KR100294162B1 - Method for producing low molecular weight of heparin by using recombinant heparinase containing a histidine oligopeptide - Google Patents

Abstract

PURPOSE: A method for producing low molecular weight of heparin by using a recombinant heparinase containing a histidine oligopeptide is provided, thereby decreasing the immobilization time, storing the heparinase stably stored for a long time and reusing it. CONSTITUTION: The low molecular weight of heparin is produced by preparing an expression vector containing the structural gene of heparinase having the nucleotide sequence of SEQ ID NO: 1, which contains a gene coding histidine oligopeptide at 5'-terminal; transforming E. coli with the expression vector to produce a E. coli transformant; cultivating the E. coli transformant to produce heparinase containing a histidine oligopeptide; and reacting the heparinase with heparin at 20 to 28 deg.C and pH 5.0 to 8.0 to produce low molecular weight of heparin, in which the expression vector is pE16b-hepa2, the E. coli transformant is E. coli BL21(DE3)(KFCC-10903), and the heparinase is immobilized on a chelating supporter.

Description

Method for preparing low molecular weight heparin using recombinant heparin degrading enzyme comprising histidine oligopeptide

1 is a diagram showing a vector having a heparin degrading enzyme gene comprising a histidine oligopeptide of the present invention, a vector having a heparin degrading enzyme gene, and a manufacturing process thereof;

2 is a photograph showing the results of SDS-PAGE (polyacrylamide gel electrophoresis) analysis of a heparin comprising a histidine oligopeptide prepared according to the present invention, a degrading enzyme (lane 2) and a heparin degrading enzyme (lane 1),

3A is a graph showing the optimal pH of heparin degrading enzyme including histidine oligopeptide,

3B is a graph showing the optimum temperature of heparin degrading enzyme including histidine oligopeptide,

3C is a graph showing pH stability of heparin degrading enzyme including histidine oligopeptide,

4 is a graph showing the results of comparing the activity of heparin degrading enzyme (●) and heparin degrading enzyme (○) immobilized on CNBr-active Sepharose 4B,

5 is a graph showing the results of comparing the activity of heparin degrading enzyme (●) including histidine oligopeptide immobilized on a chelating support and heparin degrading enzyme (○) including histidine oligopeptide.

Industrial use field

The present invention relates to a method for producing a low molecular weight heparin using a recombinant heparin degrading enzyme comprising a histidine oligopeptide, and more particularly, a recombinant vector comprising a gene of heparin degrading enzyme comprising a histidine oligopeptide, The present invention relates to a method for producing low molecular weight heparin by enzymatically decomposing heparin using a recombinant heparin degrading enzyme comprising a prepared histidine oligopeptide and a heparin degrading enzyme comprising a prepared histidine oligopeptide.

Prior art

Heparin is a glucosaminoglycan consisting of linear polysaccharides in which D-glucosamine and hexauronic acid are formed by α 1 -4 bonds, and have low molecular weights of 5,000 to 30,000, varying in size, and an average molecular weight of 12,000 to 15,000. Heparin binds to antithrombin Ⅲ and causes structural changes, and in anticoagulant action on blood coagulation mechanisms such as thrombin, FXa, FIXa, FXIa and kallikrein, which are particularly sensitive to thrombin. Inhibited (J. Hirsh et al., Blood, 79, 1-77 (1992)). The biological action of heparin has been used to prevent thromboembolism after surgery, but has been accompanied with side effects such as bleeding. Complementing this drawback is the low molecular weight heparin.

Low molecular weight heparin is a fragment of heparin with an average molecular weight of about 4,000-6,500 and has many differences from heparin. The difference in anticoagulant mechanism is that heparin has almost the same ratio of anti-IIa activity to anti-Xa activity, while low molecular weight heparin is anti-Xa anti-Xa due to its molecular weight-dependent properties when heparin inhibits thrombin. The activity of -Xa is as high as 1: 2-1: 4.

In addition, the difference in the molecular weight of heparin affects the neutralization of plasma proteins and endothelial cells. The low-molecular-weight heparin acts at a lower rate on plasma proteins, platelets, and endothelial cells than heparin. Increase the bioavailability. In particular, heparin's interaction with vWF (von Willebrand factor) inhibits vWF-dependent platelet function, leading to bleeding as a side effect of heparin, but low-molecular weight heparin may reduce bleeding due to its low affinity for vWF.

Due to the bioavailability of such low molecular weight heparin, various methods have been devised to make low molecular weight heparin from heparin. The method has been made by nitrous acid depolymerization, benzylation and alkaline depolymerization, peroxide depolymerization, enzymatic depolymerization and the like.

Double enzymatic depolymerization is a method using heparin degrading enzyme. Heparin degrading enzyme is an enzyme produced by Flavoacterium heparinum that acts on glycoside linkage between heparin's N-sulfate D-glucosamine and sulfate D-glucuronic acid (or L-iduronic acid). It is an α1-4-remolytic enzyme, first isolated by Linker and Hovingh, and its gene was cloned and expressed in E. coli in 1993 (R. sassekharah et al. Proc. Natl. Acad. Sci, USA, 90, 3660-3664 (1993). The heparin degradability of the heparin degrading enzyme is used as an enzyme for converting heparin to low molecular weight heparin.

Until now, a method of reacting heparin and heparin together to make low molecular weight heparin and immobilizing heparin by immobilizing the heparin degrading enzyme in Sepharose 4B (USP 5,106,734, H. Bernstein et al. , Methods Enzym. 137, 515-529 (1988). In particular, the method of immobilizing the heparin degrading enzyme can be easily reused to recover the enzyme after the reaction between the enzyme and heparin, and also affects the stability of the heparin degrading enzyme, thereby greatly helping to stabilize the enzyme.

To date, as a support for immobilizing heparin degrading enzymes, CNBr-active Sepharose 4B, tresyl chloride-activated Sepharose, carvidimide-, or active ester-active CM-Sepharose, CM-cellulose, polyacrylamide, epoxy Active (oxylon) acryl beads have been tried and the highest activity of immobilized heparin degrading enzyme was CNBr-activated Sepharose 4B (H. Bernstein et a1., Methods Enzym, 137, 515-529 (1988)).

However, the method of immobilizing heparin degrading enzyme in CNBr-activated Sepharose 4B takes about 40 hours, and the immobilization of heparin degrading enzyme in CNBr-activated Sepharose 4B is not limited to the active site or near the active site of heparin degrading enzyme. Due to the presence of lysine residues, a significant portion of the activity is lost (Linhart, RJ, et al., Biochim, Biophts. Acta, 702, 192-203 (1982), VCYang, et al., J. Biol. Chem. 260 , 1849-1857 (1985) There were disadvantages such as a large amount of heparin added to protect the active site with heparin and a decrease in the activity of immobilized heparin degrading enzyme.

This drawback of time, addition of heparin, and reduction of heparin degrading enzyme activity was solved by immobilizing a heparin degrading enzyme comprising histidine oligopeptide to the chelating support. In addition, heparin and heparin degrading enzymes are reacted together in a solution state, and the difficulty of removing heparin degrading enzymes when separating low molecular weight heparin is a chelating support using a heparin degrading enzyme including histidine oligo fabtide. By removing the heparin degrading enzyme containing histidine oligopeptide, thereby facilitating the separation of low molecular weight heparin.

It is an object of the present invention to provide a recombinant vector comprising a gene of a recombinant heparin degrading enzyme comprising a histidine oligopeptide and a histidine oligopeptide prepared using the same.

In addition, the present invention uses a heparin degrading enzyme to enzymatically decompose heparin to prepare a low molecular weight heparin, and then use a heparin degrading enzyme including histidine oligopeptide to terminate the reaction in solution, and then to decompose heparin containing histidine oligopeptide. Killing heparin degrading enzymes, including histidine oligopeptides, which facilitates the removal of enzymes and, in terms of time and efficiency, is superior to existing methods of immobilizing heparin degrading enzymes when immobilized heparin degrading enzymes are used. The purpose of the present invention is to prepare low molecular weight heparin by enzymatically decomposing heparin by immobilization on a rating support.

[Configuration of Invention]

The present inventors have studied a method for eliminating heparin degrading enzyme after the enzyme reaction of heparin and heparin degrading enzyme in the above solution state, and a study for overcoming the disadvantages of immobilization of heparin degrading enzyme when immobilizing heparin degrading enzyme for use in heparin decomposition. As a result, a vector having a heparin degrading enzyme gene comprising a histidine oligopeptide was prepared, and a heparin degrading enzyme comprising a histidine oligopeptide was prepared using E. coli transformed with the vector, and the prepared histidine oligopeptide was prepared. After heparin degrading enzyme was reacted with heparin in solution, heparin degrading enzyme including histidine oligopeptide was immobilized on the chelating support as a chelating support, and low molecular weight heparin was prepared by enzymatically decomposing heparin.

The present invention provides a vector (pE16b-hepa2) in which the gene of heparin degrading enzyme including histidine oligopeptide is inserted between the NcoI and BamHI recognition sites of pET16b.

The present invention also provides an E. coli transformant, wherein the vector is introduced into E. coli.

As for E. coli, BL21 (DE3) is preferable.

Escherichia coli BL21 (DE3) transformed with the pE16b-hepa2 vector was deposited with the KFCC-10903 Accession No. KFCC on July 3, 1996.

In addition, the present invention provides a method for producing a heparin degrading enzyme comprising a histidine oligopeptide using the above E. coli transformant.

And the present invention provides a method for immobilizing heparin degrading enzyme comprising histidine oligopeptide.

The present invention also provides a method for producing low molecular weight heparin by enzymatically digesting heparin using a heparin degrading enzyme comprising an immobilized histidine oligopeptide.

EXAMPLE

Hereinafter, preferred embodiments of the present invention are described. However, the following examples are only preferred examples to aid the understanding of the present invention, and the present invention is not limited to the following examples.

I. Materials and methods

1. Used strains and plasmids

The expression E. coli strain used in this study was BL21 (DE3), and the E. coli strain used for general gene manipulation and plasmid separation was DH5α (F ^_ lacZ M15 hsdR17 (r ^_ m ^_ ) gyrA36). In addition, pUC19 was used as a plasmid used for cloning the heparin degrading enzyme gene. In addition, pETlla and pET16b (No. 69436-1, 69662-1 manufactured by Novagen) were used as expression plasmids in E. coli.

2. Restriction Enzymes and Reagents

Restriction enzymes for DNA recombination, T4 DNA ligase, calf-intestinal alky phosphatase, ribonuclease and the like were purchased from NEB. Separation and purification of DNA from agarose gel was used by purchasing GENECLEAN II and MERMAID kits from Bio1O1.

3.Method

DNA recombination was prepared by Maniatis et al. (Sambrook, J. et al., Molecular Cloning A Laboratory Manul, 2nd ed (1989)), and E. coli transformation was carried out by Inoue et al. (Inoue, H. et a1. , Gene, 96, 23-28 (1990)).

In addition, the activity of heparin degrading enzyme was measured using UV 232nm method and Azur A method. In the method using UN 232nm, the temperature was fixed by connecting a constant temperature bath fixed at 25 ° C. to a Jasco V-550 spectrophotometer and the degree of change in absorbance with time was measured using UV 232 nm. Heparin was prepared in 50 nM sodium phosphate (pH 7.1) and 100 mM NaCl at a concentration of 12.5 mg / m1, and 1 ml of the enzyme solution was added to investigate the change in absorbance (H. Bernstein et al., Methods Enzym, 137, 515-529 (1988). In addition, the method using Azur A is reacted with heparin and heparin degrading enzyme, and 5 µl of the reaction solution is added to 5 ml of 0.02 mg / ml Azur A staining solution and suspended, and the absorbance is measured at 620 nm to measure 0-8 mg heparin / ml. Standard curves were made and compared to determine the amount of degradation of heparin. The activity of heparin degrading enzyme was 1 unit of the amount of heparin degrading enzyme capable of degrading 1 mg of heparin per hour (P.M. Galliner et a1., Appl. Environ. Microbiol., 41, 360-365 (1981)).

The biological activity of anti-FXa was measured by COATEST from Chromogenix using the NIBSC (National Institute for Biological Standards and Contro1s, London) international low molecular weight heparin standard. Measured with a low molecular weight heparin kit.

III. Cloning of Heparin Degrading Enzyme Gene from F. Heparinum

Gene bank database approach No. nucleotide sequence of the identified heparin degrading enzyme 5 ′ nucleotide sequence of heparin degrading enzyme (SEQ ID NO: 173) based on the DNA sequence of L12534 (SEQ ID NO: 1 of the Sequence Listing), their coding sequence (SEQ ID NO: 2), and ananoic acid sequence (SEQ ID NO: 3) Oligomer (oligomer 1) complementary to the base of NdeI and the addition of the restriction enzyme site of NdeI (oligomer 1) and oligomer (an oligomer added to the restriction enzyme site of BanlHI complementary to base sequence of 3 ′ (near base 1327 of SEQ ID NO: 1)). 2) was synthesized.

Oligomer 1: 5´-GACATATGAAAAAACAAATTCTA-3´

Oligomer 2: 5´-AGGATCCTTACTATCTGGCAGTTTC-3´

F. Heparinum was incubated with nutrient broth at 30 ° C. for 48 hours, and the culture solution was centrifuged to suspend the precipitated cells with mid-water, followed by PCR (polymerase chain reaction) reaction with 2 µl of the cell suspension and 10 µl of oligomer. PCR was carried out as a mixture, and the PCR product was obtained by electrophoresis on 0.8%, agarose gel, which was treated with NdeI and BamHI and pUC19 was treated with NdeI and BamHI, thereby connecting pU-hepa2 (first). Degree).

III. Heparin degrading enzyme expression vectors pE-llepa2 and histidine oligopeptides

Preparation of a Heparin Degrading Enzyme Expression Vector pE16b-hepa2 Containing

Heparin degrading enzyme gene was isolated from the prepared pU-hepa2 and put into the NdeI and BamHI restriction sites of pETlla and pET16b (No.69436-1, 69662-1 manufactured by Novagen) to include the recombinant heparin degrading enzyme and histidine oligopeptide. PE-hepa2 and pE16b-hepa2, which are expression vectors of recombinant heparin degrading enzymes, were prepared (FIG. 1). In the expression vector pE-Hepa2, the gene of heparin degrading enzyme is located under the control of T7 promoter and T7 transcription terminator, and in the expression vector pE16b-hepa2, the recombinant heparin degrading enzyme comprising histidine oligopeptide under the control of T7 promoter and T7 transcription terminator Is located and the histidine oligopeptide (10 histidine residues) is attached to the amino terminus of the recombinant heparin degrading enzyme.

Ⅳ. Heparin Degradation Including Heparin Degrading Enzyme and Histidine Oligopeptides

Expression of enzyme

The expression of heparin degrading enzyme was confirmed by transforming BL21 (DE3) into p21-DEpa, a recombinant heparin degrading enzyme expression vector including pE-hepa2, which is a recombinant heparin secretory enzyme derivation vector, and histidine oligopeptide. Cells with each vector were induced with IPTG (isopropyl-β-D-thiogalactoside), followed by centrifugation of the culture to precipitate the cells and Laemmli method (Laemmli, UK Nature, 227, 680-685 (1970)). According to the present invention, 12% SDS-PAGE (polyacrylamide gel electrophoresis) confirmed its expression (FIG. 2). As shown in FIG. 2, all strains having pE-hepa2 and DE16b-hepa2 expressed heparin degrading enzyme (lane 1) and heparin degrading enzyme (lane 2) including histidine oligopeptide. Lane 3 is the size marker.

Escherichia coli BL21 (DE3) transformed with the pE16b-hepa2 vector was deposited with the KFCC-10903 Accession No. KFCC on July 3, 1996.

V. Recombinant Heparin Degrading Enzyme and Dog Combination Histidine Oligopeptides

Activity of Heparin Degrading Enzyme

Strains for preparing heparin degrading enzymes containing heparin degrading enzyme and histidine oligopeptide, respectively, were incubated in 1000 ml flasks with 300 ml of unoptimized medium (Luria broth) by IPTG at 30 ° C. for 3 hours to incubate 2.4L. The culture was centrifuged and the precipitated cells were centrifuged with an ultrasonic crusher. The precipitate was rebounded with Liton-X-100 solution and rebound buffer to examine its activity. As a result of investigating the activity, the recombinant heparin degrading enzyme had about 208,000U / L, and the recombinant heparin degrading enzyme including histidine oligopeptide had about 160,000U / L. These results showed better production than 100,00 OU / L filed from F. heparanum under optimum conditions of USP 4,443,545.

Ⅵ. PH Changes of Heparin Degrading Enzyme Containing Histidine Oligopeptides

Activity change with temperature change

In order to investigate the change of activity according to the pH change of the recombinant heparin degrading enzyme containing histidine oligopeptide, the recombination of the recombinant heparin degrading enzyme containing histidine oligopeptide expressed as an inclusion body was investigated. Recombinant heparin degrading enzyme containing histidine oligopeptide was added to citrate-phosphate buffer at pH 4,5,6,7 and Tris-HC1 buffer at pH 8,9, respectively, and UV 232nm at 25 ° C. for 3 minutes. The change in absorbance was examined and its relative activity is shown in FIG. 3A. As shown in FIG. 3A, the highest activity was shown at pH6.5 and the preferred activity was at pH 5-8. This result is similar to that of Heparin degrading enzyme isolated from F. heparinum of P. Hovingh et al. (P. Hovingh, et al., J. Biol. Chem. 245, 6170-6175 (1970)). C. Yang et al. (VC Yang, et al., J. Biol. Chem. 260, 1849-1857 (1985)) showed similar results. On the other hand, the activity of recombinant heparin degrading enzymes at the same pH of citrate-phosphate buffer, Tris buffer and sodium phosphate buffer showed the highest activity in sodium phosphate buffer.

In order to investigate the change of activity according to the temperature change of the recombinant heparin degrading enzyme including histidine oligopeptide at 20 ° C., 22 ° C., 25 ° C., 28 ° C., 30 ° C., and 35 ° C., respectively, in citrate-phosphate buffer (pH 6.5). The temperature was set in a Jasco UV / VIS spectrophotometer and the activity was investigated by UV 232 nm method at each temperature and shown in FIG. 3B. As shown in FIG. 3B, the activity gradually increased from 20 ° C to the highest activity at 25 ° C, and rapidly decreased from 28 ° C. This result showed a difference in the report of P. Hovingh et al. (P. Hovingh, et al., J. Biol, Chem. 245 6170-6175 (1970)) showing the best activity between 28 and 30 ° C. ·

To determine the effect of pH on the stability of recombinant heparin degrading enzymes containing histidine oligopeptides, pH 5.0, 6.0, 6.5, 7.0 and pH 8.0 in Tris-HC1 buffers were adjusted to pH 8.0 and Recombinant heparin degrading enzyme containing histidine oligopeptide was added to each buffer according to the composition, mixed and left at 25 ° C. for 3 hours, and then placed on ice and irradiated with UV 232 nm in a predetermined amount. As shown in Figure 3C showed the highest activity at pH 6.0. Victor C. Yang et al. (VCYang, et a1, J. Biol. Chem. 260, 1849-1857 (1985)) reported that heparin degrading enzymes of F. heparinum exhibited the highest stability at pH 7.0. . On the other hand, the optimal temperature and pH stability of heparin degrading enzyme expressed cytoplasmically from E. coli transformed with pE-hepa2 showed almost the same results as heparin degrading enzyme including histidine oligopeptide.

Iii. Immobilization of Expressed Recombinant Heparin Degrading Enzyme

The recombinant heparin degrading enzyme expressed in E. coli was modulated by cation ion exchange chromatography and bound to the support. CNBr-activated Sepharose 4B lOg was washed with 1 mM HCI for about 15 minutes and washed with 0.1M NaHCO ₃ , 0.5M NaC1, pH8.3. In addition, 50 mg (137.44 U / mg) of the adjusted recombinant heparin degrading enzyme was added to 10 m1 of the support, and 40 mg of heparin per 1 mg of protein was added together and reacted while suspended at 4 ° C. for 20 hours. The buffer composition of heparin degrading enzyme was 20mM sodium phosphate, 100mM NaC1, pH7.0. The recombinant heparin degrading enzyme bound to the support was then washed with 0.1 M NaHCO ₃ , 0.5 M NaCl, pH 8.3. Blocking the residues of the support remaining 12 hours at 4 ° C. with 0.2 M lysine. And then washed with 0.25M sodium phosphate 100mM NaC1, pH7.0 and again with 25M sodium phosphate, 0.5M NaC1, pH7.0 and used as an immobilized heparin degrading enzyme (H. Bernstein et al. , Methods Enzym, 137, 515-529 (1988). The amount of protein bound to CNBr-active Sepharose 4B was shown as a 4.6 mg / ml support, with about 92% of the protein bound to the support.

In addition, the activity of the immobilized recombinant heparin degrading enzyme was compared with the heparin degrading enzyme before its immobilization by a modified 232 nm assay (H. Bernstein et al., Methods Enzym, 137, 515-52991988). It was. Immobilized heparin degrading enzyme reacted with 12.5 mg / ml of heparin in the same amount of recombinant heparin degrading enzyme at room temperature and reacted at 0, 5, 10, 15, 20, 25, When the reaction time reached 30 minutes, 40 minutes, 50 minutes, 60 minutes, and 90 minutes, 75 μl of the reaction solution was collected and mixed with 1.5 ml of 0.15N HC1 to stop the reaction. The absorbance was measured at 232 nm. Absorbance change according to the comparison was investigated (Fig. 4). As shown in Figure 4 immobilized heparin degrading enzyme ( Activity of heparin-degrading enzyme ( ) Showed about 25-35% activity.

Iii. Of recombinant heparin degrading enzyme comprising expressed histidine oligopeptide

Immobilization

Heparin degrading enzyme containing histidine oligopeptide was expressed as an inclusion body in Escherichia coli and returned back to use as a recombinant heparin degrading enzyme containing histidine oligopeptide. Heparin degrading enzyme containing a hestidine oligo peptide and heparin were reacted in solution, and after the reaction, heparin degrading enzyme containing histidine oligo peptide was removed with a chelating support. In addition, chelating nickel sulfate, copper sulfate, and zinc acetate were respectively chelated to chelating sepharose, and heparin degrading enzyme was immobilized to compare the activity of heparin degrading enzyme including immobilized histidine oligopeptide, respectively. Ratings were the most comparatively active.

Chelating Sepharose in chelating Sepharose was chelated with 25 mM zinc acetate and chelated Sepharose chelated with 10 ml (1.02 mg / ml) histidine oligopeptide and heparin degrading enzyme (106U / mg) and 2 ml zinc ions at 4 ° C. The heparin degrading enzyme activity, including the immobilized histidine oligopeptide, was washed with 50 mM sodium phosphate, 10 mM NaClm pH 7.0, and about 85% of the protein bound to the support.

The activity of recombinant heparin degrading enzymes, including immobilized histidine oligopeptides, was analyzed by a modified 232 nm assay (H. Bernstein et al., Mothods Enzym, 137, 515-529 (1988)). Heparin degrading enzyme and comparative activity were included. Recombinant heparin degrading enzyme comprising histidine oligopeptide in 10 ml of 12.5 mg / ml heparin ( Heparin degrading enzyme comprising immobilized histidine oligopeptide reacted with the same amount of enzyme as ) Were reacted while suspended at room temperature, and the reaction time was 0, 5, 10, 15, 20, 25, 30, 40, 50, 60 and 90 minutes. The solution was collected and mixed with 1.5 ml of 0.03N HC1 to stop the reaction. The absorbance was measured at 232 nm, and the change in absorbance with time was compared and investigated (FIG. 5).

Heparin degrading enzyme comprising immobilized histidine oligopeptides as shown in FIG. Activity of heparin degrading enzymes (including histidine oligopeptides before immobilization) Compared to the activity of)) showed about 60-80% activity. In the above experimental results, the method of immobilizing the chelating support using histidine oligopeptides has advantages in many ways over the method of immobilizing on a support such as CNBr-active Sepharose 4B (Table 1).

In order to prepare a low molecular weight heparin with the above-mentioned advantages, the heparin degrading enzyme 5000U containing histidine oligopeptide was immobilized in the above manner with a chelating support, and heparin degrading enzyme buffer (50 mM sodium phosphate, 10 mM) was prepared. NaCl, pH6.5) was added to 100 g of heparin dissolved at 50 mg / m1 and reacted for 3 hours while stirring at room temperature. Then, heparin degrading enzyme including histidine oligopeptide bound to the chelating support was precipitated by centrifugation, and the supernatant was precipitated with ethanol to prepare low molecular weight heparin. In this case, the average molecular weight of the prepared low molecular weight heparin was 5600 when the average molecular weight was measured using GPC-HPLC as a reference standard of NIBSC. The anti-FXa activity of the prepared low molecular weight heparin was 69 IU / mg.

TABLE 1

Comparison between immobilization of recombinant heparin degrading enzyme to CNBr-active sepharose and immobilization of recombinant heparin degrading enzyme containing histidine oligopeptide to chelating support

[Effects of the Invention]

The heparin degrading enzyme of the present invention is a heparin degrading enzyme including a histidine oligopeptide, and the immobilization time is short (10-30 minutes), which is much shorter than the conventional immobilization time of about 2,000 minutes. In addition, heparin degrading enzyme immobilization of heparin degrading enzyme, or lysine residues near the active site, heparin must be added in large amounts to protect the active site with heparin, but in the present invention, heparin degrading enzyme has histidine oligopeptide There is no need to add heparin. The activity of immobilized heparin degrading enzymes, including histidine oligopeptides, is also relatively high. In addition, the immobilized heparin degrading enzyme including the histidine oligopeptide of the present invention can be easily separated from the substrate and can be stored and reused for a long time by stabilizing the heparin degrading enzyme including the histidine oligopeptide.

[Sequence list]

Claims (6)

Histidine oligopeptides prepared using E. coli transformed with an expression vector comprising a structural gene of heparin degrading enzyme (nucleotides 173 to 1327 of SEQ ID NO: 1) comprising a nucleotide encoding a histidine oligopeptide at the 5 'end. A method of producing low molecular weight heparin by reacting a heparin degrading enzyme containing heparin. The method of claim 1, wherein the E. coli is BL21 (DE3) (KFCC-10903) transformed with the pE16b-hepa2 vector. The method of claim 1, wherein the heparin degrading enzyme comprising a histidine oligopeptide immobilized on a chelating support is reacted with heparin. The method of claim 1, wherein the heparin degrading enzyme including histidine oligopeptide is reacted with heparin and then immobilized on a chelating support to prepare a low molecular weight heparin. The method of claim 3 or 4 wherein the low molecular weight heparin is prepared under conditions wherein the pH is between 5.0 and 8.0 and the temperature is between 20 ° C. and 28 ° C. 6. The method for producing low molecular weight heparin according to claim 3 or 4, wherein the heparin degrading enzyme including histidine oligopeptide is immobilized on the chelating support using zinc, copper or nickel ions.