CN113186215B

CN113186215B - High-activity and high-heat-stability heparinase I derived from bacteroides and application thereof

Info

Publication number: CN113186215B
Application number: CN202110388232.9A
Authority: CN
Inventors: 田丰伟; 于雷雷; 张川; 翟齐啸; 王晨; 肖越; 赵建新; 张灏; 陈卫
Original assignee: Jiangnan University
Current assignee: Jiangnan University
Priority date: 2021-04-10
Filing date: 2021-04-10
Publication date: 2022-09-27
Anticipated expiration: 2041-04-10
Also published as: CN113186215A

Abstract

The invention discloses a high-activity and high-heat-stability heparinase I derived from bacteroides and application thereof, belonging to the technical field of microorganisms. The invention provides heparinase I BnHepI derived from Bacteroides nordii nordidi; the heparinase I BfHepI derived from Bacteroides finegoldii has good thermal stability, wherein the optimum reaction pH of the BnHepI is 8, and the optimum reaction temperature is 40 ℃; the enzyme has higher enzyme activity, and the highest enzyme activity under the optimal condition can reach 360.45 IU/mg. Meanwhile, the heparinase has good thermal stability, the half-life period at 50 ℃ is 10.6min, and the half-life period at 40 ℃ is 244 min. The recombinant enzyme has great industrial application potential.

Description

High-activity and high-heat-stability heparinase I derived from bacteroides and application thereof

Technical Field

The invention relates to a high-activity and high-heat-stability heparinase I derived from bacteroides and application thereof, belonging to the field of genetic engineering and enzyme engineering.

Background

Heparin (Heprain) is a specific polydisperse mixed sulfated polysaccharide widely distributed in mammalian tissues, covalently bonded to proteins. At present, commercial heparin is mainly extracted from mucous membranes of small intestines of cattle and pigs, has a complex structure and multiple important biological functions, and is generally used for treating diseases such as thrombus, cardiovascular diseases and the like in clinic. Low Molecular Weight Heparin (LMWH) is a short piece of heparin produced by the cleavage of heparin by some physicochemical methods, and has a reduced ability to bind to proteins or cells, but a significantly increased anticoagulant activity. Compared with normal heparin, the low molecular heparin can reduce the activity of anti-factor IIa, and greatly reduces the risk of bleeding. The low molecular weight heparin is prepared by physical, chemical, biological and synthetic methods at present. Among them, the biological enzymolysis method is a new method because of its advantages such as mild conditions, strong selectivity and little pollution.

Heparinase I (GenBank: AAO79780.1) is a polysaccharide lyase capable of cracking heparin structural substances and preparing low molecular heparin, has wide sources, mainly exists in prokaryotic Flavobacterium heparinum, and also comprises some bacteroides, bacillus and the like. Heparinase I was first discovered in Flavobacterium heparinum, selectively cleaving the alpha (1-4) glycosidic bond between glucosamine and uronic acid in sulfated heparosan. Heparanase I is classified into 13 families of glycoside hydrolases PLs based on its amino acid sequence from various sources and on its protein structural characteristics. Currently, heparinase I is mainly applied to the preparation of low molecular heparin, the elimination of heparin in extracorporeal circulation, the analysis of the exact structure of heparin, and the application of heparin in coagulation tests and platelet tests in the aspect of in vitro diagnostic reagents.

Different heparinase microorganisms obtained from nature have various heparinases, products obtained by enzymolysis of heparin are different, and currently, heparinase I, heparinase II and heparinase III from flavobacterium heparinum are the most widely researched and applied, and are monomer proteins with molecular weights of about 43 kDa, 78 kDa and 66kDa respectively, and the isoelectric point of the monomer proteins is about 9.0. The discovery of heparinase plays an important driving role for the structural research and quality detection of heparin, wherein enzymes I, II and III produced from Flavobacterium heparinum are already used for heparin quality detection and low-molecular heparin production. With the research on low molecular heparin, the research is continuously carried out. The low molecular heparin has wide effects on diseases such as tumors, inflammations, obstetrical pregnancy and the like besides anticoagulant activity, and has wide prospects in development of new drugs aiming at different pharmacological activities of the low molecular heparin.

However, heparin has a highly complex structure, has few active sites with a certain pharmacological effect in the whole mixture, and is easily interfered by other similar structures, thereby interfering with drug development. The enzyme cutting site of heparinase to heparin is relatively specific, so that a large number of fragments with the same structure can be separated, and drug development is facilitated.

Most studied is heparinase I, but currently, heparinase has poor thermal stability, so that the heparinase cannot be well adapted to industrial application. This has been a bottleneck that hampers the enzymatic preparation of low molecular weight heparin. Therefore, the search for a novel heparinase with good thermal stability is of great significance.

Although the invention patent of chinese publication No. CN109666666B discloses a heparinase I, it is derived from Bacteroides thetaiotaomicron (Bacteroides thetaiotaomicron), and the half-life of the original enzyme is only 6min, although the enzyme flexible analysis based on molecular dynamics of the invention provides a mutant for improving the thermostability of heparinase I, the construction of the mutant enzyme requires the use of a computer for molecular dynamics simulation and virtual mutation screening, and the required threshold is high, and it is difficult to realize wide popularization. Therefore, the search for the original enzyme heparinase I with good heat stability still has important significance.

Disclosure of Invention

The technical problem is as follows:

the technical problem to be solved by the invention is as follows: providing a heparinase I with high thermal stability and application thereof, updating strain source information of a heparinase I family, mining and screening the heparinase I with the thermal stability, and solving the bottleneck of poor thermal stability of the heparinase I in the current industry; simultaneously provides a recombinant bacterium capable of producing the heparinase I and a method for producing low molecular heparin.

Technical scheme

In order to solve the technical problems, the invention provides a gene for coding heparinase I, wherein the nucleotide sequence of the heparinase I is shown as SEQ ID NO.1 or SEQ ID NO. 2.

In one embodiment of the invention, the heparinase I disclosed by the invention has better thermal stability, has important significance for prolonging the shelf life of the heparinase I, improving the operation stability and recycling batches of the heparinase I in a catalytic process, reducing the production cost and the like, and improves the industrial application value of the heparinase I.

In one embodiment of the invention, heparinase I with the gene sequence shown in SEQ ID NO.1 or SEQ ID NO.2 is named BnHepI and BfHepI respectively.

In one embodiment of the invention, the BnHepI is derived from Bacteroides nordii FTJS11K 9; the BfHepI is derived from Bacteroides finetgoldii Bacteroides FNMHLBE3K 7.

In one embodiment of the invention, the amino acid sequence of the heparinase BnHepI is shown in SEQ ID NO.5, and the amino acid sequence of the heparinase BfHepI is shown in SEQ ID NO. 6.

In one embodiment of the invention, the heparinase I is BfHepI and BnHepI respectively, and the molecular weights are 42.3KDa and 41.8KDa respectively.

The invention also provides a recombinant vector carrying the gene of the heparinase I.

The invention also provides a recombinant cell carrying the gene of the heparinase I or the recombinant vector.

In one embodiment of the invention, the recombinant cell is a fungus or a bacterium.

The invention also provides a recombinant escherichia coli, which expresses the gene for coding the heparinase I.

In one embodiment of the invention, pET-SUMO or pUC19 is used as an expression vector.

In one embodiment, the recombinant escherichia coli genetically engineered bacterium is escherichia coli BL21 or BL21 Rosetta as an expression host, and preferably, the host is escherichia coli BL 21.

The invention also provides a method for constructing the recombinant escherichia coli, which comprises the following steps:

(1) the chemical synthesis nucleotide sequence is shown as SEQ ID NO.1 or SEQ ID NO.2, and heparinase BnHepI or heparinase BfHepI.

(2) Connecting the amplified heparinase I gene to an expression vector by taking pET-SUMO as the expression vector, constructing recombinant expression vectors pET-SUMO-BfHepI and pET-SUMO-BnHepI, transforming the recombinant expression plasmids into escherichia coli BL21, screening positive transformants and carrying out sequencing verification.

The invention provides a production method of the heparinase I, which comprises the steps of adding the recombinant escherichia coli into a seed culture medium, and culturing the recombinant escherichia coli at 37 ℃ and 220rpm until the OD600 is 0.6 to obtain a seed solution; transferring the prepared seed solution into a fermentation culture medium according to the proportion of 1% (v/v), culturing at 28 ℃, adding isoproyl beta-D-1-thiogalactopyranoside (IPTG) with the final concentration of 0.6mM, and carrying out induced expression for 10-12h at the rotating speed of 200 rpm.

The invention provides a preparation method of low molecular heparin, which comprises the following steps: adding the heparinase, the recombinant cell or the recombinant Escherichia coli into a reaction system containing heparin for reaction.

In one embodiment of the present invention, the low molecular heparin is: heparin fragments with small molecular weight obtained after heparinase cleavage; the low molecular heparin is a general name of heparin with lower molecular weight prepared by depolymerizing common heparin, the average molecular weight is 4000-6000 KDa, and the molecular weight of the heparin is over 12000 KDa.

In one embodiment of the present invention, the reaction temperature is 30 to 40 ℃.

In one embodiment of the invention, the reaction system is 50mmol/L sodium acetate, 5mmol/L calcium acetate, 5mmol/L heparin sodium, pH 7.4.

The invention also provides a heparin cracking method, which is characterized by comprising the following steps: adding the heparinase, the recombinant cell or the recombinant Escherichia coli into a reaction system containing heparin for reaction.

The invention also provides the low molecular heparin prepared by the method.

The invention also provides the application of the heparinase I, the recombinant cell or the recombinant escherichia coli in preparing a product containing low molecular heparin.

Advantageous effects

1. The invention provides a heparinase I BnHepI derived from Bacteroides nordii Nordii Bacteroides FTJS11K 9; the heparinase I BfHepI derived from Bacteroides finetgoldii Bacteroides FNMHLBE3K7 has good thermal stability, and the heparinase with good thermal stability has important significance for prolonging the shelf period of the heparinase I, improving the operation stability and recycling batches of the heparinase I in a catalytic process, reducing the production cost and the like and improving the industrial application value of the heparinase; meanwhile, the heparinase I BnHepI and the heparinase I BfHepI have higher enzyme activity, and the enzyme activity is 360.45IU/mg and 303.84IU/mg respectively; the half-lives at 50 ℃ were 10.6min and 7.9min, respectively. The half-life period at 40 ℃ is 244min and 184min respectively, and the enzyme has great application potential in industry.

2. The method of the invention utilizes bioinformatics technical means to deeply excavate the CAZymes genes of the human intestinal microorganisms, and can quickly construct a gene library of related enzymes. On the basis, a protein library can be obtained by a genetic engineering technology. Based on the method, the novel heparinase I with excellent performance can be quickly obtained, and meanwhile, a feasible idea is provided for the excavation of other novel enzymes.

3. The invention provides the heparinase I with good thermal stability, and for the production of low molecular heparin, the heparinase with the thermal stability can properly improve the reaction temperature and shorten the whole production period; in addition, the price of the heparinase is quite high, the duration of the thermostable heparinase is longer in the whole production process, the amount of the enzyme used is reduced, and the production cost is saved.

Drawings

FIG. 1: SDS-PAGE picture of the ultrasonic lysis supernatant containing four heparinase I in the invention; wherein, the molecular weight of M and protein is standard, and the size of the band from top to bottom is 170KD, 130KD, 100KD, 70KD, 55KD, 40KD, 35KD, 25KD and 15 KD; lane1, 3, 5, 7: ultrasonic lysis supernatants of BcHepI, BeHepI, BfHepI and BnHepI respectively; lane2, 4, 6, 8: the ultrasonic cleavage precipitates of BcHepI, BeHepI, BfHepI and BnHepI respectively.

FIG. 2: SDS-PAGE of the pure enzyme solutions containing the four heparanases I in the present invention; wherein, the molecular weight of the M protein is standard, and the size of the band from top to bottom is 170KD, 130KD, 100KD, 70KD, 55KD, 40KD, 35KD, 25KD and 15 KD; lane1, 3, 5, 7: ultrasonic lysis supernatants of BcHepI, BeHepI, BfHepI and BnHepI respectively; lane2, 4, 6, 8: respectively, the bands after BcHepI, BeHepI, BfHepI and BnHepI purification.

FIG. 3: the four heparinase I BcHepI, BeHepI, BfHepI and BnHepI have the optimum temperature of the enzymological properties.

FIG. 4: the four heparinase I BcHepI, BeHepI, BfHepI and BnHepI in the invention have the optimum pH value of the enzymology.

FIG. 5: the influence of the substrate concentration on the enzymatic reaction rates of the four heparinases I BcHepI, BeHepI, BfHepI and BnHepI in the invention is disclosed.

FIG. 6: the thermal stability curves of the four heparinase I BcHepI, BeHepI, BfHepI and BnHepI at 40 ℃ and 50 ℃ are shown.

Detailed Description

The invention is further illustrated below with reference to specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

The mutant primers, DNA polymerase, DpnI enzyme, DNA and protein marker and the like, which are referred to in the following examples, were purchased from Thermo. Sequencing was done by Jinzhi, Suzhou. Plasmid extraction, PCR product gel cutting recovery kit and the like were purchased from holo-type gold. Co columns for purification are available from GE.

The media involved in the following examples are as follows:

LB liquid medium: 10g/L of NaCl, 5g/L of yeast extract and 10g/L of peptone.

Fermentation medium: NaCl 10g/L, yeast extract 5g/L, peptone 10g/L, and glucose 5 g/L.

The detection methods referred to in the following examples are as follows:

the method for measuring the enzyme activity of the heparinase I comprises the following steps:

the enzyme activity determination adopts a 232nm light absorption method, heparin sodium is used as a substrate to determine the activity of heparinase I, and the reaction system is as follows: adding 100 mu L of substrate buffer solution into a 1.5mL centrifuge tube, wherein the substrate buffer solution is a mixture of 50mmol/L sodium acetate, 5mmol/L calcium acetate and 5mmol/L heparin sodium, incubating for 10min at a constant temperature of 40 ℃ in a metal bath, adding 10 mu L of diluted, purified and desalted enzyme solution with the concentration of 25ug/mL, reacting for 10min, and immediately adding 1mL of 0.06mol/L hydrochloric acid to terminate the reaction; centrifuging at 12000r/min for 5min, collecting supernatant, and measuring absorbance at 232 nm.

One unit of enzyme activity refers to the efficiency of the reaction producing 1. mu. mol of. DELTA.4, 5 unsaturated uronic acid at 30 ℃ within 1 min.

Example 1: obtaining of target Gene heparinase I

Total DNA was extracted from Bacteroides clarus (Bacteroides clepialus FFJLY22K22), Bacteroides eggerthii (Bacteroides FSDTAHCKB9), Bacteroides fintgoldii (Bacteroides subtotalis FNMHLBE3K7), and Bacteroides nordii FTJS11K9) cultured for 36 hours according to the method provided in the molecular cloning Instructions, and sent for whole genome sequencing and gene function annotation. The CAZymes genes of Bacteroides were analyzed by the techniques of student science, and the heparinase I genes BcHepI (shown in the nucleotide sequence SEQ ID NO. 3), BeHepI (shown in the nucleotide sequence SEQ ID NO. 4), BfHepI (shown in the nucleotide sequence SEQ ID NO. 2) and BnhepI (shown in the nucleotide sequence SEQ ID NO. 1) in Bacteroides clarkii (Bacteroides clepialus Cruis FFJLY22K22), Bacteroides eggerthii (Bacteroides Ex. FSDTAHCKB9), Bacteroides fintgoldii (shown in the nucleotide sequence FNMHLBE3K7) and Bacteroides nordidii (shown in the nucleotide sequence FTJS11K9) were determined according to the classification of the CAZy family.

And respectively carrying out total synthesis on nucleotide sequences of BcHepI, BeHepI, BfHepI and BnhepI to construct a pUC-19 vector, and successfully constructing sequencing recombinant sequencing plasmids pUC-19-BcHepI, pUC-19-BeHepI, pUC-19-BfHepI and pUC-19-BnhepI. The constructed plasmid is used as a template to carry out polymerase chain reaction to obtain gene sequences of target genes BcHepI, BeHepI, BfHepI and BnhepI, and primers, components and amplification conditions used in the reaction are shown in a table 1:

table 1: primer sequences

TABLE 2 PCR reaction System

Water was added to 25 μ L. The reaction system can be correspondingly enlarged as required.

Reaction conditions are as follows: pre-denaturation at 95 ℃ for 3 min; annealing at 98 deg.C for 10s, annealing at 58 deg.C for 15s, denaturing at 72 deg.C for 2min, extending, and performing 30 cycles from the second step to the fourth step; 72 ℃ for 5 min; 12 ℃ is used. The target DNA fragment was purified using a gel purification kit from omega to obtain the target gene DNA with pET-SUMO homology arms.

Example 2: construction of recombinant Escherichia coli

The method comprises the following specific steps:

(1) the plasmid pET-SUMO and the target gene prepared in example 1 were digested with Xho I and BamH I, and the digested fragments were recovered. The ligation product was prepared by homologous recombination ligation using the kit Clonexpress II One Step Cloning kit from Vazyme, and the target gene DNA with the homology arm of pET-SUMO and the double-digested pET-SUMO plasmid.

(2) And transforming the prepared ligation product into escherichia coli DH5 alpha, screening positive clones by plasmid extraction and PCR, and performing sequencing identification. The constructed Escherichia coli expression plasmids containing the heparinase I genes are named as pET-SUMO-BcHepI, pET-SUMO-BeHepI, pET-SUMO-BfHepI and pET-SUMO-BnHepI.

(3) Respectively transforming the recombinant plasmids prepared in the step (2) into escherichia coli BL21(DE3) to prepare recombinant escherichia coli: BL21(DE3) -BcHepI, BL21(DE3) -BeHepI, BL21(DE3) -BfHepI, BL21(DE3) -BnHepI.

Example 3: expression, purification and Activity assay of heparinase I

The method comprises the following specific steps:

1. preparation of crude enzyme solution of heparinase I

The recombinant E.coli prepared in example 2 was inoculated into 5mL LB medium containing kanamycin resistance (containing 50. mu.g/L kanamycin) and cultured at 37 ℃ and 220r/min for 12 hours, respectively, to prepare seed solutions.

Respectively inoculating the prepared seed liquid into 50mL of fermentation medium (250mL of shake flask) according to the inoculation amount of 1% (v/v), culturing at 37 ℃ at 220r/min until the OD600 is 0.6-0.8, adding IPTG with the final concentration of 0.6mM, and inducing at 28 ℃ at 200r/min for 10-12h to respectively prepare fermentation liquid.

The prepared fermentation liquor is respectively centrifuged at 8000r/min at 4 ℃ for 10min to collect thalli, washed twice by buffer solution (20mmol/L Tris-HCl 200mmol/L NaCl pH 7.4), and then suspended in 40mL buffer solution to carry out cell ultrasonic disruption. Centrifuging at 12000r/min and 4 deg.C for 20min, collecting supernatant, and performing SDS-PAGE analysis. The electrophoresis results are shown in FIG. 1. The results show that: the secretion expression of the four enzymes reaches about 90 percent.

2. Preparation of heparinase I pure enzyme solution

(1) Purifying by Co-NTA affinity chromatography, balancing a Co column by using 10mL of an equilibrium buffer solution (20mmol/L Tris-HCl, 300mmol/L NaCl pH 7.4), loading, washing by using 10mL of a binding buffer solution (20mmol/L Tris-HCl, 300mmol/L NaCl, 5mmol/L imidazole) to remove non-specific binding protein, eluting the recombinant target protein by using 3mL of an elution buffer solution (20mmol/L Tris-HCl, 300mmol/L NaCl, 150mmol/L imidazole), and collecting the eluent, namely the purified recombinase. The purified enzyme was desalted using a PD-10 pre-packed desalting column. After equilibration with 25mL of equilibration buffer (20mmol/L Tris-HCl, 200mmol/L NaCl pH 7.4), 2.5mL of the buffer was loaded, and 3.5mL of the buffer was eluted, and 2.5-6mL of the eluate from the loading was collected as a desalted enzyme solution.

By adopting the method, pure enzyme liquid containing BcHepI, pure enzyme liquid containing BeHepI, pure enzyme liquid containing BfHepI and pure enzyme liquid containing BnHepI are respectively prepared.

The purified enzyme was analyzed by SDS-PAGE, and the results are shown in FIG. 2, whereby gel purity was achieved.

The enzyme activity of the pure enzyme is respectively detected, and the result is as follows: the enzyme activities of the BcHepI pure enzyme, the BeHepI pure enzyme, the BfHepI pure enzyme and the BnHepI pure enzyme are 272.23IU/mg, 325.16IU/mg, 303.84IU/mg and 360.45IU/mg respectively.

Example 4: enzymatic Properties and thermal stability assay of heparinase I

1. Effect of temperature on heparinase I Activity

Temperature can change the catalytic reaction speed of the enzyme and can also lead to the activity reduction or inactivation of enzyme protein. The experiment will determine the optimal reaction temperature of heparinase I BcHepI, BeHepI, BfHepI and BnHepI.

The specific experiment is as follows:

mu.L of the pure enzyme solution prepared in example 3 was added to 100. mu.L of each reaction solution to obtain a reaction system, wherein the reaction solution was: 50mmol/L sodium acetate, 5mmol/L calcium acetate, 5mmol/L heparin sodium, pH 7.4;

respectively placing the reaction system at 25, 30, 35, 40, 45, 50, 55 and 60 ℃ for reaction; the reaction solution A was measured at regular intervals (10 min of reaction) ₂₃₂ And (3) taking the enzyme activity value measured at the optimal temperature as 100% under the condition of change, and calculating the relative enzyme activities at other temperatures. The results are shown in table 3 and fig. 3.

Table 3: specific activity of different enzymes at different temperatures

The experimental results show that the optimal reaction temperatures of BcHepI, BeHepI, BfHepI and BnHepI are 35 ℃, 30 ℃, 35 ℃ and 40 ℃ respectively.

2. Effect of pH on heparinase I Activity

The enzyme reactions have the optimal pH value range, the activity of the enzyme in catalytic reaction can Be influenced by overhigh or overlow pH value, and the optimal reaction pH values of heparinase I Bc-HepI, Be-HepI, Bf-HepI and Bn-HepI are measured in the experiment.

The specific experiment is as follows:

mu.L of the pure enzyme solution prepared in example 3 was added to 100. mu.L of each reaction solution to obtain a reaction system, wherein the reaction solution was: 50mmol/L sodium acetate, 5mmol/L calcium acetate and 5mmol/L heparin sodium; adjusting the pH value in the reaction system to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0 and 10.0, and reacting at 37 ℃; the reaction solution A was measured at regular time (reaction time 10min) ₂₃₂ And (3) taking the enzyme activity value measured under the optimal pH value as 100% under the condition of change, and calculating the relative enzyme activities under other pH values. The results are shown in table 4 and fig. 4.

Table 4: enzyme activity of different enzymes under different pH conditions

The experimental result shows that the optimal reaction pH values of Bc-HepI, Be-HepI, Bf-HepI and Bn-HepI are 7.0, 7.0, 7.0 and 8.0 respectively.

3、t _1/2 The value is the corresponding time when the residual enzyme activity is 50% after the enzyme is treated for a period of time at a specific temperature.

The specific determination method is as follows:

the activity of heparinase I which is not subjected to heat treatment is taken as 100%, the residual enzyme activity of pure enzyme liquid prepared in example 3 after being treated at 40 ℃ and 50 ℃ for different time is respectively measured and calculated, and the half-life period is calculated.

And ChBD-SUMO-Hep I enzyme, BcHepI enzyme (hereinafter referred to as BcHepI-2) disclosed in the literature as a control, the data in the literature are listed in Table 5 directly, the ChBD-SUMO-Hep I enzyme is disclosed in the "Expression and characterization of an enhanced recombinant heterologous enzyme I with binding domain" paper, and the BcHepI enzyme (hereinafter referred to as BcHepI-2) is disclosed in the "A high activity heterologous enzyme I from bacteria cells," the results are shown in Table 5:

TABLE 5 enzyme Activity at 30 ℃ and half-life data at 40 and 50 ℃ for the different enzymes

Where,/stands for no corresponding data in the published literature, it may be that the half-life cannot be detected under these conditions.

The treatment time was plotted on the abscissa and the% residual enzyme activity was plotted on the ordinate, and the results are shown in fig. 6. The experimental result shows that the BnHepI and the BfHepI have the best thermal stability.

4. Effect of substrate concentration on enzymatic reaction Rate

The substrate concentration significantly affects the efficiency of the enzymatic reaction, and the effect of the substrate concentration on the reaction rate is in rectangular hyperbolic relation under otherwise constant conditions. When the concentration of the substrate is low, the reaction speed is in direct proportion to the concentration of the substrate, and the reaction is a first-order reaction. As the substrate concentration increases, the reaction rate no longer accelerates proportionally, and the reaction is a mixed-stage reaction. When the substrate concentration reaches a certain degree, the reaction speed is not increased, and the maximum speed is reached, the reaction is zero-order reaction.

The specific experiment is as follows:

mu.L of the pure enzyme solution prepared in example 3 was added to 100. mu.L of each reaction solution to obtain a reaction system, wherein the reaction solution was: 50mmol/L sodium acetate, 5mmol/L calcium acetate, pH 7.4, wherein the concentration of heparin sodium is 0, 1, 2, 5, 10, 15, 20, 25mg/ml respectively; the reaction system is placed at 30 ℃ for reaction for 10min, and after the reaction is finished, the catalytic constant Km value and the maximum reaction rate Vmax value of the enzyme are respectively calculated through a double reciprocal curve, and the results are shown in Table 6 and figure 5.

Table 6: km value and Vmax of catalytic constants for different enzymes

Enzyme	Km(mM)	Vmax(mM/min)
			BcHepI	0.72±0.1	0.24±0.04
BeHepI	0.56±0.09	0.21±0.06
			BfHepI	0.42±0.1	0.22±0.06
BnHepI	0.25±0.07	0.24±0.05

The experimental result shows that the catalytic constants BcHepI > BeHepI > BfHepI > BnHepI, and Vmax have no obvious difference among the four enzymes. Thus indicating that the Bn-HpeI has the strongest binding capacity with the substrate.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

SEQUENCE LISTING

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<120> a highly active, thermostable heparinase I derived from bacteroides and uses thereof

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caaggaggct atcctccatt ggcattcgga ttctccggag gatggttcta tatcaaagca 720

aactctgacc gtagatggct gaccgacaaa gatgaccgtt gcaatgcaaa tgtagaaaaa 780

acacctgtca tgaaaccggt gacttctgaa tacaagtcat cgactatcgc ttacaaaatg 840

ccttttgccg atttcccgaa agattgttgg attacattcc gtatccatat tgattggact 900

gtctacggga aagaggctga gacaattgta aagccgggta tgcttgatgt acagatgaat 960

tatcaggata aaggtaaaaa ggttagtaaa caccttgtag acaatgagca gattctgatt 1020

ggacgtaacg acaaagatgg ctactatttt aaatttggta tttatagggt aggtaacagc 1080

accaaaccgg tgtgttacaa tttggcaggt tattcggaaa agtaa 1125

<210> 3

<211> 1125

<212> DNA

<213> Artificial sequence

<400> 3

actgcgcaaa ccaaaggctc tgaaacattg gttcccctga ccaaacgggt aaacgttcaa 60

gctgactcag cgcgtatcga tcaggttata gacggctgtt gggtagctgt cggcgcgaaa 120

aaggagcatg ccattcaacg ggattttaca tgtttgttca acggcaagcc gtcttatcgt 180

tttgagctgc gtgaagagga taatactttg gaagggtacg gaaagggaga gaccaaaggc 240

cgtgccgagt tctcttattg ttatgccacc tcggccgatt ttaacggatt gccggcggat 300

gcttaccgga aagcgcaaat caccaagacc gtatatcatc acggaaaagg tatttgtccg 360

caaggagcgt cacgcgacta tgagttctcc gtatatattc cgtctgcgct ggatagcaat 420

gtgtctacca tttttgccca atggcacggt atgcccgacc gtacgcttgt gcagactcct 480

gaaggtgaag tgaaaaagtt gacggttgac gagtttatag agctggataa gaccactatt 540

ttcaaaaaga acaccggaca tgaaaaagtg gcgagactgg ataagcaagg aaatccgatg 600

aaagataaaa agggaaatcc tatctataag gccggaaaga agaacggttg gttggtagag 660

caaggcggtt atccgccgtt ggctttcgga ttttccggcg gctggtttta tatcaaggcc 720

aattcggacc gccgttggct gaccgacaag actgaccgct gtaacgccaa tccggaaaaa 780

actccgataa tgaagcctgt gacttctgaa tacaagtcgt ctacaatagc ttataagatg 840

ccttttgccg atttccccaa agactgctgg gttactttcc gcattcatat cgactggact 900

acttatggca aggaagccga aaccattgtg aaaccgggca aactggacgt acaaatggag 960

tataccgaca agaagaaaac cgttaaagag catatcgtga ataatgaaga aattcagata 1020

ggccgtaatg acgatgacgg ttactatttt aaattcggca tctatcgtgt aggtaacagt 1080

acagtaccgg tatgctataa cttggccgga tataaggaag agtga 1125

<210> 4

<211> 1125

<212> DNA

<213> Artificial sequence

<400> 4

actgcacaag tcaagaacgc tgaaacactg gttcctttga ctaaacgggt aaacgttcaa 60

gctgatacag cacgtctcga tcagattata gatggctgtt gggtggctgt gggcacaaat 120

aagaaacatg caattcgacg ggatttcaca cgtttgtttg ccggcaaacc ttcttaccgc 180

ttcgaattgc gcaaagagga taacacgctg gaaggatacg gaaagggaga aaccaaaggg 240

cgtgccgagt tttcttattg ttacgccacc tcggccgatt ttaaggggtt gccggcagat 300

gcttatcgta aagcgcaaat cactaaaacc gtatatcatc atggcaaagg tatttgtccg 360

caaggagttt cacgcgatta tgagttttct gtgtatattc catctgcttt ggatagtaat 420

gtctccacga tttttgccca gtggcatggt atgcccgacc gcacgcttgt gcagactccc 480

gaaggtgaag taaagaaact gactgtcgat gagttcttgg aattggataa gaccactata 540

ttcaaaaaga atacagggca tgagaaggtt gcaaaactgg acaagcaggg aaatccgttg 600

aaggataaaa agggaaattc cgtatataag gccggaaaga agaacggctg gttggtagag 660

cagggtggtt atccgccgtt ggctttcggt ttctccggcg gttggttcta tatcaaggca 720

aattcggatc gccgttggct gacagacaaa acagaccgtt gcaacgcaag tcctgagaaa 780

acgccggtaa tgaaacccgt aacttccaag tacaagtcgt ctacaattgc ctataagatg 840

ccttttgccg atttccctaa agattgttgg gttacttttc gtgttcatat cgactggact 900

acatacggca aagaagctga aaacatagtg aagcccggta agctggatgt gcaaatggaa 960

tacaccgaca agaagaaaac cgttaaggag cacatcgtga ataatgaagt aatccagata 1020

gggcgtaatg atgatgacgg ttactatttt aagttcggca tatatcgtgt tggcaacagc 1080

acagtgccgg tatgctacaa cctggcaggg tacaaggaag agtga 1125

<210> 5

<211> 369

<212> PRT

<213> Artificial sequence

<400> 5

Gln Asn Ala Lys Leu Ile Pro Leu Thr Glu Arg Val Asn Val Gln Ala

1 5 10 15

Asp Ser Ala Arg Ile Asn Gln Val Ile Asp Gly Cys Trp Val Ala Val

20 25 30

Gly Thr Asp Lys Pro His Ser Ile Gln Arg Asp Tyr Thr Ile Pro Phe

35 40 45

Asn Gly Lys Pro Ser Tyr Arg Phe Glu Leu Lys Thr Glu Asp Asn Thr

50 55 60

Leu Glu Gly Tyr Ala Lys Gly Glu Thr Lys Gly Arg Ala Glu Phe Ser

65 70 75 80

Tyr Cys Tyr Ala Thr Ser Ala Asp Phe Lys Gly Gln Pro Ala Ser Val

85 90 95

Tyr Glu Asn Ala Gln Lys Thr Lys Thr Val Tyr His His Gly Lys Gly

100 105 110

Ile Cys Pro Gln Gly Ala Ser Arg Asp Tyr Glu Phe Ser Val Tyr Ile

115 120 125

Pro Ser Ala Leu Asn Arg Asp Val Ser Thr Ile Phe Ala Gln Trp His

130 135 140

Gly Met Pro Asp Arg Thr Leu Val Ser Thr Pro Asp Gly Glu Val Lys

145 150 155 160

Lys Leu Thr Thr Glu Glu Phe Leu Glu Leu Tyr Asp Arg Met Ile Phe

165 170 175

Lys Lys Asn Val Ala His Asp Lys Val Pro Val Leu Asp Lys Gln Gly

180 185 190

Asn Pro Lys Lys Asp Lys Asp Gly Asn Val Val Tyr Lys Ala Gly Lys

195 200 205

Ala Asn Gly Trp Leu Val Glu Gln Gly Gly Tyr Pro Pro Leu Ala Phe

210 215 220

Gly Phe Ser Gly Gly Tyr Phe Tyr Ile Lys Ala Asn Ser Asp Arg Lys

225 230 235 240

Trp Leu Thr Asp Lys Thr Asp Arg Cys Asn Ala Ser Ala Glu Lys Ser

245 250 255

Gln Ile Met Lys Pro Val Thr Ser Lys Tyr Lys Ala Ser Thr Ile Ala

260 265 270

Tyr Lys Met Pro Phe Ala Glu Phe Pro Lys Asp Cys Trp Ile Thr Phe

275 280 285

Arg Ile His Ile Asp Trp Thr Val Tyr Gly Lys Glu Thr Glu Thr Ile

290 295 300

Val Lys Pro Gly Met Leu Asp Val Gln Met Ser Tyr Thr Glu Lys Gly

305 310 315 320

Lys Gln Ile Asn Arg His Ile Val Asp Asn Glu Glu Ile Leu Ile Gly

325 330 335

Arg Asn Asp Glu Asp Gly Tyr Tyr Phe Lys Phe Gly Ile Tyr Arg Val

340 345 350

Gly Asn Ser Thr Ile Pro Val Cys Tyr Asn Leu Ala Gly Tyr Ser Glu

355 360 365

Asn

<210> 6

<211> 374

<212> PRT

<213> Artificial sequence

<400> 6

Asn Ala Gln Thr Lys Asn Thr Gln Thr Leu Val Pro Leu Thr Glu Arg

1 5 10 15

Val Asn Val Gln Ala Asp Ser Ala Arg Val Asn Gln Ile Ile Asp Gly

20 25 30

Cys Trp Val Ala Val Gly Ser Lys Lys Ser His Ala Ile Gln Arg Asp

35 40 45

Phe Thr Arg Met Phe Asn Gly Lys Pro Ser Tyr Arg Phe Glu Leu Lys

50 55 60

Glu Asp Asp Asn Thr Leu Ser Gly Tyr Ala Lys Gly Glu Thr Lys Gly

65 70 75 80

Arg Ala Glu Phe Ser Tyr Cys Tyr Ala Thr Ser Asp Asp Phe Lys Gly

85 90 95

Lys Pro Ala Asp Thr Tyr Lys Lys Ala Gln Ile Met Lys Thr Val Tyr

100 105 110

His His Gly Lys Gly Ala Cys Pro Gln Gly Ser Ser Arg Asp Tyr Glu

115 120 125

Phe Ser Val Tyr Ile Pro Ser Thr Leu Gly Ser Asp Val Ser Thr Ile

130 135 140

Phe Ala Gln Trp His Gly Met Pro Asp Arg Thr Leu Val Gln Thr Pro

145 150 155 160

Gln Gly Glu Val Lys Thr Leu Thr Val Asp Glu Phe Ile Glu Leu Glu

165 170 175

Lys Thr Thr Ile Phe Lys Lys Asn Val Gly His Glu Lys Lys Ala Lys

180 185 190

Leu Asp Lys Gln Gly Asn Pro Val Lys Asp Lys His Gly Asn Pro Val

195 200 205

Tyr Thr Ala Gly Lys Ala Asn Gly Trp Leu Val Glu Gln Gly Gly Tyr

210 215 220

Pro Pro Leu Ala Phe Gly Phe Ser Gly Gly Trp Phe Tyr Ile Lys Ala

225 230 235 240

Asn Ser Asp Arg Arg Trp Leu Thr Asp Lys Asp Asp Arg Cys Asn Ala

245 250 255

Asn Val Glu Lys Thr Pro Val Met Lys Pro Val Thr Ser Glu Tyr Lys

260 265 270

Ser Ser Thr Ile Ala Tyr Lys Met Pro Phe Ala Asp Phe Pro Lys Asp

275 280 285

Cys Trp Ile Thr Phe Arg Ile His Ile Asp Trp Thr Val Tyr Gly Lys

290 295 300

Glu Ala Glu Thr Ile Val Lys Pro Gly Met Leu Asp Val Gln Met Asn

305 310 315 320

Tyr Gln Asp Lys Gly Lys Lys Val Ser Lys His Leu Val Asp Asn Glu

325 330 335

Gln Ile Leu Ile Gly Arg Asn Asp Lys Asp Gly Tyr Tyr Phe Lys Phe

340 345 350

Gly Ile Tyr Arg Val Gly Asn Ser Thr Lys Pro Val Cys Tyr Asn Leu

355 360 365

Ala Gly Tyr Ser Glu Lys

370

Claims

1. A gene for coding heparinase I is characterized in that the nucleotide sequence of the heparinase I is shown as SEQ ID NO. 1.

2. A recombinant vector carrying the heparinase I gene according to claim 1.

3. A recombinant cell carrying the heparinase I gene of claim 1 or the recombinant vector of claim 2.

4. A recombinant Escherichia coli, which expresses the heparinase I-encoding gene of claim 1.

5. The recombinant E.coli of claim 4, wherein pET-SUMO or pUC19 is used as an expression vector.

6. The recombinant Escherichia coli according to claim 5, wherein Escherichia coli BL21 or Escherichia coli BL21 Rosetta is used as an expression host.

7. A method for producing low-molecular-weight heparin, characterized by adding the recombinant cell according to claim 3 or the recombinant Escherichia coli according to claim 4 or 5 to a reaction system containing heparin to carry out a reaction.

8. The process of claim 7, wherein the reaction temperature is 30 to 40 ℃.

9. Use of the heparinase I gene according to claim 1, or the recombinant cell according to claim 3, or the recombinant E.coli according to claim 4 or 5 for the preparation of a product containing low molecular weight heparin.