A method for constructing immobilized heparin lyase based on self-assembly strategy and preparing low molecular weight heparin

Abstract

The application discloses a kind of immobilized heparin lyase based on self-assembly strategy and its preparation low molecular weight heparin method, belong to biotechnology field.The heparin III mutant provided in the application has higher catalytic efficiency compared with wild type, and the catalytic efficiency of mutant K85A / Q95F / S471T is 1.75 times of wild type;The application also uses aggregated protein self-assembly heparin lyase III, realizes the immobilization of enzyme, further carries out screening and optimization to Linker, and improves conversion efficiency.The enzyme immobilization system constructed in the application can realize the immobilization of heparin lyase III, and the precipitate is collected by centrifugation after breaking the wall of recombinant cell, so that complex steps such as protein purification and desalting are reduced.The immobilized heparin lyase still has 50% catalytic activity after 10 cycles, realizes the efficient preparation of low molecular weight heparin, increases the utilization efficiency of enzyme, and reduces production cost.

Description

Technical Field

[0001] This invention relates to a method for constructing an immobilized heparin lyase based on a self-assembly strategy and preparing low molecular weight heparin, belonging to the field of biotechnology. Background Technology

[0002] Heparin is a naturally occurring polysaccharide, primarily produced by hepatocytes and intestinal mucosal cells. It is an important anticoagulant and anti-inflammatory drug with various biological activities and pharmacological effects. Heparin exerts its anticoagulant effect by binding to antithrombin III (ATIII), inhibiting thrombin activity. Furthermore, heparin can suppress inflammatory responses, promote cell proliferation and migration, and regulate angiogenesis, among other biological functions. Low molecular weight heparin has better anticoagulant effects and a lower bleeding risk compared to unfractionated heparin, and is therefore widely used clinically for the prevention and treatment of thrombotic diseases. The method for preparing low molecular weight heparin using heparin lyase is highly efficient, specific, and mild, and can produce low molecular weight heparin products with specific molecular weight ranges and pharmacological activities.

[0003] Heparin lyase III is an enzyme that degrades heparin. Belonging to the polysaccharide lyase family, its main function is to cleave the glycosidic bonds in the heparin molecule. Heparin lyase has significant applications in biotechnology and the pharmaceutical industry. Heparin lyase III can be used to prepare low-molecular-weight heparin, which exhibits better anticoagulant effects and a lower bleeding risk, thus finding widespread clinical use in the prevention and treatment of thrombotic diseases. Furthermore, heparin lyase III is also used for the structural analysis of heparin and the synthesis of heparin analogues.

[0004] Since free enzymes are difficult to recover and reuse after catalysis, especially since some enzymes need to be purified before use, this greatly increases the cost of use. Immobilized enzyme technology can solve this problem very well. Summary of the Invention

[0005] This invention provides a heparin lyase III mutant, which is made by mutating one or more amino acids at positions 85, 95, and 471 of heparin lyase derived from Bacteroides thetaiotaomicron; the amino acid sequence of the heparin lyase III derived from Bacteroides thetaiotaomicron is shown in SEQ ID NO.2.

[0006] In one embodiment, the mutant is obtained by mutating lysine (K) at position 85 to alanine (A) based on heparin lyase III as shown in SEQ ID NO.2, resulting in mutant K85A; or,

[0007] Mutating glutamine (Q) at position 95 to phenylalanine (F) yields the mutant Q95F; or,

[0008] Mutate serine (S) at position 471 to threonine (T) to obtain the mutant S471T; or,

[0009] The lysine at position 85 (K) is mutated to alanine (A), and the glutamine at position 95 (Q) is mutated to phenylalanine (F), resulting in the mutant K85A / Q95F; or,

[0010] Mutate the lysine (K) at position 85 to alanine (A) and the serine (S) at position 471 to threonine (T) to obtain the mutant K85A / S471T; or,

[0011] Mutate glutamine (Q) at position 95 to phenylalanine (F) and serine (S) at position 471 to threonine (T) to obtain the mutant Q95F / S471T; or,

[0012] The mutant K85A / Q95F / S471T was obtained by mutating lysine (K) at position 85 to alanine (A), glutamine (Q) at position 95 to phenylalanine (F), and serine (S) at position 471 to threonine (T).

[0013] The present invention also provides a gene encoding the mutant.

[0014] In one embodiment, the nucleotide sequence encoding the mutant K85A / Q95F / S471T is shown in SEQ ID NO.3.

[0015] The present invention also provides a recombinant plasmid carrying the said gene.

[0016] In one embodiment, the plasmid includes, but is not limited to, pET series plasmids.

[0017] The present invention also provides recombinant microbial cells expressing the mutant.

[0018] The present invention also provides a heparin lyase III immobilized enzyme having a structure shown in “heparin lyase III mutant-linker-self-assembly tag”; the self-assembly tag includes, but is not limited to, CipA, 18A or L6KD.

[0019] In one embodiment, the heparin lyase III mutant is the mutant K85A / Q95F / S471T.

[0020] In one embodiment, the linker includes, but is not limited to, G4S (i.e., GGGGS) and (G4S)3 (i.e., GGGGSGGGGSGGGGS), which links the self-assembled protein to the C-terminus of the mutant K85A / Q95F / S471T.

[0021] In one embodiment, the amino acid sequence of the self-assembled protein tag CipA is shown in SEQ ID NO.5.

[0022] The present invention also provides a method for preparing heparin lyase III mutant immobilized enzyme, wherein the method comprises expressing the heparin lyase III mutant immobilized enzyme in recombinant microbial cells, collecting and cleaving the bacterial cells, centrifuging, and collecting the precipitate to obtain the heparin lyase III mutant immobilized enzyme.

[0023] The present invention also provides the application of the heparin lyase III mutant or the immobilized enzyme in the hydrolysis of heparin.

[0024] In one embodiment, the application involves reacting the heparin lyase III mutant in a heparin-containing reaction system at 30–37°C for at least 30 min.

[0025] In one embodiment, the application involves collecting the immobilized enzyme after the reaction is complete and adding it to a new reaction system for continued use.

[0026] Beneficial effects:

[0027] (1) The present invention constructs a mutant by mutating heparin lyase, which significantly improves the catalytic efficiency. The catalytic efficiency of the mutant K85A / Q95F / S471T is 1.75 times higher than that of the original strain.

[0028] (2) This invention achieves the immobilization of heparin lyase III by screening different self-assembled protein tags and linker peptides.

[0029] (3) This invention also provides a method for preparing low molecular weight heparin using immobilized heparin lyase III, solving the problems of high purification costs and difficulty in recovery and reuse of the enzyme in the in vitro catalytic preparation of low molecular weight heparin. This invention immobilizes heparin lyase III with a self-assembled tag, enabling rapid preparation of low molecular weight heparin via centrifugation. This method exhibits better stability and is reusable. It simplifies the original enzyme purification steps and significantly reduces production costs. Attached Figure Description

[0030] Figure 1 This is a comparison of the specific enzyme activity of the heparin lyase III mutant with that of the starting enzyme.

[0031] Figure 2 SDS-PAGE electrophoresis results of inclusion bodies immobilized with different linked short peptides for heparin lyase III.

[0032] Figure 3 The unit enzyme activity results for inclusion bodies with different linked short peptides immobilized with heparin lyase III.

[0033] Figure 4 The image shows the catalytic cleavage of heparin molecules by the immobilized enzyme heparin lyase III.

[0034] Figure 5 This study compares the stability of immobilized and free heparin lyases.

[0035] Figure 6 Schematic diagram of the preparation of low molecular weight heparin using immobilized heparin lyase III.

[0036] Figure 7 The graph shows the number of cycles of 1 mL of heparin catalyzed by the immobilized enzyme heparin lyase III. Detailed Implementation

[0037] Culture medium:

[0038] LB medium: Tryptone 10 g / L, Yeast extract 5 g / L; Sodium chloride (NaCl) 10 g / L.

[0039] TB medium: Tryptone 12 g / L, Yeast extract 24 g / L; Glycerol 10 g / L, Potassium dihydrogen phosphate 2.31 g / L, Dipotassium hydrogen phosphate 12.54 g / L.

[0040] Nucleotide sequence information involved in the example:

[0041] SEQ ID NO.1 is the nucleotide sequence of the gene encoding heparin lyase III from Bacteroides proteus.

[0042] SEQ ID NO.2 is an amino acid sequence from Bacteroides mutatis mutans encoding heparin lyase III.

[0043] SEQ ID NO.3 is the nucleotide sequence of the gene encoding the mutant K85A / Q95F / S471T.

[0044] SEQ ID NO.4 is the nucleotide sequence encoding the CipA gene, a self-assembling protein derived from Photobacteria.

[0045] SEQ ID NO.5 is the amino acid sequence of CipA, a self-assembled protein from Photobacteria.

[0046] SEQ ID NO.6 is a nucleotide sequence encoding the self-assembly tag 18A.

[0047] SEQ ID NO.7 is a nucleotide sequence encoding the self-assembled tag L6KD.

[0048] Methods for determining heparin lysin activity:

[0049] Enzyme activity was measured using the UV232 method in 980 μL of preheated heparin sodium substrate solution (50 mM Tris-HCl, 20 mg / mL). -1 Add 20 μL of enzyme solution to the solution, mix quickly in a quartz cuvette, and measure the absorbance change over time at 232 nm using a spectrophotometer. The slope k of the curve yields the heparin lyase activity. The molar extinction coefficient is 3800 L·mol⁻¹. -1 ·cm -1 Enzyme activity unit is defined as the amount of enzyme required to produce 1 μmol of 4,5-unsaturated uronic acid per minute at 30°C.

[0050] The molecular weight of heparin was determined by gel permeation chromatography-high-performance liquid chromatography (GPC-HPLC). The chromatographic column used was an Ultrahydrogel™ Linear column (7.8 × 300 mm), the mobile phase was NaNO3 solution (0.1 mol·L⁻¹), the mobile phase was 0.9 mL·min⁻¹, the column temperature was 40 °C, and the injection volume was 40 μL. The elution volume of each sample was measured, and the weight-average molecular weight (Mw) of each sample was calculated using GPC software based on the standard curve between the molecular weight of the dextran standard and the elution volume.

[0051] Table 1 Primer sequences

[0052]

[0053] Table 2 Amino acid sequences of self-assembled tag proteins

[0054]

[0055] Table 3. Nucleotide sequences encoding linker peptides

[0056]

[0057] Example 1: Construction of recombinant Escherichia coli expressing a mutant of heparin lyase

[0058] To enhance the activity of heparin lyase III, saturation mutations were performed on K85, Q95, and S471, starting from SEQ ID NO1. Using the unmutated heparin lyase gene recombinant plasmid (pET-28a-BhepIII) constructed in our laboratory as a template, PCR amplification was performed. The primers used are shown in Table 1. The PCR amplification product was introduced into competent *E. coli* BL21(DE3) cells, incubated on ice for 30 min, heat-shocked at 42℃ for 90 s, then added to LB medium and incubated at 37℃ for 40 min. The cells were then plated on kanamycin-treated LB plates and incubated at 37℃ for 8–12 h. Colonies were selected for sequencing verification to obtain correct transformants. The method for culturing transformants and obtaining purified enzymes from mutants was as follows: Single clones cultured overnight were picked from plates and inoculated into 5 mL of fresh LB liquid medium containing 50 μg·mL⁻¹ kanamycin. The culture was incubated at 37°C and 220 rpm until mid-log phase (8-10 h). The seed culture was then transferred back to 50 mL of fresh LB / TB liquid medium containing 50 μg·mL⁻¹ kanamycin at a 2% (v / v) inoculation rate. The LB seed culture from test tubes was inoculated into 500 mL shake flasks at a 10% volume inoculation rate. After incubation at 37°C and 220 rpm, IPTG was added to a final concentration of 0.5 mM for induction, and the culture was continued at 25°C and 220 rpm for 20 h.

[0059] Centrifuge the fermentation broth after fermentation to collect the cells, resuspend them in phosphate buffer, break the cell walls, centrifuge again, collect the supernatant, and load the sample. Use a Ni column and equilibrate the column to 10 column volumes with phosphate buffer. Use imidazole gradient elution, with imidazole prepared in the above phosphate buffer and eluted at 10 mmol·L⁻¹, 40 mmol·L⁻¹, and 100 mmol·L⁻¹.

[0060] The obtained mutant purified enzyme was placed in Tris-HCl (20mM pH 7.2) and its enzyme activity was measured. The mutant K85A / Q95F / S471T with significantly improved enzyme activity was screened.

[0061] Specifically, such as Figure 1 As shown, the catalytic efficiency of the mutant K85A / Q95F / S471T was 1.75 times higher than that of the original strain.

[0062] Example 2: Construction and Enzyme Activity Assay of Immobilized Heparin Lyase III

[0063] pET-28a-BhepIII was linearized using primers to synthesize the encoding genes for self-assembly tags CipA, L6KD, and 18A, respectively. These fragments were then linked to the N-terminal NdeI and EcoRI restriction sites or the C-terminal XhoI and NotI sites of pET28a-BhepIII via a linker (GGGGS) to obtain different recombinant plasmids, pET-28a-BhepIII-aggregate protein (C-terminus / N-terminus).

[0064] The recombinant plasmid pET28a-BhepIII-aggregate protein (C-terminus / N-terminus) was transformed into E. coli BL21(DE3). After verification, the cells were streaked on LB agar plates containing kanamycin (50 μg / L), and single colonies were picked and inoculated into LB seed culture medium under the same conditions as in Example 1. After completion, the bacterial cells were collected, and the OD of the cells was measured. 600 The inclusion bodies were then ultrasonically broken up and used for analysis. The resulting inclusion body gel images are shown below. Figure 2 As shown in the figure. The results indicate that BhepIII is expressed in the form of intracellular precipitate.

[0065] The collected E. coli BL21(DE3) / pET28a-BhepIII bacterial cells were ultrasonically disrupted, centrifuged at high speed, and the precipitate was collected. The target protein was removed by washing three times with 20mM Tris-HCl at pH 7.5. A heparin lyase immobilized enzyme containing the aggregate protein was obtained. 50–100 mg of the heparin lyase immobilized enzyme obtained after disrupting the wet bacterial cells was added to the catalytic system, and the mixture was resuspended in Tris-HCl to 10 ml. Enzyme activity was then measured. The results are shown in Table 4. The activity of the heparin lyase immobilized enzyme with the C-terminus fused with the aggregate protein was significantly higher than that with the N-terminus, and the fusion with CipA was even more effective.

[0066] Table 4 Enzyme activity of enzymes immobilized by different self-assembled tags

[0067] Immobilization <![CDATA[Enzyme activity (U / OD 600 )]]> CipA-N terminal 44.2 CipA-C terminal 118.5 L6KD-N terminal 27.1 L6KD-C terminal 51.3 18A-N terminal 2.2 18A-C terminal 8.6

[0068] Example 3: Comparison of enzyme activities of immobilized heparin lysins containing different linking peptides

[0069] Construction of immobilized heparin lyase: The aggregate protein CipA and BhepIII from luminescent bacteria were fused into a single fragment using gene manipulation techniques. Different linker peptide sequences were used at the linker to maintain a certain spatial position between the aggregate protein and BhepIII, thus avoiding mutual interference between the proteins and affecting their catalytic efficiency on the substrate.

[0070] The specific steps are as follows: different linker peptides are linked between the heparin lyase gene and the C-terminal aggregate protein CipA gene through gene manipulation. The nucleotide sequences of the linkers are shown in Table 3, resulting in recombinant plasmids such as pET28a-BhepIII-Linker-CipA.

[0071] The constructed recombinant plasmid pET28a-BhepIII-Linker-CipA was transformed into E. coli BL21(DE3), and the strain was cultured according to the method in Example 1. The results are as follows. Figure 3 As shown, the immobilized heparin lyase constructed using linker peptide (G4S)3 exhibited the highest enzyme activity.

[0072] An immobilized enzyme, consisting of a linker peptide (G4S)3 linked to the C-terminus of heparin lyase and containing a self-assembled protein CipA, was prepared into an enzyme solution with a concentration of 1000 U / L. This solution was added in 500 μL to a catalytic system containing 20 mg / mL heparin sodium. The reaction was carried out at 37 °C for 0.5 h, 1 h, 2 h, 5 h, and 10 h, after which the molecular weight of the resulting heparin was measured. The results showed that… Figure 4 In less than 1 hour of reaction, the molecular weight of heparin can be reduced to below 10,000 Da; after 5 hours of reaction, the molecular weight can be reduced to 5,000 Da.

[0073] Example 4: Comparison of stability between immobilized and free enzymes

[0074] After the recombinant cells constructed in Example 3 were lysed and centrifuged, heparin lyase III was collected and diluted to 1000 U / L. The immobilized enzyme was resuspended to 1000 U / OD. The immobilized enzyme and the free enzyme were incubated in a metal bath at 50°C. Samples were taken at intervals and quickly placed on ice for 10 min. The residual enzyme activity was measured at 30°C.

[0075] The results are as follows Figure 5 As shown, the immobilized enzyme exhibited significantly better stability than the enzyme in its free state, with half-lives of 32 min and 370 min, respectively.

[0076] Example 5: Verification of the number of times the heparin lyase-immobilized enzyme can be reused.

[0077] The immobilized heparin lyase III constructed using linker peptide (G4S)3 in Example 3 was resuspended to 1000 U / L. 500 μL of the enzyme solution was added to 500 μL of a catalytic system containing 20 mg / mL heparin sodium. After reacting at 37°C for 30 min, the system was centrifuged, and the absorbance of the supernatant was measured to calculate enzyme activity. The resulting precipitate was resuspended in Tris-HCl (20 mL, pH = 7.2) buffer, and then added to a fresh catalytic system. The reaction was repeated for 30 min, followed by centrifugation. This process was repeated 8 times, for a total of 10 cycles. The results are as follows: Figure 7 As shown.

[0078] The results showed that the immobilized heparin lyase retained 50% of its activity after 10 cycles.

[0079] Comparative Example

[0080] The specific implementation method is the same as in Example 1, except that the mutants shown in Table 5 were also constructed. Enzyme activity was measured using the same method as in Example 1, and the results are shown in Table 5.

[0081] Table 5. Enzyme activity of different mutants

[0082] mutant Enzyme activity (U / mg) K85R 15.20 Q95D 20.29 S471W 14.35

[0083] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A heparin lyase III mutant, characterized in that it is It is obtained by mutating serine at position 471 to threonine based on heparin lyase III shown in SEQ ID NO.2; or This was obtained by mutating lysine at position 85 to alanine and serine at position 471 to threonine; or The glutamine at position 95 was mutated to phenylalanine, and the serine at position 471 was mutated to threonine; or The lysine at position 85 was mutated to alanine, the glutamine at position 95 was mutated to phenylalanine, and the serine at position 471 was mutated to threonine.

2. The gene encoding the mutant of claim 1.

3. A recombinant plasmid carrying the gene described in claim 2.

4. A recombinant microorganism expressing the mutant of claim 1.

5. A recombinant microorganism containing the gene of claim 2.

6. An immobilized enzyme, characterized in that, The C-terminus of the heparin lyase III mutant of claim 1 is linked to a self-assembled protein tag CipA derived from *Lithocarpus lucida* via a linker peptide; the linker peptide comprises G4S or (G4S)3; the amino acid sequence of the self-assembled protein tag CipA is shown in SEQ ID NO.

5.

7. The application of the heparin lyase III mutant of claim 1 or the immobilized enzyme of claim 6 in the hydrolysis of heparin.

8. The application according to claim 7, characterized in that, The applications include the preparation of low molecular weight heparin.

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