CN112626058B

CN112626058B - Circumscribed heparinase and application thereof

Info

Publication number: CN112626058B
Application number: CN202011532643.2A
Authority: CN
Inventors: 李福川; 张庆冬; 魏琳; 路丹荣
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-12-22
Filing date: 2020-12-22
Publication date: 2022-09-27
Anticipated expiration: 2040-12-22
Also published as: CN112626058A

Abstract

The invention relates to an exo-heparinase and application thereof. The exo-heparinase of the present invention tends to degrade a substrate by sequentially releasing unsaturated disaccharides having specific UV absorption at 232nm from the reducing end. The exo-heparinase provided by the invention sequentially cuts off heparin disaccharide with high sulfation from a reducing end, and the higher the sulfation degree of the heparin disaccharide, the higher the enzyme activity of the heparinase. In addition, the potential of the excision heparinase in HP/HS structure research is proved by primary sequencing of HP octasaccharide by the excision heparinase exoHep. The invention not only fills the blank of the research field of heparinase, but also provides a novel tool enzyme for the structure and function research and enzymolysis sequencing of HP/HS.

Description

Circumscribed heparinase and application thereof

Technical Field

The invention relates to an exo-type heparinase and application thereof, belonging to the technical field of biological enzymes.

Background

Heparin (HP)/Heparan Sulfate (HS) is a polyanionic linear polysaccharide, has a highly heterogeneous structure, and is an important sulfated glycosaminoglycan. They are widely found on the surface of cell membranes, in the extracellular matrix (ECM) and in the intracellular environment (e.g. mast cells). Due to the existence of a complex and changeable structure, HP/HS can interact with various protein molecules (such as growth factors, morphogen and receptors thereof) and participate in various important physiological and pathological processes such as anticoagulation, cell adhesion, inflammatory reaction, cell migration, cell differentiation, pathogenic microorganism infection, tumorigenesis and development and the like by regulating cell signal transduction, adhesion, migration and the like. These important biological effects have attracted considerable attention to HP/HS and have led to a great deal of research concerning HP/HS structure and function and clinical applications. Since its discovery in 1916, HP and its low-molecular agents have been widely used in clinical treatment as the most important anticoagulants.

The backbone of the HP/HS polysaccharide is composed of repeating disaccharide units composed of D-glucuronic acid or L-iduronic acid (GlcA/IdoA) and N-acetyl-D-glucosamine (GlcNAc). During the biosynthesis of HP/HS, a linear glycan precursor consisting of repeating disaccharide GlcA-GlcNAc units is initially synthesized and then further modified by various modifying enzymes. Wherein the N-deacetylase-N-sulfotransferase and the 3-O-and 6-O-sulfotransferases catalyze sulfation of GlcNAc residues; c5-differentially isomerase catalyzed conversion of GlcA to IdoA residues; and 2-O-sulfotransferase, catalyzing sulfation of GlcA/IdoA residues. These modifications can lead to the presence of a wide variety of disaccharide units in HP/HS polysaccharides, making HP/HS the most complex natural polymer in nature. At the same time, the highly complex structure has severely hampered the study of HP/HS structure and function.

Heparinase (Hepase) of bacterial origin is a polysaccharase that specifically catalyzes the cleavage of the glycosidic bond between GlcNAc and GlcA/IdoA residues in the HP/HS polysaccharide chain by β -elimination, resulting in the production of oligosaccharides containing 4, 5-unsaturated uronic acid residues at the non-reducing end. Heparinase is an essential tool enzyme for the study of HP/HS structure and function. To date, heparinases have been identified which can be divided into three types according to their substrate specificity: heparinase I (EC4.2.2.7), which specifically degrades the HP rich in IdoA with high sulfation; heparinase III (EC 4.2.2.8), which tends to degrade HS at low sulphur acidity and rich in GlcA; heparinase II (EC4.2.2. -) degrades HP and HS simultaneously. The heparinase I protein molecule folds into a beta-jelly roll-type three-dimensional structure, tending to cleave the 1,4 glycosidic bond linked to IdoA2S/GlcA 2S. In contrast, heparinase III consists of one N-terminus (. alpha./alpha.) ₅ Barrel-shaped domain and a C-terminal antiparallel beta sandwich domain, which tends to cleave the 1,4 glycosidic bond to GlcA/IdoA. Heparinase II adopts a topology similar to that of heparinase III and is not selective for a particular GlcA/IdoA or IdoA2S/GlcA2S structure. The three types of heparinase catalytic mechanisms are similar, and all adopt Tyr-His to serve as the catalytic mechanism

Acids and bases to catalyze the cleavage of glycosidic bonds.

It is noteworthy that all identified heparinases (HepaseI, II and III) belong to the family of endo-lyases, which randomly cleave glycosidic bonds from within the HP/HS polysaccharide chains, whereas exo-type heparinases, which are urgently needed for HP/HS sugar chain sequencing, have not been discovered and identified.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides an exo-type heparinase and application thereof, and the exo-type heparinase has high-efficiency enzymatic activity for degrading heparin substrates.

The technical scheme adopted by the invention for solving the technical problems is as follows:

an exo-heparinase for sequentially cleaving a disaccharide unit from the reducing end of a heparin-like substrate.

Preferably, according to the invention, the heparinoid substrate is heparin, heparan sulfate or HP-F _αIII 。

In the present invention, the substrate HP-F _αIII The HP polysaccharide is completely degraded by Hepase III, then 5 volumes of absolute ethyl alcohol are used for precipitation, and finally the obtained HP oligosaccharide mixture is collected by centrifugation.

Preferably, according to the invention, the disaccharide unit is heparin disaccharide with a high degree of sulfation.

Further preferably, the high-sulfation heparin disaccharide is a disulfated and higher heparin disaccharide.

Further preferably, the degree of sulfation of the tetrasaccharide unit composed of the high-sulfation heparin disaccharide and the heparin disaccharide bonded thereto is at least trisulfation.

Preferably, according to the invention, the exo-heparinase is the heparinase exoHep, GenBank: EDV07780.1, derived from the strain Bacteroides intestinalis DSM 17393.

Preferably, according to the invention, the exo-heparinase is the heparinase BTexoHep, GenBank: AAO79757.1 derived from the strain Bacteroides thetaiotaomicron VPI-5482.

Preferably, according to the invention, the exo-heparinase is the heparinase BCexoHep, GenBank: ALJ58962.1, derived from the strain Bacteroides cellulolyticus WH 2.

Preferably, according to the invention, the exo-heparinase is the heparinase BFexoHep from the strain Bacteroides finegoldii DSM 17565, GenBank: EEX 44367.1.

Preferably, according to the invention, the exo-heparinase is a heparinase PAexoHep derived from the strain Pedobacter arcticus, GenBank: WP-026063245.1.

Further preferably, the exo-heparinase exoHep, BCexohep, BTexohep, PAexohep and BFexohep is used for HP-F _αIII The enzyme activity of the enzyme is 50 plus or minus 15U/mg,62 plus or minus 18U/mg,27 plus or minus 8U/mg,24 plus or minus 7U/mg and 12 plus or minus 4U/mg.

The application of the excision heparinase in the preparation of heparin oligosaccharide.

The application of the excision heparinase in degrading heparin oligosaccharide.

The application of the excision heparinase in heparin oligosaccharide sequencing.

Has the beneficial effects that:

the exo-heparanase of the invention tends to degrade the substrate by sequential release from the reducing end of unsaturated disaccharides having a specific UV absorption at 232 nm. The exo-type heparinase sequentially cuts off the heparin disaccharide with high sulfation degree from the reducing end, and the higher the sulfation degree of the heparin disaccharide, the higher the enzyme activity of the heparinase. In addition, the potential of the exo-heparinase in the structural study of HP/HS was demonstrated by preliminary sequencing of HP octasaccharide with exo-heparinase exoHep. The invention not only fills the blank of the research field of heparinase, but also provides a novel tool enzyme for the structure and function research and enzymolysis sequencing of HP/HS.

Drawings

FIG. 1 is a polyacrylamide gel electrophoresis pattern of the expression and purification of heparinase exoHep;

wherein: lane 1, protein molecular weight markers, bands from top to bottom of 120kD, 100kD, 80kD, 60kD, 50kD, 40kD, 30kD, 20kD, 12 kD; lane 2, control bacterial liquid before wall breaking, loading amount 10 μ L, lane 3, bacterial liquid after wall breaking of recombinant bacteria, loading amount 10 μ L, lane 4, supernatant after wall breaking of recombinant bacteria, loading amount 10 μ L, lane 5, exoHep purified by nickel column, loading amount 10 μ L;

FIG. 2 is a graph showing the effect of temperature on the activity of heparinase exoHep; wherein the abscissa is temperature and the ordinate is relative activity;

FIG. 3 is a graph showing the effect of pH on the activity of heparanase exoHep; wherein the abscissa is pH, and the ordinate is relative activity;

FIG. 4 is a graph of the stability effect of temperature on heparinase exoHep; wherein, the abscissa is time, and the ordinate is relative activity;

FIG. 5 is a graph showing the effect of metal ions and chemical agents on the activity of heparinase exoHep; wherein, the abscissa is metal ions and chemical reagents, and the ordinate is relative activity;

FIG. 6 shows Ca ²⁺ And Ba ²⁺ Curve of the effect of concentration on the activity of heparinase exoHep; wherein the abscissa is Ca ²⁺ And Ba ²⁺ Concentration, ordinate is relative activity;

FIG. 7 is a High Performance Liquid Chromatography (HPLC) analysis chart of degradation products of heparinase exoHep degrading heparin (a) and heparan sulfate (b); wherein: di is unsaturated heparin disaccharide;

FIG. 8 is a High Performance Liquid Chromatography (HPLC) analysis of degradation products of exo-heparinase exoHep degrading heparin at different times; wherein: di is unsaturated heparin disaccharide;

FIG. 9 is a High Performance Liquid Chromatography (HPLC) analysis of degradation products of saturated heparin 13 sugar by exo-heparinase exoHep at different times; wherein: di is unsaturated heparin disaccharide;

FIG. 10 is a High Performance Liquid Chromatography (HPLC) analysis chart of degradation products of the exo-heparinase exoHep to degrade unsaturated heparin hexaose (a) and octaose (b); wherein: the ratio of Octa: unsaturated heparin octasaccharides; hexa: unsaturated heparin hexasaccharide; di: unsaturated heparin disaccharide;

FIG. 11 is a HPLC analysis chart of re-separation of heparin octasaccharide;

FIG. 12 is a flowchart of the enzymatic sequencing of exo-heparinase exoHep;

FIG. 13 is a sequence result presentation graph of P8-1(a), P8-2(b), P8-3(c), P8-4(d) and P8-5(e), (f) is a disaccharide standard chromatogram; wherein: 3S: Δ UA1-4GlcNAc 3S; 2 SNS: Δ UA2S1-4 GlcNS; NS 6S: Δ UA1-4GlcNS 6S; 2SNS 6S: delta UA2S1-4GlcNS 6S.

Detailed Description

The following examples are set forth so as to provide a thorough disclosure of some of the commonly used techniques of how the present invention may be practiced, and are not intended to limit the scope of the invention. The inventors have made the best effort to ensure accuracy with respect to parameters (e.g., amounts, temperature, etc.) used in the examples, but some experimental errors and deviations should be accounted for. Unless otherwise indicated, molecular weight in the present invention refers to average molecular weight and temperature to degrees celsius.

The source of the biological material is as follows:

the strain of Enterobacter (Bacteroides intestinalis DSM 17393) was purchased from the German Collection of microorganisms and cell cultures.

Substrate HP-F _αIII The HP polysaccharide is completely degraded by using Hepase III, then 5 volumes of absolute ethyl alcohol is used for precipitation, and finally, the obtained HP oligosaccharide mixture is collected by centrifugation.

Example 1 extraction of genomic DNA of a Strain of Bacteroides intestinalis DSM 17393

A strain of Bacteroides intestinalis DSM 17393 was inoculated into a liquid medium and cultured with shaking to OD at 37 ℃ and 200rpm in the absence of oxygen ₆₀₀ Is 1.2; 10mL of the culture broth was centrifuged at 12,000rmp for 25min, and the pellet was collected, washed with 10mL of lysozyme buffer (10mM Tris-HCl pH 8.0), centrifuged at 12,000rpm for 25min, and the pellet was collected.

The liquid culture medium comprises the following components per liter:

5g of tryptone, 5g of peptone, 10g of yeast extract, 5g of beef extract, 5g of glucose and K ₂ HPO ₄ 2g, Tween-801 mL, cysteine hydrochloride H ₂ O0.5 g, Resazurin 1mg, ddH ₂ O950 mL, vitamin K solution 0.2mL, hemin solution 10mL, buffer S40 mL;

the buffer S comprises the following components per liter:

CaCl ₂ ·2H ₂ O 0.25g，MgSO ₄ ·7H ₂ O 0.5g，K ₂ HPO ₄ 1g，KH ₂ PO ₄ 1g，NaHCO ₃ 10g，NaCl 2g，ddH ₂ O 1000mL。

in the thallus precipitation, 6.0mL of lysozyme buffer solution is added into each tube to obtain about 7.0mL of bacterial liquid, 280 mu L of lysozyme with the concentration of 20mg/mL is respectively added, and the final concentration is about 800 mu g/mL; after ice bath for 1.0h, warm bath is carried out for 2h at 37 ℃ until the solution is viscous; adding 0.41mL of SDS (sodium dodecyl sulfate) of 100mg/mL and 30 mu L of proteinase K solution of 100mg/mL, and carrying out water bath at 52 ℃ for 1.0 h; adding 7.5mL of Tris-balanced phenol/chloroform/isoamyl alcohol (volume ratio is 25: 24: 1), and slightly inverting and mixing until full emulsification; centrifuging at 4 deg.C for 10min at 10,000g, transferring supernatant, adding 1.0mL NaAc-HAc (pH 5.2, 3.0M) buffer solution and 8.50mL absolute ethanol, and mixing; picking out filamentous DNA with a 1.0mL pipette tip, transferring to a 1.5mL EP centrifuge tube, washing for 2 times with a 70% ethanol solution (stored at-20 ℃) by volume percentage, and discarding the supernatant after microcentrifugation; centrifuging at 4 deg.C for 3min at 10,000g, and thoroughly discarding supernatant; drying the sample in a sterile workbench by blowing air under an alcohol lamp; and (4) resuspending and dissolving the DNA sample by using sterile deionized water, and standing overnight at 4 ℃ to obtain the high molecular weight genome DNA.

Example 2 heparinase-encoding Gene sequences

The genome sequence (GenBank: ABJL02000001.1) of the Enterobacter intestinalis DSM 17393 (Bacteroides intestinalis) already exists in the NCBI database, so that the gene sequences encoding the heparinase exoHep (GenBank: EDV07780.1) are downloaded from the NCBI database without genome resequencing. In addition, we screened some genes with higher similarity to the exoHep from NCBI database and carried out whole gene synthesis, namely heparinase BTexohep derived from strain Bacteroides thetaiotaomicron VPI-5482 (GenBank: AAO79757.1), heparinase BCexohep derived from strain Bacteroides cellulolyticus WH2 (GenBank: ALJ58962.1), heparinase BFexohep derived from strain Bacteroides finegoldii DSM 17565 (GenBank: EEX44367.1) and heparinase PAexohep derived from strain Pedobacter arcticus (GenBank: WP _ 026063245.1).

Example 3 recombinant expression of the ExoHep Gene in E.coli

PCR amplification was performed using the high molecular weight genomic DNA prepared in example 1 as a template.

The PCR primers were as follows:

forward primer 30 aexoHep-F: 5' -CATATGGCCGTGATAAAGGTTTCC-3’(Nde I)；

Forward primer 30 aexoHep-R: 5' -CTCGAGTATTTCAGGTTCCG-3’(Xho I)；

The restriction sites for restriction enzyme Nde I are underlined for the forward primer, and the restriction sites for restriction enzyme Xho I are underlined for the reverse primer. Primerstar HS DNA polymerase was purchased from Takara Shuzo and the PCR reaction system was performed according to the manufacturer's instructions.

And (3) PCR reaction conditions: pre-denaturation at 95 ℃ for 4 min; denaturation at 95 ℃ for 40s, annealing at 60 ℃ for 40s, extension at 72 ℃ for 2min, and 30 cycles; extension at 72 deg.C for 10min, and stabilization at 4 deg.C for 15 min.

The PCR product was double digested with Nde I and Xho I, and the digested PCR product was recovered by agarose gel electrophoresis. The pET-30a vector purchased from Novagen, USA, was double-digested with Nde I and Xho I, and the large fragment of the digested vector was recovered by agarose gel electrophoresis. Nde I and Xho I are both available from baozoia, and the system, temperature and time of reaction of the enzyme with the substrate are all operated according to the product instructions provided by the company.

Connecting the PCR product subjected to double enzyme digestion with a pET-30a carrier subjected to double enzyme digestion, converting the connection product into an escherichia coli DH5 alpha strain, coating the strain on a Luria-Bertani culture medium solid plate (prepared according to the conventional technology) containing 50 mu g/mL kanamycin, culturing for 14h at 37 ℃, and selecting a monoclonal antibody; inoculating the monoclone into a liquid Luria-Bertani culture medium (prepared according to the conventional technology) containing 50 mu g/mL kanamycin to culture, and extracting plasmids; carrying out bacteria liquid PCR verification on the plasmid by using a forward primer 30aexoHep-F and a reverse primer 30aexoHep-R, obtaining an amplification product with correct size as a result, and preliminarily proving that the constructed recombinant plasmid is correct; the sequencing result of the recombinant plasmid shows that the gene exoHep is inserted between Nde I and Xho I enzyme cutting sites of pET-30a, and the insertion direction is correct, so that the constructed recombinant plasmid is further proved to be correct, and the recombinant plasmid is named as pET30 a-exoHep.

pET30a-exoHep was transformed into E.coli strain BL21(DE3) (available from Novagen, USA), and then inducible expression of recombinant heparinase exoHep was performed according to the procedures provided by the company. And exoHep was purified on a Ni Sepharose 6Fast Flow (GE) gel under the conditions according to the product manual of GE. The purification condition of the recombinant heparinase exoHep is detected by 13.2% polyacrylamide gel electrophoresis, and the result is shown in figure 1, the purified recombinant heparinase exoHep is in a single band on the electrophoresis gel, and the position of the recombinant heparinase exoHep is matched with the predicted molecular weight and is 99 kDa.

Example 4 enzymatic Properties analysis of recombinant heparinase exoHep

1. Effect of pH and temperature on enzyme Activity

Heparin with the mass concentration of 1%, exoHep enzyme solution and 150mM NaH ₂ PO ₄ -Na ₂ HPO ₄ (pH 8.0) buffer and water 1: 1: 3: 5 (volume ratio), reacting at different temperatures (0-70 ℃) for 30min, measuring the enzyme activity by an ultraviolet spectrophotometry, and defining the optimal enzyme activity as 100 percent relative activity. The results showed that the exoHep reached maximum activity at 30 ℃, indicating that the optimum reaction temperature for the exoHep was 30 ℃ (fig. 2).

At the optimum temperature of 30 ℃, heparin with the mass concentration of 1 percent, exoHep enzyme solution, 150mM HAc-Naac and NaH with different pH values ₂ PO ₄ -Na ₂ HPO ₄ Tris-HCl buffer solution (with the pH range of 5.0-10.0) and water, wherein the weight ratio of Tris-HCl buffer solution to water is 1: 1: 3: 5 (volume ratio), reacting for 60min, measuring enzyme activity by ultraviolet spectrophotometry, and defining the optimal enzyme activity as 100% relative activity. The results showed that the exoHep reached maximum activity at pH 6.0, indicating that the optimum reaction pH for the exoHep was 6.0 (see fig. 3).

The ultraviolet spectrophotometry measures the optical density value at the position with the wavelength of 232nm, and determines the enzyme activity unit U by taking the increase of 0.1 per minute of the optical density value as the enzyme activity unit U.

2. Effect of temperature on enzyme stability

The exoHep enzyme solution after heat treatment at 0 ℃, 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃ and 70 ℃ for different times and the heparin substrate solution with the mass concentration of 1 percent are mixed according to the ratio of 2:3 (volume ratio), and then measuring the residual enzyme activity at the optimal temperature and the optimal pH, wherein the enzyme activity of the enzyme solution without heat treatment is defined as 100 percent relative activity (relative activity), and the result shows that the exoHep still has more than 80 percent of activity after 24 hours at the temperature of 30 ℃ (as shown in figure 4) and has certain thermal stability.

3. Effect of Metal ions on ExoHep Activity

Heparin at a mass concentration of 1%, exoHep enzyme solution, 150mM MAc-NaAc (pH 6.0) buffer solution, and water were mixed in a ratio of 1: 1: 3: 5 (volume ratio), then adding different metal ions into the reaction system, wherein the final concentration of the added ions is 5mM, then reacting for 30min at 30 ℃, and measuring the enzyme activity according to the ultraviolet spectrophotometry. The control group shows the activity of exoHep without any metal ion (set to 100%), and the results are shown in fig. 5. The experimental results show that Ca ²⁺ And Ba ²⁺ Promoting enzyme activity, and Ag ⁺ ,Co ²⁺ ,Hg ²⁺ ,Pb ²⁺ ,Ni ²⁺ ,Cu ²⁺ ,Zn ²⁺ ,Fe ³⁺ ,Cr ³⁺ The plasma showed a significant inhibitory effect on enzyme activity (figure 5).

4、Ca ²⁺ And Ba ²⁺ Effect of concentration on exoHep Activity

Heparin with a mass concentration of 1%, exoHep enzyme solution, 150mM HAc-Naac (pH 6.0) buffer solution, and water were mixed in a ratio of 1: 1: 3: 5 (volume ratio), and then adding metal ions Ca of different concentrations to the reaction system ²⁺ And Ba ²⁺ The final concentrations of the added ions were 0mM, 1mM, 2mM, 5mM, 10mM, and 25mM, and the reaction was carried out at 30 ℃ for 30min, and the enzyme activity was measured by the aforementioned UV spectrophotometry. The control group shows the activity of exoHep without any metal ion (set to 100%), and the results are shown in fig. 6. The results of the experiment show that 5mM Ca ²⁺ Can promote 144.69% enzyme activity, and 2mM Ba ²⁺ Can promote 122.37% of enzyme activity (figure 6).

Example 5 determination of enzyme Activity of exoHep

Heparin, heparan sulfate or HP-F with the mass concentration of 1 percent _αIII exoHep enzyme solution, 150mM MAc-NaAc (pH 6.0) buffer, and water were mixed at a ratio of 1: 1: 3: 5 (volume ratio), reacting for 1-5 min at the optimum temperature and the optimum pH, measuring the enzyme activity by the ultraviolet spectrophotometry, and simultaneously measuring the protein content of the exoHep enzyme solution by using a protein quantitative kit purchased from Kangji corporationThe results show that the specific activity of exoHep to heparin is 1 +/-0.6U/mg for HP-F _αIII The specific activity is 50 +/-15U/mg, the heparan sulfate enzyme activity is extremely low, and the detection is difficult, and the exoHep is preliminarily proved to be more prone to degrade the heparin substrate with higher sulfation degree.

Furthermore heparinases BCexohep, BTexohep, PAexohep and BFexohep on HP-F _αIII The enzyme activity of the enzyme is respectively 62 plus or minus 18U/mg,27 plus or minus 8U/mg,24 plus or minus 7U/mg and 12 plus or minus 4U/mg.

Example 6 high Performance liquid phase (HPLC) analysis of recombinant heparinase exoHep degradation heparin and heparan sulfate degradation products

Mixing 3mg/mL heparin or heparan sulfate, 150mM HA-NaAc (pH 6.0) buffer solution, exoHep enzyme solution and deionized water according to the ratio of 10:10:3:7 (volume ratio), reacting for 12h under the optimal condition, and carrying out HPLC analysis on degradation products, wherein the HPLC analysis conditions are that a gel column: superdex peptide 10/300GL (GE); mobile phase: 0.2M ammonium bicarbonate; flow rate: 0.4 mL/min; detection conditions are as follows: UV232 nm.

As shown in fig. 7, as described in example 5, the recombinant heparinase exoHep has low activity on heparin and heparan sulfate, but under the condition of sufficient reaction, the recombinant heparinase exoHep can still degrade the heparin and heparan sulfate and only generate unsaturated disaccharide, and the activity on the heparin is obviously higher than that of the heparan sulfate.

Example 7 degradation Pattern of recombinant heparinase exoHep

3mg/mL of heparin, exoHep enzyme solution, 150mM MAc-NaAc (pH 6.0) buffer, and water were mixed in 10:3: 10: 7 (volume ratio), carrying out reaction under the optimal condition, selecting degradation products with different enzymolysis time for HPLC analysis, wherein the HPLC analysis conditions are that a gel column: superdex peptide 10/300GL (GE); mobile phase: 0.2M ammonium bicarbonate; flow rate: 0.4 mL/min; detection conditions are as follows: ex 330nm, Em 420 nm.

The results of the detection are shown in fig. 8, when the exoHep degrades the heparin, only unsaturated disaccharide is always generated, no obvious oligosaccharide with other molecular weight appears, and a very small part of oligosaccharide appears in the final product, and the result shows that the recombinant heparinase exoHep should be an exonuclease.

Example 8 degradation Direction of recombinant heparinase exoHep

3mg/mL of saturated heparin 13 sugar, exoHep enzyme solution, 150mM MAc-NaAc (pH 6.0) buffer solution and water were mixed in a 10:3: 10: 7 (volume ratio), reacting under the optimal condition, selecting degradation products with different enzymolysis time for HPLC analysis, wherein the HPLC analysis conditions are that a gel column: superdex peptide 10/300gl (ge); mobile phase: 0.2M ammonium bicarbonate; flow rate: 0.4 mL/min; detection conditions are as follows: ex 330nm, Em 420 nm.

The detection results are shown in FIG. 9, when the exoHep degrades the saturated heparin 13 sugar, only unsaturated disaccharide is always generated, and no oligosaccharide with other molecular weight appears, and the results show that the recombinant heparinase exoHep is an exonuclease and degrades the substrate from the reduction end.

Example 9 recombinant heparinase exoHep oligosaccharide degradation assay

2mg/mL of substrate (heparin octaose, heparin hexaose), 150mM HA-NaAc (pH 6.0) buffer, exoHep enzyme solution and deionized water were mixed at a ratio of 10:2:3:15 (by volume), reacted overnight under optimum conditions, and the degradation products were analyzed by HPLC. HPLC analysis conditions were gel column: superdex peptide 10/300GL (GE); mobile phase: 0.2M ammonium bicarbonate; flow rate: 0.4 mL/min; detection conditions are as follows: UV232 nm.

As shown in FIG. 10, the recombinant heparinase exoHep can degrade heparin hexaose (a) and heparin octaose (b), and can completely degrade heparin hexaose (a) and heparin octaose (b) into disaccharides, and the residual small part of tetrasaccharide cannot degrade, presumably because the residual small part of tetrasaccharide is a resistant structure, such as low sulfation, 3-O-sulfation modification and the like.

Example 10 substrate tendency of recombinant heparinase exoHep

Mu.g of heparin tetrasaccharide substrates of different sulfation patterns (P4-4 (. DELTA. UA1-4GlcNAc6S1-4GlcA1-4GlcNS6S), P4-5 (. DELTA. UA1-4GlcNS1-4GlcA1-4GlcNS6S), P4-6 (. DELTA. UA1-4GlcNS6S1-4GlcA1-4GlcNS6S), P4-7 (. DELTA. UA2S1-4GlcNS6S1-4GlcA1-4GlcNS6S), P4-8 (. DELTA. UA2S1-4GlcNS6S1-4IdoA2S1-4GlcNS6S)), 150 mAc-NaAc (pH 6.0) buffer, exoHep enzyme solution, and deionized water were subjected to HPLC analysis under the conditions of enzyme activity ratio at a volume ratio of mixed enzyme volume ratio of 10:2:3:15, and degradation products were calculated. HPLC analysis conditions were gel column: superdex peptide 10/300GL (GE); mobile phase: 0.2M ammonium bicarbonate; flow rate: 0.4 mL/min; detection conditions are as follows: UV232 nm.

The results show that the enzyme activities of the recombinant heparinase exoHep on P4-4 (trisulfated), P4-5 (trisulfated), P4-6 (tetrasulfated), P4-7 (pentasulfated) and P4-8 (hexasulfated) are respectively 0.5U/mg, 3.89U/mg, 24.22U/mg, 49.12U/mg and 79.49U/mg. This indicates that the enzyme activity of the recombinant heparinase exoHep is gradually increased with the increase of the sulfation degree of the substrate. In contrast, when the total degree of sulfation of heparin tetrasaccharide is either disulfated or monosulfated or even not sulfated, the enzyme activity of the recombinant heparinase exoHep is lower or even non-reactive. This is consistent with the results of the previous example five.

Example 11 sequencing application of recombinant heparinase exoHep

From HP-F _αIII The prepared HP octaose of defined size was further separated with an ion column and the main peak was recovered. The HPLC separation conditions were: ion column: propack PA 1; mobile phase: 0.5-1.5M NaCl; flow rate: 1 mL/min; separation time: 70 min; detection conditions are as follows: UV232 nm. The main peak was collected and desalted by gel filtration chromatography on a G10 chromatography column.

HPLC separation pattern is shown in FIG. 11, and HP octaose is further divided into five octaose components, P8-1, P8-2, P8-3, P8-4 and P8-5.

Based on the exonuclease characteristic of the recombinant heparinase exoHep, the invention establishes an enzymatic sequencing method, and takes the five octasaccharides as examples, and the steps (figure 12) are as follows:

step 1: 500pmol of five octasaccharides (P8-1, P8-2, P8-3, P8-4 and P8-5) were digested with 50, 9 and 1.5mU of exoHep for 5 minutes, respectively, and UDP6 and UDP4 products were collected and lyophilized repeatedly to remove NH ₄ HCO ₃ Obtaining purified oligosaccharide; wherein, according to the enzyme activities of the exoHep to different substrates, the appropriate enzyme amount corresponding to the corresponding substrate is selected for substrate degradation;

step 2: the UDP8, UDP6 and UDP4 components of each octasaccharide were degraded by heparinase (I, II), labeled with product 2-AB, and then analyzed by HPLC; HPLC analytical conditions were ion column: YMC Pack polyamine II (YMC); mobile phase: 16-550mM sodium dihydrogen phosphate; flow rate: 1 mL/min; analysis time: 60 min; detection conditions are as follows: a fluorescence detector, excitation 330nm, emission 420 nm;

step 3: the non-reducing end of each octasaccharide UDP4 was treated with O ₃ Treatment eliminated non-reducing terminal unsaturated residues, which were then degraded and analyzed as described in Step 2.

The results of HPLC analysis of the above five octasaccharides are shown in FIG. 13. The sequencing analysis process is detailed by taking the sequencing of P8-4 as an example: first, 500pmol of P8-4 was partially degraded with 9mU of exoHep to prepare its non-reducing terminal hexasaccharide (UDP 6) _NE-P8-4 ) And tetrasaccharide (UDP 4) _NE-P8-4 ). HPLC analysis of P8-4(UDP 8) _NE-P8-4 )，UDP6 _NE-P8-4 And UDP4 _NE-P8-4 The disaccharide composition of (2) is shown in FIG. 13d, UDP8 _NE-P8-4 Consists of two unsaturated disaccharides of Delta UA1-4GlcNS6S and Delta UA2S1-4GlcNS6S in a molar ratio of 1: 3; these two disaccharides are UDP6 _NE-P8-4 And UDP4 _NE-P8-4 In the molar ratio of (1): 2 and 1: 1. by comparing UDP8 _NE-P8-4 And UDP6 _NE-P8-4 The change in the molar ratio of the middle disaccharides can be concluded that the disaccharide (D) at the reducing end of P8-4 is HexUA2S1-4GlcNS6S, and similarly, the disaccharide at the C-position in P8-4 is determined by comparing UDP6 _NE-P8-4 And UDP4 _NE-P8-4 The disaccharide composition of (a) can be determined as HexUA2S1-4GlcNS 6S. To determine the type of disaccharide at position B in P8-4, O was used ₃ Processing UDP4 _NE-P8-4 To remove the unsaturated uronic acid at the non-reducing end and further treated with heparinase (I, II) for disaccharide composition analysis, as shown in FIG. 13d, a single disaccharide peak corresponding to Δ UA2S1-4GlcNS6S was detected, indicating that the disaccharide at the B-site is also HexUA2S1-4GlcNS 6S. Finally, based on UDP4 _NE-P8-4 The non-reducing terminal disaccharide (A) of P8-4 was assumed to be. DELTA.UA 1-4GlcNS 6S. In summary, the base sequence of P8-4 can be deduced as Δ UA1-4GlcNS6S1-4HexUA2S1-4GlcNS6S1-4HexUA2S1-4GlcNS6S1-4HexUA2S1-4GlcNS 6S.

By the same method, the base sequences of P-1, P-2, P-3 and P-5 were determined to be Δ UA-4 GlcNS6S-4HexA 2S-4 GlcNS 6-4 HexA 2S-4 GlcNS6S-4HexA 2S-4 GlcNS, Δ UA-4 GlcNS6S-4HexA 2S-4 GlcNS-4HexA 2S-4 GlcNS6S-4HexA 2S-4 GlcNS6, Δ UA-4 GlcNAc 3S-4 HexA 2S-4 GlcNS 6-4 HexA 2S-4 GlcNS6 and Δ UA-4 GlcNAc 3S-4 HexA 2S-4 GlcNS 6-4 HexA 2S-4 GlcNS6S-4 GlcNS 6.

Heparinase BTexoHep (GenBank: AAO79757.1) derived from strain Bacteroides thetaiotaomicron VPI-5482, heparinase BCexoHep (GenBank: ALJ58962.1) derived from strain Bacteroides cellulolyticus WH2, heparinase BFexoHep (GenBank: EEX44367.1) derived from strain Bacteroides finegoldii DSM 17565 and heparinase PAexoHep (GenBank: WP _026063245.1) derived from strain Pedobacter arcus were verified as described in examples 7 and 8 to be exo-type heparinases and all degrade the substrates from the reducing end. According to the method of example 9 and example 10, the exo-type heparinase can degrade the substrate with higher sulfation degree like exoHep, and the enzyme activity of the heparinase is gradually improved along with the improvement of the sulfation degree of the substrate. The excision heparinase can be applied to heparin sequencing.

Claims

1. The protein is used as an externally tangent heparinase to be applied to heparin oligosaccharide sequencing, the externally tangent heparinase degrades heparin oligosaccharides from a reducing end, degradation products are heparin disaccharide with the disulfidation degree and above, and the sulfation degree of a tetrasaccharide unit consisting of the heparin disaccharide and the heparin disaccharide connected with the heparin disaccharide is within the trisulfation degree and above; the protein is derived from bacterial strainBacteroides intestinalisThe heparinase exoHep of DSM 17393, GenBank: EDV07780.1, or from a strainBacteroides thetaiotaomicronVPI-5482 heparinase BTexoHep, GenBank: AAO79757.1, or from a strainBacteroides cellulosilyticusWH2 heparinase BCexoHep, GenBank ALJ58962.1, or from a strainBacteroides finegoldiiHeparinase BFexoHep, GenBank: EEX44367.1 of DSM 17565 or derived from a strainPedobacter arcticusThe heparinase PAexoHep, GenBank: WP _ 026063245.1.