Nuclease mutant for heparin purification and use thereof

Abstract

This invention discloses a heparin purification nuclease mutant and its application. It is derived from the nuclease with the amino acid sequence shown in SEQ ID NO.1 as the parent, modified by amino acid site mutation. The mutation scheme is selected from one or more combinations of T77S, Q111K, L126F, N127M, D138V, Q150F, K153Q, Y164Q, I197F, T235I, K254Q, and G263P. Compared with the parent, this invention significantly improves enzyme activity, can efficiently degrade residual nucleic acid impurities in crude heparin, and is simple to operate and low in cost.

Description

Technical Field

[0001] This invention relates to the field of enzyme engineering technology, and in particular to a nuclease mutant for heparin purification and its application. Background Technology

[0002] Nucleases are hydrolases that can directly cleave phosphodiester bonds, degrading ribonucleic acid (RNA) or deoxyribonucleic acid (DNA). Based on their site of action, nucleases can be classified into exonucleases and endonucleases. Exonucleases hydrolyze RNA chains one end at a time, while endonucleases act on the phosphodiester bonds within polynucleotides. Nucleases currently have a wide range of applications in many fields.

[0003] Heparin is a sulfated mucopolysaccharide composed of alternating uronic acid and glucosamine linked by glycosidic bonds, with a molecular weight ranging from 1 kDa to 20 kDa. A unique pentose sequence on its molecular chain gives it anticoagulant activity both in vivo and in vitro, making it an important anticoagulant. Natural heparin is a mixture of polysaccharides, but only heparin containing the specific pentose sequence fragment can efficiently bind to antithrombin III; other fragments have very low or no anticoagulant activity. Based on this characteristic, current mainstream artificial heparin preparations often use natural heparin as a raw material, modifying it to degrade high-molecular-weight natural heparin into low-molecular-weight heparin.

[0004] Natural heparin is mainly extracted from animal tissues such as pig intestinal mucosa and bovine lungs, which easily leave behind impurities such as nucleic acids, posing a potential risk of animal disease contamination and insufficient purity in subsequent artificial refining of heparin, thus creating certain risks during clinical use. Residual nucleic acids can be removed using chemical, physical, or biological methods. Traditional chemical methods, such as perchloric acid precipitation, hot ethanol precipitation (CN 104530261A), and surfactant precipitation, suffer from problems such as reduced yield due to heparin co-precipitation, loss of heparin activity, and difficulty in removing residual surfactants, all of which affect product yield and safety. Single physical methods, such as membrane filtration, have limited processing capacity and high operating costs, making it difficult to simultaneously achieve both high heparin yield and purity. In the biological preparation of heparin, a pepsin and trypsin complex enzyme system is often used to degrade nucleic acid impurities in crude heparin. However, this method is not ideal for nucleic acid degradation and still requires subsequent flocculation and precipitation steps to achieve impurity removal (CN103030715B).

[0005] In contrast, nucleases can specifically recognize and cleave phosphodiester bonds in nucleic acid molecules, achieving efficient degradation of nucleic acid impurities. This method can maximize the preservation of heparin's potency and safety, and improve product yield; at the same time, the process is more streamlined and leaves no harmful reagent residues, aligning with the concept of green and environmentally friendly development. However, heparin is a highly sulfated glycosaminoglycan with a very strong negative charge. It can non-specifically bind to the active site or domain of nucleases, like an "ion exchange resin," leading to conformational changes or blockage of active sites, significantly affecting the nuclease's activity. This means that several times the amount of enzyme is required, greatly increasing raw material costs.

[0006] Therefore, significantly improving the enzyme activity of nucleases is of great significance for removing nucleic acid impurities from crude heparin, improving heparin quality, simplifying the process, and reducing costs. Summary of the Invention

[0007] The purpose of this invention is to provide a nuclease mutant for heparin purification and its application. Compared with the parent, it significantly improves enzyme activity, can efficiently degrade residual nucleic acid impurities in crude heparin, and is simple to operate and low in cost.

[0008] The technical solution adopted by this invention to solve its technical problem is: A heparin purification nuclease mutant is derived by modifying the nuclease with the amino acid sequence shown in SEQ ID NO.1 as the parent by mutating the amino acid sites. The mutation scheme is selected from one or more combinations of T77S, Q111K, L126F, N127M, D138V, Q150F, K153Q, Y164Q, I197F, T235I, K254Q, and G263P: T77S: The threonine T at position 77 is mutated to serine S. Q111K: The glutamine Q at position 111 is mutated to lysine K. L126F: The leucine L at position 126 is mutated to phenylalanine F. N127M: The asparagine N at position 127 is mutated to methionine M. D138V: The aspartic acid D at position 138 is mutated to valine V. Q150F: The glutamine Q at position 150 is mutated to phenylalanine F. K153Q: The lysine K at position 153 is mutated to glutamine Q. Y164Q: Tyrosine Y at position 164 is mutated to glutamine Q. I197F: The isoleucine I at position 197 is mutated to phenylalanine F. T235I: The threonine T mutation at position 235 is replaced by isoleucine I. K254Q: The lysine K at position 254 is mutated to glutamine Q. G263P: The glycine G at position 263 is mutated to proline P.

[0009] The nuclease mutant of the present invention can not only significantly improve enzyme activity, but also improve substrate tolerance.

[0010] The nuclease with the amino acid sequence shown in SEQ ID NO.1 is derived from Serratia marcescens.

[0011] Preferably, the mutation scheme is selected from one of the following multi-site mutation schemes: Q111K / L126F, N127M / Q150F, D138V / K153Q, Y164Q / G263P, K153Q / G263P, L126F / D138V / K153Q, N127M / K153Q / Y164Q, D138V / K153Q / G263P, N127M / K153Q / Y164Q / G263P, D138V / K153Q / Y164Q / G263P, L126F / D138V / K153Q / Y164Q / G263P.

[0012] A polynucleotide sequence, wherein the nuclease mutant is described.

[0013] A recombinant vector comprising the aforementioned polynucleotide sequence.

[0014] A host cell comprising the recombinant vector. The host cell is preferably *Escherichia coli*.

[0015] A method for purifying nucleic acid impurities in heparin involves using crude heparin as a substrate, employing the aforementioned nuclease mutant for enzyme-catalyzed reaction, inactivating the enzyme after the reaction, centrifuging to collect the supernatant, collecting the precipitate by alcohol precipitation, washing the precipitate, drying it, and obtaining the purified product.

[0016] Preferably, the enzyme catalytic reaction system consists of: 150-300 g / L substrate, 5-20 g / L of the nuclease mutant enzyme solution, and the remainder being PB buffer. The enzyme solution is prepared as follows: the nuclease mutant is dissolved in PB buffer to a concentration of 100 mg / mL, and the mixture is disrupted using a cell disruptor under ice bath conditions. The supernatant is collected by centrifugation as the crude enzyme solution. The crude enzyme solution is then concentrated 10-fold by rotary evaporation at 35°C and 100 rpm to obtain the final enzyme solution.

[0017] Preferably, the concentration of the PB buffer solution is 0.01-0.1 M and the pH value is 6.0-7.0.

[0018] The beneficial effects of this invention are: This invention involves site-directed mutagenesis of specific nucleases. High-activity nuclease mutant strains were obtained through enzyme activity screening. A recombinant expression vector carrying the nucleotide sequence encoding the mutant was then transformed into *E. coli* BL21(DE3) to obtain recombinant strains. The mutant recombinant strains exhibited significantly improved enzyme activity and substrate tolerance. In the removal of nucleic acid impurities from crude heparin, this invention demonstrates superior enzymatic hydrolysis performance, significantly improving the efficiency of nucleic acid impurity treatment and reducing industrial production costs, thus possessing broad prospects for industrial application. Detailed Implementation

[0019] The technical solution of the present invention will be further described in detail below through specific embodiments.

[0020] All reagents, raw materials, and equipment used in this invention are commercially available unless otherwise specified. In the following examples, unless otherwise specified, various molecular biology operations, including PCR system conditions, enzyme digestion system conditions, competent cell transformation, bacterial culture, and enzyme production, are performed in accordance with the instructions of the commercial kits or conventional methods in the art.

[0021] In this invention, the LB liquid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, and 5 g / L NaCl; the LB solid culture medium formula is: 10 g / L tryptone, 5 g / L yeast extract, 5 g / L NaCl, and 15 g / L agar.

[0022] Nuclease activity assay This nuclease can hydrolyze the phosphodiester bonds of ribonucleic acid and deoxyribonucleic acid. The degradation products have a characteristic absorption peak at a wavelength of 260 nm, which leads to an increase in absorbance. Therefore, the difference in absorbance of the reaction solution can be measured by spectrophotometry to compare the activity of the nuclease.

[0023] Using yeast RNA as a substrate, at 50°C and pH 5.0, an increase of 0.001 in absorbance at 260 nm was defined as one Kunitz unit of enzyme activity. The relative enzyme activities of different nuclease mutants were calculated using parental nuclease activities as controls.

[0024] Example 1 Obtaining engineered bacteria expressing parental nucleases The nuclease gene sequence (GenBank ID: WP_442990766.1) derived from *Serratia marcescens* was used as the target gene. The pET-28a(+) expression vector was constructed by Beijing Qingke Biotechnology Co., Ltd., and named pET28a(+)-E6. The amino acid sequence of the parental nuclease is shown in SEQ ID NO.1. MRFNNKMLALAALLFAAQASADTLESIDNCAVGCPTGGSSNVSIVRHAYTLNNNSTTKFANWVAYHITKDTPASGKTRNWKTDPALNPADTLAPADYTGANAALKVDRGHQAPLASLAGVSDWESLNYLSNITPQ KSDLNQGAWARLEDQERKLIDRADISSVYTVTGPLYERDMGKLPGTQKAHTIPSAYWKVIFINNSPAVNHYAAFLFDQNTPKGADFCQFRVTVDEIEKRTGLIIWAGLPDDVQASLKSKPGVLPELMGCKN (SEQ ID NO.1).

[0025] The expression vector was transformed into *E. coli* BL21(DE3), and positive strains were screened using LB agar plates containing the resistant strain. The specific implementation steps are as follows: The constructed vector plasmid was transformed into Escherichia coli BL21(DE3) (purchased from Shanghai Sangon Biotech) by heat shock method. The bacterial culture was spread on LB plates with kanamycin resistance (kanamycin concentration of 50 mg / L) and cultured at 37℃ for 14 h to obtain positive engineered bacteria E6 that can express parental nuclease.

[0026] Example 2 Expression and preparation of parental nucleases The engineered strain E6 obtained in Example 1 was inoculated into 10 mL of LB broth (with kanamycin resistance, concentration 50 mg / L) and cultured at 37°C with a shaker at 180 rpm for 8-10 h. Then, 1% of the inoculum was transferred to 100 mL of LB broth and cultured at 37°C with a shaker at 180 rpm until OD (outlet count) was reached. 600 When the bacterial growth rate reaches approximately 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, and induce culture for 16 h at 25°C and 180 rpm on a shaker. Collect the bacterial cells by centrifugation at 4°C and 8000 rpm for 10 min, and set aside for later use.

[0027] Example 3 Construction of nuclease single mutant libraries Site-directed mutagenesis was performed using the expression vector pET28a(+)-E6 from Example 1 as a template. Primer design is shown in Table 1. Polymerase chain reaction (PCR) was performed based on the original nuclease sequence. The PCR product was then digested with restriction endonuclease DpnI. After digestion, the cells were transformed into Escherichia coli BL21(DE3) competent cells, and the bacterial culture was plated on LB plates with kanamycin resistance and cultured at 37°C for 14 h.

[0028] Table 1 Primer Information

[0029] PCR amplification system (25 µL): 10 ng plasmid template, 0.5 µL each of forward and reverse primers (10 μM), 12.5 µL of 2×Phanta buffer (Novizan, China), 0.5 µL of dNTP mixture (10 mM each), 0.5 µL of DNA polymerase Phanta (Novizan, China), and ddH2O to bring the total volume to 25 µL.

[0030] PCR amplification conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 4 min, for a total of 34 cycles, 72℃ complete extension for 10 min, and incubation at 12℃.

[0031] Enzyme activity detection screening Positive clones on the plates were randomly selected and inoculated into 10 mL of LB broth (containing kanamycin resistance at a concentration of 50 mg / L) and cultured at 37°C with a shaker at 180 rpm for 8–10 h. Then, the bacterial culture was transferred at a 1% inoculum to 100 mL of LB broth and cultured at 37°C with a shaker at 180 rpm until OD (outcome limit) was reached. 600 When the bacterial growth rate reaches approximately 0.6-0.8, add isopropyl-β-D-thiogalactoside (IPTG) to a final concentration of 0.5 mM, and induce culture at 25°C and 180 rpm for 16 h. Collect the bacterial cells by centrifugation at 4°C and 8000 rpm for 10 min, and set aside for later use.

[0032] Equal amounts of nuclease parent and mutant cells were weighed and dissolved in 0.1M pH 7.0 PB buffer to a concentration of 10 mg / mL. The cells were disrupted using a cell disruptor under ice bath conditions, and the supernatant was collected by centrifugation. The relative enzyme activity was calculated by enzyme activity detection, and dominant mutants were screened to obtain the results.

[0033] The dominant mutants were sent to Hangzhou Qingke Biotechnology Co., Ltd. for sequencing, and the cultures were preserved at -80℃ with an equal volume of 30% sterile glycerol. A total of 205 mutants were obtained, and the screening results of the dominant mutants are shown in Table 2. The final dominant mutants were E6-1 (T77S), E6-2 (Q111K), E6-3 (L126F), E6-4 (N127M), E6-5 (D138V), E6-6 (Q150F), E6-7 (K153Q), E6-8 (Y164Q), E6-9 (I197F), E6-10 (T235I), E6-11 (K254Q), and E6-12 (G263P).

[0034] Table 2. Library of Dominant Single Mutants .

[0035] Example 4 Iterative mutation screening Using the dominant mutant gene sequence in Example 3 as a template, iterative mutations were performed. The primer design was the same as in Table 1. PCR reaction was carried out, and the PCR amplification system and conditions, transformation steps, and enzyme activity detection methods were the same as in Example 3.

[0036] The results of enzyme activity detection screening are shown in Table 3: Table 3. Library of Advantageous Multi-Point Mutants .

[0037] The following mutants were obtained: E6-13 (Q111K / L126F), E6-14 (N127M / Q150F), E6-15 (D138V / K153Q), E6-16 (Y164Q / G263P), E6-17 (K153Q / G263P), E6-18 (L126F / D138V / K153Q), and E6-19 (N127). The five mutant strains were M / K153Q / Y164Q, E6-20 (D138V / K153Q / G263P), E6-21 (N127M / K153Q / Y164Q / G263P), E6-22 (D138V / K153Q / Y164Q / G263P), and E6-23 (L126F / D138V / K153Q / Y164Q / G263P). Among these, the five-site mutant L126F / D138V / K153Q / Y164Q / G263P showed the greatest increase in enzyme activity compared to the parental nuclease strain.

[0038] Example 5: Investigation of Optimal Reaction Conditions Optimal substrate concentration investigation Weigh the mutant cells and dissolve them in 0.01M pH 7.0 PB buffer to a concentration of 100 mg / mL. Disrupt the cells using a cell disruptor under ice bath conditions. Centrifuge and retain the supernatant enzyme solution as crude enzyme solution. Concentrate the crude enzyme solution 10 times by rotary evaporation at 35℃ water bath temperature and 100 rpm as the enzyme solution raw material for subsequent reactions.

[0039] A 1 mL reaction system was selected, and crude heparin (commercially available) derived from porcine small intestinal mucosa and 0.01 g of enzyme solution were added. The reaction buffer was 0.1 M pH 7.0 PB. The reaction was carried out at 37℃ with shaking at 800 rpm for 24 h. After the reaction, the temperature was raised to 90℃ and heated for 10 min (to terminate the reaction). The supernatant was collected by centrifugation at 8000 rpm for 2 min. Four times the volume of anhydrous ethanol was added to the collected supernatant for alcohol precipitation for 14 h. The precipitate was collected by centrifugation at 12000 rpm for 1 min. The precipitate was washed with 1 mL of anhydrous ethanol, centrifuged again, and dried in a 60℃ oven to obtain a solid powder. According to the relevant methods for nucleic acid determination in the 2020 edition of the Chinese Pharmacopoeia, the dried powder was weighed and dissolved in pure water to a concentration of 4 g / L. The absorbance and OD were measured at a wavelength of 260 nm. 260 <0.10 is considered acceptable.

[0040] Based on the above enzymatic hydrolysis reaction detection method, substrate concentration gradients of 150 g / L, 175 g / L, 200 g / L, 225 g / L, 250 g / L, 275 g / L, and 300 g / L were set. 0.1 M PB buffer with pH 7.0 was used as the reaction medium. Enzyme solution with a final concentration of 10 g / L was added for enzymatic hydrolysis reaction. The reaction results are shown in Table 4.

[0041] Table 4 Results of the investigation into optimal substrate concentration .

[0042] Based on the above reaction test results, compared with the parent strain, the optimal mutant strain E6-23 has a significantly improved tolerance to the substrate, and the optimal substrate concentration is 225 g / L.

[0043] Exploring the optimal enzyme concentration Based on the optimal substrate concentration, enzyme concentration gradients of 5 g / L, 7.5 g / L, 10 g / L, 12.5 g / L, 15 g / L, 17.5 g / L, and 20 g / L were set up, and substrate at a concentration of 225 g / L was added for enzymatic hydrolysis. The reaction results are shown in Table 5.

[0044] Table 5 Results of the investigation into optimal enzyme concentration .

[0045] Based on the above reaction test results, the optimal mutant strain showed a significant degradation effect on nucleic acid impurities in the substrate at an enzyme concentration of only 10 g / L, and had a significant increase in enzyme activity compared to the parent strain. The optimal enzyme concentration was 10 g / L.

[0046] Investigation of the optimal reaction temperature Based on the above enzymatic hydrolysis reaction detection method, reaction temperature gradients of 27℃, 32℃, 37℃, 42℃, 47℃, 52℃, and 57℃ were set, with an enzyme concentration of 10 g / L and a substrate concentration of 225 g / L for enzymatic hydrolysis reaction. The reaction results are shown in Table 6.

[0047] Table 6 Results of the investigation into the optimal reaction temperature .

[0048] Based on the above reaction test results, the optimal reaction temperature for both the mutant enzyme expressed by the optimal mutant strain and the wild-type enzyme expressed by the parent strain is 37℃, with no significant change, maintaining the same temperature adaptability as the parent strain.

[0049] Example 6 Methods and applications for degrading nucleic acid impurities in crude heparin In this embodiment, enzyme solution from the dominant strain E6-23 was used as a catalyst to catalyze the hydrolysis of nucleic acid content in crude heparin and to detect OD. 260 Absorbance values ​​compare catalytic activity, OD values 260 The lower the absorbance value, the better the enzyme activity and the more thoroughly nucleic acid impurities are degraded.

[0050] The specific implementation steps are as follows: A 1 L reaction system was selected, and 225 g of crude heparin (commercially available) derived from porcine small intestinal mucosa and 10 g of enzyme solution were added. The reaction buffer was 0.1 M pH 7.0 PB. The reaction was carried out at 37℃ with thorough stirring at 800 rpm for 24 h. After the reaction was completed, the temperature was raised to 90℃ and heated for 10 min (to terminate the reaction). The supernatant was collected by centrifugation at 8000 rpm for 2 min. Four times the volume of anhydrous ethanol was added to precipitate the supernatant for 14 h. The precipitate was collected by centrifugation at 8000 rpm for 10 min. The precipitate was washed with 100 mL of anhydrous ethanol, centrifuged again, and dried in a 60℃ oven to obtain a solid powder. The comparative results are as follows: Table 7 Comparison of Applications of Parental Lines and Mutant E6-23 .

[0051] The above examples demonstrate the technical results of the optimal nuclease mutant in degrading nucleic acid impurities in crude heparin. The reaction results show that, compared with the parental nuclease, the nuclease mutant provided by this invention has significantly improved enzyme activity in degrading nucleic acids and tolerance to substrates without changing the optimal reaction temperature, which has significant advantages in industrial applications.

[0052] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Other variations and modifications are possible without departing from the technical solutions described in the claims.

Claims

1. A heparin purification nuclease mutant, characterized in that, It was modified by mutating amino acid sites using a nuclease with the amino acid sequence shown in SEQ ID NO.1 as the parent. The mutation scheme is selected from one or more combinations of T77S, Q111K, L126F, N127M, D138V, Q150F, K153Q, Y164Q, I197F, T235I, K254Q, and G263P: T77S: The threonine T at position 77 is mutated to serine S. Q111K: The glutamine Q at position 111 is mutated to lysine K. L126F: The leucine L at position 126 is mutated to phenylalanine F. N127M: The asparagine N at position 127 is mutated to methionine M. D138V: The aspartic acid D at position 138 is mutated to valine V. Q150F: The glutamine Q at position 150 is mutated to phenylalanine F. K153Q: The lysine K at position 153 is mutated to glutamine Q. Y164Q: Tyrosine Y at position 164 is mutated to glutamine Q. I197F: The isoleucine I at position 197 is mutated to phenylalanine F. T235I: The threonine T mutation at position 235 is replaced by isoleucine I. K254Q: The lysine K at position 254 is mutated to glutamine Q. G263P: The glycine G at position 263 is mutated to proline P.

2. The nuclease mutant according to claim 1, characterized in that, The nuclease with the amino acid sequence shown in SEQ ID NO.1 is derived from Serratia marcescens.

3. The nuclease mutant according to claim 1, characterized in that, The mutation scheme is selected from one of the following multi-site mutation schemes: Q111K / L126F, N127M / Q150F, D138V / K153Q, Y164Q / G263P, K153Q / G263P, L126F / D138V / K153Q, N127M / K153Q / Y164Q, D138V / K153Q / G263P, N127M / K153Q / Y164Q / G263P, D138V / K153Q / Y164Q / G263P, L126F / D138V / K153Q / Y164Q / G263P.

4. A polynucleotide sequence, characterized in that, It encodes the nuclease mutant of claim 1.

5. A recombinant vector, characterized in that, It contains the polynucleotide sequence as described in claim 4.

6. A host cell, characterized in that, It comprises the recombinant vector as described in claim 5.

7. A method for purifying nucleic acid impurities in heparin, characterized in that, Using crude heparin as a substrate, an enzyme-catalyzed reaction was carried out using the nuclease mutant described in claim 1. After the reaction, the enzyme activity was inactivated, the supernatant was collected by centrifugation, the supernatant was collected by alcohol precipitation, the precipitate was collected after washing, dried, and the purified product was obtained.

8. The purification method according to claim 1, characterized in that, The enzyme catalytic reaction system consists of: 150-300 g / L substrate, 5-20 g / L enzyme solution of the nuclease mutant according to claim 1, and the remainder is PB buffer.

9. The purification method according to claim 8, characterized in that, The concentration of the PB buffer solution is 0.01-0.1M, and the pH value is 6.0-7.0.

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