Immobilized heparin lyase constructed based on self-assembly strategy and method for preparing low-molecular-weight heparin by using immobilized heparin lyase

Abstract

The invention discloses a method for constructing immobilized heparin lyase based on a self-assembly strategy and preparing low-molecular-weight heparin by using the immobilized heparin lyase, and belongs to the technical field of biology. Compared with a wild type, the heparin III mutant provided by the invention has higher catalytic efficiency, and the catalytic efficiency of the mutant K85A / Q95F / S471T is 1.75 times that of the wild type; according to the method disclosed by the invention, the heparin lyase III is self-assembled by utilizing aggregation protein, so that the enzyme immobilization is realized, the Linker is further screened and optimized, and the conversion efficiency is improved. The enzyme immobilization system constructed by the invention can realize immobilization of heparin lyase III, and complex steps such as protein purification and desalination are reduced by centrifugally collecting precipitates after reconstitution cells are subjected to wall breaking. The immobilized heparin lyase still has 50% of catalytic activity after circulation for 10 times, so that efficient preparation of the low-molecular-weight heparin is realized, the utilization efficiency of the enzyme is increased, and the production cost is reduced.

Description

Technical Field

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

[0002] Heparin is a naturally occurring polysaccharide that is mainly produced by hepatocytes and intestinal mucosal cells. It is an important anticoagulant and anti-inflammatory drug with multiple biological activities and pharmacological effects. Heparin can inhibit the activity of thrombin by binding to antithrombin III (ATⅢ), thereby exerting an anticoagulant effect. In addition, heparin can also inhibit inflammatory responses, promote cell proliferation and migration, regulate angiogenesis and other biological functions. Compared with ordinary heparin, low molecular weight heparin has better anticoagulant effect and lower bleeding risk, so it is widely used in clinical practice to prevent and treat thrombotic diseases. The method of preparing low molecular weight heparin by heparin lyase is characterized by high efficiency, specificity and mildness, and can prepare low molecular weight heparin products with a specific molecular weight range and pharmacological activity.

[0003] Heparinase III is an enzyme that can degrade heparin. It belongs to the polysaccharide lyase family and its main function is to cut the glycosidic bonds in the heparin molecule. Heparinase has important application value in biotechnology and the pharmaceutical industry. Heparinase III can be used to prepare low molecular weight heparin. This heparin fragment has better anticoagulant effect and lower bleeding risk, so it is widely used in the prevention and treatment of thrombotic diseases in clinical practice. In addition, heparinase III is also used for structural analysis of heparin and synthesis research of heparin analogs.

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

[0005] The present invention provides a heparin lyase III mutant, which is obtained by mutating one or more amino acids at positions 85, 95 and 471 of a 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 the lysine (K) at position 85 to alanine (A) based on the heparin lyase III shown in SEQ ID NO.2, thereby obtaining the mutant K85A; or

[0007] Mutate the glutamine (Q) at position 95 to phenylalanine (F) to obtain the mutant Q95F; or

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

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

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

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

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

[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 as SEQ ID NO.3.

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

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

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

[0018] The present invention also provides a heparin lyase III immobilized enzyme, which has a structure shown as "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 G 4 S (i.e. GGGGS), (G 4 S) 3(i.e., GGGGSGGGGSGGGGS), the linker connects the self-assembling protein to the C-terminus of the mutant K85A / Q95F / S471T.

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

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

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

[0024] In one embodiment, the use is to react the heparin lyase III mutant in a reaction system containing heparin at 30-37° C. for at least 30 minutes.

[0025] In one embodiment, the application is to collect the immobilized enzyme after the reaction is completed and put it into a new reaction system for continued use.

[0026] Beneficial effects:

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

[0028] (2) The present invention achieves the immobilization of heparin lyase III by screening different self-assembling protein tags and connecting short peptides.

[0029] (3) The present invention also provides a method for preparing low molecular weight heparin using immobilized heparin lyase III, which solves the problem in the prior art that the enzyme needs to be purified for in vitro catalytic preparation of low molecular weight heparin, resulting in high purification costs and difficulty in recovery and reuse after the reaction. The present invention immobilizes heparin lyase III via a self-assembly tag, and can quickly prepare low molecular weight heparin by centrifugation, has better stability, and can be reused. The original enzyme purification steps are simplified, greatly reducing production costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 Comparison of the enzymatic activity of the heparin lyase III mutant with that of the original enzyme.

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

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

[0033] Figure 4 This is the result of the molecular weight cleavage of heparin catalyzed by the immobilized enzyme heparin lyase III.

[0034] Figure 5 Comparison of the stability of immobilized heparin lyase and free enzyme.

[0035] Figure 6 Schematic diagram of the cyclic use of immobilized enzyme heparin lyase III to prepare low molecular weight heparin

[0036] Figure 7 This is a graph showing the number of cycles of 1 mL of heparin catalyzed by the immobilized enzyme heparin lyase III. DETAILED DESCRIPTION

[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, potassium dihydrogen phosphate: 12.54 g / L.

[0040] The nucleotide sequence information involved in the example is:

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

[0042] SEQ ID NO. 2 is the amino acid sequence encoding heparin lyase III derived from Bacteroides proteus.

[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 self-assembly protein CipA gene from Photorhabdus sp.

[0045] SEQ ID NO.5 is the amino acid sequence of the self-assembling protein CipA from Photorhabdus sp.

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

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

[0048] Heparin lyase activity assay method:

[0049] The enzyme activity was determined by UV232 method in 980 μL preheated heparin sodium substrate solution (50 mM Tris-HCl, 20 mg mL -1 ) was added with 20 μL of enzyme solution, mixed quickly in a quartz cuvette, and placed in a spectrophotometer to measure the absorbance curve at 232 nm wavelength versus time. The slope k of the curve was calculated to obtain the heparin lyase activity. The molar extinction coefficient is 3800 L·mol -1 cm -1 The 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] Heparin molecular weight detection method: Gel permeation chromatography-liquid chromatography (GPC-HPLC) was used. The chromatographic column used was Ultrahydrogel TM Linear column 7.8×300mm, the mobile phase was NaNO3 solution (0.1mol·L-1), 0.9mL·min-1, column temperature 40℃, injection volume 40μL. The elution volume of each sample was measured, and the weight average molecular weight (Mw) of each sample was calculated using the GPC software based on the standard curve between the molecular weight and elution volume of the dextran standard sample.

[0051] Table 1 Primer sequences

[0052]

[0053] Table 2 Amino acid sequence of self-assembly tag protein

[0054]

[0055] Table 3 Nucleotide sequences encoding connecting peptides

[0056]

[0057] Example 1 Construction of recombinant Escherichia coli expressing mutants of heparin lyase

[0058] In order to improve the enzymatic activity of heparin lyase III, SEQ ID. NO1 was used as the starting sequence, and K85, Q95, and S471 were saturated mutated respectively. The recombinant plasmid (pET-28a-BhepIII) of the heparin lyase gene constructed in this laboratory without mutation was used as a template, and amplified by PCR program, and the primers used are shown in Table 1. The PCR amplification product was introduced into the competent cells of Escherichia coli BL21 (DE3), ice bathed for 30 minutes, and heat-shocked at 42°C for 90 seconds, and then LB medium was added, cultured at 37°C for 40 minutes, coated on LB plates containing kanamycin, and cultured at 37°C for 8-12 hours. The colonies were selected for sequencing verification to obtain the correct transformants. The method for culturing the transformant and obtaining the pure enzyme of the mutant is as follows: picking a single clone cultured overnight in a plate, inoculating it in 5 mL of fresh LB liquid medium containing 50 μg·mL-1 kanamycin, culturing it at 37°C and 220 rpm to mid-logarithmic phase (8-10 h), and re-transferring the seed liquid to 50 mL of fresh LB / TB liquid medium containing 50 μg·mL-1 kanamycin at an inoculum volume of 2% (v / v). The LB seed liquid cultured in a test tube is inoculated in a 500 mL shaking flask at an inoculum volume of 10%, and after culturing at 37°C and 220 rpm, IPTG with a final concentration of 0.5 mM is added for induction, and the culture is continued at 25°C and 220 rpm for 20 h.

[0059] The fermentation broth at the end of fermentation was centrifuged, the bacteria were collected, resuspended with phosphate buffer, broken, centrifuged, the supernatant was collected, and the sample was loaded; a Ni column was used, and the column was balanced with phosphate buffer for 10 column volumes; imidazole gradient elution was adopted, and imidazole was prepared with the above-mentioned phosphate buffer, and the gradient elution was carried out according to 10mmol·L-1, 40mmol·L-1, and 100mmol·L-1.

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

[0061] Specific as Figure 1 As shown, the catalytic efficiency of the mutant K85A / Q95F / S471T was increased 1.75 times compared with the original strain.

[0062] Example 2 Construction of immobilized heparin lyase III and enzyme activity determination

[0063] pET-28a-BhepIII was linearized by primers, and the coding genes of self-assembly tags CipA, L6KD, and 18A were synthesized respectively. The above fragments were connected to the N-terminal NdeI and EcoRI restriction enzyme sites or the C-terminal XhoI and NotI of pET28a-BhepIII through linker (GGGGS) to obtain different recombinant plasmids, pET-28a-BhepIII-aggregating protein (C-terminus / N-terminus).

[0064] The recombinant plasmid pET28a-BhepIII-aggregin (C-terminus / N-terminus) was transferred into E. coli BL21 (DE3) respectively, and after verification, it was streaked on an LB plate containing kanamycin (50 μg / L), and a single colony was picked and inoculated into LB seed medium. The culture conditions were the same as those in Example 1. After completion, the cells were collected and the cell OD was measured. 600 The inclusion bodies were then broken by ultrasound for analysis. Figure 2 The results showed that BhepIII was expressed in the form of intracellular precipitation.

[0065] The collected bacteria E.coli BL21 (DE3) / pET28a-BhepIII was ultrasonically crushed and then centrifuged at high speed to collect the precipitate, and the target protein was washed three times with 20mM Tris-HCl at pH 7.5 to remove impurities; the heparin lyase immobilized enzyme containing aggregated protein was obtained; 50-100mg of the heparin lyase immobilized enzyme obtained after the wet bacteria were crushed was added to the catalytic system, and Tris-HCl was added to resuspend it to 10ml, and the enzyme activity was measured. The results are shown in Table 4. The activity of the heparin lyase immobilized enzyme after the C-terminus fused with aggregated protein is much higher than that of the N-terminus, and the effect of fusion with CipA is better.

[0066] Table 4 Enzyme activities of enzymes immobilized with different self-assembly 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 Lyases Containing Different Connecting Peptides

[0069] Construction of fused immobilized heparin lyase: The aggregation proteins CipA and BhepIII from Photorhabditis elegans were fused into one fragment by genetic manipulation, and different connecting peptide sequences were used in the linker to maintain a certain spatial position between the aggregation protein and BhepIII to avoid mutual interference between the proteins, thereby affecting their catalytic efficiency on the substrate.

[0070] The specific operation steps are: different linker peptides are connected between the heparin lyase gene and the C-terminal aggregation protein CipA gene by genetic manipulation; the nucleotide sequences of the linkers are shown in Table 3, and the recombinant plasmids shown in pET28a-BhepIII-Linker-CipA are obtained.

[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 of Example 1. The results are as follows: Figure 3 As shown, with a connecting peptide (G 4 S) 3 The constructed immobilized heparin lyase has the highest enzymatic activity.

[0072] The connecting peptide (G 4 S) 3 The immobilized enzyme with the self-assembling protein CipA connected to the C-terminus of the heparin lyase was prepared into an enzyme solution with a concentration of 1000U / L, and 500μL of the enzyme solution was added to a catalytic system with a concentration of 500μL of 20mg / mL heparin sodium. The molecular weight of heparin after the reaction was detected after 0.5h, 1h, 2h, 5h, and 10h at 37℃. The results showed that ( Figure 4 ), within 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 the stability of immobilized enzyme and free enzyme

[0074] After the recombinant cells constructed in Example 3 were crushed 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 kept in a 50°C metal bath, respectively, samples were taken at intervals, and the samples were quickly placed on ice for 10 minutes, and 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 free enzyme, with half-lives of 32 min and 370 min, respectively.

[0076] Example 5 Verification of the number of times the immobilized heparin lyase enzyme is reused

[0077] The connecting peptide (G 4 S) 3The constructed immobilized heparin lyase III was resuspended to 1000U / L, 500μL of enzyme solution was added to 500μL of 20mg / mL heparin sodium catalytic system, reacted at 37°C for 30min, and then the system was centrifuged. The change in the absorbance of the supernatant was measured to calculate the enzyme activity. The obtained precipitate was added to Tris-HCl (20mm, pH=7.2) buffer and resuspended, and then added to the new catalytic system. After reacting for 30min, centrifuged again, the above steps were repeated, and the cycle was continued for 8 times, a total of 10 times. The results are as follows: Figure 7 shown.

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

[0079] Comparative Example

[0080] Specific implementation method The same as Example 1, except that the mutants shown in Table 5 were also constructed. The enzyme activity was determined in the same manner as Example 1, and the results are shown in Table 5.

[0081] Table 5 Specific enzyme activities of different mutants

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

[0083] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A heparin lyase III mutant, characterized in that: Based on the heparin lyase parent, it has one or more amino acid mutations at positions 85, 95 and 471; the amino acid sequence of the heparin lyase III parent is shown in SEQ ID NO.

2.

2. The heparin lyase III mutant according to claim 1, characterized in that The mutant is based on the heparin lyase III shown in SEQ ID NO.2, with the lysine at position 85 mutated to alanine; or Mutate glutamine at position 95 to phenylalanine; or Mutate serine at position 471 to threonine; or mutating the lysine at position 85 to alanine and the glutamine at position 95 to phenylalanine; or mutating the lysine at position 85 to alanine and the serine at position 471 to threonine; or Mutating glutamine at position 95 to phenylalanine and serine at position 471 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.

3. A gene encoding the mutant according to claim 1 or 2.

4. A recombinant plasmid carrying the gene according to claim 3.

5. A recombinant microorganism expressing the mutant according to claim 1 or 2, or containing the gene according to claim 3.

6. An immobilized enzyme, characterized in that The self-assembling protein tag CipA derived from Photorhabdus is connected to the C-terminus or N-terminus of the heparin lyase III mutant described in claim 1 or 2 via a connecting peptide.

7. The immobilized enzyme according to claim 6, characterized in that The connecting peptide includes but is not limited to GGGGS, GGGGSGGGGSGGGGS.

8. The immobilized enzyme according to claim 6 or 7, characterized in that The amino acid sequence of the self-assembling protein tag CipA is shown in SEQ ID NO.

5.

9. Use of the heparin lyase III mutant according to claim 1 or 2 or the immobilized enzyme according to any one of claims 6 to 8 in hydrolyzing heparin.

10. The use according to claim 9, characterized in that: The applications include, but are not limited to, the preparation of low molecular weight heparin.

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