Heparin C5-epimerase mutant with high expression quantity, high activity and high stability and application of heparin C5-epimerase mutant

Abstract

The invention relates to a heparin C5-epimerase mutant with high expression quantity, high activity and high stability and application of the heparin C5-epimerase mutant. Wherein the sequence information of the heparin C5-epimerase mutant CbC5epi is as shown in SEQ ID NO. 1 to SEQ ID NO. 2. And the sequence information of the CbC5epi mutant (A418P), the CbC5epi mutant (F453Y), the CbC5epi mutant (L591I) and the CbC5epi mutant (V170R-V260L-L276V) is as shown in SEQ ID NO. 3-10. The invention also discloses a method for preparing the CbC5epi mutant (A418P), the CbC5epi mutant (F453Y) and the CbC5epi mutant (L591I). The heparin C5-epimerase mutant CbC5epi provided by the invention is obviously improved in activity, reversible reaction forward proceeding degree and stability. The expression quantity of the CbC5epi mutant (V170R-V260L-L276V) which is further mutated on the basis of CbC5epi is about 31 mg / L and is 3.6 times that of HsC5epi, the activity is 1.36 mu mol / (mg.h) and is increased by nearly 20 times, and the residual enzyme activity can still be kept at 1.11 mu mol / (mg.h) after the CbC5epi mutant is incubated at 37 DEG C for 72 hours.

Description

Technical Field

[0001] The present invention relates to the discovery and modification of a truncated heparin C5-epimerase with high expression level, high activity and high stability derived from the genus Caenorhabditis briggsae Caenorhabditis brenneri ), and its application in heparin synthesis, belonging to the field of biotechnology. Background Art

[0002] Heparin (HP) is a linear polysaccharide composed of D -glucuronic acid ( D -glucuronic acid, GlcA) or L -iduronic acid ( L -iduronic acid, IdoA) and N -acetylglucosamine ( N -acetylglucosamine, GlcNAc) or N -sulfoglucosamine ( N -sulfoglucosamine, GlcNS) are alternately linked. Heparin has a wide range of clinical applications and is the first choice drug for treating thrombotic diseases, playing an auxiliary role in the treatment of various diseases such as cardiovascular diseases, hemodialysis, anti-inflammatory treatment, antiviral treatment, etc. In addition, heparin also plays an important role in cell signal transduction, tissue repair, etc.

[0003] Currently, heparin is mainly obtained by extraction from animal tissues (porcine intestinal mucosa). The extracted heparin has a natural structure and high biological activity. However, there are risks of contamination and adulteration, and the raw material supply is affected by animal diseases (such as African swine fever). In addition, a small part of heparin is synthesized by chemical methods. Such heparin products have uniform quality and controllable synthesis processes. However, their synthesis costs are high, the length of the synthesized sugar chains is limited, and it is difficult to produce on a large scale. In recent years, the developed chemoenzymatic synthesis uses enzymatic catalytic reactions to synthesize heparin, combining chemical modification and enzymatic reactions, with high biological activity, capable of synthesizing heparin with specific sulfation modifications, and having broad application prospects.

[0004] The pentasaccharide structural sequence (GlcNAc / NS6S-GlcA-GlcNS3S6S-IdoA2S-GlcNS6S) in the HP structure has been confirmed as the smallest structural unit that can specifically bind to antithrombin. Among them, IdoA2S is first converted from GlcA by heparin C5-epimerase (C5-epimerase, C5-epi), and then through 2- O -sulfotransferase (2- O-sulfotransferase, 2OST) catalyzes the formation of IdoA2S from IdoA. C5-epi is the only enzyme that catalyzes the isomerization reaction in the heparin synthesis process. It can only recognize GlcA in GlcNS-GlcA-GlcNS and catalyze the formation of IdoA, which provides internal flexibility to heparin and forms protein-binding sites.

[0005] However, the reaction catalyzed by C5-epi is reversible and thermodynamically tends to form the more stable GlcA, which is not conducive to the synthesis of anticoagulant heparin. In addition, most of the C5-epi used in current chemoenzymatic synthesis is derived from Homo sapiens ( Homo sapiens ), and this enzyme from higher animals is recombinantly expressed in the Escherichia coli expression system, lacking the correct folding and post-translational modification processes, showing problems such as low soluble expression level and poor activity. The present invention modifies C5-epi through exploration and protein engineering means, and develops a C5-epimerase mutant with high expression level, high activity and high stability from a new species. Summary of the Invention

[0006] Aiming at the problems existing in the prior art, the present invention provides a heparin C5-epimerase mutant with high expression level, high activity and high stability and its application. The present invention explores C5-epi from other sources, constructs truncated C5-epi of the corresponding source according to the amino acid sequence alignment result with the truncated body of C5-epi from Homo sapiens (hereinafter referred to as HsC5epi), successfully obtains the truncated body of C5-epi from Caenorhabditis briggsae (hereinafter referred to as CbC5epi), and performs protein engineering modification on CbC5epi to improve its expression level, activity and stability.

[0007] Term Explanation: GlcA-GlcNS-GlcA-GlcNS-GlcA- p NP: The full Chinese name is p-nitrophenyl-glucuronic acid- N -sulfoglucosamine-glucuronic acid- N -sulfoglucosamine-glucuronic acid, which is the substrate of C5-epi and the receptor substrate GlcA-GlcNS-IdoA-GlcNS-GlcA- for catalyzing the formation of 2OST p NP.

[0008] PAPS: The full Chinese name is 3′-phosphoadenosine-5′-phosphosulfate, which is the sulfate donor.

[0009] The technical solution of the present invention is as follows: In the first aspect of the present invention, there is provided a heparin C5-epimerase mutant CbC5epi, whose amino acid sequence is as shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.2; The heparin C5-epimerase mutant CbC5epi has truncated 49 amino acids at the N-terminus based on the wild-type heparin C5-epimerase; the wild-type heparin C5-epimerase is derived from Caenorhabditis briggsae ( Caenorhabditis brenneri ), NCBI ACCESSION: EGT51961. That is, the heparin C5-epimerase mutant CbC5epi is a truncated form of the wild-type heparin C5-epimerase.

[0010] In the second aspect of the present invention, protein engineering modification is carried out on the above heparin C5-epimerase mutant CbC5epi to obtain a CbC5epi mutant with high expression level, high activity and high stability.

[0011] A CbC5epi mutant (A418P), whose amino acid sequence is as shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.4; it is obtained by first site-directed mutagenesis of alanine at position 418 in the amino acid sequence of the wild-type heparin C5-epimerase to proline, and then truncating 49 amino acids at the N-terminus according to the pattern of the heparin C5-epimerase mutant CbC5epi.

[0012] A CbC5epi mutant (F453Y), whose amino acid sequence is as shown in SEQ ID NO.5, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.6; it is obtained by first site-directed mutagenesis of phenylalanine at position 453 in the amino acid sequence of the wild-type heparin C5-epimerase to tyrosine, and then truncating 49 amino acids at the N-terminus according to the pattern of the heparin C5-epimerase mutant CbC5epi.

[0013] A CbC5epi mutant (L591I), whose amino acid sequence is as shown in SEQ ID NO.7, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.8; it is obtained by first site-directed mutagenesis of leucine at position 591 in the amino acid sequence of the wild-type heparin C5-epimerase to isoleucine, and then truncating 49 amino acids at the N-terminus according to the pattern of the heparin C5-epimerase mutant CbC5epi.

[0014] A CbC5epi mutant (V170R-V260L-L276V) has an amino acid sequence as shown in SEQ ID NO.9, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.10; first, the valine at the 170th position in the amino acid sequence of wild-type heparin C5-epimerase is site-directed mutated to arginine, the valine at the 260th position is site-directed mutated to leucine, and the leucine at the 276th position is site-directed mutated to valine, and then 49 amino acids at the N-terminus are truncated according to the pattern of heparin C5-epimerase mutant CbC5epi.

[0015] In the third aspect of the present invention, a recombinant vector is provided. The recombinant vector is constructed by inserting the nucleotide sequence of the above-mentioned heparin C5-epimerase mutant CbC5epi or the CbC5epi mutant into a plasmid vector. There is no particular limitation on the starting vector, and it can be any vector known in the art as long as it can replicate in the host. For example, the vector includes but is not limited to plasmids and phages. Once transformed into a suitable host, the vector can replicate and function independently of the host genome, or in some cases, integrate into the genome itself.

[0016] Further preferably, the plasmid vector is pMAL-c5x.

[0017] In the fourth aspect of the present invention, a recombinant cell is provided. The recombinant cell is obtained by transforming the above-mentioned recombinant vector into a host cell. The "host cell" herein has the meaning commonly understood in the art, which is a host cell capable of introducing the encoding gene of the mutant of the present invention, and is called a recombinant host cell after introduction. The strain of the present invention can be a prokaryotic cell or a eukaryotic cell, preferably a prokaryotic cell, and more preferably Escherichia coli OrigamiB (DE3) containing the molecular chaperone pGro7.

[0018] In the fifth aspect of the present invention, a method for fermentatively preparing heparin C5-epimerase mutant CbC5epi or the CbC5epi mutant is provided. The method includes culturing the above-mentioned host cell and separating and obtaining heparin C5-epimerase mutant CbC5epi or the CbC5epi mutant.

[0019] Preferably according to the present invention, the specific steps of the preparation method are: culturing the recombinant host cell containing the encoding gene of heparin C5-epimerase mutant CbC5epi or the CbC5epi mutant in LB liquid medium until the OD600 is 0.6 - 0.8, adding IPTG for induction expression for 16 - 18 h; collecting the thalli, ultrasonically disrupting, centrifuging and filtering, and purifying through the MBP tag to obtain heparin C5-epimerase mutant CbC5epi or the CbC5epi mutant.

[0020] The sixth aspect of the present invention provides the use of the above-mentioned heparin C5-epimerase mutant CbC5epi or the CbC5epi mutant in heparin oligosaccharides containing iduronic acid.

[0021] Preferably according to the present invention, the application uses the heparin pentasaccharide of GlcA-GlcNS-GlcA-GlcNS-GlcA- p NP as a substrate, uses PAPS as a sulfate donor, uses the heparin C5-epimerase mutant CbC5epi or the CbC5epi mutant, and 2- O -sulfotransferase as a catalytic enzyme, and after the reaction, a heparin oligosaccharide containing iduronic acid with the structure of GlcA-GlcNS-IdoA2S-GlcNS-GlcA- p NP is generated.

[0022] The experimental operations not described in detail in the present invention can be carried out according to the conventional experimental operations in the technical field.

[0023] Beneficial effects 1. The heparin C5-epimerase mutant CbC5epi disclosed in the present invention is a brand-new artificial heparin C5-epimerase derived from Caenorhabditis briggsae, and has the activity of converting GlcA in the structure of GlcNS-GlcA-GlcNS of heparin into IdoA. Compared with the existing HsC5epi derived from Homo sapiens ( Homo sapiens ), the CbC5epi provided by the present invention has a higher expression level, higher activity, a higher degree of the catalytic reaction proceeding in the forward direction, and better stability.

[0024] 2. In order to improve the enzyme performance, the present invention further performs protein engineering modification on the heparin C5-epimerase mutant CbC5epi to obtain a CbC5epi mutant with higher expression level, activity and stability. The activity of this CbC5epi mutant is increased to more than 16 times that of HsC5epi. The CbC5epi single-site mutant can still maintain more than 65% of the enzyme activity after incubation at 37 °C for 72 h, and the expression level is slightly increased, and there are significant improvements in both activity and stability. In particular, the CbC5epi mutant (V170R-V260L-L276V) has an expression level of about 31 mg / L, which is 3.6 times that of HsC5epi, and the activity is 1.36 μmol / (mg·h), which is increased by nearly 20 times. The remaining enzyme activity after incubation at 37 °C for 72 h can still be maintained at 1.11 μmol / (mg·h).

[0025] 3. The heparin C5-epimerase mutant CbC5epi and the CbC5epi mutant provided by the present invention have higher expression levels, activities and stabilities. When applied in biosynthesis, the efficiency of chemoenzymatic synthesis of anticoagulant heparin is effectively improved. At the same time, the types of heparin C5-epimerase are expanded, providing experience for the discovery and modification of other glycosaminoglycan synthesis-related enzymes. Description of the Drawings

[0026] Figure 1 SDS-PAGE results of heparin C5-epi truncates from different sources.

[0027] Figure 2 Expression level results of heparin C5-epi truncates from different sources; In the figure: The abscissa represents heparin C5-epi truncates from different sources, and the ordinate represents the protein expression level.

[0028] Figure 3 Activity results of heparin C5-epi truncates from different sources; In the figure: The abscissa represents heparin C5-epi truncates from different sources, and the ordinate represents the enzyme activity.

[0029] Figure 4 Equilibrium state of the reversible reaction catalyzed by heparin C5-epi truncates from different sources; In the figure: The abscissa represents heparin C5-epi truncates from different sources, and the ordinate represents the ratio of IdoA / GlcA.

[0030] Figure 5 Change of enzyme activity of heparin C5-epi truncates from different sources with incubation time at 37°C; In the figure: The abscissa represents the incubation time, and the ordinate represents the enzyme activity.

[0031] Figure 6 Change of product conversion rate of HsC5epi, DrBC5epi and CbC5epi catalyzing the reaction at different reaction times; In the figure: The abscissa represents the reaction time, and the ordinate represents the product conversion rate.

[0032] Figure 7 Comparison of the expression level, enzyme activity and remaining enzyme activity after incubation at 37°C for 72 h of single-site mutants of heparin CbC5epi; In the figure: The abscissa represents single-site mutants of heparin CbC5epi. The left ordinate represents the enzyme activity relative to CbC5epi, and the right ordinate represents the protein expression level.

[0033] Figure 8Comparison of the heparin CbC5epi multi-site mutants in terms of expression level, enzyme activity, and remaining enzyme activity after incubation at 37 °C for 72 h; In the figure: The abscissa represents the CbC5epi multi-site mutants, the left ordinate represents the enzyme activity relative to CbC5epi, and the right ordinate represents the protein expression level. Specific implementation manners

[0034] The technical solutions of the present invention will be further described below in conjunction with the embodiments and the accompanying drawings of the specification, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the present invention are all methods well known to those skilled in the art.

[0035] Example 1. Screening of heparin C5-epimerase mutant CbC5epi 1. The inventors searched for heparin C5-epimerase (C5-epi) in the PDB database and obtained C5-epi from Caenorhabditis briggsae ( Caenorhabditis brenneri ), with NCBI ACCESSION: EGT51961, denoted as CbC5epi; type A C5-epi from Danio rerio ( Danio rerio ), with NCBI ACCESSION: AAQ90466, denoted as DrAC5epi; type B C5-epi from Danio rerio ( Danio rerio ), with NCBI ACCESSION: AAQ90467, denoted as DrBC5epi; C5-epi from Caenorhabditis elegans ( Caenorhabditis elegans ), with NCBI ACCESSION: NP_497876, denoted as CeC5epi.

[0036] The amino acid sequences of the above 4 types of C5-epi from 3 sources were aligned with the truncated form of C5-epi (HsC5epi) from Homo sapiens ( Homo sapiens ), and the N-terminus was truncated accordingly according to the situation of HsC5epi to obtain CbC5epi, DrAC5epi, DrBC5epi, and CeC5epi respectively.

[0037] Among them, the NCBI ACCESSION of the HsC5epi is NP_001311023; the amino acid sequence of CbC5epi is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0038] The sequences of DrAC5epi, DrBC5epi, and CeC5epi not protected by the present invention will not be shown, but the methods for obtaining their sequences are the same as those of the present invention.

[0039] 2. Heterologous expression of different heparin C5-epimerase truncates Entrusted Nanjing Genscript Co., Ltd. to artificially synthesize the gene according to the nucleotide sequence shown in SEQ ID NO.1 CbC5epi and clone it onto the pMAL-c5x vector, then chemically transform it into OrigamiB (DE3) competent cells containing the molecular chaperone pGro7 E.coli and culture on LB solid medium containing carbenicillin (50 μg / mL), kanamycin (15 μg / mL), and chloramphenicol (37 μg / mL) for 20 h, screen for transformants (negative control experiments were also carried out simultaneously), and obtain positive transformants of heparin C5-epimerase truncate CbC5epi

[0040] Pick a single colony of the positive transformant of heparin C5-epimerase truncate CbC5epi and inoculate it into 25 mL of sterilized LB liquid medium (containing 50 μg / mL carbenicillin, 15 μg / mL kanamycin, and 37 μg / mL chloramphenicol), and culture at 37 °C and 225 rpm for 16 h. Inoculate the activated culture broth into 1 L of sterilized LB liquid medium (containing 50 μg / mL carbenicillin, 15 μg / mL kanamycin, and 37 μg / mL chloramphenicol) for scale-up culture at an inoculation amount of 1%, and culture at 37 °C and 225 rpm for 4 h until the OD600 is about 0.8, then add IPTG with a final concentration of 0.2 mM and arabinose with a concentration of 1 mg / L, and induce expression at 20 °C and 225 rpm for 20 h

[0041] After the culture is completed, centrifuge at 8000 rpm for 10 min to collect the cell precipitate, resuspend it with 1×PBS buffer, and break the resuspended solution with an ultrasonic instrument. Approximately 15 mL of solution is used to resuspend every 1 L of cell precipitate, and the effective breaking time is 2.5 min. The parameters of the ultrasonic instrument are set as follows: ultrasonic for 15 s, pause for 45 s, amplitude 60%, and energy 1500 kJ. After ultrasonic treatment, centrifuge the broken solution at 12000 rpm for 25 min and filter the supernatant with a 0.45 μm filter membrane. Purify the supernatant with an MBP affinity chromatography column. The purification process is as follows: equilibration, equilibrate the MBP affinity chromatography column with 1×PBS buffer for 4 column volumes; loading, load the filtered supernatant onto the MBP column; washing to remove impurities, wash the column with 1×PBS solution to remove impurity proteins, and the Bradford protein concentration assay kit can be used to detect whether the impurity proteins are completely removed; elution, elute with Elution buffer (1×PBS solution containing 20 mM maltose) to obtain the target protein heparin C5-epimerase truncate CbC5epi

[0042] According to the same heterologous expression method, HsC5epi, CeC5epi, DrBC5epi, and DrAC5epi were obtained respectively

[0043] CbC5epi, HsC5epi, CeC5epi, DrBC5epi, and DrAC5epi after purification were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and the results are as Figure 1 shown.

[0044] As Figure 1 can be seen, a clear band appeared in the lane of the heparin C5-epimerase truncation body CbC5epi. The molecular weight of CbC5epi fused with the MBP tag was approximately 100 kDa, which was easily cleaved after heating and boiling. The molecular weight of the MBP tag was about 40 kDa, and the molecular weight of CbC5epi was approximately 60 kDa. The molecular weight of the molecular chaperone pGro7 was about 55 kDa. Since the latter two had close molecular weights, the upper band in the figure was slightly wider. The size and position of the band were in line with expectations, indicating the successful heterologous expression of the heparin C5-epimerase truncation body CbC5epi.

[0045] 3. Comparison of the expression levels of different heparin C5-epimerase truncation bodies The protein concentration was measured using a Bradford protein concentration assay kit. According to the instructions, a protein standard was prepared. 5 μL of protein standards with different concentrations was added to the protein standard wells of a 96-well plate, and 5 μL of the sample was added to the sample wells of the 96-well plate (if the sample was less than 5 μL, the standard diluent needed to be added to make up to 5 μL. Please note the sample volume). 250 μL of G250 staining solution was added to each well, and the A595 was immediately measured using a microplate reader. The protein concentration in the sample was calculated based on the standard curve and the volume of the sample used. The expression level of the protein could be obtained by multiplying the measured protein concentration by the volume of the purified protein.

[0046] The expression levels of CbC5epi, HsC5epi, CeC5epi, DrBC5epi, and DrAC5epi were measured according to the above method, and the results are as Figure 2 shown.

[0047] As Figure 2 can be seen, the expression level of CbC5epi was the highest, approximately 15 mg / L, followed by the protein expression level of DrBC5epi, approximately 12.2 mg / L, both of which were higher than the expression level of HsC5epi.

[0048] 4. Comparison of the activities of different heparin C5-epimerase truncation bodies Using GlcA-GlcNS-GlcA-GlcNS-GlcA- pThe heparin backbone pentasaccharide of NP (5mer-NS, final concentration 0.2 mM) was used as the substrate, and HsC5epi, CeC5epi, CbC5epi, DrBC5epi, and DrAC5epi were used as catalytic enzymes for the reaction respectively. The reaction system is shown in Table 1. The reaction system was reacted at 37 °C for 4 h, and the reaction was terminated by heating and boiling for 5 min. After the reaction solution was filtered through a 0.22 μm filter membrane, HPLC detection was carried out according to the method described in Table 2. An amino column of YMC was used, and the detection wavelengths were 310 nm and 254 nm for UV detection. The flow rate of the mobile phase was 0.5 mL / min. The production rate of the IdoA product catalyzed by the enzyme was calculated according to the liquid phase results, and the obtained results were the activities of different C5-epimerase truncations. The results are as Figure 3 shown.

[0049] Table 1. Reaction systems of different heparin C5epi truncations.

[0050]

[0051] Table 2. HPLC elution program for detecting heparin oligosaccharides

[0052] It can be Figure 3 seen that except for DrAC5epi, the activities of the other three truncations are higher than that of HsC5epi. Among them, the enzyme activities of DrBC5epi and CbC5epi can reach 0.2 μmol / (mg·h).

[0053] 5. Comparison of the forward reaction progress of different heparin C5-epimerase truncations The reaction of C5-epi catalyzing GlcA-GlcNS-GlcA-GlcNS-GlcA- p NP to generate GlcA-GlcNS-IdoA-GlcNS-GlcA- p NP is a reversible reaction. IdoA2S largely determines the anticoagulant effect of heparin. Therefore, the applicant hopes that the reversible reaction catalyzed by C5-epi proceeds as much as possible in the forward direction to generate more IdoA products, which is convenient for the subsequent 2- O -sulfotransferase to carry out 2- O -sulfation reaction. According to the liquid phase results of the activity determination in point 4, the value of IdoA / GlcA in the reaction system was calculated. The results are as Figure 4 shown.

[0054] It can be Figure 4 seen that in the reversible reactions catalyzed by DrBC5epi and CbC5epi, the IdoA / GlcA can reach above 0.6, while in the reversible reaction catalyzed by HsC5epi, the IdoA / GlcA is only 0.18.

[0055] 6. Comparison of the Stability of Different Heparin C5-Epimerase Truncates First, HsC5epi, DrBC5epi, DrAC5epi, CbC5epi, and CeC5epi were incubated at 37 °C for 72 h, and samples were taken every 6 h or 12 h. Then, using the heparin backbone pentasaccharide (5mer-NS, final concentration 0.2 mM) of GlcA-GlcNS-GlcA-GlcNS-GlcA- p NP as the substrate, after different incubation times, HsC5epi, DrBC5epi, DrAC5epi, CbC5epi, and CeC5epi were used as catalytic enzymes for the reaction, and the reaction system is shown in Table 1. The reaction system was reacted at 37 °C for 4 h, and the reaction was terminated by heating and boiling for 5 min. After the reaction solution was filtered through a 0.22 μm filter membrane, HPLC detection was carried out according to the method described in Table 2. An amino column of YMC was used, the detection wavelengths were 310 nm and 254 nm, ultraviolet detection was performed, the mobile phase flow rate was 0.5 mL / min, and a line graph of the activity results of different heparin C5-epimerase truncates at different incubation times was drawn based on the HPLC chromatogram results. The results are as Figure 5 shown.

[0056] As Figure 5 can be seen, CbC5epi has the highest stability at 37 °C, with a half-life of approximately 48 h, while the activities of HsC5epi and DrBC5epi are lost after incubation at 37 °C for 6 h.

[0057] 7. Comparison of the Product Conversion Rates of Different Heparin C5-Epimerase Truncates in Catalytic Reactions at Different Reaction Times Using the heparin backbone pentasaccharide (5mer-NS, final concentration 0.2 mM) of GlcA-GlcNS-GlcA-GlcNS-GlcA- p NP as the substrate, HsC5epi, CbC5epi, and DrBC5epi were used as catalytic enzymes for the reaction, and the reaction system is shown in Table 1. The reaction system was reacted at 37 °C for 0.5 h, 1 h, 2 h, 3 h, and 4 h respectively, and the reaction was terminated by heating and boiling for 5 min. After the reaction solution was filtered through a 0.22 μm filter membrane, HPLC detection was carried out according to the method described in Table 2. An amino column of YMC was used, the detection wavelengths were 310 nm and 254 nm, ultraviolet detection was performed, the mobile phase flow rate was 0.5 mL / min, and the conversion rate of the enzyme-catalyzed IdoA product was calculated based on the liquid phase results. The results are as Figure 6 shown.

[0058] As Figure 6It can be seen that the reversible reaction catalyzed by HsC5epi reaches equilibrium at about 4 h, and the conversion rate is about 15%. The reversible reactions catalyzed by DrBC5epi and CbC5epi reach equilibrium at about 1 h. The conversion rate of CbC5epi is slightly higher than that of DrBC5epi, at about 41%, indicating that the reversible isomerization reaction catalyzed by CbC5epi is more inclined to the forward reaction, and the proportion of IdoA is higher when the reaction reaches equilibrium.

[0059] Based on the above data, it can be found that compared with HsC5epi, DrAC5epi, DrBC5epi, and CeC5epi, the heparin C5-epimerase mutant CbC5epi has higher expression, higher activity, a higher degree of catalyzing the forward reaction, and better stability. That is to say, the present invention successfully screened and provided a novel heparin C5-epimerase mutant CbC5epi derived from Caenorhabditis briggsae, which has the activity of converting GlcA in GlcNS-GlcA-GlcNS with heparin structure into IdoA.

[0060] Example 2. Screening of single-site mutants of CbC5epi 1. Input the amino acid sequence of CbC5epi (shown as SEQ ID NO.1) into the Swiss-Model website to predict the crystal structure of CbC5epi. At the same time, perform molecular docking of CbC5epi with the substrate. Combining various strategies such as PROSS engineering, multiple sequence alignment, and virtual saturation mutagenesis, analyze the hot-spot amino acid sites, and design 17 single-site mutants of CbC5epi, namely CbC5epi mutant (V54E), CbC5epi mutant (S56K), CbC5epi mutant (A57Q), CbC5epi mutant (S145N), CbC5epi mutant (V170R), CbC5epi mutant (M189Y), CbC5epi mutant (K190V), CbC5epi mutant (V260L), CbC5epi mutant (L276V), CbC5epi mutant (V413E), CbC5epi mutant (A418P), CbC5epi mutant (F453Y), CbC5epi mutant (K454H), CbC5epi mutant (D457K), CbC5epi mutant (I588L), CbC5epi mutant (L591I), CbC5epi mutant (D595P).

[0061] Among them, the amino acid sequence of the CbC5epi mutant (A418P) is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.4; the amino acid sequence of the CbC5epi mutant (F453Y) is shown in SEQ ID NO.5, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.6; the amino acid sequence of the CbC5epi mutant (L591I) is shown in SEQ ID NO.7, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.8.

[0062] 2. Construct single-site mutants by PCR. Design mutant primers, and hand over the mutant primers to Qingdao Ruibo and Shanghai Sangon for synthesis according to the sequence information. Using the nucleotide sequence of CbC5epi (SEQ ID NO.2) as a template and the sequences at the corresponding sites in Table 3 as primers for PCR amplification, the nucleotide sequences of the above-mentioned CbC5epi mutants (V54E), CbC5epi mutant (S56K), CbC5epi mutant (A57Q), CbC5epi mutant (S145N), CbC5epi mutant (V170R), CbC5epi mutant (M189Y), CbC5epi mutant (K190V), CbC5epi mutant (V260L), CbC5epi mutant (L276V), CbC5epi mutant (V413E), CbC5epi mutant (A418P), CbC5epi mutant (F453Y), CbC5epi mutant (K454H), CbC5epi mutant (D457K), CbC5epi mutant (I588L), CbC5epi mutant (L591I), and CbC5epi mutant (D595P) were obtained.

[0063] The reaction system of PCR: 20 μL of ddH2O, 25 μL of 2×Phanta Max Master Mix, 2 μL of F-primer (10 μM), 2 μL of R-primer (10 μM), and 1 μL of template, with a total volume of 50 μL.

[0064] The PCR amplification program: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, extension at 72°C for 60 s / kb, 30 cycles; extension at 72°C for 5 min; finally, keep at 4°C.

[0065] Table 3. Primers required for constructing single-site mutants

[0066] Then, according to the heterologous expression and purification method described in point 2 of Example 1, CbC5epi mutants (V54E), CbC5epi mutants (S56K), CbC5epi mutants (A57Q), CbC5epi mutants (S145N), CbC5epi mutants (V170R), CbC5epi mutants (M189Y), CbC5epi mutants (K190V), CbC5epi mutants (V260L), CbC5epi mutants (L276V), CbC5epi mutants (V413E), CbC5epi mutants (A418P), CbC5epi mutants (F453Y), CbC5epi mutants (K454H), CbC5epi mutants (D457K), CbC5epi mutants (I588L), CbC5epi mutants (L591I), and CbC5epi mutants (D595P) were prepared. Then, according to the methods described in points 3, 4, and 6 of Example 1, the expression levels, activities, and stabilities of the purified CbC5epi mutants (V54E), CbC5epi mutants (S56K), CbC5epi mutants (A57Q), CbC5epi mutants (S145N), CbC5epi mutants (V170R), CbC5epi mutants (M189Y), CbC5epi mutants (K190V), CbC5epi mutants (V260L), CbC5epi mutants (L276V), CbC5epi mutants (V413E), CbC5epi mutants (A418P), CbC5epi mutants (F453Y), CbC5epi mutants (K454H), CbC5epi mutants (D457K), CbC5epi mutants (I588L), CbC5epi mutants (L591I), and CbC5epi mutants (D595P) were detected and compared. Using heparin C5-epimerase mutant CbC5epi as a control, the measurement results are as Figure 7 shown.

[0067] Among them, for the stability of single-site mutants, only the activity of the enzyme after incubation at 37 °C for 72 h was measured as an index to measure stability. The activity and stability were measured according to the methods described in points 4 and 6 of Example 1, with the reaction time changed to 1 h and the enzyme concentration changed to 0.05 mg / mL, and the other conditions remained unchanged.

[0068] From Figure 7It can be seen that there are 5 single-site mutants with increased expression levels, namely CbC5epi mutant (S145N), CbC5epi mutant (V170R), CbC5epi mutant (A418P), CbC5epi mutant (F453Y), and CbC5epi mutant (L591I). Among them, the CbC5epi mutant (A418P) has the highest expression level, approximately 19.5 mg / L. There are 16 mutants with increased activity. Among them, the activity of the CbC5epi mutant (V170R) is increased to 1.38 times that of CbC5epi, the activity of the CbC5epi mutant (A418P) is increased to 1.35 times, the activity of the CbC5epi mutant (F453Y) is increased to 1.40 times that of CbC5epi, and the activity of the CbC5epi mutant (L591I) is increased to 1.41 times. Compared with CbC5epi, the residual enzyme activity of each mutant after incubation at 37°C for 72 h has been significantly improved. The residual enzyme activity of 11 mutants is basically the same as the original enzyme activity, and the stability is increased to more than 6 times that of CbC5epi.

[0069] Example 3. Screening of CbC5epi multi-site mutants Compared with CbC5epi, the above 17 single-site mutants have been improved in two or three aspects of expression level, activity, or stability. Therefore, based on these 17 sites, site combinations are carried out, and these 17 sites are grouped according to spatial positions as follows: V413E and A418P are the first group; V54E, S56K, and A57Q are the second group; S145N, M189Y, and K190V are the third group; V170R, V260L, and L276V are the fourth group; F453Y, K454H, and D457K are the fifth group; I588L, L591I, and D595P are the sixth group.

[0070] The specific construction process is as follows: For the first group, using the CbC5epi mutant (V413E) as a template and C5-418new-F and C5-418new-R as primers for amplification, the nucleotide sequence of the CbC5epi mutant (V413E-A418P) is obtained; For the second group, using the CbC5epi mutant (A57Q) as a template and C5-54-56-57-F and C5-54-56-57-R as primers for amplification, the nucleotide sequence of the CbC5epi mutant (V54E-S56K-A57Q) is obtained; Group 3: Using the CbC5epi mutant (S145N) as a template, and C5-189-190-F and C5-189-190-R as primers for amplification, the nucleotide sequence of the CbC5epi mutant (S145N-M189Y-K190V) was obtained; Group 4: Using the CbC5epi mutant (V170R) as a template, and C5-V260L-F and C5-V260L-R as primers for amplification, the nucleotide sequence of the CbC5epi mutant (V170R-V260L) was obtained. Continuing to use this as a template, and C5-L276V-F and C5-L276V-R as primers for amplification, the nucleotide sequence of the CbC5epi mutant (V170R-V260L-L276V) was obtained; Group 5: Using the CbC5epi mutant (D457K) as a template, and C5-453-454-457-F and C5-453-454-457-R as primers for amplification, the nucleotide sequence of the CbC5epi mutant (F453Y-K454H-D457K) was obtained; Group 6: Using the CbC5epi mutant (D595P) as a template, and C5-588-591-F and C5-588-591-R as primers for amplification, the nucleotide sequence of the CbC5epi mutant (I588L-L591I-D595P) was obtained. The required primer sequences are shown in Tables 3 and 4.

[0071] Among them, the amino acid sequence of the CbC5epi mutant (V170R-V260L-L276V) is as shown in SEQ ID NO.9, and the nucleotide sequence of the encoding gene is as shown in SEQ ID NO.10.

[0072] Table 4. Primers required for constructing multi-site mutants

[0073] Then, according to the heterologous expression and purification method described in point 2 of Example 1, CbC5epi mutants (V413E-A418P), CbC5epi mutants (V54E-S56K-A57Q), CbC5epi mutants (S145N-M189Y-K190V), CbC5epi mutants (V170R-V260L-L276V), CbC5epi mutants (F453Y-K454H-D457K) and CbC5epi mutants (I588L-L591I-D595P) were prepared. Then, according to the methods described in points 3, 4, and 6 of Example 1, the expression levels, activities, and stabilities of the purified CbC5epi mutants (V413E-A418P), CbC5epi mutants (V54E-S56K-A57Q), CbC5epi mutants (S145N-M189Y-K190V), CbC5epi mutants (V170R-V260L-L276V), CbC5epi mutants (F453Y-K454H-D457K) and CbC5epi mutants (I588L-L591I-D595P) were detected and compared. The measurement results are as Figure 8 shown.

[0074] Among them, the stability of the multi-site mutants was measured by the activity of the enzyme after incubation at 37 °C for 72 h as an index to measure stability. The activity and stability were measured according to the methods described in points 4 and 6 of Example 1, with the reaction time changed to 1 h and the enzyme concentration changed to 0.05 mg / mL, and the other conditions remained unchanged.

[0075] It can be Figure 8 seen that the expression level of the CbC5epi mutant (V170R-V260L-L276V) is about 31 mg / L, which is twice that of CbC5epi. However, in combination with Figure 7 comparison, the stability of the CbC5epi mutant (V170R-V260L-L276V) decreased by more than 30% compared with the corresponding single-site mutant, and the stabilities of V413E-A418P and F453Y-K454H-D457K decreased by more than 60%. The activities of V54E-S56K-A57Q, S145N-M189Y-K190V and I588L-L591I-D595P decreased by more than 40%. In contrast, the expression levels of the combined mutants increased.

[0076] From the above data, it can be found that the expression level of the CbC5epi mutant (V170R-V260L-L276V) is about 31 mg / L, which is 3.6 times that of HsC5epi. Its activity is 1.36 μmol / (mg·h), which is nearly 20 times higher. The residual enzyme activity after incubation at 37 °C for 72 h is 1.11 μmol / (mg·h). It is a CbC5epi mutant with good expression level, activity and stability.

Claims

1. A heparin C5-epimerase mutant CbC5epi, characterized in that, Its amino acid sequence is shown in SEQ ID NO.1, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

2.

2. A CbC5epi mutant (A418P), characterized in that, Its amino acid sequence is shown in SEQ ID NO.3, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

4.

3. A CbC5epi mutant (F453Y), characterized in that, Its amino acid sequence is shown in SEQ ID NO.5, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

6.

4. A CbC5epi mutant (L591I), characterized in that, Its amino acid sequence is shown in SEQ ID NO.7, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

8.

5. A CbC5epi mutant (V170R-V260L-L276V), characterized in that, Its amino acid sequence is shown in SEQ IDNO.9, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.

10.

6. A recombinant vector, characterized in that, The recombinant vector is constructed by inserting the nucleotide sequence of any one of heparin C5-epimerase mutant CbC5epi or CbC5epi mutant of claims 1 to 5 into a plasmid vector.

7. A recombinant cell, characterized in that, The recombinant cell is obtained by transforming the recombinant vector of claim 6 into a host cell.

8. A method for fermentatively preparing heparin C5-epimerase mutant CbC5epi or a mutant of CbC5epi, characterized in that, The method includes culturing the host cell of claim 7 and isolating and obtaining heparin C5-epimerase mutant CbC5epi or CbC5epi mutant.

9. Use of the heparin C5-epimerase mutant CbC5epi or CbC5epi mutant of any one of claims 1 to 5 in heparin oligosaccharides containing iduronic acid.

10. The application obtained as claimed in claim 9, characterized in that, The application uses heparin pentasaccharide of GlcA-GlcNS-GlcA-GlcNS-GlcA- p NP as a substrate, PAPS as a sulfate donor, heparin C5-epimerase mutant CbC5epi or CbC5epi mutant, and 2- O sulfotransferase as catalytic enzymes, and after the reaction, heparin oligosaccharide containing iduronic acid with the structure of GlcA-GlcNS-IdoA2S-GlcNS-GlcA- p NP is produced.

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