A method for constructing a beta-nicotinamide adenine dinucleotide kinase mutant

Abstract

This invention discloses a method for constructing a β-nicotinamide adenine dinucleotide kinase mutant, relating to the field of biocatalysis technology, comprising the following steps: constructing an NADK mutant library through homology modeling and result analysis to obtain key energy-contributing residues; performing PCR amplification on the wild-type NADK gene to obtain PCR amplification products; double digesting the PCR amplification products with the expression vector pET-28a(+), followed by purification and ligation, transforming them into E. coli BL21(DE3), screening to obtain NADK mutant strains; inducing expression in the NADK mutant strains, collecting the crude NADK mutant enzyme solution, heating and centrifuging, and passing the supernatant through a Ni-NTA column to obtain the β-nicotinamide adenine dinucleotide kinase mutant.

Description

Technical Field

[0001] This invention relates to the field of biocatalysis technology, specifically a method for constructing a β-nicotinamide adenine dinucleotide kinase mutant. Background Technology

[0002] Nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP) participate in intracellular redox metabolism and are key cofactors in organisms. In organisms, NAD can be synthesized via the kynurenic acid pathway, the Preiss-Handler pathway, and the rescue pathway, while NADP can only be obtained by catalyzing NAD through β-nicotinamide adenine dinucleotide kinase (NADK). Therefore, a lack of NADK affects the conversion of NAD to NADP. Since NADP plays a crucial role in biological processes such as energy conversion, protein synthesis, and DNA repair, a lack of NADK can severely impact the survival of organisms.

[0003] In industry, NADP is a commonly used enzyme catalyst, widely applied in biomanufacturing and biofuel production. In agriculture, NADP enhances crop resistance to stress. In food production, NADP is involved in the production of natural food additives. In healthcare, NADP is not only a key cofactor in many drug synthesis reactions but also an important component of numerous antioxidant drugs and health products. Furthermore, with the increasing demand for health and beauty, the market for NADP continues to expand. However, the large-scale industrial production of NADP currently faces many challenges.

[0004] Currently, the main methods for synthesizing NADP include chemical synthesis, bio-fermentation, and enzymatic methods. Chemical synthesis involves reacting NAD and phosphorus oxychloride under strongly acidic conditions. This reaction requires high temperature and pH < 2, making the conditions harsh and prone to NAD decomposition, resulting in a yield of only 30-40%. Furthermore, the synthesis process generates large amounts of phosphorus-containing wastewater, easily causing environmental pollution. The reaction also readily produces byproducts, making product purification difficult, requiring multiple column chromatography analyses, which is costly and hinders industrial-scale production. Traditional bio-fermentation methods often use fermentation or other microbial culture media to extract NADP from fermenting microorganisms. This method suffers from high raw material consumption, long reaction times, low synthesis yields, and high production costs, and the high price of the product limits the widespread application of NADP. In the process of bioenzymatic synthesis, wild-type Escherichia coli NADK is commonly used to synthesize NADP using NAD and ATP as substrates. This method has mild synthesis conditions, but the inhibition constant of the product NADP is only 0.2 mM, which makes the wild-type enzyme NADK severely inhibited by NADP. Moreover, the conversion rate can only be achieved in the gram range. In addition, this method requires the presence of ATP, which is expensive, increasing the production cost by more than 50%, making it difficult to achieve economical production.

[0005] Therefore, it is of great significance to develop a NADK with high stability and low product inhibition. Summary of the Invention

[0006] The purpose of this invention is to provide a method for constructing a β-nicotinamide adenine dinucleotide kinase mutant to solve the problems raised in the prior art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: A method for constructing a β-nicotinamide adenine dinucleotide kinase mutant includes the following steps: S1: A NADK mutation library was constructed through homology modeling and result analysis to obtain key energy-contributing residues; S2: The wild-type NADK gene was amplified by PCR to obtain the PCR amplification product; the PCR amplification product was double-digested with the expression vector pET-28a(+), then purified and ligated, and transformed into E. coli BL21(DE3). After screening, NADK mutant strains were obtained. S3: NADK mutant strains were induced to express the enzyme, and the crude NADK mutant enzyme solution was collected. After heating and centrifugation, the supernatant was passed through a Ni-NTA column to obtain the β-nicotinamide adenine dinucleotide kinase mutant.

[0008] Furthermore, the DNA sequence of the NADK is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2.

[0009] Furthermore, the NADK mutant was obtained through saturation mutation screening.

[0010] Furthermore, the β-nicotinamide adenine dinucleotide kinase mutant combination is to mutate glycine G at position 127 of the NADK gene sequence to arginine R, and valine V at position 239 to proline P.

[0011] Further, in step S2, during the PCR amplification process, the amplification program includes initial denaturation, cycling, and final extension; the initial denaturation condition is 94℃ for 5 min; the cycling stage includes three phases: denaturation, annealing, and extension, with corresponding temperatures of 94℃, 55℃, and 72℃, and times of 30s, 30s, and 1 min, respectively; the cycling stage is repeated 30 times; the final extension condition is 72℃ for 10 min.

[0012] Furthermore, in step S2, during the PCR amplification process, the forward and reverse primers are designed based on the corresponding genomic DNA sequences.

[0013] Further, in step S3, the elution buffer of the Ni-NTA column is composed of Tris-HCl, sodium chloride and imidazole, and the molar ratio of Tris-HCl, sodium chloride and imidazole is (15-25):(250-350):(200-300).

[0014] Compared with the prior art, the beneficial effects of the present invention are: 1. The NADK mutant constructed in this invention mutates glycine at position 127 to arginine. The positively charged guanidino side chain on arginine can adjust the conformation of the NADP binding site, enhancing the affinity of NADK for the substrate and thus improving the reaction conversion rate of NADK. Simultaneously, valine at position 239 is mutated to proline. The pyrrolidine ring on proline restricts chain movement, enhancing the conformational rigidity of NADK and thus improving the enzyme's thermostability. Furthermore, proline disrupts the α3-helix structure in the heat-sensitive region of NADK, enhancing the hydrophobicity of the enzyme's internal structure and further improving its thermostability.

[0015] 2. The G127R V239P mutant obtained in this invention still exhibits good thermal stability at 65℃. This mutant can synthesize NADP using NAD and sodium tripolyphosphate as substrates via a bioenzymatic method. The synthesis conditions are mild, and the conversion rate is ≥97% when the product concentration is 100 g / L, while the product inhibition effect is significantly reduced. Therefore, this technology provides a possibility for the large-scale industrial production of NADP. Detailed Implementation

[0016] The technical solutions in the embodiments of the present invention will be clearly and completely described below. 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.

[0017] In the examples, test methods without specific conditions are generally performed under conventional conditions.

[0018] Example 1: A method for constructing a β-nicotinamide adenine dinucleotide kinase mutant: Step 1: Constructing an NADK mutant library: S1: Obtain the NADK sequence of wild-type Escherichia coli from UniProt; S2: By comparing the PDB database with the SWISS-MODEL server, the best template 3Q0L is obtained by using resolution and sequence consistency as screening criteria. S3: An initial model was constructed on Modeller 10.4 using the wild-type NADK sequence and 3Q0L structural plate. The model quality was evaluated using Ramachandran plots and QMEAN scores generated by PROCHECK. By comparing and analyzing the hydrogen bond network using LIGPLOT, the key functional domains were identified as follows: ATP binding pocket: G10-G13 loop (Gly10-Gly13); NADP binding site: D36-R39-G127; and heat-sensitive region: α3 helix (V239-V210). S4: Substrate / product molecular docking was performed and free energy was calculated using AutoDock Vina 1.2.3 software. The binding free energy was decomposed by calculating the MM / GBSA method, and the key energy contributing residues were found to be G127 and V239. Step 2: Gene cloning and strain construction of NADK mutants: S1: Nucleotide sequence optimization was performed based on the amino acid sequence. 1 mL of TOP10 E. coli bacterial culture was cultured overnight, centrifuged at 12,000 rpm for 1 min to collect the bacterial cells, and 200 μL of lysis buffer (10 mM Tris-HCl, 1 mM EDTA, 0.1% Triton X-100) was added. The mixture was heated at 95℃ for 10 min, then placed on ice for 5 min. After centrifugation at 12,000 rpm for 5 min, the supernatant was collected to obtain the template DNA. S2: Amplify the template DNA by PCR to obtain the PCR amplification product; PCR amplification system: 10×PCR buffer: 5 μL; dNTPs (2.5 mM): 4 μL; Forward primer (10 μM): 2 μL; Reverse primer (10 μM): 2 μL; Template DNA: 1 μL; Taq DNA polymerase: 0.5 μL; ddH2O: 35.5 μL.

[0019] Specifically, the corresponding PCR forward and reverse primers were designed based on the corresponding genomic DNA sequence (SEQ ID NO.1); S3: Wild-type NADK gene and pET-28a(+) vector plasmid DNA were digested with NdeI and XhoI under the following conditions: 37℃ water bath for 3 h to obtain the digestion products. S4: The enzyme digestion products were electrophoresed on a 1% agarose gel at 100V for 30 min. The target band was excised and purified using a gel extraction kit. The purified product was ligated with T4 DNA ligase at 16℃ for 12 h to obtain the ligation product. S5: Inoculate a single colony of Escherichia coli BL21(DE3) into 5 mL of LB medium and incubate at 37°C with shaking for 12 h. Then, transfer the colony to 50 mL of LB medium at a 1% ratio. When the OD... 600 When the concentration of calcium chloride (CLC50) is 0.6, incubate on ice for 30 min, centrifuge to collect the bacterial cells, resuspend in 10 mL of pre-cooled 0.1 M calcium chloride, incubate on ice for 30 min, centrifuge, aliquot into 100 μL, and store at -80 °C to obtain competent cells. S6: Take 100 μL of competent cells, add 10 μL of ligation product, incubate on ice for 30 min, heat shock at 42℃ for 90 s, incubate on ice for 2 min, then add 900 μL of LB medium, revive at 37℃ for 1 h, and then spread on LB plates containing 50 μg / mL kanamycin to obtain BL21 strain containing pET-28a(+)-NADK recombinant plasmid; Step 3: Preparation and purification of NADK enzyme solution: S1: Inoculate 1% of the bacterial culture into LB medium containing 50 mg / L kanamycin and incubate overnight at 37°C. The next day, inoculate 5% of the bacterial culture into LB medium containing 50 mg / L kanamycin and incubate at 37°C with shaking at 200 rpm for 2 hours. When the OD600 reaches approximately 0.6, add 0.2 mM IPTG and induce at 30°C with 200 rpm for 4 hours. After induction, centrifuge the bacterial culture at 12000 rpm for 2 minutes at 4°C, discard the supernatant, invert the culture to drain, and then resuspend the cells in a 1:4 (w / w) solution of lysis buffer (pH 7.4). Disrupt the bacterial culture using an ultrasonic homogenizer for 2 minutes. Centrifuge again, and the resulting supernatant is the crude enzyme solution. Collect the crude enzyme solution of the mutant using this method. S2: After heat-treating the crude mutant enzyme solution at 65℃ for 30 min, centrifuge and pass the supernatant through a Ni-NTA column (elution buffer: 20 mM Tris-HCl, 300 mM NaCl, 250 mM imidazole) to obtain the purified mutant enzyme solution.

[0020] Detection experiment: Conversion rate detection at high concentration: Reaction system: 50 mM Tris-HCl (pH 70), 150 mM NAD, 150 mM STP, 20 mM MgCl2, 0.5 mg / mL enzyme, reaction at 37℃ for 10 h; HPLC conditions: C18 column (4.6 × 250 mm), mobile phase 10 mM potassium phosphate (pH 6.5) / methanol (95:5), detection wavelength 260 nm. Single-point saturation mutation experimental data are shown in Table 1 below.

[0021] Table 1 Results of G127 mutant Conclusion: Compared with other site-directed mutations of amino acids, the mutation of glycine (G) to arginine (R) at position 127 of the NADK sequence resulted in the best reaction conversion rate, 85.4%. In this invention, amino acid position 127 is located at the NADP binding site. Compared to the short side chain of glycine with only hydrogen atoms, the long side chain of arginine with a positively charged guanidinyl group can reshape the local structure of the NADP binding site on the enzyme, optimize the geometry of the catalytic pocket, enhance the strong electrostatic interaction between the active site and NADP 2'-phosphate, increase the affinity of NADK for the substrate, and thus improve the reaction conversion rate of NADK. Therefore, further combined mutations were performed based on the mutant at position 127.

[0022] Table 2 Results of G127V239 mutant Conclusion: Based on the optimal mutation in the first round, the mutation of valine at position 239 to proline in this round resulted in the highest substrate conversion rate, 98.24%. Compared to the β-carbon branched side chain on valine, the pyrrolidine ring in proline not only restricts chain movement, reduces the local flexibility of the heat-sensitive region, and stabilizes the conformation of the NADK enzyme, but also lowers the free energy during NADK folding, thus improving the enzyme's thermal stability. Furthermore, proline disrupts the α3-helix structure on the NADK heat-sensitive region, enhancing the hydrophobicity of the enzyme's internal structure, thereby improving its thermal stability.

[0023] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, it is intended that all variations falling within the meaning and scope of equivalents of the claims be included within the present invention.

Claims

1. A β-nicotinamide adenine dinucleotide kinase mutant, characterized in that: The mutation combination of the β-nicotinamide adenine dinucleotide kinase mutant is to mutate glycine G at position 127 of the NADK amino acid sequence to arginine R and valine V at position 239 to proline P. The NADK was obtained through saturation mutation screening; the DNA sequence of the NADK is shown in SEQ ID NO. 1, and the amino acid sequence is shown in SEQ ID NO. 2.

Images