Formic acid dehydrogenase mutant and application thereof in preparation of reduced nicotinamide adenine dinucleotide

Abstract

The application relates to the technical field of enzyme catalysis, in particular to a formate dehydrogenase mutant and application thereof in preparation of reduced nicotinamide adenine dinucleotide, the amino acid sequence of the formate dehydrogenase mutant is shown as SEQ ID NO: 3, and the formate dehydrogenase mutant can be used for preparing reduced nicotinamide adenine dinucleotide. Compared with wild-type formate dehydrogenase, the formate dehydrogenase mutant has higher enzyme activity, faster reaction speed and higher substrate conversion rate under specific reaction conditions (45 DEG C, pH 8.5), less substrate waste, and is favorable for efficient separation of products.

Description

Technical Field

[0001] This invention relates to the field of enzyme catalysis technology, specifically to a formate dehydrogenase mutant and its application in the preparation of reduced nicotinamide adenine dinucleotide. Background Technology

[0002] The regeneration cycle of NADH is achieved by reducing NAD to NADH. Currently, the main enzymatic methods for reducing NAD to NADH include alcohol dehydrogenase, formate dehydrogenase, glucose dehydrogenase, and glucose-6-phosphate dehydrogenase, as well as alcohol dehydrogenase and ethanol. The ammonium formate process involves incubating formic acid, an alkali metal, and formate dehydrogenase (FDH) together to remove most of the formed carbonate, enzymatically preparing reduced nicotinamide adenine dinucleotide or its phosphate, and using at least one alkali metal formate, ultimately eliminating the formed alkali metal carbonate. The alcohol dehydrogenase scheme requires the use of excess ethanol and continuous removal of the generated volatile acetaldehyde. Its biggest drawback is incomplete NADH conversion and the production of toxic acetaldehyde during the reaction. In comparison, the formate dehydrogenase reaction system produces only one byproduct—carbon dioxide—which has no adverse effect on enzyme activity, making it more attractive for industrial manufacturing.

[0003] The existing ammonium formate process has some inherent defects. The sources of formate dehydrogenase are relatively limited, mainly concentrated in heat-sensitive microorganisms, resulting in poor thermal stability of the currently mainstream FDH. The optimal reaction temperature is mostly around 30℃, while it is rapidly deactivated between 45℃ and 60℃.

[0004] Therefore, this invention seeks to find a formate dehydrogenase mutant to solve the above problems, which can both allow formate dehydrogenase to withstand high temperatures and maintain high enzyme activity, and at the same time achieve stable regeneration of NADH, thereby expanding the scope of application. Summary of the Invention

[0005] The purpose of this invention is to provide a formate dehydrogenase mutant and its application in the preparation of reduced nicotinamide adenine dinucleotide.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A formate dehydrogenase mutant, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0008] The formate dehydrogenase mutant of the present invention can be used to prepare reduced nicotinamide adenine dinucleotide. The method of use is as follows: weigh ammonium formate and NAD into a flask, add pure water, stir and dissolve in a 45°C water bath, and adjust the pH to 8.5 with sodium hydroxide. Add the crude enzyme solution of the formate dehydrogenase mutant to react, and maintain the pH at 8.5 with sodium hydroxide.

[0009] The method for preparing the crude enzyme solution includes the following steps:

[0010] (1) The corresponding coding polynucleotide sequence of the protein shown in SEQ ID NO:3 was assembled by primer splicing and cloned into a prokaryotic expression vector to achieve high expression in Escherichia coli;

[0011] (2) Shake flask fermentation or fed-batch fermentation are adopted.

[0012] ① Shake flask fermentation

[0013] A single colony of Escherichia coli containing the expression vector was picked and inoculated into 3 mL of autoclaved medium A and cultured overnight at 30°C and 250 rpm.

[0014] The next day, take a 1L Erlenmeyer flask and inoculate it into 250mL of autoclaved culture medium B at an inoculation ratio of 1:100. Incubate at 37℃ until the bacterial cell OD reaches 5-6. Immediately place the Erlenmeyer flask in a 25℃ shaker and incubate at 250rpm for 1 hour. Add IPTG to a final concentration of 0.1mM and continue incubating at 25℃ and 180rpm for 12 hours.

[0015] After the culture was completed, the culture medium was centrifuged at 12000g for 20 minutes at 4℃ to collect the wet bacterial cells; then the bacterial precipitate was washed once with distilled water, the bacterial cells were collected and stored at -70℃; at the same time, a small amount of bacterial cells were taken for SDS-PAGE detection.

[0016] ② Batch feeding fermentation

[0017] Fed-batch fermentation was carried out in a computer-controlled bioreactor. A single colony of *E. coli* containing the expression vector was prepared into a 200 mL culture, and inoculated into the bioreactor when the OD600 reached 2.0. Throughout the fermentation process, the temperature was maintained at 37°C, and the dissolved oxygen concentration was automatically controlled at 30% by a cascade of stirring and aeration. The pH of the culture medium was maintained at 7.0 using 50% v / v orthophosphate and 30% v / v ammonia. During fermentation, when dissolved oxygen levels rebounded, feeding was initiated. The feed solution contained 9% w / v peptone, 9% w / v yeast extract, and 14% w / v glycerol. When the OD600 reached 50.0, the temperature was controlled at 25°C, and expression was induced with 0.1 mM IPTG for 16 hours. The cells were then collected by centrifugation and stored at -25°C.

[0018] The culture medium A consisted of: 10 g / L tryptone, 5 g / L yeast extract, 3.55 g / L disodium hydrogen phosphate, 3.4 g / L potassium dihydrogen phosphate, 2.68 g / L ammonium chloride, 0.71 g / L sodium sulfate, 0.493 g / L magnesium sulfate heptahydrate, 0.027 g / L ferric chloride hexahydrate, 5 g / L glycerol, 0.8 g / L glucose, with kanamycin added to bring the concentration to 50 mg / L.

[0019] The culture medium B consists of: 10 g / L tryptone, 5 g / L yeast extract, 3.55 g / L disodium hydrogen phosphate, 3.4 g / L potassium dihydrogen phosphate, 2.68 g / L ammonium chloride, 0.71 g / L sodium sulfate, 0.493 g / L magnesium sulfate heptahydrate, 0.027 g / L ferric chloride hexahydrate, 5 g / L glycerol, 0.3 g / L glucose, with kanamycin added to a concentration of 50 mg / L.

[0020] The culture medium used for fed-batch fermentation consisted of 24 g / L yeast extract, 12 g / L peptone, 0.4% w / v glucose, 2.31 g / L dihydrogen phosphate enzyme, and 12.54 g / L dipotassium hydrogen phosphate, with a pH of 7.0.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] Compared with wild-type formate dehydrogenase, the formate dehydrogenase mutant of the present invention has higher enzyme activity and exhibits faster reaction rate and higher substrate conversion rate under specific reaction conditions (45°C, pH 8.5), with less substrate waste, which is beneficial for efficient product separation.

[0023] The formate dehydrogenase of the present invention has significant advantages in the preparation of NADH at higher pH and higher temperature. The formate dehydrogenation reaction is irreversible and produces only one byproduct, carbon dioxide, which can be directly separated from the product. Since no carbonate is produced or substrate residue is left after the reaction, the purification cost is reduced. The shorter reaction time also helps to reduce energy consumption. Attached Figure Description

[0024] Figure 1 The detection chromatogram for NAD (substrate, oxidized nicotinamide adenine dinucleotide) standard;

[0025] Figure 2 The detection chromatogram for NADH (product, reduced nicotinamide adenine dinucleotide) standard;

[0026] Figure 3 The results are for detecting the mutant after 60 minutes of reaction;

[0027] Figure 4 The results are for detecting the mutant after 180 minutes of reaction.

[0028] Figure 5 The spectrum of the wild-type protein after 180 min of reaction;

[0029] Figure 6 The results are from Example 7. Detailed Implementation

[0030] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the instruments and reagents used in this embodiment are all commercially available products.

[0031] Example 1: Obtaining the wild-type formate dehydrogenase gene sequence

[0032] The secondary structure and codon bias of the formate dehydrogenase gene from *Sinorhizobium terangae* were modified using a whole-genome synthesis method to achieve high expression in *E. coli*. Primers were designed using Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ), ensuring that the annealing temperature (Tm) difference was controlled within 3°C and the primer length within 60 bases. The obtained primers were dissolved in double-distilled water and added to the reaction system to achieve a final primer concentration of 30 nM and a final concentration of 0.6 μM for the first and last primers.

[0033] 10×Pfu buffer 5μL Pfu DNA polymerase (10 U / μL) 0.5μL <![CDATA[ddH2O]]> This brings the total volume of the reaction system to 50 μL.

[0034] Place the prepared PCR reaction system in the Borge XP cycler gene amplification instrument and amplify according to the following program: 98℃ for 30s, 55℃ for 45s, 72℃ for 120s, 35x.

[0035] The DNA fragment obtained by PCR was purified by gel extraction and cloned into the NdeI / XhoI site of pET30a using homologous recombination. Single clones were selected for sequencing. The successfully sequenced DNA sequence is SEQ ID NO: 2, named STFwt, and its corresponding amino acid sequence is SEQ ID NO: 1.

[0036] Example 2: Obtaining the gene sequence of the formate dehydrogenase mutant

[0037] The formate dehydrogenase mutant of this embodiment is derived from the wild-type formate dehydrogenase of Example 1. It catalyzes the conversion of nicotinamide adenine dinucleotide (NADP) to reduced NADP, and can be used for the conversion of NADP to NADP. This formate dehydrogenase mutant exhibits higher catalytic activity compared to the wild-type formate dehydrogenase. The formate dehydrogenase mutant and the polynucleotide encoding this mutant can be prepared using methods commonly used by those skilled in the art. The mutant can be obtained through in vitro recombination of the enzyme, polynucleotide mutagenesis, DNA shuffling, error-prone PCR, and directed evolution methods.

[0038] High expression of the gene in *E. coli* was achieved by modifying its secondary structure and codon bias using whole-genome synthesis. Primers were designed using Primer Premier (http: / / primer3.ut.ee / ) and OPTIMIZER (http: / / genomes.urv.es / OPTIMIZER / ), ensuring the annealing temperature (Tm) difference was controlled within 3°C and the primer length within 60 bases. The obtained primers were dissolved in double-distilled water and added to the following reaction system, resulting in a final primer concentration of 30 nM and a final concentration of 0.6 μM for the first and last primers.

[0039] 10×Pfu buffer 5μL Pfu DNA polymerase (10 U / μL) 0.5μL <![CDATA[ddH2O]]> This brings the total volume of the reaction system to 50 μL.

[0040] Place the prepared PCR reaction system in the Borge XP cycler gene amplification instrument and amplify according to the following program: 98℃ for 30s, 55℃ for 45s, 72℃ for 120s, 35x.

[0041] The DNA fragments obtained by PCR were purified by gel extraction and cloned into the NdeI / XhoI sites of pET30a using homologous recombination. Single clones were selected for sequencing. The successfully sequenced DNA sequence is SEQ ID NO: 4, named STF, and its corresponding amino acid sequence is SEQ ID NO: 3. The mutation sites are E92D, T205L, and A256C.

[0042] Example 3: Shaking Bottle Expression Test

[0043] A single colony of *E. coli* containing the expression vector was picked and inoculated into 3 mL of autoclaved medium containing: 10 g / L tryptone, 5 g / L yeast extract, 3.55 g / L disodium hydrogen phosphate, 3.4 g / L potassium dihydrogen phosphate, 2.68 g / L ammonium chloride, 0.71 g / L sodium sulfate, 0.493 g / L magnesium sulfate heptahydrate, 0.027 g / L ferric chloride hexahydrate, 5 g / L glycerol, 0.8 g / L glucose, and kanamycin to a final concentration of 50 mg / L. The medium was incubated overnight at 30°C and 250 rpm. The following day, take a 1L Erlenmeyer flask and inoculate it into 250mL of autoclaved culture medium at a 1:100 ratio: tryptone 10g / L, yeast extract 5g / L, disodium hydrogen phosphate 3.55g / L, potassium dihydrogen phosphate 3.4g / L, ammonium chloride 2.68g / L, sodium sulfate 0.71g / L, magnesium sulfate heptahydrate 0.493g / L, ferric chloride hexahydrate 0.027g / L, glycerol 5g / L, glucose 0.3g / L, and add kanamycin to a final concentration of 50mg / L. Incubate at 37℃ until the bacterial OD reaches 5-6, then immediately place the Erlenmeyer flask in a 25℃ shaker at 250rpm for 1 hour. Add IPTG to a final concentration of 0.1mM and continue incubating at 25℃ and 180rpm for 12 hours. After incubation, centrifuge the culture at 4℃ and 12000g for 20 minutes to collect the wet bacterial cells. The bacterial precipitate was then washed once with distilled water, and the bacterial cells were collected and stored at -70℃. A small amount of the bacterial cells was also taken for SDS-PAGE analysis.

[0044] Example 4: Batch Feeding Fermentation

[0045] Fed-batch fermentation was conducted in a computer-controlled bioreactor (Beijing Hols) with a capacity of 5L and a working volume of 3.5L. The culture medium used consisted of 24g / L yeast extract, 12g / L peptone, 0.4% glucose, 2.31g / L dihydrogen phosphate enzyme, and 12.54g / L dipotassium hydrogen phosphate, at pH 7.0. A 200mL culture was prepared as the primary inoculum and inoculated at OD 2.0. Throughout the fermentation process, the temperature was maintained at 37℃. Dissolved oxygen concentration was automatically controlled at 30% by a cascade of agitation rate (rpm) and aeration. The pH of the culture medium was maintained at 7.0 by 50% (v / v) orthophosphate and 30% (v / v) ammonia. Fed-batch fermentation was initiated when a significant increase in dissolved oxygen was observed. The fed-batch solution contained 9% w / v peptone, 9% w / v yeast extract, and 14% w / v glycerol. When the OD600 was approximately 50.0 (wet weight approximately 100 g / L), the temperature was controlled at 25 °C, and expression was induced with 0.1 mM IPTG.

[0046] Example 5: Comparison of enzyme activity assays

[0047] Determination of formate dehydrogenase activity:

[0048] Since NADH has an absorption peak at 340 nm, while NAD does not, the activity of formate dehydrogenase can be calculated by detecting the change in NADH absorbance during the reaction. First, adjust the pH of the reaction system to 7.5. Add 0.5 mL of 100 mM pH 7.5 PBS buffer to a 2 mL reaction system, then add 1 mM NAD and 20 mM ammonium formate to a final concentration. Add double-distilled water to a final volume of 1.9 mL, mix thoroughly, and place in a 25°C water bath. Add 100 μL of sample to the reaction system, mix well, and measure the change in absorbance per minute at 340 nm. Calculate the formate dehydrogenase activity using the NADH standard curve. Unit enzyme activity (U) is defined as the amount of enzyme required to generate 1 μmol of NAD per minute. The activity of wild-type formate dehydrogenase was detected using the same method. Compare the results.

[0049] Enzyme activity 155.3U 60.7U

[0050] Example 6: Application of formate dehydrogenase mutant in the preparation of NADH

[0051] Enzyme activity assays are only one method to assess the level of reactivity under specific conditions and cannot accurately reflect the performance under actual production conditions. Therefore, further reaction verification is required based on reaction conditions that conform to reality.

[0052] Weigh 0.7 g of ammonium formate and 2 g of NAD into a 250 mL flask, add 16 mL of pure water, stir to dissolve in a 45 °C water bath, and adjust the pH to 8.5 using 5 M sodium hydroxide. Bring the total volume to 20 mL; the initial substrate concentration is approximately 100 g / L. Finally, add 1 g of enzyme solution to initiate the reaction, and maintain the pH at 8.5 using 5 M sodium hydroxide. Samples were taken at 30, 60, and 180 minutes for analysis. Some detection chromatograms are shown below. Figures 3-5 The test results are shown in the table below:

[0053] 30min 90.11 38.64 60min 95.24 42.59 180min 96.52 57.85

[0054] Example 7: Large-scale preparation of NADH

[0055] Weigh 85 g of ammonium formate and 200 g of NAD powder into a 5 L bioreactor, add 1.6 L of tap water, stir and dissolve in a 45 °C water bath, and adjust the pH to 8.5 with ammonia water. Bring the total volume to 2 L; the initial substrate concentration is approximately 100 g / L. Finally, add 20 g of enzyme solution to begin the reaction, and maintain the pH at 8.5 with ammonia water. Samples were taken and analyzed after 60 minutes of reaction. The results are as follows: Figure 6 As shown, the NADH concentration is approximately 95.6 g / L.

[0056] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A mutant formate dehydrogenase enzyme, characterized in that: Its amino acid sequence is shown in SEQ ID NO:

3.

2. The use of the formate dehydrogenase mutant of claim 1 in the preparation of reduced nicotinamide adenine dinucleotide.

3. Use of the formate dehydrogenase mutant according to claim 2 for the preparation of reduced nicotinamide adenine dinucleotide, characterized in that: Weigh out ammonium formate and NAD into a flask, add pure water, stir and dissolve in a 45°C water bath, and adjust the pH to 8.5 with sodium hydroxide. Add the crude enzyme solution of formate dehydrogenase mutant to react, and maintain the pH at 8.5 with sodium hydroxide.

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