CN114752588B

CN114752588B - Heparinase II

Info

Publication number: CN114752588B
Application number: CN202210356343.6A
Authority: CN
Inventors: 刘颖
Original assignee: Beijing Ed Hauck International Technology Co ltd
Current assignee: Beijing Ed Hauck International Technology Co ltd
Priority date: 2022-04-06
Filing date: 2022-04-06
Publication date: 2023-07-25
Anticipated expiration: 2042-04-06
Also published as: CN114752588A

Abstract

The application discloses a heparanase II, a coding nucleotide sequence thereof, a recombinant vector comprising the nucleotide sequence, a host cell and application thereof, wherein the heparanase II has better stability compared with the prior heparanase II under the condition that the activity of the heparanase II is not influenced by carrying out site-directed mutation on the prior heparanase II amino acid sequence, specifically, carrying out site-directed mutation on three sites of glutamine (Q) into alanine (A) or valine (V).

Description

Heparinase II

Technical Field

The invention relates to the field of biological gene engineering and fermentation engineering. Within this field, the invention relates to heparanase II and genes encoding the same, and the invention further provides a method for preparing heparanase II using recombinant vectors and host cells.

Background

Heparinase (heparin) is a polysaccharide lyase acting on heparin (heparin) or heparan sulfate (heparin sulfate), is used for researching the interaction between heparinase and a substrate polysaccharide heparin thereof, is helpful for elucidating the action mechanism of the polysaccharide lyase, and has important application in analyzing the structure and biological functions of complex mucopolysaccharides such as heparin and the like, analyzing the coagulation and anticoagulation mechanism in human bodies, and the aspects of anticoagulation medicine low molecular heparin which is only subjected to low molecular anticoagulation medicine low molecular weight, clinical and the like. Heparanase is found in many microorganisms, of which there are mainly three heparanases from Flavobacterium heparinum, heparanase II (EC 4.2.2.7), heparanase II (No EC code) and heparanase III (EC 4.2.2.8), respectively.

The natural heparinase II is usually obtained by purifying fermentation liquid of Flavobacterium heparinum, and the heparinase II is produced by the Flavobacterium heparinum and simultaneously produces the heparinase I, III and four chondroitinases (chondratinase B, C, ABC and AC), so that the separation and purification of the heparinase II become complicated, multi-step chromatographic purification is usually needed, the activity loss of the enzyme is extremely high, the yield is low, and the induced additive heparin sodium can only be extracted from small intestinal mucosa of animals (mainly pigs and cows) at present, so that the process is complicated, the cost is high, and the yield and the application of the heparinase II are severely limited.

Disclosure of Invention

The heparin enzyme II prepared by the existing method has poor stability, the activity of the heparin enzyme II prepared by the existing method is reduced to half of that of the original heparin enzyme II when the heparin enzyme II is stored at a low temperature in a short time, and the activity of the heparin enzyme II can cause serious loss of enzyme activity after one freeze thawing and one freeze drying. Accordingly, there is a need in the art for improvements in the current heparanase II and methods of making the same.

In view of this, the inventors of the present application have conducted intensive studies and have specifically proposed the present invention.

The invention provides heparinase II and a coding gene thereof, and compared with original heparinase II, the heparinase II provided by the invention has stronger stability under the condition of not influencing enzyme activity, and the invention also provides a method for preparing the heparinase II.

In order to achieve the aim of the invention, the invention adopts the following technical scheme:

in one aspect, the invention provides a heparanase II comprising the amino acid sequence as set forth in SEQ ID NO:02, wherein the amino acid sequence is obtained by site-directed mutagenesis of glutamine (Q) at a plurality of positions of the amino acid sequence of original heparanase II (SEQ ID NO: 01) into alanine (A), and in order to improve the protein purification efficiency, a strep II tag sequence is added at the C-terminal of heparanase II, and the crude cell extract can pass through a desulfating biotin purification column to realize the purification of target heparanase II by means of the interaction of strep II tags between biotin.

The invention also provides a nucleotide sequence for encoding the heparanase II.

Preferably, the nucleotide sequence is shown as SEQ ID NO: shown at 04.

In a preferred embodiment, the invention also provides a heparanase II comprising a polypeptide as set forth in SEQ ID NO:03, wherein the amino acid sequence is obtained by site-directed mutagenesis of glutamine (Q) at a plurality of positions of the amino acid sequence of original heparanase II (SEQ ID NO: 01) into alanine (A) or valine (V), and in order to improve the protein purification efficiency, a strep II tag sequence is added at the C end of heparanase II, and the crude cell extract can pass through a desulfurization biotin purification column to realize the purification of target heparanase II by means of the interaction of strep II tags between biotin.

The invention also provides a method for encoding the amino acid sequence shown in SEQ ID NO:03, and a heparanase II nucleotide sequence shown in SEQ ID NO.

Preferably, the nucleotide sequence is shown as SEQ ID NO: 05.

In another aspect, the present invention provides a recombinant vector comprising the nucleotide sequence described above.

Further, the recombinant vector comprises a eukaryotic cell recombinant expression vector.

Further, the eukaryotic cell recombinant expression vector comprises any one of pPink-HC, pPICZaA and pPICZ A;

as a preferred method, the eukaryotic recombinant expression vector is pPink-HC.

The invention also provides a host cell comprising the recombinant vector.

Further, the host cell is one of pichia pastoris or saccharomyces cerevisiae.

Still further, the host cell is pichia pastoris.

In one aspect, the present invention provides a method for preparing the heparanase II, comprising the steps of:

firstly, synthesizing a nucleotide sequence of the heparanase II, and then combining the nucleotide sequence with a eukaryotic cell recombinant expression vector to obtain a recombinant vector;

transferring the recombinant vector into a host cell, then inducing expression, and purifying to obtain the heparinase II.

Preferably, the host cell is one of pPink-HC, pPICZaA, pPICZA, and further preferred, the eukaryotic recombinant expression vector is pPink-HC.

Preferably, in the above preparation method, the step of combining the nucleotide sequence with a eukaryotic recombinant expression vector and the recombinant vector is performed according to the instructions of PichiaPink system kit.

Preferably, in the above preparation method, the step of inducing expression comprises: the recombinant expression vector is used for transforming saccharomycetes, peptone and YNB, water and phosphate buffer solution are added, BMMY is subpackaged, glycerol is added into the rest culture medium, BMGY is subpackaged, positive transformants are selected and inoculated into shake flasks of the BMGY culture medium, supernatant is removed through culture and centrifugation, BMMY re-suspension thalli are taken, the BMMY re-suspension thalli are added into shake flasks with 20-25mL of BMMY culture medium, initial OD600 is controlled to be about 1, culture is continued, sampling is carried out at regular time, methanol is added, OD600 and exogenous protein expression quantity are measured, and fermentation broth is collected through centrifugation after fermentation is completed.

Preferably, in the above preparation method, the purification step comprises: the bacterial cells are collected (3-6 ℃), 1-2 ml Buffer W (precooled at 3-6 ℃ in advance) suspension is used for each 100ml of the collected bacterial cells, protease inhibitor is added, and cells are crushed on an ice-water mixture by ultrasonic waves to obtain lysate. Then purifying, specifically, cleaning the purifying column by using Buffer W, taking the lysate (3-6 ℃) of 0.5-10CVs, slowly loading the lysate into the column, after the sample completely enters the column, washing the column by using Buffer W, collecting the eluent of each part, adding 4-7 times of Buffer E, collecting the eluent in each section, and operating in a low-temperature chromatography cabinet in the whole process.

Further, the purification column is a desulfur biotin purification column.

Compared with the prior art, the invention has the beneficial effects that:

the heparanase II provided by the invention comprises a polypeptide shown as SEQ ID NO:02 or an amino acid sequence as set forth in SEQ ID NO: 03. The invention mutates protease enzyme cutting sites which possibly influence the stability of the heparanase II in the amino acid sequence of the original heparanase II (shown as SEQ ID NO: 01), and specifically mutates glutamine (Q) of a plurality of positions of the amino acid sequence of the natural heparanase II (shown as SEQ ID NO: 01) into alanine (A) or valine (V) in a fixed-point manner. The enzyme activity of the heparanase II obtained after the point mutation is not obviously reduced, and compared with the original heparanase II, the enzyme activity stability of the two heparanase II is obviously improved under the condition of 30 degrees.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present invention, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art. Wherein:

FIG. 1 shows the results of the purification electrophoresis assay provided in example 4 of the present invention;

FIG. 2 is an analysis of the stability of enzyme activity provided in example 6 of the present invention.

Detailed Description

The following description of the embodiments of the present invention will be made apparent and fully in view of the accompanying drawings, in which some, but not all embodiments of the invention are shown. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

The amino acid sequence of the original heparanase II is shown as SEQ ID NO:01, from the published heparanase II sequences in the NCBI database, the NCBI database website is: https:// www.ncbi.nlm.nih.gov/protein/ACB38160.1.

Heparanase II cleaves heparin and heparan mainly at the 1-4 linkage sites of hexosamine and uronic acid (glucuronic acid and iduronic acid), the product is mainly disaccharide, and has good degradation effect on heparin substances, but the stability of original heparanase II is poor, and in general, the purified heparanase II needs multi-step operation, in the process, the activity loss of enzyme is huge, and the low yield of less than 10% limits the application of heparanase II. Although certain substances can be used in the purification process to improve the service life of the enzyme, the problem of instability of the enzyme is fundamentally solved, and the structure and the catalytic mechanism of the enzyme are further studied.

The invention provides a heparanase II, which is prepared by performing site-directed mutagenesis on glutamine (Q) at 77, 261 and 276 sites in an amino acid sequence of original heparanase II to obtain alanine (A) or valine (V), wherein the obtained heparanase II comprises a sequence shown in SEQ ID NO:02 or SEQ ID NO: 03.

According to the reported crystal structure sequence information of the heparinase II, three sites 77, 261 and 276 in the amino acid sequence of the original heparinase II are the sites which are positioned at the outer negative charge aggregation region of the protein and are weaker in the structure of the heparinase II, when the heparinase II is expressed, the heparinase II can be attacked by protease to be degraded, particularly, a large amount of protease is released after thalli are cracked in the purifying process of the heparinase, and the heparinase II is very easy to be degraded, so that the stability of the heparinase II is influenced. Therefore, the invention respectively carries out amino acid substitution and compound mutation on three sites 77, 261 and 276, specifically, the invention changes the glutamine (Q) of the three sites into alanine (A) or valine (V) by site-directed mutation, and can obviously improve the enzyme activity stability of heparanase II. Meanwhile, through activity measurement, the mutation modification of the site is proved to not influence the activity of the heparanase II, and has better stability compared with the original heparanase II.

Wherein, the heparin enzyme II obtained by site-directed mutagenesis of 77 and 261 glutamine (Q) in the amino acid sequence of original heparin enzyme II into alanine (A) and site-directed mutagenesis of 276-site glutamine (Q) into valine (V) has stronger enzyme activity stability than that obtained by site-directed mutagenesis of three-site glutamine (Q) into alanine (A), and the activity of the heparin enzyme II is not obviously reduced compared with that of the original heparin enzyme II.

The nucleotide sequence for encoding the heparanase II is any nucleotide sequence capable of expressing the heparanase II, and in a preferred embodiment, the nucleotide sequence is shown as SEQ ID NO:04 or SEQ ID NO: 05.

The recombinant vector provided by the invention comprises a copy of any one of the nucleotide sequences, and is used for expressing heparinase II.

The recombinant vector refers to a DNA sequence which can be inserted into an exogenous DNA sequence and autonomously replicated, and the intentionally modified amino acid sequence or nucleotide sequence generally comprises a prokaryotic expression vector and a eukaryotic cell recombinant expression vector, and the recombinant vector provided by the invention can be any recombinant vector for encoding the heparinase I, and in a preferred embodiment of the invention, the recombinant vector is a eukaryotic cell recombinant expression vector.

Eukaryotic recombinant expression vectors can produce glycosylated soluble expressed heparanase II, while prokaryotic expression vectors express heparanase II, proteins cannot be glycosylated, most of the proteins are inactive inclusion body forms, and active heparanase II cannot be purified and obtained. Therefore, compared with the prokaryotic expression vector, the heparanase II obtained by the eukaryotic cell recombinant expression vector has higher activity. The species include pPIC9K, pPICZaA, pGAPZaA, pPIC3.5K, pPink-HC, pPICZaA, pPICZA, etc.

As a preferred embodiment, the eukaryotic recombinant expression vector comprises one of pPink-HC, pPICZaA, pPICZA, and further preferred embodiment, the eukaryotic recombinant expression vector is pPink-HC.

The invention provides a host cell, which comprises the recombinant vector and is used for expressing heparinase II.

Such host cells include single-cell prokaryotes and eukaryotes (e.g., bacteria, yeast, and actinomycetes) and single cells derived from higher plants or animals when grown in cell culture, and Flavobacterium heparinum has been commonly used as a host cell for expression of heparinase II, and in recent years, escherichia coli, yeast, and the like have also been used for expression of heparinase II.

The host cell provided by the invention can be any host cell for expressing the heparinase II, and as a preferred embodiment of the invention, the host cell is one of pichia pastoris or saccharomyces cerevisiae, and preferably, the host cell is pichia pastoris.

Pichia pastoris as host cell to express foreign protein is one new efficient expression system, which contains specific powerful alcohol oxidase gene promoter, methanol can regulate the expression of foreign gene strictly, and the foreign protein gene is genetically stable, and is integrated into Pichia pastoris genome in high copy number, so that it is not easy to lose and high expression strain can be obtained. The culture cost of pichia pastoris is very low, the fermentation culture medium is very cheap, the common carbon source is glycerol or glucose and methanol, the rest is inorganic salt, and the culture medium does not contain protein, thereby being beneficial to the separation and purification of downstream products and the easy separation of the products.

The invention provides a preparation method of heparanase II, which comprises the following steps: firstly synthesizing a nucleotide sequence for encoding heparanase II related in the text, and then combining the nucleotide sequence with a eukaryotic cell recombinant expression vector to obtain a recombinant vector; transferring the recombinant vector into a host cell, then inducing expression, and purifying to obtain the heparinase II.

In a preferred embodiment of the present invention, in the above preparation method, the synthesis of the nucleotide sequence encoding heparanase II referred to herein comprises the following steps:

firstly, inversely translating an amino acid sequence of original heparanase II into a nucleotide sequence, preferably, the obtained nucleotide sequence is favored by a selected host cell, wherein the translated nucleotide sequence is favored by pichia pastoris, and then, carrying out site-directed mutagenesis on the translated nucleotide sequence to obtain the nucleotide sequence of target heparanase II after mutation.

In a preferred embodiment of the present invention, the nucleotide sequence of heparanase II is combined with a eukaryotic recombinant expression vector to obtain a recombinant vector as follows:

firstly, cutting enzyme cutting sites of connecting points on a recombinant expression vector, and then connecting heparanase II nucleotide with the cut expression vector to construct the recombinant expression vector.

In a preferred embodiment of the present invention, in the above preparation method, the host cell is one of pPink-HC, pPICZaA, pPICZA, and in a further preferred embodiment, the eukaryotic recombinant expression vector is pPink-HC.

In a preferred embodiment of the present invention, in the above preparation method, the nucleotide sequence and eukaryotic recombinant expression vector binding recombinant vector step is performed according to the instructions of PichiaPink system kit.

In a preferred embodiment of the present invention, in the above preparation method, the induced expression process comprises the steps of:

the recombinant expression vector is used for transforming saccharomycetes, peptone and YNB, water and phosphate buffer solution are added, BMMY is subpackaged, glycerol is added into the rest culture medium, BMGY is subpackaged, positive transformants are selected and inoculated into shake flasks of the BMGY culture medium, supernatant is removed through culture and centrifugation, BMMY re-suspension thalli are taken, the BMMY re-suspension thalli are added into shake flasks with 20-25mL of BMMY culture medium, initial OD600 is controlled to be about 1, culture is continued, sampling is carried out at regular time, methanol is added, OD600 and exogenous protein expression quantity are measured, and fermentation broth is collected through centrifugation after fermentation is completed.

The YNB culture medium is also called as an amino-free yeast nitrogen culture medium and is used for fermentation culture of saccharomycetes.

The BMMY culture medium is a seed liquid culture medium, namely an induction expression culture medium, and is used for inducing the pichia pastoris recombinant strain to secrete and express target proteins by methanol.

The peptone is light yellow powder with the appearance of light yellow formed by hydrolyzing meat, casein or gelatin with acid or protease and drying, and provides a required carbon source for yeast cells.

In a preferred embodiment of the present invention, in the above preparation method, the purification step is as follows:

the bacterial cells are collected (3-6 ℃), 1-2 ml Buffer W (precooled at 3-6 ℃ in advance) suspension is used for each 100ml of the collected bacterial cells, protease inhibitor is added, and cells are crushed on an ice-water mixture by ultrasonic waves to obtain lysate. Then purifying, specifically, cleaning the purifying column by using Buffer W, taking the lysate (3-6 ℃) of 0.5-10CVs, slowly loading the lysate into the column, after the sample completely enters the column, washing the column by using Buffer W, collecting the eluent of each part, adding 4-7 times of Buffer E, collecting the eluent in each section, and operating in a low-temperature chromatography cabinet in the whole process.

The Buffer W (wash Buffer) rinse solution is used for washing off the impurity protein which is specifically bound with the purification column

The Buffer E (elution Buffer) is used for eluting the target protein specifically bound with the purification column, namely heparinase.

As a preferred scheme of the invention, the purification column is a desulfurization biotin purification column, and in order to improve the protein purification efficiency, a strep II tag sequence is added at the C end of the heparinase II, and better purification effect of target protein heparinase II is realized by means of interaction between strep II tags and biotin in the desulfurization biotin purification column.

The beneficial effects are that:

compared with the original heparinase II, the enzyme activity stability of the heparinase II is obviously improved under the condition of 30 degrees without affecting the enzyme activity, the enzyme activity half-life is nearly doubled, and the stability is stronger.

The following examples of the invention are merely illustrative of specific embodiments for carrying out the invention and are not to be construed as limiting the invention. Any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principles of the invention are intended to be equivalent arrangements which are within the scope of the invention.

Examples

Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Experimental materials used in table 1

Raw material name	Model/purity	Manufacturing factories
			PichiaPink system kit	A11152	Thermo Fisher Co
YNB medium	Y8040-100g	Solarbio
			Peptone	LP0042	OXOID
Strep-Tactin column (cysteine-containing column)	BTR211Q	Bo's western medicine
			Heparin sodium	NHS200803	Hebei Changshan Biochemical pharmaceutical Co Ltd

Example 1 improvement of nucleotide sequence optimization of heparanase II and construction of expression vector

(a) The amino acid sequence of original heparanase II is a published heparanase II sequence from NCBI database, with the NCBI database website: https:// www.ncbi.nlm.nih.gov/protein/ACB38160.1.

(b) The protein sequence was reverse translated into a DNA sequence by the company Jin Weizhi biotechnology, su, according to the codon usage preference of pichia pastoris in the pichia pastoris codon preference data table, such that the codons of the DNA sequence were all pichia pastoris-preferred.

(c) The potential protease cleavage site is subjected to mutation transformation, specifically, glutamine (Q) at 77, 261 and 276 sites of an amino acid sequence of original heparanase II is replaced by alanine (A) to obtain heparanase II-1, glutamine (Q) at 77 and 261 sites of the amino acid sequence of original heparanase II is replaced by alanine (A), glutamine (Q) at 276 site is replaced by valine (V) to obtain heparanase II-2, and the two modified sequences of heparanase II-1 and heparanase II-2 are respectively subjected to full sequence synthesis to obtain a heparanase II nucleotide sequence.

(d) On a pPink-HC recombinant expression vector, selecting two enzyme cutting sites of EcoRI and EcoRV to disconnect the expression vector, and connecting heparinase II with the disconnected expression vector to construct the HepI-pPink-HC recombinant expression vector.

Example 2 competent preparation and electrotransformation

The procedure was as per the instructions of PichiaPink system kit.

Example 3 inducible expression of heparanase II

1. Medium configuration:

(1) BMGY liquid medium: 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB,1% (w/v) glycerol, 10% (v/v) 1M phosphate buffer pH 6.0. Sterilizing at 115 ℃ for 20min;

(2) BMMY liquid medium: 1% (w/v) yeast extract, 2% (w/v) peptone, 1.34% (w/v) YNB,1% (v/v) methanol, 10% (v/v) 1M phosphate buffer pH 6.0. Sterilizing at 115 deg.C for 20min.

2. Induction of expression: weighing yeast extract, peptone and YNB, adding water and phosphate buffer, packaging BMMY, adding glycerol into the rest culture medium, and packaging BMGY.

3. The fermentation process comprises the following steps:

three positive transformants were picked up and inoculated into 50mL shake flasks containing 5-10mL of BMGY medium, cultured at 30 ℃ at 250rpm until OD600 (optical density value measured at 600 nm) =4 (16-18 hours), centrifuged off supernatant at 3000g for 3min, 1mL of BMMY resuspended cells were taken, added into 250mL shake flasks containing 23mL of BMMY medium, the initial OD600 was controlled to be about 1, then cultured at 30 ℃ at 250rpm, sampled every 24 hours (-200 uL) and 1% methanol was added, OD600 and foreign protein expression were measured, fermentation was stopped after 96 hours, and the fermentation broth was collected by centrifugation.

EXAMPLE 4 purification

1. Treatment of cell disruption lysate before purification

Buffer W solution ratio: 20mM Na2HPO4,0.28M NaCl,6mM KCl,pH7.4.

Buffer E solution ratio: 20mM Na2HPO4,0.28M NaCl,6mM KCl,2.5mM desulphated biotin, pH7.4.

The cells (4500 g,15min,4 ℃) were collected, and 1ml Buffer W (pre-chilled at 4 ℃) suspension was used for each 100ml of the collected cells, protease inhibitors were added, and the cells were disrupted by sonication on an ice-water mixture to obtain a lysate.

2. Purification and identification

The Strep-Tactin column (cysteine-containing column) was washed with Buffer W of 2CVs (the binding material of the column was cysteine and the elution was desthiobiotin), the lysate of 5CVs (4 ℃) was slowly applied to the column, after the sample had completely entered the column, the column was washed with Buffer W of 5CVs and the eluate of each fraction was collected, 6 times of Buffer E of 0.5CVs was added and collected in each section (0.5 CVs). Each fraction was collected and identified by 20. Mu.l SDS-PAGE, fusion tag proteins were typically 2 ^nd And 5 ^th Part(s). The whole process is operated in a low-temperature chromatography cabinet at the temperature of 4 ℃.

The result of SDS-PAGE electrophoresis analysis shows that the method can successfully obtain heparinase II protein with purity of more than 90 percent, and the molecular weight is consistent with that expected.

EXAMPLE 5 protein Activity assay

The substrate was heparin sodium (Hebei Changshan Biochemical pharmaceutical Co., ltd.) and the absorbance change with time was measured using an ultraviolet-visible spectrophotometer (Shanghai Ling Guang technology Co., ltd. GOLDS 54). The scanning wavelength was 232nm for 3min. Taking reaction buffer solution (20mM Tris,200mM NaCl, fully dissolving, adjusting pH=7.4 by using 6M hydrochloric acid, preserving at 4 ℃), adding about 1000 mu L of the reaction buffer solution and a certain amount of enzyme solution (the specific proportion is required to be adjusted according to the activity of the enzyme solution, and the buffer solution is generally 1ml, the enzyme solution is 2 mu L), 500 mu L of substrate solution (17mM Tris,44mM NaCl,3.5mM CaCl2, 25g/L heparin sodium, preserving at 7.0,4 ℃ by using 6M hydrochloric acid after fully stirring), placing the mixture into a quartz cuvette after uniform mixing, immediately placing the mixture into a spectrophotometer for scanning (the reaction buffer solution and the substrate solution are preheated to constant temperature in a water bath at 30 ℃ for at least 30min before uniform mixing), taking data of 40-60s, and calculating the slope k (min-1) of a curve after the completion, so that the enzyme activity (IU/L) of the heparin enzyme is calculated by the following formula and the deduction process:

according to beer's law, absorbance A=εc, where ε=3800M-1.cm-1, so the total enzyme activity in 1500. Mu.L reaction system is 15/38k (min-1) IU, and if the volume of enzyme solution added in 1500. Mu.L reaction system is V (. Mu.L), the enzyme activity of the added enzyme solution is calculated as follows:

the 3 mutation sites selected by the invention are 77, 261 and 276 respectively, amino acid substitution and compound mutation are carried out one by one, and the data of enzyme activity and stability are tested. In the heparinase II structure, all the 3 sites are positioned in a negative charge aggregation region outside the protein and are weaker in structure, so that the activity and stability of the enzyme can be greatly influenced.

Under the same conditions of fermentation, crushing and purification, the enzyme activity data of the mutants are shown in Table 2, and as can be seen from Table 2, the enzyme activity of the mutant Q77A is reduced to a certain extent relative to that of the original heparanase II; whereas the enzyme activities of the mutants of Q261A, Q276A, Q261A & Q276A decreased slightly, with the enzyme activities of heparanase II-1 and the mutants of heparanase closest to the original enzyme activity of heparanase I, the decrease was minimal.

Table 2: enzyme activity measurement results

Mutation site	Enzyme Activity (IU/L)
		Unmutated	882.02
Q77A	655.12
		Q261A	1133.95
Q276A	1032.55
		Q261A&Q276A	1311.54

Example 6 thermal stability analysis

The purified heparinase II-1, heparinase II-2 and heparinase II of the original sequence are respectively placed on ice, enzyme activity is immediately detected, and the time at the moment is recorded as 0, and the enzyme activity value is taken as 100%. The enzyme was then incubated at 30℃and samples were taken at 10min intervals to determine the enzyme activity, and the ratio of the enzyme activity at this time to the enzyme activity value at time 0 was recorded. The assay is timed until one of the heparanase II reaches half-life stop. When comparing the stability of different enzymes, the determination is made based on the inactivation rate of the enzyme at the same concentration, under the same solution conditions, and under the same incubation conditions. (the enzyme activities were measured in parallel for 3 times, and the average value was taken as the enzyme activity at that time)

The analysis results are shown in fig. 2, and it can be seen from fig. 2 that under the same warm-bath condition at 30 ℃, compared with the heparinase II of the original sequence, the improved heat stability of the heparinase II is obviously improved, the enzyme activity half-life of the heparinase II-1 is improved from about 11 hours to about 50 hours, and the stability of the heparinase II-2 is further improved, so that 276 is more critical for resisting intracellular protease of the heparinase II, and the stability of the heparinase II is more beneficial to improvement when valine is arranged at the 276.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

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Tyr Asp Pro Lys Glu Thr Phe Ser Ser Ser Gly Tyr Gly Gly Ser Asp

435 440 445

His Thr Asp Phe Ala Ala Asn Asp Gly Gly Gln Arg Leu Pro Gly Lys

450 455 460

Gly Trp Ile Ala Pro Arg Asp Leu Lys Glu Met Leu Ala Gly Asp Phe

465 470 475 480

Arg Thr Gly Lys Ile Leu Ala Gln Gly Phe Gly Pro Asp Asn Gln Thr

485 490 495

Pro Asp Tyr Thr Tyr Leu Lys Gly Asp Ile Thr Ala Ala Tyr Ser Ala

500 505 510

Lys Val Lys Glu Val Lys Arg Ser Phe Leu Phe Leu Asn Leu Lys Asp

515 520 525

Ala Lys Val Pro Ala Ala Met Ile Val Phe Asp Lys Val Val Ala Ser

530 535 540

Asn Pro Asp Phe Lys Lys Phe Trp Leu Leu His Ser Ile Glu Gln Pro

545 550 555 560

Glu Ile Lys Gly Asn Gln Ile Thr Ile Lys Arg Thr Lys Asn Gly Asp

565 570 575

Ser Gly Met Leu Val Asn Thr Ala Leu Leu Pro Asp Ala Ala Asn Ser

580 585 590

Asn Ile Thr Ser Ile Gly Gly Lys Gly Lys Asp Phe Trp Val Phe Gly

595 600 605

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

610 615 620

Arg Gly Glu Trp Arg Val Glu Ile Thr Pro Lys Lys Ala Ala Ala Glu

625 630 635 640

Asp Tyr Tyr Leu Asn Val Ile Gln Ile Ala Asp Asn Thr Gln Gln Lys

645 650 655

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

660 665 670

Leu Ala Asp Arg Ile Val Thr Phe Ser Lys Thr Ser Glu Thr Val Asp

675 680 685

Arg Pro Phe Gly Phe Ser Val Val Gly Lys Gly Thr Phe Lys Phe Val

690 695 700

Met Thr Asp Leu Leu Pro Gly Thr Trp Gln Val Leu Lys Asp Gly Lys

705 710 715 720

Ile Leu Tyr Pro Ala Leu Ser Ala Lys Gly Asp Asp Gly Pro Leu Tyr

725 730 735

Phe Glu Gly Thr Glu Gly Thr Tyr Arg Phe Leu Arg Trp Ser His Pro

740 745 750

Gln Phe Glu Lys

755

<210> 2

<211> 756

<212> PRT

<213> artificial sequence

<220>

<223> artificial sequence description: synthetic sequences

<400> 2

Met Gln Thr Lys Ala Asp Val Val Trp Lys Asp Val Asp Gly Val Ser

1 5 10 15

Met Pro Ile Pro Pro Lys Thr His Pro Arg Leu Tyr Leu Arg Glu Gln

20 25 30

Gln Val Pro Asp Leu Lys Asn Arg Met Asn Asp Pro Lys Leu Lys Lys

35 40 45

Val Trp Ala Asp Met Ile Lys Met Gln Glu Asp Trp Lys Pro Ala Asp

50 55 60

Ile Pro Glu Val Lys Asp Phe Arg Phe Tyr Phe Asn Ala Lys Gly Leu

65 70 75 80

Thr Val Arg Val Glu Leu Met Ala Leu Asn Tyr Leu Met Thr Lys Asp

85 90 95

Pro Lys Val Gly Arg Glu Ala Ile Thr Ser Ile Ile Asp Thr Leu Glu

100 105 110

Thr Ala Thr Phe Lys Pro Ala Gly Asp Ile Ser Arg Gly Ile Gly Leu

115 120 125

Phe Met Val Thr Gly Ala Ile Val Tyr Asp Trp Cys Tyr Asp Gln Leu

130 135 140

Lys Pro Glu Glu Lys Thr Arg Phe Val Lys Ala Phe Val Arg Leu Ala

145 150 155 160

Lys Met Leu Glu Cys Gly Tyr Pro Pro Val Lys Asp Lys Ser Ile Val

165 170 175

Gly His Ala Ser Glu Trp Met Ile Met Arg Asp Leu Leu Ser Val Gly

180 185 190

Ile Ala Ile Tyr Asp Glu Phe Pro Glu Met Tyr Asn Leu Ala Ala Gly

195 200 205

Arg Phe Phe Lys Glu His Leu Val Ala Arg Asn Trp Phe Tyr Pro Ser

210 215 220

His Asn Tyr His Gln Gly Met Ser Tyr Leu Asn Val Arg Phe Thr Asn

225 230 235 240

Asp Leu Phe Ala Leu Trp Ile Leu Asp Arg Met Gly Ala Gly Asn Val

245 250 255

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

260 265 270

Arg Pro Asp Gly Ala Ile Leu Ala Gly Gly Asp Val Asp Tyr Ser Arg

275 280 285

Lys Lys Pro Lys Tyr Tyr Thr Met Pro Ala Leu Leu Ala Gly Ser Tyr

290 295 300

Tyr Lys Asp Glu Tyr Leu Asn Tyr Glu Phe Leu Lys Asp Pro Asn Val

305 310 315 320

Glu Pro His Cys Lys Leu Phe Glu Phe Leu Trp Arg Asp Thr Gln Leu

325 330 335

Gly Ser Arg Lys Pro Asp Asp Leu Pro Leu Ser Arg Tyr Ser Gly Ser

340 345 350

Pro Phe Gly Trp Met Ile Ala Arg Thr Gly Trp Gly Pro Glu Ser Val

355 360 365

Ile Ala Glu Met Lys Val Asn Glu Tyr Ser Phe Leu Asn His Gln His

370 375 380

Gln Asp Ala Gly Ala Phe Gln Ile Tyr Tyr Lys Gly Pro Leu Ala Ile

385 390 395 400

Asp Ala Gly Ser Tyr Thr Gly Ser Ser Gly Gly Tyr Asn Ser Pro His

405 410 415

Asn Lys Asn Phe Phe Lys Arg Thr Ile Ala His Asn Ser Leu Leu Ile

420 425 430

Tyr Asp Pro Lys Glu Thr Phe Ser Ser Ser Gly Tyr Gly Gly Ser Asp

435 440 445

His Thr Asp Phe Ala Ala Asn Asp Gly Gly Gln Arg Leu Pro Gly Lys

450 455 460

Gly Trp Ile Ala Pro Arg Asp Leu Lys Glu Met Leu Ala Gly Asp Phe

465 470 475 480

Arg Thr Gly Lys Ile Leu Ala Gln Gly Phe Gly Pro Asp Asn Gln Thr

485 490 495

Pro Asp Tyr Thr Tyr Leu Lys Gly Asp Ile Thr Ala Ala Tyr Ser Ala

500 505 510

Lys Val Lys Glu Val Lys Arg Ser Phe Leu Phe Leu Asn Leu Lys Asp

515 520 525

Ala Lys Val Pro Ala Ala Met Ile Val Phe Asp Lys Val Val Ala Ser

530 535 540

Asn Pro Asp Phe Lys Lys Phe Trp Leu Leu His Ser Ile Glu Gln Pro

545 550 555 560

Glu Ile Lys Gly Asn Gln Ile Thr Ile Lys Arg Thr Lys Asn Gly Asp

565 570 575

Ser Gly Met Leu Val Asn Thr Ala Leu Leu Pro Asp Ala Ala Asn Ser

580 585 590

Asn Ile Thr Ser Ile Gly Gly Lys Gly Lys Asp Phe Trp Val Phe Gly

595 600 605

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

610 615 620

Arg Gly Glu Trp Arg Val Glu Ile Thr Pro Lys Lys Ala Ala Ala Glu

625 630 635 640

Asp Tyr Tyr Leu Asn Val Ile Gln Ile Ala Asp Asn Thr Gln Gln Lys

645 650 655

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

660 665 670

Leu Ala Asp Arg Ile Val Thr Phe Ser Lys Thr Ser Glu Thr Val Asp

675 680 685

Arg Pro Phe Gly Phe Ser Val Val Gly Lys Gly Thr Phe Lys Phe Val

690 695 700

Met Thr Asp Leu Leu Pro Gly Thr Trp Gln Val Leu Lys Asp Gly Lys

705 710 715 720

Ile Leu Tyr Pro Ala Leu Ser Ala Lys Gly Asp Asp Gly Pro Leu Tyr

725 730 735

Phe Glu Gly Thr Glu Gly Thr Tyr Arg Phe Leu Arg Trp Ser His Pro

740 745 750

Gln Phe Glu Lys

755

<210> 3

<211> 756

<212> PRT

<213> artificial sequence

<220>

<223> artificial sequence description: synthetic sequences

<400> 3

Met Gln Thr Lys Ala Asp Val Val Trp Lys Asp Val Asp Gly Val Ser

1 5 10 15

Met Pro Ile Pro Pro Lys Thr His Pro Arg Leu Tyr Leu Arg Glu Gln

20 25 30

Gln Val Pro Asp Leu Lys Asn Arg Met Asn Asp Pro Lys Leu Lys Lys

35 40 45

Val Trp Ala Asp Met Ile Lys Met Gln Glu Asp Trp Lys Pro Ala Asp

50 55 60

Ile Pro Glu Val Lys Asp Phe Arg Phe Tyr Phe Asn Ala Lys Gly Leu

65 70 75 80

Thr Val Arg Val Glu Leu Met Ala Leu Asn Tyr Leu Met Thr Lys Asp

85 90 95

Pro Lys Val Gly Arg Glu Ala Ile Thr Ser Ile Ile Asp Thr Leu Glu

100 105 110

Thr Ala Thr Phe Lys Pro Ala Gly Asp Ile Ser Arg Gly Ile Gly Leu

115 120 125

Phe Met Val Thr Gly Ala Ile Val Tyr Asp Trp Cys Tyr Asp Gln Leu

130 135 140

Lys Pro Glu Glu Lys Thr Arg Phe Val Lys Ala Phe Val Arg Leu Ala

145 150 155 160

Lys Met Leu Glu Cys Gly Tyr Pro Pro Val Lys Asp Lys Ser Ile Val

165 170 175

Gly His Ala Ser Glu Trp Met Ile Met Arg Asp Leu Leu Ser Val Gly

180 185 190

Ile Ala Ile Tyr Asp Glu Phe Pro Glu Met Tyr Asn Leu Ala Ala Gly

195 200 205

Arg Phe Phe Lys Glu His Leu Val Ala Arg Asn Trp Phe Tyr Pro Ser

210 215 220

His Asn Tyr His Gln Gly Met Ser Tyr Leu Asn Val Arg Phe Thr Asn

225 230 235 240

Asp Leu Phe Ala Leu Trp Ile Leu Asp Arg Met Gly Ala Gly Asn Val

245 250 255

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

260 265 270

Arg Pro Asp Gly Val Ile Leu Ala Gly Gly Asp Val Asp Tyr Ser Arg

275 280 285

Lys Lys Pro Lys Tyr Tyr Thr Met Pro Ala Leu Leu Ala Gly Ser Tyr

290 295 300

Tyr Lys Asp Glu Tyr Leu Asn Tyr Glu Phe Leu Lys Asp Pro Asn Val

305 310 315 320

Glu Pro His Cys Lys Leu Phe Glu Phe Leu Trp Arg Asp Thr Gln Leu

325 330 335

Gly Ser Arg Lys Pro Asp Asp Leu Pro Leu Ser Arg Tyr Ser Gly Ser

340 345 350

Pro Phe Gly Trp Met Ile Ala Arg Thr Gly Trp Gly Pro Glu Ser Val

355 360 365

Ile Ala Glu Met Lys Val Asn Glu Tyr Ser Phe Leu Asn His Gln His

370 375 380

Gln Asp Ala Gly Ala Phe Gln Ile Tyr Tyr Lys Gly Pro Leu Ala Ile

385 390 395 400

Asp Ala Gly Ser Tyr Thr Gly Ser Ser Gly Gly Tyr Asn Ser Pro His

405 410 415

Asn Lys Asn Phe Phe Lys Arg Thr Ile Ala His Asn Ser Leu Leu Ile

420 425 430

Tyr Asp Pro Lys Glu Thr Phe Ser Ser Ser Gly Tyr Gly Gly Ser Asp

435 440 445

His Thr Asp Phe Ala Ala Asn Asp Gly Gly Gln Arg Leu Pro Gly Lys

450 455 460

Gly Trp Ile Ala Pro Arg Asp Leu Lys Glu Met Leu Ala Gly Asp Phe

465 470 475 480

Arg Thr Gly Lys Ile Leu Ala Gln Gly Phe Gly Pro Asp Asn Gln Thr

485 490 495

Pro Asp Tyr Thr Tyr Leu Lys Gly Asp Ile Thr Ala Ala Tyr Ser Ala

500 505 510

Lys Val Lys Glu Val Lys Arg Ser Phe Leu Phe Leu Asn Leu Lys Asp

515 520 525

Ala Lys Val Pro Ala Ala Met Ile Val Phe Asp Lys Val Val Ala Ser

530 535 540

Asn Pro Asp Phe Lys Lys Phe Trp Leu Leu His Ser Ile Glu Gln Pro

545 550 555 560

Glu Ile Lys Gly Asn Gln Ile Thr Ile Lys Arg Thr Lys Asn Gly Asp

565 570 575

Ser Gly Met Leu Val Asn Thr Ala Leu Leu Pro Asp Ala Ala Asn Ser

580 585 590

Asn Ile Thr Ser Ile Gly Gly Lys Gly Lys Asp Phe Trp Val Phe Gly

595 600 605

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

610 615 620

Arg Gly Glu Trp Arg Val Glu Ile Thr Pro Lys Lys Ala Ala Ala Glu

625 630 635 640

Asp Tyr Tyr Leu Asn Val Ile Gln Ile Ala Asp Asn Thr Gln Gln Lys

645 650 655

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

660 665 670

Leu Ala Asp Arg Ile Val Thr Phe Ser Lys Thr Ser Glu Thr Val Asp

675 680 685

Arg Pro Phe Gly Phe Ser Val Val Gly Lys Gly Thr Phe Lys Phe Val

690 695 700

Met Thr Asp Leu Leu Pro Gly Thr Trp Gln Val Leu Lys Asp Gly Lys

705 710 715 720

Ile Leu Tyr Pro Ala Leu Ser Ala Lys Gly Asp Asp Gly Pro Leu Tyr

725 730 735

Phe Glu Gly Thr Glu Gly Thr Tyr Arg Phe Leu Arg Trp Ser His Pro

740 745 750

Gln Phe Glu Lys

755

<210> 4

<211> 2271

<212> DNA

<213> artificial sequence

<220>

<223> artificial sequence description: synthetic sequences

<400> 4

atgcaaacta aggctgatgt tgtttggaaa gatgttgatg gtgtttctat gccaattcca 60

cctaagactc atcctagatt gtacttgaga gaacaacaag ttccagattt gaagaacaga 120

atgaacgatc ctaaattgaa gaaagtttgg gctgatatga ttaagatgca agaagattgg 180

aaaccagctg atattcctga ggttaaggat ttcagattct acttcaacgc taagggtttg 240

actgttagag ttgagttgat ggctttgaac tatttgatga ctaaagatcc aaaagttggt 300

agagaagcta tcacttctat catcgatact ttggagactg ctactttcaa accagctgga 360

gatatttcca gaggtattgg tttgtttatg gttactggtg ctatcgttta cgattggtgt 420

tacgatcaat tgaagcctga agagaaaact agattcgtta aggcttttgt tagattggct 480

aaaatgttgg aatgtggtta tccacctgtt aaggataaat ctattgttgg tcatgcttct 540

gagtggatga ttatgagaga tttgttgtct gttggtattg ctatctacga tgaatttcct 600

gagatgtata atttggctgc tggtagattt ttcaaggaac acttggttgc tagaaactgg 660

ttttacccat ctcataatta tcaccaagga atgtcttact tgaacgttag attcactaac 720

gatttgttcg ctttgtggat tttggataga atgggtgctg gtaacgtttt caatcctggt 780

caagctttca ttttgtacga tgctatctac aagagaagac cagatggtgc tattttggct 840

ggtggagatg ttgattattc cagaaagaag ccaaagtact atactatgcc tgctttgttg 900

gctggttctt actacaagga tgagtacttg aactacgaat ttttgaaaga tccaaacgtt 960

gaacctcact gtaaattgtt cgagtttttg tggagagata ctcaattggg ttccagaaag 1020

ccagatgatt tgcctttgtc cagatactct ggttctcctt ttggttggat gattgctaga 1080

actggttggg gtcctgagtc tgttattgct gaaatgaagg ttaacgagta ctctttcttg 1140

aaccatcaac atcaagatgc tggtgctttt caaatctact ataaaggtcc tttggctatt 1200

gatgctggtt cttacactgg ttcttctggt ggttacaact ctccacataa caagaacttt 1260

ttcaagagaa ctatcgctca caactctttg ttgatctacg atccaaagga aactttttct 1320

tcttctggtt atggtggttc tgatcatact gattttgctg ctaatgatgg tggtcaaaga 1380

ttgcctggta aaggttggat tgctcctaga gatttgaaag agatgttggc tggagatttc 1440

agaactggta aaattttggc tcaaggtttt ggtccagata accaaactcc tgattacact 1500

tatttgaagg gagatattac tgctgcttac tctgctaagg ttaaggaagt taagagatct 1560

ttcttgtttt tgaacttgaa ggatgctaaa gttccagctg ctatgatcgt tttcgataag 1620

gttgttgctt ctaaccctga tttcaagaaa ttctggttgt tgcactctat tgaacaacca 1680

gagattaaag gtaaccaaat cactattaag agaactaaaa acggagattc tggaatgttg 1740

gttaatactg ctttgttgcc tgatgctgct aactctaaca tcacttctat cggtggtaaa 1800

ggtaaagatt tctgggtttt cggtactaac tacactaacg atccaaagcc tggtactgat 1860

gaggctttgg aaagaggtga atggagagtt gaaattactc caaagaaagc tgctgctgag 1920

gattactatt tgaacgttat ccaaatcgct gataacactc aacaaaagtt gcatgaagtt 1980

aagagaattg atggagataa ggttgttggt gttcaattgg ctgatagaat cgttactttc 2040

tctaagactt ctgagactgt tgatagacct ttcggttttt ctgttgttgg taaaggtact 2100

ttcaaattcg ttatgactga tttgttgcca ggtacttggc aagttttgaa ggatggtaaa 2160

attttgtacc cagctttgtc tgctaaggga gatgatggtc ctttgtactt tgagggtact 2220

gaaggtactt atagattctt gagatggtct cacccacaat ttgaaaaata a 2271

<210> 5

<211> 2271

<212> DNA

<213> artificial sequence

<220>

<223> artificial sequence description: synthetic sequences

<400> 5

atgcaaacta aggctgatgt tgtttggaaa gatgttgatg gtgtttctat gccaattcca 60

cctaagactc atcctagatt gtacttgaga gaacaacaag ttccagattt gaagaacaga 120

atgaacgatc ctaaattgaa gaaagtttgg gctgatatga ttaagatgca agaagattgg 180

aaaccagctg atattcctga ggttaaggat ttcagattct acttcaacgc taagggtttg 240

actgttagag ttgagttgat ggctttgaac tatttgatga ctaaagatcc aaaagttggt 300

agagaagcta tcacttctat catcgatact ttggagactg ctactttcaa accagctgga 360

gatatttcca gaggtattgg tttgtttatg gttactggtg ctatcgttta cgattggtgt 420

tacgatcaat tgaagcctga agagaaaact agattcgtta aggcttttgt tagattggct 480

aaaatgttgg aatgtggtta tccacctgtt aaggataaat ctattgttgg tcatgcttct 540

gagtggatga ttatgagaga tttgttgtct gttggtattg ctatctacga tgaatttcct 600

gagatgtata atttggctgc tggtagattt ttcaaggaac acttggttgc tagaaactgg 660

ttttacccat ctcataatta tcaccaagga atgtcttact tgaacgttag attcactaac 720

gatttgttcg ctttgtggat tttggataga atgggtgctg gtaacgtttt caatcctggt 780

caagctttca ttttgtacga tgctatctac aagagaagac cagatggtgt tattttggct 840

ggtggagatg ttgattattc cagaaagaag ccaaagtact atactatgcc tgctttgttg 900

gctggttctt actacaagga tgagtacttg aactacgaat ttttgaaaga tccaaacgtt 960

gaacctcact gtaaattgtt cgagtttttg tggagagata ctcaattggg ttccagaaag 1020

ccagatgatt tgcctttgtc cagatactct ggttctcctt ttggttggat gattgctaga 1080

actggttggg gtcctgagtc tgttattgct gaaatgaagg ttaacgagta ctctttcttg 1140

aaccatcaac atcaagatgc tggtgctttt caaatctact ataaaggtcc tttggctatt 1200

gatgctggtt cttacactgg ttcttctggt ggttacaact ctccacataa caagaacttt 1260

ttcaagagaa ctatcgctca caactctttg ttgatctacg atccaaagga aactttttct 1320

tcttctggtt atggtggttc tgatcatact gattttgctg ctaatgatgg tggtcaaaga 1380

ttgcctggta aaggttggat tgctcctaga gatttgaaag agatgttggc tggagatttc 1440

agaactggta aaattttggc tcaaggtttt ggtccagata accaaactcc tgattacact 1500

tatttgaagg gagatattac tgctgcttac tctgctaagg ttaaggaagt taagagatct 1560

ttcttgtttt tgaacttgaa ggatgctaaa gttccagctg ctatgatcgt tttcgataag 1620

gttgttgctt ctaaccctga tttcaagaaa ttctggttgt tgcactctat tgaacaacca 1680

gagattaaag gtaaccaaat cactattaag agaactaaaa acggagattc tggaatgttg 1740

gttaatactg ctttgttgcc tgatgctgct aactctaaca tcacttctat cggtggtaaa 1800

ggtaaagatt tctgggtttt cggtactaac tacactaacg atccaaagcc tggtactgat 1860

gaggctttgg aaagaggtga atggagagtt gaaattactc caaagaaagc tgctgctgag 1920

gattactatt tgaacgttat ccaaatcgct gataacactc aacaaaagtt gcatgaagtt 1980

aagagaattg atggagataa ggttgttggt gttcaattgg ctgatagaat cgttactttc 2040

tctaagactt ctgagactgt tgatagacct ttcggttttt ctgttgttgg taaaggtact 2100

ttcaaattcg ttatgactga tttgttgcca ggtacttggc aagttttgaa ggatggtaaa 2160

attttgtacc cagctttgtc tgctaaggga gatgatggtc ctttgtactt tgagggtact 2220

gaaggtactt atagattctt gagatggtct cacccacaat ttgaaaaata a 2271

Claims

1. A heparanase II, wherein the heparanase II is the polypeptide as set forth in SEQ ID NO:02 or SEQ ID NO: 03.

2. A nucleotide sequence encoding the heparanase II of claim 1.

3. The nucleotide sequence according to claim 2, wherein the nucleotide sequence encoding heparanase II according to claim 1 is set forth in SEQ ID NO:04 or SEQ ID NO: 05.

4. A recombinant vector comprising the nucleotide sequence of claim 2 or 3.

5. The recombinant vector according to claim 4, wherein the recombinant vector is a eukaryotic recombinant expression vector.

6. The recombinant vector according to claim 5, wherein the eukaryotic cell recombinant expression vector is any one selected from the group consisting of pPink-HC, pPICZaA, pPICZA.

7. The recombinant vector of claim 6, wherein the eukaryotic recombinant expression vector is pPink-HC.

8. A host cell comprising the recombinant vector of any one of claims 4-6.

9. The host cell of claim 8, wherein the host cell is pichia or saccharomyces cerevisiae.

10. The host cell of claim 9, wherein the host cell is pichia pastoris.

11. A process for preparing heparanase II according to claim 1, characterized in that the preparation process comprises the following steps:

firstly, synthesizing a nucleotide sequence for encoding the heparanase II of claim 1, and then combining the nucleotide sequence with a eukaryotic cell recombinant expression vector to obtain a recombinant vector;

transferring the recombinant vector into a host cell, then inducing expression, and purifying to obtain heparinase II.

12. The method of claim 11, further comprising:

the purification is performed by using a desulphated biotin purification column.