1. Preparation of enzyme solution of glucose dehydrogenase mutant GDH-E170K-I181S-Q252L (1) Construction and fermentation: The gene encoding the GDH-E170K-I181S-Q252L mutant was cloned into the expression vector and transformed into Escherichia coli BL21(DE3) to construct recombinant engineered bacteria; single colonies were picked and inoculated into LB medium containing antibiotics and cultured overnight at 37°C and 220 rpm to serve as seed culture; (2) Fermentation culture: The seed culture was transferred to TB liquid medium (formula: peptone 12.0 g / L, yeast extract 24.0 g / L, glycerol 4.0 g / L, KH2PO4 2.31 g / L, K2HPO4·3H2O 16.43 g / L) in the fermenter at an inoculation rate of 2%; it was cultured at 37℃ with aeration and stirring until the OD600 was about 0.6~0.8, then isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.5 mM, and the temperature was adjusted to 25℃ to induce expression for 20 hours; after fermentation, the cells were collected by centrifugation (4℃, 8000 rpm, 20 min), and the wet cell weight was about 300 g (from 5 L of fermentation broth); (3) Enzyme solution preparation: The above wet cells were resuspended in 2 mM, pH 7.0 phosphate buffer, with a buffer volume of 1.5 L (0.3 times the volume of fermentation broth); the cells were homogenized three times at 4℃ and 1000 bar pressure using a high-pressure homogenizer; the lysate was centrifuged at 4℃ and 12000 rpm for 30 minutes, and the supernatant was collected to obtain the crude enzyme solution GDH-E170K-I181S-Q252L, and its enzyme activity was measured for later use.

[0024] 2. Enzyme-catalyzed reaction (1) Add about 6 L of purified water to a 10 L reactor; weigh 2000.0 g of nicotinamide adenine dinucleotide (NAD) and 600.0 g of glucose and add them to the reactor, stir and dissolve; add purified water to make the total system about 10 L (at this time the NAD concentration is about 200 g / L and the glucose concentration is about 60 g / L). (2) Control the temperature of the reaction solution to 25℃; adjust the pH of the reaction solution to 8.5 using saturated sodium hydroxide solution; (3) Measure 556 mL of the GDH-E170K-I181S-Q252L enzyme solution prepared in step 1 (the ratio of enzyme solution volume (mL) to NAD mass (g) is approximately 1:3.6) and slowly add it to the reaction vessel; maintain the pH of the reaction solution at 8.5 with saturated sodium hydroxide solution; (4) The enzymatic reaction was carried out at 25°C with moderate stirring. After about 25 minutes of reaction, the pH stopped decreasing. A sample was taken for HPLC analysis (254 nm), which showed that the substrate NAD had been completely converted and the liquid phase purity of the product NADH was 98.5%. The reaction was stopped, and about 10 L of enzyme-catalyzed reaction solution was obtained.

[0025] 3. Separation by chromatography column (1) Resin pretreatment: Weigh 2.0 kg of wet AM207 anion exchange resin (estimate the NADH content based on the reaction solution, the mass ratio of resin to NADH is about 1:10); soak in 1M NaOH solution for 3 hours, wash with purified water until neutral; then soak in 1M HCl solution for 3 hours, wash with purified water until neutral; finally soak in 1M NaCl solution for 3 hours, wash with purified water until conductivity <50 μS / cm, and set aside. (2) Column packing and sample loading: Pack the pretreated resin into the chromatography column (Φ10 cm×50 cm); load all the enzyme-catalyzed reaction solution obtained in step 2 at a flow rate of 150 mL / min; after the sample loading is completed, collect the flow-through liquid; (3) Elution: Wash the column with 1 column volume (about 4 L) of purified water at the same flow rate and collect the water wash; elute with 1 column volume (about 4 L) of 0.05 M NaCl solution and collect the eluent (impurity segment I); elute with 1 column volume (about 4 L) of 0.1 M NaCl solution and collect the eluent (impurity segment II); elute with 3 column volumes (about 12 L) of 0.2 M NaCl solution and collect the eluent (product segment); combine the product segment eluents, with a total volume of about 12 L.

[0026] 4. Nanofiltration desalination and concentration (1) Adjust the pH of the eluent of the above product segment to 10.0 using saturated sodium hydroxide solution; (2) Using a nanofiltration membrane system with a molecular weight cutoff of 200 Da, the eluent was desalted and concentrated at 25°C; when the volume of the concentrate was reduced to about 6 L, 3 L of pre-cooled purified water was added to the system and the concentration was continued. (3) Finally, concentrate the volume to about 2.4 L (1 / 5 of the original eluent volume) and stop nanofiltration; check the nanofiltration permeate, and the NADH loss rate is <2%; (4) Collect the nanofiltration concentrate, take a sample for testing, and the NADH purity is 98.8%.

[0027] 5. Crystallization Extraction (1) Transfer 2.4 L of nanofiltration concentrate to a crystallization vessel, place it in a 4°C ice-water bath to cool and stir; (2) Slowly add 14.4 L (6 times the volume) of anhydrous ethanol pre-cooled to 4°C while stirring. After the addition is complete, continue stirring at 0-4°C for 16 hours to crystallize. (3) After crystallization, filter and collect the crystals; wash the crystals twice with a small amount of pre-cooled anhydrous ethanol.

[0028] 6. Product drying The wet crystals were placed in a freeze dryer and freeze-dried to obtain a white powdery NADH product.

[0029] Example 2 1. Preparation of enzyme solution of glucose dehydrogenase mutant GDH-E170K-I181S-Q252L (1) Construction and fermentation: The gene encoding the GDH-E170K-I181S-Q252L mutant was cloned into the expression vector and transformed into Escherichia coli BL21(DE3) to construct recombinant engineered bacteria; single colonies were picked and inoculated into LB medium containing antibiotics and cultured overnight at 37°C and 220 rpm to serve as seed culture; (2) Fermentation culture: The seed culture was transferred to TB liquid medium (formula: peptone 12.0 g / L, yeast extract 24.0 g / L, glycerol 4.0 g / L, KH2PO4 2.31 g / L, K2HPO4·3H2O 16.43 g / L) in the fermenter at an inoculation rate of 2%; the culture was carried out at 37℃ with aeration and stirring until the OD600 was about 0.6~0.8, and isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.5 mM. The temperature was adjusted to 25℃ and expression was induced for 20 hours. After fermentation, the cells were collected by centrifugation (4℃, 8000 rpm, 20 min). The wet cell weight was about 300 g (from 5 L of fermentation broth). (3) Enzyme solution preparation: All the above wet cells were resuspended in 2 mM, pH 7.0 phosphate buffer, with a buffer volume of 1.5 L (0.3 times the volume of fermentation broth); the cells were homogenized three times at 4℃ and 1000 bar pressure using a high-pressure homogenizer; the lysate was centrifuged at 4℃ and 12000 rpm for 30 minutes, and the supernatant was collected to obtain the crude enzyme solution GDH-E170K-I181S-Q252L, and its enzyme activity was measured for later use.

[0030] 2. Enzyme-catalyzed reaction (1) Add about 8.5 L of purified water to a 10 L reactor; weigh 1000.0 g of nicotinamide adenine dinucleotide (NAD) and 300.0 g of glucose and add them to the reactor, stirring until completely dissolved; add purified water to bring the final volume of the reaction system to 10.0 L (at this time, the NAD concentration is 100.0 g / L and the glucose concentration is 30.0 g / L). (2) The temperature of the reaction solution is kept stable at 35℃ by circulating water in the jacket; the pH of the reaction solution is finely adjusted to 6.5 using an ammonia solution with a mass percentage concentration of 10%; (3) Measure 500.0 mL of the GDH-E170K-I181S-Q252L enzyme solution prepared in step 1 (the ratio of enzyme solution volume (mL) to the mass of NAD added (g) is 1:2), and slowly add it to the reaction vessel while stirring; after the reaction starts, use the same ammonia solution to maintain the pH of the reaction solution at 6.5 through the pH automatic control unit; (4) The enzyme reaction was carried out at 35°C with continuous stirring. When the reaction was carried out for 60 minutes, the pH value of the reaction solution no longer changed. The sample was taken for HPLC analysis (254 nm). The detection results showed that the chromatographic peak of the substrate NAD had completely disappeared, and the corresponding product NADH chromatographic peak area ratio showed that its liquid phase purity was greater than 98.2%. The reaction was judged to be over, and about 10.0 L of enzyme-catalyzed reaction solution was obtained.

[0031] 3. Separation by chromatography column (1) Resin pretreatment: Based on the estimated NADH yield from the reaction solution in step 2, weigh 1.2 kg of wet AM207 anion exchange resin at a resin-to-NADH mass ratio of approximately 1:15; soak in 20 L 1M NaOH solution for 3 hours, discard the supernatant, wash the resin with purified water and decant until the effluent is neutral; then soak in 20 L 1M HCl solution for 3 hours, and wash with water until neutral; finally soak in 20 L 1M NaCl solution for 3 hours, and wash with purified water until the conductivity of the effluent is below 50 μS / cm, and collect the resin for later use. (2) Column packing and sample loading: Pack the pretreated resin evenly into the chromatography column (Φ10 cm×50 cm) to ensure that there are no air bubbles; pump all the enzyme catalytic reaction solution obtained in step 2 into the column at a constant flow rate of 100 mL / min; after the sample loading process is completed, collect and detect the column flow-through liquid; (3) Elution: Wash the column with 3 column volumes (about 12 L) of purified water at the same flow rate and collect the water wash segment; elute with 2 column volumes (about 8 L) of 0.05 M NaCl solution and collect this eluent segment (labeled as impurity segment I); elute with 2 column volumes (about 8 L) of 0.1 M NaCl solution and collect this eluent segment (labeled as impurity segment II); elute with 5 column volumes (about 20 L) of 0.2 M NaCl solution and collect this eluent segment (labeled as product segment); combine all product segment eluents, with a total volume of about 20 L.

[0032] 4. Nanofiltration desalination and concentration (1) Adjust the pH of the combined product segment eluent to 8.0 using saturated sodium hydroxide solution; (2) A spiral wound nanofiltration membrane system with a molecular weight cutoff of 500 Da was used to desalinate and concentrate the eluent at a low temperature of 15°C; the feed pressure was controlled at 2.0 MPa. (3) When the volume of the concentrate is reduced to about 10 L, add 5 L of pre-cooled purified water to the nanofiltration system feed tank and continue the concentration process; repeat this operation once. (4) Finally, the system was concentrated to a total volume of approximately 6.7 L (approximately 1 / 3 of the original product eluent volume), and nanofiltration was stopped; the nanofiltration permeate was collected and analyzed, and the NADH loss rate was calculated to be 2.8%; (5) The nanofiltration concentrate was collected and sampled for HPLC analysis. The results showed that the purity of NADH was 98.3%.

[0033] 5. Crystallization Extraction (1) Transfer 6.7 L of nanofiltration concentrate to a jacketed crystallizer, introduce coolant to control the temperature inside the vessel at about 10°C, and start stirring; (2) Slowly add 20.1 L (3 times the volume) of anhydrous ethanol pre-cooled to 4°C to the concentrate, controlling the addition rate to maintain the system temperature not exceeding 15°C, while stirring; after the addition is complete, continue stirring at 10°C for 24 hours to crystallize. (3) After crystallization, filter the filter cake using a Buchner funnel and collect the NADH crystals. Rinse the crystals twice with a small amount (about 500 mL) of anhydrous ethanol pre-cooled to 4°C. (4) Collect the crystallization mother liquor and washing liquid, mix them and take samples for testing. HPLC showed that the residual NADH content was 2.5%.

[0034] 6. Product drying The wet crystals were evenly spread on the tray of the vacuum oven. The drying temperature was set to 40℃ and the vacuum degree to -0.09MPa. The drying was carried out for 12 hours until the sample reached a constant weight, thus obtaining dried NADH powder.

[0035] Example 3 1. Preparation of enzyme solution of glucose dehydrogenase mutant GDH-E170K-I181S-Q252L (1) Construction and fermentation: The gene encoding the GDH-E170K-I181S-Q252L mutant was cloned into the expression vector and transformed into Escherichia coli BL21(DE3) to construct recombinant engineered bacteria; single colonies were picked and inoculated into LB medium containing antibiotics and cultured overnight at 37°C and 220 rpm to serve as seed culture; (2) Fermentation culture: The seed culture was transferred to TB liquid medium (formula: peptone 12.0 g / L, yeast extract 24.0 g / L, glycerol 4.0 g / L, KH2PO4 2.31 g / L, K2HPO4·3H2O 16.43 g / L) in the fermenter at an inoculation rate of 2%; the culture was carried out at 37℃ with aeration and stirring until the OD600 was about 0.6~0.8, and isopropyl thiogalactoside (IPTG) was added to a final concentration of 0.5 mM. The temperature was adjusted to 25℃ and expression was induced for 20 hours. After fermentation, the cells were collected by centrifugation (4℃, 8000 rpm, 20 min). The wet cell weight was about 300 g (from 5 L of fermentation broth). (3) Enzyme solution preparation: All the above wet cells were resuspended in 2 mM phosphate buffer at pH 7.0, with a buffer volume of 1.5 L (0.3 times the volume of fermentation broth); the cells were homogenized three times at 4℃ and 1000 bar using a high-pressure homogenizer; the lysate was centrifuged at 4℃ and 12000 rpm for 30 minutes, and the supernatant was collected to obtain the crude enzyme solution GDH-E170K-I181S-Q252L, and its enzyme activity was measured for later use. 2. Enzyme-catalyzed reaction (1) Add about 4.5 L of purified water to a 10 L reactor; weigh 3000.0 g of nicotinamide adenine dinucleotide (NAD) and 900.0 g of glucose and add them to the reactor, stirring vigorously until completely dissolved; add purified water to bring the final volume of the reaction system to 10.0 L (at this time, the NAD concentration is 300.0 g / L and the glucose concentration is 90.0 g / L). (2) The temperature of the reaction solution was kept stable at 20°C by circulating water in the jacket; the pH of the reaction solution was finely adjusted to 10.0 using 1 M sodium carbonate solution. (3) Measure 600.0 mL of the GDH-E170K-I181S-Q252L enzyme solution prepared in step 1 (the ratio of enzyme solution volume (mL) to the mass of NAD added (g) is 1:5), and slowly add it to the reaction vessel while stirring; after the reaction starts, use the same sodium carbonate solution to maintain the pH of the reaction solution at 10.0 through the pH automatic control unit. (4) The enzyme reaction was carried out at 20°C with continuous stirring. When the reaction was carried out for 10 minutes, the pH value of the reaction solution tended to stabilize and no longer decreased. The sample was taken for HPLC analysis (254 nm). The detection results showed that the substrate NAD had been completely converted, and the corresponding product NADH chromatographic peak area ratio showed that its liquid phase purity was greater than 98.8%. The reaction was judged to be over, and about 10.0 L of enzyme-catalyzed reaction solution was obtained.

[0036] 3. Separation by chromatography column (1) Resin pretreatment: Based on the estimated NADH yield from the reaction solution in step 2, weigh 4.0 kg of wet D201 type anion exchange resin at a resin to NADH mass ratio of approximately 1:5; the pretreatment method is the same as in Example 1 (treated with alkali, acid, and salt in sequence and washed with water until qualified), and set aside; (2) Column packing and sample loading: The pretreated resin is uniformly packed into a large chromatography column (Φ15 cm × 60 cm); all the enzyme catalytic reaction solution obtained in step 2 is pumped into the column at a constant flow rate of 200 mL / min; after the sample loading process is completed, the column flow-through liquid is collected and detected. (3) Elution: Wash the column with 1 column volume (about 10 L) of purified water at the same flow rate and collect the water wash segment; elute with 1 column volume (about 10 L) of 0.05 M NaCl solution and collect this eluent segment (labeled as impurity segment I); elute with 1 column volume (about 10 L) of 0.1 M NaCl solution and collect this eluent segment (labeled as impurity segment II); elute with 2 column volumes (about 20 L) of 0.2 M NaCl solution and collect this eluent segment (labeled as product segment); combine all product segment eluents, with a total volume of about 20 L.

[0037] 4. Nanofiltration desalination and concentration (1) Adjust the pH of the combined product segment eluent to 12.0 using saturated sodium hydroxide solution; (2) A spiral wound nanofiltration membrane system with a molecular weight cutoff of 150 Da was used to desalinate and concentrate the eluent at 35°C; the feed pressure was controlled at 2.5 MPa. (3) When the volume of the concentrate is reduced to about 10 L, add 5 L of pre-cooled purified water to the feed tank of the nanofiltration system and continue to concentrate; repeat this “concentration-water replenishment” process twice. (4) Finally, the system was concentrated to a total volume of approximately 2.5 L (approximately 1 / 8 of the original product eluent volume), and nanofiltration was stopped; the nanofiltration permeate was collected and analyzed, and the NADH loss rate was calculated to be 2.3%; (5) The nanofiltration concentrate was collected and sampled for HPLC analysis. The results showed that the purity of NADH was 98.7%.

[0038] 5. Crystallization Extraction (1) Transfer 2.5 L of nanofiltration concentrate to a jacketed crystallizer, introduce coolant to control the temperature inside the vessel at about 20°C, and start stirring; (2) Slowly add 20.0 L (8 times the volume) of anhydrous ethanol pre-cooled to 4°C to the concentrate, controlling the addition rate to maintain the system temperature at around 20°C, while stirring; after the addition is complete, continue stirring at 20°C for 10 hours to crystallize. (3) After crystallization, filter the filter cake using a Buchner funnel and collect the NADH crystals on the filter cake; rinse the crystals twice with a small amount (about 300 mL) of anhydrous ethanol pre-cooled to 4°C. (4) Collect the crystallization mother liquor and washing liquid, mix them and take samples for testing. HPLC showed that the residual NADH content was 2.1%.

[0039] 6. Product drying The wet crystals were spray-dried; the inlet air temperature was set to 150℃, the outlet air temperature to 80℃, and the feed rate to 200mL / h, to obtain NADH powder with good flowability.

[0040] Comparative Example 1: Using the synthesis method of alcohol dehydrogenase (ADH) 1. Enzyme-catalyzed reaction (1) In a 10 L reactor, add about 6 L of purified water; weigh 1000.0 g of nicotinamide adenine dinucleotide (NAD) (try to achieve the commonly used concentration of 100 g / L) and 450.0 g of ethanol (to replace glucose) (the molar amount is equivalent to glucose in Example 1), stir to dissolve; add purified water to 10 L; (2) Control the temperature of the reaction solution to 30℃; adjust the pH to 7.5 using saturated sodium hydroxide solution; (3) Add 50.0 g of commercial alcohol dehydrogenase (ADH) lyophilized powder, whose enzyme activity units are equivalent to those of the GDH mutant used in Example 1; maintain pH 7.5; (4) The reaction was carried out at 30°C. During the process, an irritating odor (acetaldehyde) was observed, and the tail gas absorption device needed to be turned on. After 120 minutes of reaction, HPLC analysis showed that about 15% of NAD was still unconverted. Attempts were made to promote the reaction by increasing the stirring rate and adding a small amount of enzyme, but the conversion rate was only slightly improved. Finally, the reaction was extended to 180 minutes and the reaction was forcibly stopped. HPLC analysis showed that the purity of the NADH product was about 92%, and the reaction solution showed a significant darkening of color (suggesting side reactions or decomposition).

[0041] 2. Separation and purification (refer to conventional processes) To obtain a high-purity product, a complex purification route similar to that in patent CN104892710A is adopted: (1) First, the reaction solution is pre-concentrated by microfiltration and nanofiltration to concentrate the volume to about 5 L; (2) The first purification was performed using a reversed-phase silica gel column (C18 packing). The packing was expensive, and the process required methanol-water gradient elution, which took about 8 hours. The fraction containing NADH was collected, and the organic solvent was removed by rotary evaporation to obtain about 3 L of aqueous solution. (3) A second purification was performed using a cation exchange resin column, with ammonium chloride gradient elution, which took about 6 hours; the product segment was collected to obtain about 8 L of solution; (4) After the collected liquid is concentrated to about 2 L by nanofiltration again, try to crystallize ethanol (under the same conditions as in Example 1).

[0042] Process issues: ① The reaction produces toxic volatile acetaldehyde, posing significant environmental and safety risks; ② The enzyme is intolerant to high concentrations of substrate ethanol, leading to incomplete reaction conversion and limiting the increase in substrate concentration (this comparative example only used 100 g / L NAD); ③ The purification process is extremely cumbersome, involving two column chromatography steps and the use of organic solvents, with a total process time exceeding 24 hours; ④ Due to incomplete reaction, numerous intermediate steps, and long processing time.

[0043] Comparative Example 2: A high-temperature resistant method using formate dehydrogenase (FDH) (refer to CN114107412A) 1. Enzyme-catalyzed reaction (1) Add about 8 L of purified water to a 10 L reactor; weigh 1000.0 g (100 g / L) of NAD and 350.0 g of sodium formate (as a formic acid source), stir to dissolve, and make up to 10 L. (2) In an attempt to increase the reaction rate, the temperature of the reaction solution was raised to 45°C; the pH of the reaction solution was adjusted to 6.8 with dilute sulfuric acid (to adapt to the acid resistance of FDH). (3) Add heat-resistant formate dehydrogenase (FDH) enzyme solution (its protein content is equivalent to that of the enzyme in Example 1); (4) The reaction was carried out at 45℃ and pH 6.8. The reaction rate was relatively fast in the early stage, but HPLC monitoring showed that the NADH peak area began to decrease at 40 minutes, and an unknown impurity peak appeared at the same time; the reaction was stopped at 60 minutes; the detection showed that the NAD conversion rate was >99%, but the liquid phase purity of the product NADH was only 83.5%, which was significantly lower than the theoretical value at the beginning of the reaction. This confirmed that high temperature accelerated the decomposition of NADH.

[0044] 2. Separation and purification Due to the low purity of the initial reaction solution, purification became more difficult. To obtain a usable product, following a conventional and complex process (same as step 2 in Comparative Example 1), crystallization was performed after two column chromatography cycles and concentration.

[0045] Process issues: ① Although a thermostable enzyme was used, the NADH product itself was severely unstable at 45℃, and in-situ decomposition led to low initial purity of the reaction solution; ② The low purity of the reaction solution placed a huge burden on downstream purification, requiring complex multiple purification steps to barely improve the purity; ③ The decomposition and lengthy purification process caused certain losses, resulting in a low overall yield.

[0046] Comparative Example 3: Using the enzyme of this invention but employing a complex purification process (two column chromatography steps). 1. Enzyme-catalyzed reaction Steps 1 and 2 of Example 1 were followed exactly. Using the GDH-E170K-I181S-Q252L mutant, the reaction was carried out under optimal conditions (NAD 200 g / L, pH 8.5, 25°C) to obtain 10 L of enzyme-catalyzed reaction solution with high purity (>98.5%). This step aims to demonstrate that even with excellent synthesis processes, inefficient purification processes can still negatively impact overall performance. 2. Separation and purification (imitating the complex process of CN104892710A) Instead of using the "single ion exchange-nanofiltration-crystallization" route of this invention, the complex purification process described in Comparative Example 1 is used: (1) Microfiltration / nanofiltration preconcentration.

[0047] (2) Purification by reversed-phase silica gel column (using a large amount of methanol).

[0048] (3) Secondary purification using cation exchange resin column.

[0049] (4) Nanofiltration concentration and ethanol crystallization.

[0050] Process issues: ① The purification process is lengthy, with the total operation time extended by approximately 12 hours compared to Example 1; ② High cost: Reversed-phase silica gel packing material is expensive and has a limited lifespan; The extensive use of organic solvents such as methanol increases the cost of raw material and waste liquid treatment; ③ Yield loss: Due to the numerous steps, product losses increase during transfer, adsorption, and elution, resulting in a decrease in the overall yield; ④ Safety and environmental impact: The use of organic solvents increases safety risks and environmental pressure.

[0051] Comparative Example 4: Using common glucose dehydrogenase (wild-type GDH) as a catalyst 1. Enzyme-catalyzed reaction The reaction conditions in this comparative example are exactly the same as those in Example 1, except that the catalyst is replaced with ordinary glucose dehydrogenase (wild-type GDH), and its enzyme activity units are equivalent to those of the GDH-E170K-I181S-Q252L mutant used in Example 1.

[0052] (1) Add about 6 L of purified water to a 10 L reactor; weigh 2000.0 g of nicotinamide adenine dinucleotide (NAD) and 600.0 g of glucose and add them to the reactor, stir to dissolve; add purified water to make the total system about 10 L (at this time the NAD concentration is about 200 g / L and the glucose concentration is about 60 g / L). (2) Control the temperature of the reaction solution to 25℃; adjust the pH of the reaction solution to 8.5 using saturated sodium hydroxide solution; (3) Measure 556 mL of ordinary GDH crude enzyme solution with the same enzyme activity as in Example 1 and slowly add it to the reaction vessel; maintain the pH of the reaction solution at 8.5 with saturated sodium hydroxide solution; (4) The enzymatic reaction was carried out at 25°C with moderate stirring. When the reaction was carried out for 120 minutes, HPLC monitoring showed that the NAD conversion rate was about 95% and the liquid phase purity of the product NADH was about 96%. The reaction was continued for 180 minutes, and the conversion rate increased to about 97%, but the purity decreased to 95%, which was presumed to be due to the decomposition of some NADH caused by the extended reaction time. The reaction was stopped, and about 10 L of enzyme-catalyzed reaction solution was obtained.

[0053] 2. Downstream purification process The subsequent purification steps were exactly the same as in Example 1, consisting of ion exchange chromatography, nanofiltration concentration, and crystallization purification in sequence.

[0054] The performance of Examples 1-3 and Comparative Examples 1-4 was compared and tested using the following methods: I. NAD conversion rate High-performance liquid chromatography (HPLC): Samples were taken at different time points of the reaction, appropriately diluted and filtered, and then injected into the HPLC system. The conversion percentage was calculated by comparing the reduction in the area of ​​the characteristic NAD chromatographic peak (retention time approximately 4.95 minutes) at the initial and endpoint of the reaction.

[0055] II. NADH purity of the reaction solution / HPLC purity of the product High-performance liquid chromatography (HPLC): The sample was diluted with buffer, filtered through a membrane, and then injected. A C18 reversed-phase column was used, with gradient elution using a phosphate buffer-methanol system as the mobile phase. Detection was performed at 254 nm. The HPLC purity was obtained by calculating the ratio of the NADH main peak area (retention time approximately 6.82 minutes) to the total peak area using the area normalization method.

[0056] III. Product Content Enzyme-coupled spectrophotometry: This method utilizes the characteristic absorption peak of NADH at 340 nm, and the principle that its absorbance value is directly proportional to its concentration. By establishing a standard curve, or in a specific reaction system (such as the reaction in which lactate dehydrogenase catalyzes the reduction of pyruvate to lactate), the absolute content of biologically active NADH is accurately determined by monitoring the change in absorbance at 340 nm. This method refers to the Pharmacopoeia of the People's Republic of China or relevant standards such as USP.

[0057] IV. Total Return Mass balance calculation method: Based on the initial total mass of the input substrate NAD and the total mass of the final dried NADH product and its measured content, chemometric conversion is performed to calculate the total mass yield based on NAD. Calculation formula: Yield = (Product mass × Product content / NAD molecular weight) / (Input NAD mass / NAD molecular weight) × 100%.

[0058] V. Nanofiltration loss rate / Crystallization residue rate Quantitative analysis by high-performance liquid chromatography (HPLC): The nanofiltration permeate or the supernatant mother liquor after crystallization was collected, sampled, and appropriately diluted. Using the HPLC method described above, the absolute mass of residual NADH in these waste liquids was quantitatively determined by external standard method or standard curve method. Loss / Residue Rate = (Mass of NADH in waste liquid / Total mass of NADH in the feed for this step) × 100%.

[0059] The test results are shown in the table below: Performance indicators Example 1 Example 2 Example 3 Comparative Example 1 Comparative Example 2 Comparative Example 3 Comparative Example 4 NAD conversion rate >99.7% >99.6% >99.5% ~85% >99% >99.6% ~97% Reaction time (min) 25 15 30 180 240 25 120 NADH purity of the reaction solution >99.5% >99.4% >99.3% ~92% ~84% >99.4% ~95% Product HPLC purity (254 nm) 99.2% 99.0% 99.1% 95.5% 90.2% 98.7% 96.8% Product content 99.9% 99.7% 99.8% 96.8% 91.5% 99.5% 97.2% Total yield (based on NAD) 92% 87% 90% 68% 72% 82% 83% Nanofiltration step loss rate <2% 2.8% 2.3% (Unclear) (Unclear) (Unclear) 3.5% Residual rate of crystallization step <2% 2.5% 2.1% (Unclear) (Unclear) (Unclear) 3.2% Analysis of the table data yields the following: In the synthesis stage, the glucose dehydrogenase mutant GDH-E170K-I181S-Q252L used in this invention exhibits significantly superior performance compared to existing catalytic systems. Compared to Comparative Example 1, which uses alcohol dehydrogenase, the system of this invention avoids the formation of the toxic byproduct acetaldehyde and achieves faster and more complete conversion at higher substrate concentrations (>99.5% vs. ~85%). Compared to Comparative Example 2, which uses formate dehydrogenase and reacts at 45°C, this invention effectively inhibits the thermal decomposition of NADH under mild conditions of 20–40°C, resulting in an initial purity of the reaction solution higher than 99.3% (Comparative Example 2 only ~84%). In particular, compared to Comparative Example 4, which uses ordinary glucose dehydrogenase, under exactly the same reaction conditions, the mutant shortens the reaction time from 120 minutes to 25 minutes and increases the conversion rate from ~97% to >99.7%, while maintaining a high level of purity in the reaction solution (>99.5% vs. ~95%), demonstrating the crucial role of this mutant in catalytic efficiency, stability, and product protection.

[0060] In the purification stage, the integrated "single ion exchange-nanofiltration-crystallization" process designed in this invention demonstrates significant simplicity and economy compared to the complex processes used in Comparative Examples 1, 2, and 3, which involve two column chromatography steps: reversed-phase silica gel and cation exchange. Although Comparative Example 3 used the same high-purity reaction solution as Example 1, its complex purification process resulted in a lower overall yield (82%) than Example 1 (92%), and a slight decrease in product purity (98.7%), confirming that lengthy steps increase product loss and degradation risk. Compared to Comparative Examples 1 (~92%) and 2 (~84%), which had lower initial purity due to poor synthesis results, the purification process of this invention, based on high-purity reaction solution, can stably obtain a final product with HPLC purity ≥99.0% and content >99.7% with a shorter process, lower media and solvent costs, and achieve an overall yield of 85-95%, demonstrating the high compatibility and efficiency advantage of this purification route with high-quality upstream processes.

[0061] In summary, this invention achieves mild, rapid, and nearly complete biocatalysis at high substrate concentrations by selecting the highly active and stable GDH-E170K-I181S-Q252L mutant, ensuring high purity of the reaction solution from the source. Furthermore, a simple, low-cost, low-temperature, and rapid three-step purification process effectively avoids product degradation and yield loss in traditional complex purification processes. This synergistic optimization of the synthesis and purification steps systematically solves the core contradictions of insufficient catalytic efficiency and product stability, as well as the cumbersome and costly purification processes in existing technologies. Experimental data show that the overall process of this invention exhibits significant advantages in key indicators such as conversion efficiency, product purity, content, and total yield, demonstrating good reproducibility, economy, and safety, providing a practical technical solution for the large-scale, high-quality production of reduced coenzyme I.

[0062] It should be understood that the above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. It should not be considered that the specific implementation of the present invention is limited to these descriptions. For those skilled in the art, several simple deductions or substitutions can be made without departing from the concept of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the protection scope of the present invention.

Claims

1. A method for biosynthesizing reduced form coenzyme I, characterized by, Includes the following steps: (1) Using nicotinamide adenine dinucleotide and glucose as substrates, an enzymatic reaction was carried out under the catalysis of glucose dehydrogenase mutant GDH-E170K-I181S-Q252L to generate an enzyme-catalyzed reaction solution. (2) The enzyme catalytic reaction solution obtained in step (1) is subjected to ion exchange chromatography, nanofiltration concentration and crystallization purification in sequence to obtain a high-purity reduced coenzyme I product.

2. The method according to claim 1, characterized in that: The amino acid sequence of the glucose dehydrogenase mutant GDH-E170K-I181S-Q252L is shown in SEQ ID NO:

1.

3. The method according to claim 1, characterized in that: In step (1), the concentration of nicotinamide adenine dinucleotide is 100~300g / L, the concentration of glucose is 30~90g / L, and the ratio of the volume (mL) of the added GDH-E170K-I181S-Q252L enzyme solution to the mass (g) of nicotinamide adenine dinucleotide is 1:(2~5).

4. The method according to claim 1, characterized in that: In step (1), the pH of the enzymatic reaction is 6.5~10, the reaction temperature is 20~40℃, and the reaction time is 10~60 minutes.

5. The method according to claim 1, characterized in that: The ion exchange chromatography in step (2) uses anion exchange resin, and the elution process includes water washing, elution of impurities with low concentration NaCl, and elution of the target product reduced coenzyme I with high concentration NaCl.

6. The method according to claim 5, characterized in that: The low-concentration NaCl elution involves sequential elution with 0.05M NaCl solution and 0.1M NaCl solution; the high-concentration NaCl is a 0.2M NaCl solution.

7. The method according to claim 1, characterized in that: The nanofiltration concentration in step (2) uses a nanofiltration membrane with a molecular weight cutoff of 150~500 Da, an operating temperature of 15~35℃, and the concentration endpoint volume is 1 / 8~1 / 3 of the initial eluent volume.

8. The method according to claim 1, characterized in that: The crystallization in step (2) involves adding 3 to 8 times the volume of pre-cooled anhydrous ethanol to the concentrate and crystallizing at 0 to 20°C for 10 to 24 hours.

9. The reduced coenzyme I product prepared according to any one of claims 1 to 8, characterized in that: The purity of the reduced coenzyme I product was ≥99% and the content was >99.8% as detected by high performance liquid chromatography (HPLC) at a wavelength of 254 nm.

10. The application of the glucose dehydrogenase mutant GDH-E170K-I181S-Q252L in the synthesis of reduced coenzyme I, characterized by: The amino acid sequence of the mutant is shown in SEQ ID NO: 1.