ACID PHOSPHATASE MUTANT, USE THEREOF AND METHOD FOR PREPARING NICOTINAMIDE RIBOSIDE BY SAME

Abstract

An acid phosphatase mutant, and method for preparing nicotinamide riboside by same. The mutant is a protein of the following (a), (b) or (c): (a) a protein, having an amino acid sequence shown in SEQ ID NO: 3; (b) a protein, derived from (a), having catalytic activity higher than an acid phosphatase parent having an amino acid sequence shown in SEQ ID NO: 2, obtained by substituting, deleting or adding several amino acids in the amino acid sequence shown in SEQ ID NO: 3, using nicotinamide mononucleotide as a substrate; (c) a protein, having catalytic activity higher than the acid phosphatase shown in SEQ ID NO: 2, having 90% or more homology with the amino acid sequence of the protein defined by (a) or (b), and using nicotinamide mononucleotide as a substrate. It is used to prepare nicotinamide riboside and the conversion rate can be 99%.

Claims

1. An acid phosphatase mutant, wherein the mutant is a protein of the following (a), (b) or (c): (a) a protein, having an amino acid sequence shown in SEQ ID NO: 3; (b) a protein, derived from (a), having catalytic activity higher than an acid phosphatase parent having an amino acid sequence shown in SEQ ID NO: 2, obtained by substituting, deleting or adding one or several amino acids in the amino acid sequence shown in SEQ ID NO: 3, and using nicotinamide mononucleotide as a substrate; and (c) a protein, having catalytic activity higher than the acid phosphatase parent shown in SEQ ID NO: 2, having 90% or more homology with the amino acid sequence of the protein defined by (a) or (b), and using nicotinamide mononucleotide as a substrate.

2. The acid phosphatase mutant according to claim 1, wherein compared with the amino acid sequence of the acid phosphatase parent as shown in SEQ ID NO: 2, the mutant has at least one mutation at at least one of the following sites: 44.sup.th site, 115.sup.th site, 206.sup.th site and 240.sup.th site.

3. The acid phosphatase mutant according to claim 1, wherein compared with the amino acid sequence of the acid phosphatase parent as shown in SEQ ID NO: 2, the mutant has at least one of the following mutations: K44C, K115F, K115I, D206I and S240E.

4. A method for preparing nicotinamide riboside, wherein the method comprises: catalyzing nicotinamide mononucleotide by acid phosphatase to convert into nicotinamide riboside in the presence of KCl and anhydrous MgCl.sub.2, the acid phosphatase being an acid phosphatase parent having an amino acid sequence shown in SEQ ID NO: 2 or an acid phosphatase mutant, and the acid phosphatase mutant being a protein of the following (a), (b) or (c): (a) a protein, having an amino acid sequence shown in SEQ ID NO: 3; (b) a protein, derived from (a), having catalytic activity higher than an acid phosphatase parent having an amino acid sequence shown in SEQ ID NO: 2, obtained by substituting, deleting or adding one or several amino acids in the amino acid sequence shown in SEQ ID NO: 3, and using nicotinamide mononucleotide as a substrate; and (c) a protein, having catalytic activity higher than the acid phosphatase parent shown in SEQ ID NO: 2, having 90% or more homology with the amino acid sequence of the protein defined by (a) or (b), and using nicotinamide mononucleotide as a substrate.

5. The method for preparing the nicotinamide riboside according to claim 4, wherein the method sequentially comprises the following steps: 1) dissolving KCl and anhydrous MgCl.sub.2 in water to obtain a mixed solution of KCl and MgCl.sub.2; 2) cooling to room temperature; 3) adding the nicotinamide mononucleotide to the mixed solution of KCl and MgCl.sub.2, dissolving and adjusting a pH value to 6 to 7; 4) adding the acid phosphatase and adjusting the pH value to 6.8 to 7.3; and 5) reacting at a temperature of 375 C. for at least 1.5 hours.

6. The method for preparing the nicotinamide riboside according to claim 4, wherein the method also comprises: adding konjac glucomannan and/or rebaudioside A to a reaction system before an enzyme reaction that the acid phosphatase catalyzes the nicotinamide mononucleotide to convert into the nicotinamide riboside starts.

7. The method for preparing the nicotinamide riboside according to claim 4, wherein the method also comprises: the acid phosphatase is added in a form of a pure enzyme solution, and the pure enzyme solution refers to a solution containing the acid phosphatase, obtained by purifying, by a nickel column, a crude enzyme solution obtained after being induced by a microorganism containing the acid phosphatase gene to express, and then subjected to cell disruption, and centrifugation to remove a precipitate.

8. The method for preparing the nicotinamide riboside according to claim 4, wherein the acid phosphatase is an immobilized enzyme, and the immobilized enzyme is prepared by the following method: adding 0.2 to 0.8 M K.sub.2HPO.sub.4 to the acid phosphatase, adjusting the pH value to 7.5 to 9.5, adding a solid phase resin to stir at room temperature for 12 to 24 h, and then filtering to remove a filtrate to obtain the immobilized enzyme.

9. The method for preparing the nicotinamide riboside according to claim 4, wherein the method also comprises a post-treatment process for obtaining a nicotinamide riboside refined product by purifying an enzyme reaction solution in which the nicotinamide mononucleotide is catalyzed by the acid phosphatase to convert into the nicotinamide riboside, and the post-treatment process comprises the following steps: 1) filtering the enzyme reaction solution to collect a filtrate; 2) adjusting the pH value of the filtrate to 5.00.5; 3) microfiltering the filtrate of which the pH value is adjusted in step 2) to collect a filtrate; 4) purifying a microfiltrate obtained by microfiltering in step 3) with a preparative liquid phase, the preparative liquid phase using polystyrene as a filler of a preparation column, using ethanol and water as a mobile phase, performing gradient elution, and collecting an eluate of a peak of interest; and 5) lyophilizing the eluate collected in step 4) to obtain the nicotinamide riboside refined product.

10. (canceled)

11. A biological material, comprising a recombinant vector, a recombinant cell or a recombinant microorganism, wherein the biological material comprises a gene encoding the acid phosphatase mutant according to claim 1, 2 or 3.

12. (canceled)

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0063] FIG. 1 is a construction process diagram of a recombinant plasmid pET22b-NS3 in

[0064] Embodiment 1 of the present invention;

[0065] FIG. 2 is an HPLC chromatogram of an enzyme reaction solution of a mutant K1151 in Embodiment 7;

[0066] FIG. 3 is an HPLC chromatogram of an enzyme reaction solution of a parent in Embodiment 8; and

[0067] FIG. 4 is an HPLC chromatogram of an enzyme reaction solution of a mutant K1151 in Embodiment 9.

DETAILED DESCRIPTION

[0068] The present invention will be further described in detail below with reference to the specific embodiments and the accompanying drawings. The following embodiments illustrate the present invention. The present invention is not limited to the following embodiments, and the embodiments in which specific conditions are not specified are implemented according to conventional conditions or conditions recommended by manufacturers. Unless otherwise stated, the raw materials and other chemical reagents used in the embodiments of the present invention are commercially available goods.

Embodiment 1

[0069] Preparation of Acid Phosphatase Parent.

[0070] An acid phosphatase gene NS-3 (the amino acid sequence thereof is shown in SEQ ID NO: 2) shown in SEQ ID NO: 1 and derived from Saccharomyces cerevisiae was amplified by a PCR amplification technology using the following primer pair 5-GGAATTCCATATGATGACCATTGCGAAGGATTACCGT-3 and 5-CCGGAATT CTTAGTGGTGGTGGTGGTGGT-3, an obtained PCR amplification product was digested and simultaneously inserted into Ndel and EcoR I sites of an expression vector pET22b(+) to obtain a recombinant plasmid pET22b-NS3, as shown in FIG. 1. After being validated by sequencing, the recombinant plasmid was transferred into E. coli Rosetta (de3). The obtained recombinant E. coli was inoculated into a small volume of LB medium (containing 100 g/mL of Amp), cultured at a temperature of 30 to 37 C. overnight, and then transferred to a certain volume of LB medium (containing 100 g/mL of Amp) at an inoculated dose of 1 to 5%, continuously cultured at a temperature of 30 to 37 C. until OD600 reaches 0.6 to 1.0, added with isopropyl--D-thiogalactoside (IPTG) having a final concentration of 0.1 mM to 1 mM, and induced to express at a temperature of 20 to 37 C. for 10 to 20 hours, and then a thallus was collected by centrifugation.

Embodiment 2

[0071] Preparation of Acid Phosphatase Mutant

[0072] The following mutation sites: K44C, K115F, K115I, D206I, S240E, K115I/S240E, K115F/K44C/D206I, and K115I/S240E/D206I were designed, mutant primer sequences shown in Table 1 were utilized to perform reverse PCR by using the recombinant plasmid pET22b-NS3 constructed in Embodiment 1 as a template. The PCR system and PCR procedure were as follows:

[0073] PCR system:

TABLE-US-00001 PrimeSTAR GXL DNA Polymerase 0.5 ul 5PrimeSTAR GXL Buffer (Mg2+ plus)*2 0 ul Template plasmid 1 ul dNTP (2.5 mM each) 4 ul Mutant primer 1 1 ul Mutant primer 2 ul Sterile water up to 50 ul

[0074] PCR procedure:

TABLE-US-00002 98 C. 3 min 98 C. 10 s 30 50-65 C. 15 s 72 C. 7 min Cycle 30 times 72 C. 10 min

[0075] A PCR product was purified by a gel extraction kit, and then digested with DpnI. After digestion, the product was transferred into E. coli DH5a. After screening by an Amp plate, colonies were picked and sent for sequencing. It was confirmed that the mutant recombinant plasmid with successful mutation at K44C, K115F, K115I, D206I. S240E, K115I/S240E, K115F/K44C/D206I, and K115I/S240E/D206I sites was transferred into E. coli Rosetta (de3). The obtained mutant recombinant E. coli was inoculated into a small volume of LB medium (containing 100 g/mL of Amp), cultured at a temperature of 30 to 37 C. overnight, and then transferred to a certain volume of LB medium (containing 100 g/mL of Amp) at an inoculated dose of 1 to 5%, continuously cultured at a temperature of 30 to 37 C. until OD600 reaches 0.6 to 1.0, added with isopropyl--D-thiogalactoside (IPTG) having a final concentration of 0.1 mM to 1 mM, and induced to express at a temperature of 20 to 37 C. for 10 to 20 h, and then a thallus was collected by centrifugation.

TABLE-US-00003 TABLE1 Primer name Primersequence(5to3) D206I_F 5-TTCATCTGCAAGCCGATTCCGAAATTTTTCGAG-3 D206I_R 5-CTCGAAAAATTTCGGAATCGGCTTGCAGATGAA-3 K115I_F 5-GGCCTGATTAAGAACATTCAGATCGATGACGTT-3 K115I_R 5-AACGTCATCGATCTGAATGTTCTTAATCAGGCC-3 K115F_F 5-GGCCTGATTAAGAACTTCCAGATCGATGACGTT-3 K115F_R 5-AACGTCATCGATCTGGAAGTTCTTAATCAGGCC-3 S240E_F 5-GTGCGTAGCGCGCTGGAAATGGGTATGGGCCAC-3 S240E_R 5-GTGGCCCATACCCATTTCCAGCGCGCTACGCAC-3 K44C_F 5-GAAGTGGACCCGCCGTGCCTGCCGGATCCGGAC-3 K44C_R 5-GTCCGGATCCGGCAGGCACGGCGGGTCCACTTC-3

Embodiment 3

[0076] Preparation of Crude Enzyme Solution of Acid Phosphatase

[0077] A thallus collected was resuspended and fermented using a 1 PBS+10 mM imidazole solution of which the mass is 4 times the mass of the thallus, and then the thallus was disrupted by a high-pressure homogenizer; after the homogenization was completed, 0.5 to 1% of the total volume of polyethyleneimine (PEI) was added under stirring, and the pH was adjusted to 7.0 to 8.0; and a supernatant clarified enzyme solution was collected by centrifugation, the enzyme solution was filtered through a 0.45 L filter membrane, and a filtrate was collected to obtain a crude enzyme solution of acid phosphatase.

Embodiment 4

[0078] Preparation of Nickel Column

[0079] a) after a column was cleaned, agarose gel was used as a nickel carrier, the agarose gel was taken according to a ratio of nickel filler (ml) to thallus (g) of 1 to 2, and the agarose gel was washed by successively using ddH2O and 1 PBS with 5 column volumes separately;

[0080] b) after a 0.2 mol/L nickel chloride solution was prepared, nickel was hung, and in order to fully hang the nickel, the hanging may be repeated; and

[0081] c) unbound Ni.sup.2+ was washed away with 5 column volumes of ddH.sub.2O, and the nickel column was equilibrated with 5 to 10 column volumes of a 1PBS+10 mM imidazole solution (pH=8.0).

Embodiment 5

[0082] Loading and Elution

[0083] a) The crude enzyme solution of the acid phosphatase obtained in Embodiment 3 was uniformly mixed with the nickel filler, then transferred to a conical flask, and incubated in a ice bath on a shaker for 1 to 2 h at a rotation speed of 100 rm;

[0084] b) a heteroprotein was eluted with a 1 PBS+10 mM imidazole solution (pH=8.0), and a target protein was fully eluted with a 1 PBS+200 mM imidazole solution (pH=8.0), and an eluate was collected to obtain a pure enzyme solution of acid phosphatase.

Embodiment 6

[0085] Enzyme Activity Comparison Experiment

[0086] Determination method of enzyme activity: KCl having a final concentration of 200 mM and MgCl.sub.2 having a final concentration of 400 mM were used as cofactors, NMN having a final concentration of 2 mmol was added to a 200 uL reaction system, then 10 uL of the diluted pure enzyme solution was added, and the pure enzyme solution was replaced with a corresponding buffer in a blank control; the solutions were uniformly mixed to react for 10 min under the conditions that the temperature was 37 C. and the pH was 7.0, then 200 L of a 10% trichloroacetic acid solution was immediately added to be uniformly mixed, the reaction was terminated, a sample was taken and diluted with methanol by 50 times, then the amount of the product was determined by HPLC, and the enzyme activity unit U was calculated.

[0087] Definition of enzyme activity U of acid phosphatase: the amount of enzyme required to hydrolyze the substrate NMN in 1 minute to release 1 umol under the conditions that the temperature is 37 C. and the pH is 7.0 is called one enzyme activity international unit (U).

[0088] The comparison experiment results of the enzyme activity and stability of the acid phosphatase parent and the acid phosphatase mutant of the present invention are shown in Table 2, where the temperature stability and pH stability mean that the target enzyme is in a storage range that after the target enzyme is stored for 12h under the corresponding storage conditions, the residual activity is more than or equal to 90%.

TABLE-US-00004 TABLE 2 Enzyme activity Temperature pH Type U stability stability Parent 365.8 8.5 4-35 C. 6.0-8.0 D206I 413.7 5.3 4-35 C. 6.0-8.0 K115I 826.8 6.3 4-50 C. 5.0-9.0 K115F 441.5 9.6 4-40 C. 6.0-8.5 S240E 502.9 4.8 4-35 C. 5.5-8.0 K44C 392.6 7.4 4-35 C. 6.0-8.0 K115I/S240E 931.7 3.1 4-50 C. 4.5-9.0 K115F/K44C/D206I 964.5 5.2 4-50 C. 5.0-9.0 K115I/S240E/D206I 1038.8 6.7 4-55 C. 5.0-9.5

[0089] The results show that compared with the acid phosphatase parent, the acid phosphatase mutant of the present invention is more obviously improved in enzyme activity, temperature stability and pH stability.

Embodiment 7

[0090] Preparation of Nicotinamide Riboside

[0091] A 2 total system was designed, the final concentration of a substrate was set to 100 g/L, a clean 3 three-necked bottle was prepared, 1 of ddH.sub.2O was added, then 30 g of KCl and 80 g of anhydrous MgCl.sub.2were added to be stirred and dissolved, after the solution temperature drops to room temperature, 200 g of NMN was added, and the pH was adjusted to 6 to 7 with 5 M NaOH so that the dissolution was complete. The reaction temperature was set to 37 C., 900 ml of the pure enzyme solution of the acid phosphatase mutant K115I obtained in Embodiment 5 was added, and a sodium phosphate buffer of pH 7.0 was used to be supplemented to the 2 system, and the reaction system was adjusted to pH 7.2 to start reaction. After reacting for 2 hours, a reaction solution was taken and diluted by 50 to 100 times with pure water, and the results of the reaction were analyzed by high performance liquid chromatography after millipore filtration. The analysis method is shown in Table 3, and the HPLC chromatogram is shown in FIG. 2. The detection results show that the substrate NMN is degraded more obviously, about 42% of the substrate is degraded, and the conversion rate of the remaining NMN to NR is 99.9%.

TABLE-US-00005 TABLE 3 Chromatographic Welch Xtimate C18 5 m 250 4.6 mm column Mobile phase A: Sodium dihydrogen phosphate buffer (a 100 mM aqueous solution of sodium dihydrogen phosphate, pH adjusted to 6.6 with 6 mol of sodium hydroxide) Mobile phase B: Methanol (chromatographic grade) Instrument model Thermo Fisher U3000 Detector UV260 nm Flow rate: 1.0 ml/min Column 25 C. temperature: Injection volume: 20 l Sample treatment Dissolve in water Gradient: Time/min Phase A(%) Phase B(%) 0 99 1 4 99 1 5 95 5 10 85 15 12 80 20 12.1 99 1 16 99 1 Remarks: Calculated by volume ratio

Embodiment 8

[0092] A 2 total system was designed, the final concentration of the substrate was set to 100 g/L, a clean 3 three-necked bottle was prepared, 1 of ddH.sub.2O was added, then 30 g of KCl and 80 g of anhydrous MgCl.sub.2were added to be stirred and dissolved, after the solution temperature drops to room temperature, 20 g of konjac glucomannan was added, then 200 g of NMN was added and the pH was adjusted to 6 to 7 with 5 M NaOH so that the dissolution was complete. The reaction temperature was set to 37 C., 900 ml of the pure enzyme solution of the acid phosphatase mutant K115I obtained in Embodiment 5 was added, and a sodium phosphate buffer of pH 7.0 was used to be supplemented to the 2 system, and the reaction system was adjusted to pH 7.2 to start reaction. After reacting for 2 hours, a reaction solution was taken and diluted by 50 to 100 times with pure water, and the results of the reaction were analyzed by high performance liquid chromatography after millipore filtration. The analysis method is shown in Table 3, and the HPLC chromatogram is shown in FIG. 3 The detection results show that the substrate NMN is not degraded obviously, and the conversion rate of NMN to NR is 99.8%.

Embodiment 9

[0093] A 2 total system was designed, the final concentration of the substrate was set to 100 g/L, a clean 3 three-necked bottle was prepared, 1 of ddH.sub.2O was added, then 30 g of KCl and 80 g of anhydrous MgCl.sub.2were added to be stirred and dissolved, after the solution temperature drops to room temperature, 20 g of konjac glucomannan was added, then 200 g of NMN was added and the pH was adjusted to 6 to 7 with 5 M NaOH so that the dissolution was complete. The reaction temperature was set to 37 C., 900 ml of the pure enzyme solution of the acid phosphatase mutant K115I obtained in Embodiment 5 was added, and a sodium phosphate buffer of pH 7.0 was used to be supplemented to the 2 system, and the reaction system was adjusted to pH 7.2 to start reaction. After reacting for 2 hours, a reaction solution was taken and diluted by 50 to 100 times with pure water, and the results of the reaction were analyzed by high performance liquid chromatography after millipore filtration. The analysis method is shown in Table 3, and the HPLC chromatogram is shown in FIG. 4. The detection results show that the substrate NMN is not degraded obviously, and the conversion rate of NMN to NR is 72.5%.

Embodiment 10

[0094] After a catalytic reaction for preparing nicotinamide ribose was completed, an enzyme reaction solution was filtered through 200-400 mesh filter cloth to remove the acid phosphatase therein, 2 to 6 M hydrochloric acid was added to an obtained filtrate to adjust the pH to about 5.0, then microfiltration was performed by a 0.45 m microfiltration membrane, and a microfiltrate was collected; the microfiltrate was purified by a preparative liquid phase, the control conditions of the preparative liquid phase being shown in Table 4, and an eluate of an NR segment was collected to obtain a nicotinamide riboside aqueous solution having a purity greater than 99%; then, the obtained nicotinamide riboside aqueous solution having the purity greater than 99% is subjected to nanofiltration concentration at a temperature of 25 C. or below to increase the nicotinamide riboside concentration to 50 to 60 g/L, and then the hydrochloric acid was added to adjust the pH to 3 to 4; the nicotinamide riboside aqueous solution having a concentration of 90 g/L was added with the konjac glucomannan, and fully dissolved, stirred and mixed uniformly; and drying treatment was performed in a lyophilizer, the sublimation temperature in the drying process was controlled to 25 C. or below, and a white powdered nicotinamide riboside refined product could be obtained after 24 hours, and has a purity of 99% or more. The yield and purity of the nicotinamide riboside refined product obtained after separating and purifying the enzyme reaction solutions in the above-mentioned Embodiments 7, 8, and 9 are shown in Table 5.

TABLE-US-00006 TABLE 4 Preparation column Polystyrene preparation column Mobile phase A: Absolute ethanol Mobile phase B: Pure water Instrument Model Beijing Chuangxin Tongheng LC6000 Detector UV260 nm Flow rate: 40 ml/min Column 25 C. temperature: Injection volume: NR10 g (volume: not more than 200 mL) Sample treatment Microfiltration membrane Gradient: Time/min Phase A(%) Phase B(%) 0 0 100 13 1 99 23 5 95 31 20 80 43 20 80 45 0 100 60 0 100 Remarks: Calculated by volume ratio

TABLE-US-00007 TABLE 5 Embodiment7 Embodiment8 Embodiment9 Weight of 226.05 g 389.78 g 282.59 g lyophilized powder Purity of 99.31% 99.70% 99.62% lyophilized powder