A method for producing purines and ribose

Abstract

The present disclosure provides a purine nucleoside enzyme variant, a nucleic acid sequence encoding the purine nucleoside enzyme variant, and further discloses a composition, a vector and a host cell comprising the purine nucleoside enzyme variant, and further provides a method for improving the efficiency of purine and / or D-ribose production.

Description

Technical Field

[0001] This disclosure pertains to the field of protein engineering, and in particular relates to a highly active nucleoside hydrolase. Background Technology

[0002] Purines are an important component of nucleic acids and are currently used primarily as intermediates in the production of nucleic acid drugs. The current market demand for adenine and guanine is approximately 3,000 tons annually, used as pharmaceutical intermediates. Adenine, also known as 6-aminopurine, has the chemical formula C5H5N5 and is a fused-ring system composed of pyrimidine and imidazole rings. In vivo, adenine is mainly synthesized from amino acids and small molecules through a series of enzymatic reactions. Adenine is not only an important component of DNA and RNA in organisms but also serves as a pharmaceutical intermediate in the production of antiviral drugs and plant growth regulators, and its global consumption has been steadily increasing in recent years.

[0003] Currently, the main methods for producing purines include chemical synthesis, natural raw material extraction, and microbial fermentation. Chemical synthesis primarily refers to the acetyl hypoxanthine method. First, acetyl hypoxanthine and phosphorus oxychloride undergo a chlorination reaction under catalytic conditions to obtain 6-chloropurine. Then, ammonia gas is passed through the 6-chloropurine in an autoclave until saturation, the reactor is sealed, and the temperature is increased until the reaction is complete. After cooling, adenine is precipitated. Besides the acetyl hypoxanthine method, other chemical synthesis processes exist, but their yields are generally low, and the reaction times are long and the reaction conditions are harsh. The acetyl hypoxanthine method generates a large amount of waste liquid after using phosphorus oxychloride, making subsequent environmental treatment difficult. Natural raw material extraction mainly extracts adenine enriched in natural substances, but this method has low yields and high costs, making it unsuitable for industrial production. Microbial fermentation offers mild production conditions, a simple process, and a reaction conversion rate exceeding 99%, making it suitable for industrial production. The specific process involves the enzymatic hydrolysis of adenosine or guanosine by hydrolytic enzymes to produce adenine or guanine and D-ribose. Adenosine or guanosine is mixed with water, and the pH of the reaction system is adjusted to 6.5-7.0. Nucleoside hydrolase is added, and the mixture is heated to 35-37°C and maintained at this temperature for reaction. After the reaction, solid-liquid separation is performed. The liquid phase is concentrated to obtain D-ribose, and the solid phase is washed to obtain adenine and guanine. Currently, most companies in the industry use chemical hydrolysis to hydrolyze nucleosides to produce the corresponding purines, while only a few companies are producing purines using nucleoside hydrolase hydrolysis.

[0004] Currently, the enzymatic hydrolysis of nucleosides to produce purines and D-ribose typically uses crude enzyme solutions derived from cell disruption. This process results in a large amount of intracellular proteins in the supernatant and purine precipitate after hydrolysis, increasing the cost of subsequent separation and purification. Furthermore, using crude enzyme solutions requires prior enzyme disruption, increasing production costs. Additionally, the disrupted enzyme solution is difficult to store and is prone to inactivation. Summary of the Invention

[0005] On the one hand, this disclosure provides a purine nucleoside enzyme variant that, compared to wild-type purine nucleoside enzyme, includes the following modifications: A11H substitution, A12K substitution, S13R substitution, T14R substitution, L15K substitution, L16R substitution, S13K substitution, or T14H substitution.

[0006] In some embodiments, the wild-type purine nucleoside enzyme comprises an amino acid sequence as shown in SEQ ID NO: 1.

[0007] In some embodiments, the purine nucleoside enzyme variant comprises an amino acid sequence as shown in SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9, or an amino acid sequence having at least 80%, 85%, 90%, 95%, or 99% identity with the sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 9.

[0008] On the other hand, this disclosure provides a nucleic acid molecule comprising a nucleic acid sequence encoding a purine nucleoside enzyme variant as described above.

[0009] This disclosure also provides a composition comprising the purine nucleoside enzyme variant as described above.

[0010] This disclosure also provides a vector comprising the nucleic acid molecule as described above. The vector is an expression vector, preferably selected from pET-22b, pET-30a, or pPIC-9k, and more preferably, the expression vector is pET-30a.

[0011] This disclosure also provides a host cell comprising the purine nucleoside enzyme variant, nucleic acid molecule, or composition as described above, wherein the host cell is a fungal cell or a prokaryotic cell.

[0012] In some embodiments, the fungal cells are selected from yeast cells, preferably Pichia pastoris cells or Saccharomyces cerevisiae cells.

[0013] In some embodiments, the prokaryotic cell is a bacterial cell, preferably selected from Escherichia coli, Bacillus, or Corynebacterium.

[0014] The present invention also provides the use of the purine nucleoside enzyme variants, nucleic acid molecules, compositions, carriers, and host cells described above in the preparation of products that produce purines.

[0015] The present invention also provides a method for producing adenine and / or D-ribose, comprising: (1) contacting a purine nucleoside enzyme variant or host cell as described above with adenosine to react; and (2) separating adenine and / or D-ribose from the reaction solution.

[0016] The present invention also provides a method for producing guanine and / or D-ribose, comprising: (1) contacting a purine nucleoside enzyme variant or host cell as described above with guanine to react; and (2) separating guanine and / or D-ribose from the reaction solution.

[0017] In some embodiments, the host cell is selected from Pichia pastoris cells, Saccharomyces cerevisiae cells, Escherichia coli, Bacillus, or Corynebacterium cells, and preferably, the purine nuclease is expressed on the cell membrane. Attached Figure Description

[0018] This disclosure can be more fully understood with reference to the following figures.

[0019] Figure 1 The electrophoresis diagrams of BL21(DE3) / pET 30a-PN and mutant strains are shown. 1, 2: BL21(DE3) / pET30a-PN, crushing supernatant, precipitate; 3, 4: BL21(DE3) / pET30a-PN1(A11H), crushing supernatant, precipitate; 5, 6: BL21(DE3) / pET30a-PN2(A12K), crushing supernatant, precipitate; 7, 8: BL21(DE3) / pET30a-PN3(S13R), crushing supernatant, precipitate; 9, 10: BL21(DE3) / pET30a-PN4(T14R), crushing supernatant, precipitate; 11, 12: BL21(DE3) / pET30a-PN5(L15K), crushing supernatant, precipitate; 13, 14: BL21(DE3) / pET30a-PN6(L16R), crushing supernatant, precipitate; 15, 16: BL21(DE3) / pET 30a-PN7(S13K) crushed supernatant and precipitate; 17, 18: BL21(DE3) / pET 30a-PN8(T14H) crushed supernatant and precipitate. Detailed Implementation

[0020] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0021] To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0022] Table 1. Strains and Plasmids

[0023] Table 2. Primers

[0024] Table 3. Sequence Information

[0025] Culture media and reagents: (1) LB: 0.5% Yeast extract, 1% Tryptone, 1% NaCl, dissolved in deionized water, sterilized at 121 °C for 20 min, solid culture medium with 1.5~2 g agar powder, dispensed and sterilized at 121 °C for 20 min.

[0026] (2) Galactose: Dissolve 10 g of galactose in 36 mL of deionized water, seal in a glass bottle, and sterilize at 115 °C for 20 min.

[0027] (3) Kanamycin: Weigh 10 g of kanamycin, dissolve it in ddH2O and bring the volume to 100 mL. Filter it through a 0.22 μm sterile microporous membrane to remove bacteria, dispense it into 1.5 mL EP tubes, and store it in a -20 °C refrigerator for later use.

[0028] Example Example 1: Construction and expression of nucleoside hydrolase expression vector 1.1 Construction of recombinant plasmids To address the challenge of using crude enzyme solutions for catalysis, we employed a whole-cell catalysis approach. The principle involves adding approximately 20 membrane-friendly signal peptides before the enzyme sequence to guide the target enzyme expression into the periplasmic space of the cell, thereby imparting catalytic activity. Therefore, highly active membrane-friendly signal peptides are crucial for high enzyme expression activity. We selected a nucleoside hydrolase gene, purine nucleosidase (PN), and increased whole-cell enzyme activity by optimizing the amino acid sites in the signal peptide region.

[0029] First, we performed eight site mutations on the signal peptide region MKRILAAVCIAASTLLALPAQADTEK of purine nucleosidase (PN), namely A11H, A12K, S13R, T14R, L15K, L16R, S13K, and T14H. Primers were designed according to homologous recombination to construct the wild-type nucleoside hydrolase gene and the nucleoside hydrolase gene containing the optimized signal peptide sequence into the pET-30a vector for expression. The recombinant plasmids pET30a-PN, pET30a-PN1, pET30a-PN2, pET30a-PN3, pET30a-PN4, pET30a-PN5, pET30a-PN6, pET30a-PN7, and pET30a-PN8 were constructed as follows: (1) Using pET 30a-PN plasmid as a template, the pET 30a vector backbone (5337 bp) was amplified by PCR using pET30a-PNX-SF / pET30a-PNX-SR as primers; using pET 30a-PN plasmid as a template, the genes PN1~PN8 (1085 bp) were amplified by PCR using PN1-IF / PNX-IR, PN2-IF / PNX-IR, PN3-IF / PNX-IR, PN4-IF / PNX-IR, PN5-IF / PNX-IR, PN6-IF / PNX-IR, PN7-IF / PNX-IR, and PN8-IF / PNX-IR as primers, respectively. The amplification system and amplification program are shown in the table below: Table 4. PCR amplification system of PN1~PN8 genes and pET 30a backbone

[0030] Table 5. PCR amplification program for PN1~PN8 genes and pET 30a backbone

[0031] (2) DpnI. Digest the plasmid template and purify the pET 30a backbone and genes PN1-PN8 using a column PCR product purification kit (purchased from Sangon Biotech Co., Ltd.). The plasmid digestion system is shown in the table below: Table 6. Dpn I. Digestion Plasmid System

[0032] (3) The concentrations of the purified backbone and genes PN1~PN8 were measured, and seamless cloning was performed according to the instructions. The system is shown in the table below: Table 7. Seamless Cloning System

[0033] (4) The ligation products were respectively converted to Escherichia coli DH5α competent cells were spread on LB (Kan resistant) plates and incubated overnight at 37°C.

[0034] (5) Pick pET 30a-PN1~ pET 30a-PN8 ( Escherichia coli DH5α transformants were inoculated into LB (Kan resistant) test tubes and cultured overnight at 37°C.

[0035] (6) Using pET 30a-PN1~ pET 30a-PN8 respectively Escherichia coli Using DH5α bacterial culture as a template, PCR verification was performed using pET30a-Verify-F / pET30a-Verify-R primers. The band size was 1245 bp. The verification system and procedure are shown in the table below: Table 8. PCR validation system for pET 30a-PN1 to pET 30a-PN8 transformants

[0036] Table 9. PCR validation procedure for pET 30a-PN1 to pET 30a-PN8 transformants

[0037] (7) PCR products with correct band size should be sent for sequencing, and glycerol tubes with correct sequencing results should be kept in a -80°C refrigerator for sterilization.

[0038] 1.2 Expression of purine nucleosidase variants in recombinant strains (1) Transfer strains pET 30a-PN1~ pET 30a-PN8 (Escherichia coli DH5α) from glycerol tubes to LB (Kan resistant) tubes and activate them overnight at 37°C. The next day, plasmids pET 30a-PN1~ pET 30a-PN8 were extracted using a plasmid mini-prep kit (purchased from Tiangen Biotech Co., Ltd.).

[0039] (2) Preparation Escherichia coli BL21(DE3) competent cells.

[0040] (3) The control plasmid pET 30a-PN and the recombinant plasmids pET 30a-PN1~pET 30a-PN8 were transformed into the following plasmids respectively: Escherichia coli BL21(DE3) was coated onto LB (Kan resistance) plates.

[0041] (4) Select transformants and transfer them to LB (Kan resistant) test tubes, and incubate overnight at 37°C. The next day, transfer 1 mL of bacterial culture to a 100 mL LB shake flask and incubate until OD500. 600 When the concentration reached approximately 0.6, IPTG was added and the target protein expression was induced at 16°C. SDS-PAGE was used to verify the protein expression.

[0042] Depend on Figure 1 As shown, the purine nucleoside enzyme variant was expressed in all recombinant strains, and the total expression level of the mutant protein was increased compared with that of the control strain. Furthermore, the proteins expressed by the BL21(DE3) / pET 30a-PN3(S13R) mutant strain were all active proteins, and no obvious protein bands were observed in the precipitate, indicating that the mutation at this site further promoted the secretion and expression of the target protease into the periplasmic space of the bacterial cell.

[0043] Example 2: Whole-cell catalysis of nucleoside hydrolase expression strain. Whole-cell enzymatic digestion was performed using guanosine or adenosine as substrates to verify the whole-cell expression effect.

[0044] Reaction system: 100 mL reaction system, 300 g / L adenosine or guanosine as substrate, with whole-cell OD... 600 =3 for reaction, and samples are taken every hour to determine the purine and D-ribose content.

[0045] All mutant and control strains were induced under the same conditions, with approximately 1-2 g of bacterial cells added to 100 mL of water, and the substrate being 300 g / L of adenosine or guanosine. For strains with mutation sites 2, 3, 5, 6, and 7, the reaction rate was significantly better than the control strain before 2 hours. The S13K mutation to a membrane-loving amino acid significantly increased intracellular enzyme activity, indicating that these mutations further promoted the secretion and expression of the target protease into the periplasmic space of the bacterial cell.

[0046] Table 10. Data related to the catalytic production of ribose from adenosine by control and mutant strains

[0047] Table 11. Data related to the catalytic production of ribose from guanosine by control and mutant strains

[0048] By incorporating references The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0049] Equivalence This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. A purine nucleoside enzyme variant comprising, compared to wild-type purine nucleoside enzyme, the following modifications: A11H substitution, A12K substitution, S13R substitution, T14R substitution, L15K substitution, L16R substitution, S13K substitution, or T14H substitution.

2. The purine nucleoside enzyme variant of claim 1, wherein the wild-type purine nucleoside enzyme comprises the amino acid sequence shown in SEQ ID NO:

1.

3. The purine nucleoside enzyme variant of claim 1 or 2, comprising an amino acid sequence as shown in any one of SEQ ID NO: 2-9 or an amino acid sequence having at least 80%, 85%, 90%, 95% or 99% identity with the sequence shown in any one of SEQ ID NO: 2-9.

4. A nucleic acid molecule comprising a nucleic acid sequence encoding a purine nucleoside enzyme variant as described in any one of claims 1-3.

5. A composition comprising a purine nucleoside enzyme variant as described in any one of claims 1-3.

6. A vector comprising the nucleic acid molecule as described in claim 4.

7. The vector as claimed in claim 6, wherein the vector is an expression vector, preferably, the expression vector is selected from pET-22b, pET-30a or pPIC-9k, preferably, the expression vector is pET-30a.

8. A host cell comprising a purine nucleoside enzyme variant as described in any one of claims 1-3, a nucleic acid molecule as described in claim 4, or a composition as described in claim 5.

9. The host cell of claim 8, wherein the host cell is selected from yeast cells, preferably, the yeast cells are Pichia pastoris cells or Saccharomyces cerevisiae cells.

10. The host cell of claim 8, wherein the host cell is a prokaryotic cell, preferably, the prokaryotic cell is selected from Escherichia coli, Bacillus, or Corynebacterium.

11. Use of the purine nucleoside enzyme variant of any one of claims 1-3, the nucleic acid molecule of any one of claims 4, the composition of any one of claims 5, the vector of any one of claims 6 or 7, or the host cell of any one of claims 8-10 in the preparation of a product producing purines and D-ribose.

12. A method for producing adenine and / or D-ribose, comprising: (1) The purine nucleoside enzyme variant of any one of claims 1-3 or the host cell of any one of claims 8-10 is contacted with adenosine to carry out the reaction; (2) Separate adenine and / or D-ribose from the reaction solution.

13. A method for producing guanine and / or D-ribose, comprising: (1) The purine nucleoside enzyme variant of any one of claims 1-3 or the host cell of any one of claims 8-10 is contacted with guanosine to carry out the reaction; (2) Separate guanine and / or D-ribose from the reaction solution.

14. The method of claim 12 or 13, wherein the host cell is selected from Pichia pastoris cells, Saccharomyces cerevisiae cells, Escherichia coli cells, Bacillus or Corynebacterium.

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