WO2021128254A1

WO2021128254A1 - Method for preparing heparinase iii

Info

Publication number: WO2021128254A1
Application number: PCT/CN2019/129111
Authority: WO
Inventors: 李锂; 林森茂; 邓利容
Original assignee: 深圳市天道医药有限公司
Priority date: 2019-12-27
Filing date: 2019-12-27
Publication date: 2021-07-01
Also published as: CN114787195B; CN114787195A

Abstract

A method for preparing heparinase III, comprising the following steps: crushing and centrifuging Flavobacterium heparinum, enabling precipitation from a resulting supernatant using ammonium sulfate, dissolving precipitate, and performing dialysis; loading an SP column, enabling equilibrium using a 20-30 mM Tris-HCl buffer solution, performing linear elution using 0-0.5 M NaCl in the same buffer solution, collecting heparinase active components, and performing dialysis; loading the SP column, performing elution using a 50 mM Tris-HCl buffer solution containing 0.05-0.2 M NaCl, collecting heparinase active components, and performing dialysis; loading the SP column, performing isocratic elution using a 50 mM Tris-HCl buffer solution containing 0.3% heparin, collecting heparinase active components, and performing dialysis; and loading the SP column, performing isocratic elution using a 50 mM Tris-HCl solution containing 0.055 M NaCl, collecting heparinase active components, and performing concentration to obtain heparinase III.

Description

A kind of preparation method of heparinase III

Technical field

This application belongs to the technical field of biological materials, and relates to a method for preparing heparinase III.

Background technique

Heparinase was originally discovered and isolated from Flavobacterium heparinum (Flavobacterium heparinum). Flavobacterium heparinum is a type of gram-negative bacteria derived from soil. At present, three types of heparinase have been isolated and purified from it: heparinase I , Heparinase II and Heparinase III. The main differences between the three heparinases are relative molecular mass, charge properties and substrate specificity.

Heparinase I has a relative molecular weight of 43800 and an isoelectric point of 9.3, which can specifically cut the bond between heparin molecules 6SGlcNS(1→4)2SIdoA. The relative molecular weight of heparinase II is 84500, the isoelectric point is about 8.9, and the substrate specificity is the widest among the three heparinases. It can cut the connection sites of heparin and heparan sulfate. Heparinase III has a relative molecular weight of 73000 and an isoelectric point of about 10, which can specifically cleave the GlcNS/GlcNAc(1→4)GlcA linkage in heparan sulfate molecules.

Heparinase has important applications in the preparation of low molecular weight heparin, elimination of heparin anticoagulants in cardiopulmonary bypass, and determination of the precise structure of heparin. However, heparinase is poor in stability and difficult to purify, so it is expensive, which limits its wide application in the pharmaceutical industry.

There are many research reports on the purification of heparinase. Yang et al. (Purification and characterization of heparinase from Flavobacterium heparinum, J Biol Chem, 1985, 260(3): 1849-1857) used hydroxyapatite adsorption/elution treatment, QAE -Sepharose column chromatography, HA-HPLC, Mono-S FPLC, GPC-HPLC and other five-step purification, and finally obtain pure heparinase III, the specific activity reaches 63.5IU/mg, and the activity yield is 2.74%.

At present, the Flavobacterium heparinase fermentation method is still the main method for the production research of heparinase I, II, and III, and there have been many research reports on the preparation of heparinase. Ma Xiaolai et al. (CN102286448B) used combined column chromatography to simultaneously extract heparinase I, II and III from the cell crushing liquid of Flavobacterium heparinum. The final yield of heparinase I was 35% and the specific activity was 416.67IU. /mg, the yield of heparinase II is 22%, the specific activity is 15.33IU/mg, the yield of heparinase III is 4%, the specific activity is 235IU/mg, but the yield of heparinase III activity is too low .

Therefore, in this field, it is desired to develop a preparation method that can significantly improve the yield of heparinase III activity.

Summary of the invention

In view of the shortcomings of the prior art, the purpose of this application is to provide a method for preparing heparinase III. The method described in this application enables the activity yield of heparinase III to be significantly improved compared to the prior art.

In order to achieve the purpose of this application, this application adopts the following technical solutions:

This application provides a method for preparing heparinase III, which includes the following steps:

(1) The Flavobacterium heparinum was crushed and centrifuged, ammonium sulfate was added to the supernatant for precipitation, and the precipitate was dissolved in a buffer solution and then dialyzed;

(2) Load the dialyzed enzyme solution onto the SP column, equilibrate with 20-30mM Tris-HCl buffer, linearly elute with 0-0.5M NaCl solution in the same buffer, and collect the second group with heparinase activity Points, and then dialysis;

(3) Load the dialyzed enzyme solution from step (2) on the SP column, eluting with 50mM Tris-HCl buffer containing 0.05-0.2M NaCl, and collect the first component with heparinase activity. Then undergo dialysis;

(4) Load the dialysis enzyme solution from step (3) on the SP column, and perform isocratic elution with 50mM Tris-HCl buffer, which contains 0.3% heparin, 10mM CaCl ₂ and 25mM by mass. NaCl, the pH of which is 7.0, collect the fractions with heparinase activity, and then conduct dialysis; and

(5) Load the dialyzed enzyme solution from step (4) on the SP column, and elution isocratically with a 50mM Tris-HCl solution containing 0.055M NaCl, collect the fractions with heparinase activity, and then concentrate to obtain heparin Enzyme III.

In this application, the use of the preparation method can improve the activity yield of heparinase III; currently the highest activity yield of heparinase III in the prior art is 4%, and the activity yield of heparinase III in this application reaches 15.1% , Which is nearly four times that of the existing technology.

Preferably, the crushing in step (1) is carried out at 4-8°C (for example, 4°C, 5°C, 6°C, 7°C or 8°C).

Preferably, the rotation speed of the centrifugation in step (1) is 10000-20000rpm, such as 10000rpm, 11000rpm, 12000rpm, 13000rpm, 14000rpm, 15000rpm, 16000rpm, 17000rpm, 18000rpm, 19000rpm or 20000rpm.

Preferably, the specific method of adding ammonium sulfate for precipitation in step (1) is: adding ammonium sulfate to the supernatant so that the final concentration of ammonium sulfate is 50% saturation, and stirring for 30-45min (for example, 30min, 33min, 35min, 38min, 40min, 43min or 45min), and then 4-8℃ (for example 4℃, 5℃, 6℃, 7℃ or 8℃) at 10000-20000rpm (for example 10000rpm, 11000rpm, 12000rpm, 13000rpm, 14000rpm, 15000rpm, Centrifuge at 16000rpm, 17000rpm, 18000rpm, 19000rpm or 20000rpm for 20-40min (such as 20min, 25min, 28min, 30min, 35min, 38min or 40min), take the supernatant, and add ammonium sulfate to the supernatant to make the final concentration For 80% saturation, continue to stir for 30-45min (for example, 30min, 33min, 35min, 38min, 40min, 43min or 45min) at 4-8℃ (for example 4℃, 5℃, 6℃, 7℃ or 8℃) Centrifuge at 10000-20000rpm (e.g. 10000rpm, 11000rpm, 12000rpm, 13000rpm, 14000rpm, 15000rpm, 16000rpm, 17000rpm, 18000rpm, 19000rpm or 20000rpm) for 20-40min (e.g. 20min, 25min, 28min, 30min, 35min, 38min or 40min), Collect the sediment.

Preferably, the buffer in step (1) is 20-30mM (20mM, 22mM, 25mM, 28mM or 30mM) Tris-HCl buffer, which contains 5-10mM (for example, 5mM, 6mM, 7mM, 8mM, 9mM or 10mM). ) CaCl ₂ , pH value is 7.0.

Preferably, the dialysis in step (1) is performed in a dialysis bag with a molecular weight cut-off of 10 kDa.

Preferably, the temperature of the dialysis in step (1) is 4-8°C, for example 4°C, 5°C, 6°C, 7°C or 8°C.

Preferably, the dialysis solvent used for the dialysis in step (1) is the same as the buffer used for dissolving the precipitate in step (1).

Preferably, the Tris-HCl buffer in step (2) further contains 5-10 mM (for example, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, or 10 mM) CaCl ₂ with a pH of 7.0.

Preferably, the dialysis in step (2) is performed in a dialysis bag with a molecular weight cut-off of 10 kDa.

Preferably, the dialysis solvent used in the dialysis in step (2) is 50 mM Tris-HCl buffer, which also contains 10 mM CaCl ₂ , and its pH value is 7.0.

Preferably, the temperature of the dialysis in step (2) is 4-8°C, for example, 4°C, 5°C, 6°C, 7°C or 8°C.

Preferably, the 50 mM Tris-HCl buffer containing 0.05-0.2 M NaCl in step (3) also contains 10 mM CaCl ₂ with a pH value of 7.0.

Preferably, the dialysis in step (3) is performed in a dialysis bag with a molecular weight cut-off of 10 kDa.

Preferably, the dialysis solvent used in the dialysis in step (3) is 50 mM Tris-HCl buffer, which also contains 10 mM CaCl ₂ , and its pH value is 7.0.

Preferably, the temperature of the dialysis in step (3) is 4-8°C, for example 4°C, 5°C, 6°C, 7°C or 8°C.

Preferably, the dialysis in step (4) is performed in a dialysis bag with a molecular weight cut-off of 10 kDa.

Preferably, the dialysis solvent used in the dialysis in step (4) is 50 mM Tris-HCl buffer, which also contains 10 mM CaCl ₂ , and its pH value is 7.0.

Preferably, the temperature of the dialysis in step (4) is 4-8°C, for example 4°C, 5°C, 6°C, 7°C or 8°C.

Preferably, the 50 mM Tris-HCl solution containing 0.055 M NaCl in step (5) also contains 10 mM CaCl ₂ with a pH value of 7.0.

Preferably, the concentration is performed using a 30kDa ultrafiltration centrifuge tube.

Compared with the prior art, this application has the following beneficial effects:

In this application, the use of the preparation method can increase the activity yield of heparinase III; based on the purified enzyme activity, the highest activity yield of heparinase III in the prior art is 4%. The activity yield of III reaches 15.1%, which is nearly four times that of the prior art.

Description of the drawings

Figure 1 is the SDS-PAGE electrophoresis diagram of the crude enzyme obtained after the F. heparinum is disrupted and centrifuged and the prepared heparinase III in Example 1, where 1 is the crude enzyme obtained after the F. heparinum is disrupted and centrifuged; 2 is the purified enzyme After heparinase III; 3 is Marker.

Detailed ways

The technical solutions of the present application will be further explained through specific implementations below. It should be understood by those skilled in the art that the described embodiments are merely to help understand the application and should not be regarded as specific limitations to the application.

Example 1

The Flavobacterium heparinus used in this embodiment can be cultured by the prior art culture method, for example, it can be cultured by the following method:

Seed culture medium: Weigh 5g beef extract, 10g peptone, 5g yeast powder, 5g NaCl, dissolve in 1L purified water, adjust pH to 7.0 with 6M NaOH, divide into 500mL Erlenmeyer flasks, sterilize at 121°C for 20 minutes , Cool down and spare.

Fermentation medium: Take 80g of heparin, _{25g of KH 2} PO _4, 25g of Na ₂ HPO ₄ ·12H ₂ O, _{5g of MgSO 4} ·7H ₂ O, and dissolve 100g of peptone in 10L of pure water, adjust the pH to 7.0 with 6M NaOH, and divide Sterilize in a 5L Erlenmeyer flask at 121°C for 20 minutes, then cool for later use.

Seed culture: In a clean environment, scrape Flavobacterium heparinum from a plate or a slope to a seed culture medium (50 mL), culture at 23°C, 150 rpm, and culture for 1 day. Then, the bacteria solution (25 mL) was transferred to seed culture medium (375 mL), and cultured at 23° C., 150 rpm, for 1 day.

Fermentation culture: The seed culture solution (375 mL) was inoculated into the fermentation medium (5 L) at a volume ratio of 7.5%, 23° C., 150 rpm, and shaking culture for 24-36 hours.

Collect bacteria: Centrifuge the fermentation broth obtained from fermentation culture at 3800 rpm and 4°C for 60 minutes, collect the precipitate, weigh, package and store in a clean centrifuge tube, and store frozen at -20°C.

After the above-mentioned cultivation process, the Flavobacterium heparinum is obtained, and then the preparation of heparinase III is carried out, which specifically includes the following steps:

(1) Crude enzyme preparation

Add 500 mL of 25 mM Tris-HCl (10 mM CaCl ₂ , pH 7.0) buffer to Flavobacterium heparinum, stir, and then pulverize with a high-pressure homogenizer at a pressure of 800 bar and 4°C. Centrifuge the cell crushing solution at 4°C and 18000 rpm for 30 minutes, and the supernatant obtained is the crude enzyme solution.

(2) Ammonium sulfate precipitation

Place the crude enzyme solution obtained in step (1) in an ice bath, add dry ammonium sulfate solid powder, the final concentration of ammonium sulfate is 50% saturation, continue to stir for 30-45 minutes, and then centrifuge at 18000 rpm and 4°C for 30 minutes; take Supernatant and slowly add dry ammonium sulfate powder to it. The final concentration of ammonium sulfate is 80% saturation. Continue to stir for 30-45 minutes, then centrifuge at 18000 rpm and 4°C for 30 minutes. Discard the supernatant and convert the precipitate to 25 mM. Dissolve in 150 mL of Tris-HCl (containing 10mM CaCl ₂ , pH 7.0) buffer solution, put it into a dialysis bag (MWCO: 10kDa), and then place the dialysis bag in 50 times the volume of 25mM Tris-HCl (containing 10mM CaCl ₂ , pH 7.0) ) In the buffer, dialyze overnight at 4-8°C.

(3) The first rough separation of heparinase III

The enzyme solution obtained from the dialysis step (2) is loaded onto the SP column, and _{equilibrated with a 25mM Tris-HCl (containing 10mM CaCl 2} , pH 7.0) buffer, and then 0-0.5M NaCl in the same buffer is linearly eluted. Collect with a partial collector, detect the heparin degradation ability and HS degradation ability of each tube of eluate, and detect the absorbance value of each tube sample at 280nm. Collect the eluate corresponding to the second activity peak, put the collected eluate into a dialysis bag (MWCO: 10kDa), and place it in 2L 50mM Tris-HCl (containing 10mM CaCl ₂ , pH 7.0) buffer at 4 Dialysis overnight at -8°C.

(4) The second rough separation of heparinase III

The enzyme solution obtained from the dialysis in step (3) was loaded onto the SP column, and then equilibrated with 50mM Tris-HCl (containing 10mM CaCl ₂ , 0.05M NaCl, pH 7.0) buffer, and then 50mM Tris-HCl containing 0.05-0.2M NaCl (Containing 10mM CaCl ₂ , pH 7.0) solution was linearly eluted. Collect with a partial collector, detect the heparin degradation ability and HS degradation ability of each tube of eluate and the absorbance value of each tube sample at 280nm, collect the eluate corresponding to the first activity peak, and load the obtained eluate The dialysis bag (MWCO: 10kDa) is placed in 2L of 50mM Tris-HCl (containing 10mM CaCl ₂ , pH 7.0) buffer, and fully dialyzed overnight at a temperature of 4-8°C.

(5) The first purification of heparinase III

The enzyme solution obtained from the dialysis in step (4) was loaded onto the SP column, _{and eluted isocratically with 50 mM Tris-HCl (containing 10 mM CaCl 2} , pH 7.0, 25 mM NaCl, 0.3% heparin) buffer, and collected with a partial collector for detection The HS degradation capacity of each tube of eluent and the absorbance value at 280nm are collected, and the eluent corresponding to the activity peak is collected. The eluate was loaded into a dialysis bag (MWCO: 10kDa), in 2L 50mM Tris-HCl (containing 10mM CaCl _2, pH 7.0) buffer at a temperature sufficiently dialyzed overnight at 4-8 deg.] C.

(6) The second refinement of heparinase III

The enzyme solution obtained from the dialysis step (5) was loaded onto the SP column, and eluted isocratically with a 50mM Tris-HCl (containing 10mM CaCl ₂ , pH 7.0) solution containing 0.055M NaCl, collected by a part of the collector, and tested for the HS degradation ability of each tube , Collect the eluate corresponding to the activity peak, and concentrate it with a 30kDa ultrafiltration centrifuge tube to obtain purified heparinase III.

SDS-PAGE electrophoresis analysis was performed on the crude enzyme obtained after the crushing and centrifugation of Flavobacterium heparinum and the prepared heparinase III, and the results shown in Figure 1 were obtained, where 1 is the crude enzyme obtained after the crushing and centrifugation of Flavobacterium heparin; 2 is The purified heparinase III; 3 is a Marker. It can be seen from FIG. 1 that the heparinase III obtained by the preparation method of the present application has high purity.

Example 2

Referring to Example 1, the heparin isocratic elution in step (5) of Example 1 was changed to linear elution, and the specific conditions were: 50mM Tris-HCl (containing 10mM CaCl ₂ , pH 7.0) buffer containing NaCl and heparin was used Perform linear elution with 5～75mM NaCl and 0.1%～0.5% heparin, collect with a partial collector, detect the HS degradation ability of each tube of eluate and absorbance at 280nm, and collect the eluate corresponding to the activity peak.

Finally, purified heparinase III is obtained.

Example 3

In this example, the enzyme activity test was performed on the heparinase III prepared in the above example, and the heparin substrate and HS substrate used in the test were prepared by the following method:

Heparin substrate: Weigh 100 mg of heparin, dissolve it with 25mM Tris-HCl (containing 10mM CaCl2, pH 7.0) buffer, and dilute to 100mL to prepare a 1mg/mL heparin substrate solution.

HS substrate: Weigh 25mg HS, dissolve it with 25mM Tris-HCl (containing 10mM CaCl2, pH 7.0) buffer, and dilute to 25mL to prepare a 1mg/mL HS substrate solution.

The enzyme activity test method is as follows:

Add 2.5mL HS substrate solution preheated at 37℃ in 5mL quartz cuvette, pipette 20μL crude enzyme solution or heparinase III enzyme solution, shake well and measure the absorbance at 232nm within two minutes. Absorbance change value, and calculate the average change value per minute. Use the molar extinction coefficient of the HS double bond ε=3800 to calculate its formation, and convert it to the international unit of enzyme in the enzyme solution. The conversion coefficient is 32.9, which is △OD232nm/min. The value of ×32.9 is the unit enzyme activity (IU/mL).

The activity yield of heparinase III obtained through the test is shown in Table 1 below.

Table 1:

It can be seen from the results in Table 1 that the heparinase III activity yield obtained by the preparation method described in this application reaches 15.1%, and if the step (5) is changed from moderate elution to linear elution, heparinase III The activity yield will decrease significantly.

This application uses the foregoing embodiments to illustrate the process method of the present application, but the application is not limited to the foregoing process steps, which does not mean that the application must rely on the foregoing process steps to be implemented. Those skilled in the art should understand that any improvement to this application, the equivalent replacement of raw materials selected in this application, the addition of auxiliary components, the selection of specific methods, etc., fall within the scope of protection and disclosure of this application.

Claims

A preparation method of heparinase III, which comprises the following steps:

(1) The Flavobacterium heparinum was crushed and centrifuged, ammonium sulfate was added to the supernatant for precipitation, and the precipitate was dissolved in a buffer solution and then dialyzed;

(2) Load the dialyzed enzyme solution onto the SP column, equilibrate with 20-30mM Tris-HCl buffer, linearly elute with 0-0.5M NaCl solution in the same buffer, and collect the second group with heparinase activity Points, and then dialysis;

(3) Load the dialyzed enzyme solution from step (2) on the SP column, eluting with 50mM Tris-HCl buffer containing 0.05-0.2M NaCl, and collect the first component with heparinase activity. Then undergo dialysis;

(4) Load the dialysis enzyme solution from step (3) on the SP column, and perform isocratic elution with 50mM Tris-HCl buffer, which contains 0.3% heparin, 10mM CaCl 2 and 25mM by mass. NaCl, the pH of which is 7.0, collect the fractions with heparinase activity, and then conduct dialysis; and

(5) Load the dialyzed enzyme solution from step (4) on the SP column, and elution isocratically with a 50mM Tris-HCl solution containing 0.055M NaCl, collect the fractions with heparinase activity, and then concentrate to obtain heparin Enzyme III.
The preparation method according to claim 1, wherein the crushing in step (1) is 4-8°C, and the rotation speed of the centrifugation is 10000-20000 rpm.
The preparation method according to claim 1 or 2, wherein the specific method of adding ammonium sulfate for precipitation in step (1) is: adding ammonium sulfate to the supernatant so that the final concentration of ammonium sulfate is 50% saturation, and stirring 30-45min, then centrifuge at 10000-20000rpm at 4-8℃ for 20-40min, take the supernatant, add ammonium sulfate to the supernatant, make the final concentration of ammonium sulfate 80% saturation, continue to stir for 30-45min, Centrifuge at 10000-20000rpm for 20-40min at 4-8℃, and collect the sediment.
The preparation method according to any one of claims 1 to 3, wherein the buffer in step (1) is a 20-30 mM Tris-HCl buffer, which contains 5-10 mM CaCl 2 and has a pH of 7.0.
The preparation method according to any one of claims 1 to 4, wherein the dialysis in step (1) is performed in a dialysis bag with a molecular weight cut-off of 10kDa;

Preferably, the temperature of the dialysis in step (1) is 4-8°C.
The preparation method according to any one of claims 1 to 5, wherein the dialysis solvent used in the dialysis step (1) is the same as the buffer used in the step (1) to dissolve the precipitate.
The preparation method according to any one of claims 1 to 6, wherein the Tris-HCl buffer in step (2) contains 5-10 mM CaCl 2 and has a pH of 7.0.
The preparation method according to any one of claims 1-7, wherein the dialysis in step (2) is performed in a dialysis bag with a molecular weight cut-off of 10 kDa.
The production method as claimed in any one of the preceding claims, wherein, in step (2) the solvent is a dialysis using dialysis 50mM Tris-HCl buffer, wherein further comprising 10mM CaCl 2, a pH of 7.0;

Preferably, the temperature of the dialysis in step (2) is 4-8°C.
The preparation method according to any one of claims 1-9, wherein the 50 mM Tris-HCl buffer containing 0.05-0.2 M NaCl in step (3) further contains 10 mM CaCl 2 with a pH value of 7.0.
The preparation method according to any one of claims 1-10, wherein the dialysis in step (3) is performed in a dialysis bag with a molecular weight cut-off of 10 kDa.
The production method according to any one of 1-11 claims, wherein, in step (3) the solvent is a dialysis using dialysis 50mM Tris-HCl buffer, wherein further comprising 10mM CaCl 2, a pH of 7.0;

Preferably, the temperature of the dialysis in step (3) is 4-8°C.
The preparation method according to any one of claims 1-12, wherein the dialysis in step (4) is performed in a dialysis bag with a molecular weight cut-off of 10kDa;

Preferably, the dialysis solvent used in the dialysis in step (4) is 50mM Tris-HCl buffer, which also contains 10mM CaCl 2 , and its pH value is 7.0;

Preferably, the temperature of the dialysis in step (4) is 4-8°C.
The preparation method according to any one of claims 1-13, wherein the 50 mM Tris-HCl solution containing 0.055 M NaCl in step (5) further contains 10 mM CaCl 2 with a pH value of 7.0.
The preparation method according to any one of claims 1-14, wherein the concentration in step (5) is performed using a 30kDa ultrafiltration centrifuge tube.