A heparinase mutant H1-6 and its engineered strain and application

Abstract

The present invention provides a heparinase mutant H1-6 and its engineering strain and application, belonging to the field of protein mutation technology. The heparinase mutant H1-6 provided by the present invention is subjected to the following mutations on the basis of the wild-type heparinase amino acid sequence: P10R, V45A and L160P. The present invention uses the error-prone PCR technology combined with CepPCR and MegawhopPCR to perform random mutations on the heparinase gene derived from Raoultellasp., and high-throughput screening is performed to obtain a heparinase mutant with high enzyme activity. The experimental results show that the enzyme activity of the recombinantly expressed heparinase mutant strain is more than 3726U / L, which is more than 97.6% higher than the wild-type heparinase enzyme activity, and has a high application value.

Description

Technical Field

[0001] The invention belongs to the technical field of protein mutation, and specifically relates to a heparinase mutant H1-6 and an engineering strain and application thereof. Background Art

[0002] In 1922, Howell extracted and separated a sugar substance from animal liver and formally named it heparin. [1] (unfractionated heparin, UFH), heparin is a sulphated aminodextran composed of iduronic acid and glucuronic acid. [2] It belongs to mucopolysaccharide, with a molecular weight of about 14000Da. It has anticoagulant and antithrombotic functions. [3] , and is currently one of the largest exported pharmaceutical raw materials in my country. Heparin has been used clinically to prevent and treat thromboembolism since 1935. [4] However, heparin can easily cause hypotension and shock during treatment. [5] Therefore, researchers have developed a new type of anticoagulant drug, low molecular weight heparin (LMWH), which has a smaller average molecular weight than unfractionated heparin, about 5000 Da. More importantly, its antithrombotic effect is better than heparin, and it has the advantages of good absorption after subcutaneous injection, high bioavailability, long half-life in the body, and low bleeding tendency. [6] Among the global heparin products, only one-third of heparin APIs are made into standard heparin preparations, while two-thirds of heparin is used as raw material to produce low molecular weight heparin or ultra-low molecular weight heparin. The market sales of low molecular weight heparin are increasing year by year. [7] At present, we are also developing new heparins with lower molecular weights - ultra low molecular weight heparin (ULMWH), with a molecular weight below 2500Da. [8] , its anticoagulant effect is better than the previous two, and the possibility of causing complications is lower. Therefore, ultra-low molecular weight heparin has become a hot spot in the new round of anticoagulant drug research and development.

[0003] The existing methods for preparing low molecular weight heparin include chemical depolymerization, physical preparation and bioenzyme. The chemical depolymerization method mainly uses nitrite to break the heparin glycosidic bond; the physical preparation method mainly uses ultrasonic degradation. [9]However, both chemical and physical methods generally have problems such as high preparation cost, low efficiency, high production cost, poor controllability of the degradation process, easily damaged drug functional structure heparin active functional groups, poor uniformity between products, and easy residues of reagents in the manufacturing process. Relatively speaking, the preparation of low molecular weight heparin by bioenzymatic method can avoid environmental pollution problems and has a higher yield. Therefore, the enzymatic degradation technology of using heparinase to prepare small molecular weight heparin is currently a hot topic in the field of preparing small molecular weight heparin. Nowadays, the key problem that needs to be solved in the production of heparin drugs by heparinase cleavage method is how to improve the enzymatic activity of heparinase.

[0004] References

[0005] [1]Wardrop D,Keeling D.The story of the discovery of heparin andwarfarin[J].British Journal of Haematology,2008,141(6):757-763.

[0006] [2]Jaques LB,Cho M H.The preparation of heparinase[J].TheBiochemical Journal,1954,58(2):25-26.

[0007] [3]Jordan RE, Oosta GM, Gardner WT, et al. The kinetics of hemostaticenzyme-antithrombin interactions in the presence of low molecular weight heparin[J]. The Journal of Biological Chemistry, 1980, 255(21):81-90.

[0008] [4]Vineeta S, Shafiul H, Vibha K, et al. Isolation, purification, and characterization of heparinase from streptomyces variabilis MTCC 12266[J]. Scientific Reports, 2019, 9(1):6482.

[0009] [5]Yilmazel UE,Metin A,Omer A,et al.Comparison of LMWH versus UFH for hemorrhage and hospital mortality in the treatment of acute massivepulmonary thromboembolism after thrombolytic treatment:randomizedcontrolledparallel group study[J].Lung,2015,193(1):121-127.

[0010] [6] He Yueyue. Research on the process of enoxaparin sodium[D]. Hebei University of Science and Technology, 2020.

[0011] [7] Ren Yonghai, Li Xiaoyan, Huang Lei, et al. Expression and purification of recombinant heparinase I in Escherichia coli [J]. Pharmaceutical Biotechnology, 2012, 19(05): 377-380.

[0012] [8]Tolga G, Zubeyde AP, Eyup Y, et al. Effects of low molecular weight heparins and unfractionated heparin on viability of human umbilical veinendothelial cells[J]. Archives of Gynecology and Obstetrics, 2013, 287(2):217-222.

[0013] [9]Miyazaki T, Yomota C, Okada S. Ultrasonic depolymerization ofhyaluronic acid[J]. Polymer Degradation and Stability, 2001, 74(1):77-85. Summary of the invention

[0014] In view of this, the purpose of the present invention is to provide a heparinase mutant H1-6, which randomly mutates the heparinase gene at the molecular biology level and screens out a heparinase mutant with significantly improved enzyme activity, which is more than 97.6% higher than that of wild-type heparinase.

[0015] The present invention provides a heparinase mutant H1-6, the amino acid sequence of the heparinase mutant is shown in SEQ ID NO: 1, and the following mutations are performed on the basis of the amino acid sequence of wild-type heparinase: P10R, V45A and L160P.

[0016] The present invention provides a gene encoding the heparinase mutant H1-6, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO:3.

[0017] The present invention provides a recombinant vector, in which the coding gene or the nucleotide sequence encoding the heparinase mutant H1-6 is inserted into a backbone vector.

[0018] The present invention provides an engineering strain for recombinantly expressing the heparinase mutant, comprising a sequence encoding the heparinase mutant H1-6, the encoding gene or the recombinant vector.

[0019] The invention provides an immobilized enzyme, which uses calcium alginate gel as a carrier to immobilize the heparinase mutant H1-6.

[0020] The present invention provides a method for preparing the immobilized enzyme, comprising the following steps:

[0021] 1) recombinantly expressing the heparinase mutant H1-6 to obtain a solution containing the heparinase mutant recombinant protein;

[0022] 2) Mixing the solution containing the heparinase mutant recombinant protein with a sodium alginate solution, dripping the obtained mixed solution into a CaCl2 solution, cooling and solidifying, and obtaining an immobilized enzyme.

[0023] Preferably, the method for recombinant expression in step 1) is to culture the engineered strain in a medium containing antibiotics, induce expression under 0.1 mmol / L IPTG conditions, lyse the bacteria, and collect the supernatant.

[0024] Preferably, the volume ratio of the solution containing the heparinase mutant recombinant protein to the sodium alginate solution in step 2) is 0.8 to 1.2:1;

[0025] The mass percentage of the sodium alginate solution is 8%;

[0026] The concentration of the CaCl2 solution is 0.1 mol / L.

[0027] The invention provides the use of the heparinase mutant H1-6, the engineered strain or the immobilized enzyme in catalytic production of heparin.

[0028] The present invention provides a heparinase mutant H1-6, the amino acid sequence of which is SEQ ID NO: 1, and the following mutations are carried out on the basis of the amino acid sequence of wild-type heparinase: P10R, V45A and L160P. The present invention uses the error-prone PCR technology combined with CepPCR and MegawhopPCR to carry out random mutation on the heparinase gene derived from Raoultella sp., and obtains a heparinase mutant with high enzyme activity through high-throughput screening. The experimental results show that the enzyme activity of the recombinantly expressed heparinase mutant strain is more than 3726U / L, while the enzyme activity of the wild-type strain is only 1886U / L. The heparinase mutant provided by the present invention has an enzyme activity increased by more than 97.6% compared with the wild-type heparinase, and has a high application value.

[0029] The present invention provides an immobilized enzyme, which uses calcium alginate gel as a carrier to immobilize the heparinase mutant H1-6. The immobilized enzyme has improved temperature stability, pH stability and storage stability compared to free heparinase. Experimental results show that in the study of thermal stability, the free enzyme begins to inactivate at 55°C, and has almost no enzyme activity at 65°C, while the immobilized enzyme has relatively stable enzyme activity between 35 and 55°C, and at 65°C, the enzyme activity remains above 65%, and has good heat resistance. In the study of acid-base stability, the enzyme activity of the free enzyme decreases at pH 7 to 9, while the immobilized enzyme maintains relatively stable enzyme activity at pH 5 to 9, and the acid-base stability is significantly improved relative to the free enzyme. The storage stability study of the free enzyme and the immobilized enzyme shows that after the free enzyme is stored at 4°C for 3 days, the enzyme activity drops to about 40% of the original, and the enzyme activity is close to 0 after the 9th day, while the storage stability of the immobilized enzyme is improved, and the enzyme activity can still maintain the original 40% on the 9th day. The immobilized enzyme was used repeatedly for 10 times under the same conditions. As the number of uses increased, the enzyme activity decreased first quickly and then slowly. After 6 uses, the enzyme activity was about 50% of the original. In general, compared with free enzymes, the thermal stability, acid-base stability and storage stability of immobilized enzymes are improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 A flow chart of the screening experiment of the heparinase mutant H1-6 provided by the present invention;

[0031] Figure 2 The results of CepPCR gel imaging under different manganese ion concentrations, M: DL 5000 DNA marker (1: P1, C (Mn 2+ )=0.10mmol / L; 2: P1, C(Mn2+)=0.30mmol / L; 3: P1, C(Mn 2+ )=0.50mmol / L; 4: P1, C(Mn 2 +)=0.70mmol / L; 5: P1, C(Mn 2+ )=0.90mmol / L; 6: P2, C(Mn 2+ )=0.10mmol / L; 7: P2, C(Mn 2+ )=0.30mmol / L; 8: P2, C(Mn 2+ )=0.50mmol / L; 9: P2, C(Mn 2+ )=0.70mmol / L; 10: P2, C(Mn 2+ )=0.90mmol / L; 11: P3, C(Mn 2+ )=0.10mmol / L; 12: P3, C(Mn 2+ )=0.30mmol / L; 13: P3, C(Mn 2+ )=0.50mmol / L; 14: P3, C(Mn 2+ )=0.70mmol / L; 15: P3, C(Mn 2+ )=0.90mmol / L);

[0032] Figure 3 Agarose gel electrophoresis results of CepPCR products, M: DL5000 DNA Marker; 1: P4 PCR product; 2: P5 PCR product;

[0033] Figure 4 This is the gel electrophoresis of Megawhop PCR products, M: DL 15000 Marker; 1: amplified by primers of fragment 1; 2: amplified by primers of fragment 2; 3: amplified by primers of fragment 3;

[0034] Figure 5 This is the gel electrophoresis of Megawhop PCR products, M: DL 5000Marker; 1: amplified by primers for fragment 4; 2: amplified by primers for fragment 5;

[0035] Figure 6 Agarose gel electrophoresis of colony PCR products, M: DL 5000DNAMarker; 1-13: target gene fragment mutant strains; 14: positive control;

[0036] Figure 7 Agarose gel electrophoresis of colony PCR products, 1-9: target gene fragment mutant strain; 10: positive control; M: DL 5000DNAMarker;

[0037] Figure 8 It is a monoclonal colony image;

[0038] Fig. 9is the heparin standard curve;

[0039] Fig.10 Enzyme activity was tested repeatedly for one round of mutagenesis;

[0040] Fig.11 This is the result of comparison of amino acid mutation sites of heparinase;

[0041] Fig.12 The active site diagram of the three-dimensional model of heparinase docking with the lowest binding energy molecule;

[0042] Fig.13 This is the heparin standard curve measured in Example 2 of the present invention. DETAILED DESCRIPTION

[0043] The invention provides a heparinase mutant H1-6. The heparinase mutant H1-6 is subjected to the following mutations based on the wild-type heparinase amino acid sequence SEQ ID NO: 1 (MRMKQYLIARSILSADFARLGEDTAKALAAGADVVHFDVMDNHYAPNLTIGPMVLKALRNYGITAPIDVHLMVKPVDRIIPDFAAAGASIITFHPEASEHVDRSLQLIKEHGCKAGLVFNPATPLSYLDYVMDKLDVILLMSVNPGFGGQSFIPQTLDKPREVRQRIDASGYDIRLEVDGGVKVSNIADIAAAGADMFVAGSAIFDRPDYKEVIDQMRSELAKVSHG*): P10R, V45A and L160P.

[0044] In the present invention, homology modeling and molecular docking results show that the docking binding energy of the heparinase mutant H1-6 and the substrate docking is -3.4kcal / mol, and its root mean square deviation is 0.73, while the docking binding energy of the wild-type heparinase is -5.7kcal / mol; the potential binding sites of the heparinase mutant H1-6 configuration are SER11, ASP38, GLY146GLY149, ASP179, GLY181, VAL182, VAL199, GLY201, SER202, while the potential binding sites of the wild-type heparinase mutant H1-6 include SER14, ASP179, VAL182, SER202 and ALA203. It can be seen that the mutation of heparinase increases the number of amino acid residues bound to the active site, thereby ensuring that the enzyme activity of the heparinase mutant is significantly improved compared with the wild-type heparinase.

[0045] The present invention provides a gene encoding the heparinase mutant H1-6, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO:3().

[0046] The present invention provides a recombinant vector, in which the coding gene or the nucleotide sequence encoding the heparinase mutant H1-6 is inserted into a backbone vector.

[0047] The present invention has no particular restrictions on the type of the backbone vector, and a backbone vector well known in the art can be used, such as a soluble expression vector PRHis-MBP. The insertion position is preferably at the Xho I / BamHI multiple cloning site of the backbone vector. The present invention has no particular restrictions on the insertion method, and a double enzyme digestion and ligation method well known in the art can be used.

[0048] The present invention provides an engineering strain for recombinantly expressing the heparinase mutant H1-6, comprising a sequence encoding the heparinase mutant H1-6, the encoding gene or the recombinant vector.

[0049] The initial strain of the engineering strain of the present invention is preferably E. coli BL21 (DE3). The present invention has no special restrictions on the preparation method of the engineering strain, and a transformation method known in the art can be used.

[0050] The invention provides an immobilized enzyme, which uses calcium alginate gel as a carrier to immobilize the heparinase mutant H1-6.

[0051] The present invention provides a method for preparing the immobilized enzyme, comprising the following steps:

[0052] 1) recombinantly expressing the heparinase mutant H1-6 to obtain a solution containing the heparinase mutant recombinant protein;

[0053] 2) Mixing the solution containing the heparinase mutant recombinant protein with a sodium alginate solution, dripping the obtained mixed solution into a CaCl2 solution, cooling and solidifying, and obtaining an immobilized enzyme.

[0054] The present invention recombinantly expresses the heparinase mutant H1-6 to obtain a solution containing the heparinase mutant recombinant protein.

[0055] In the present invention, the recombinant expression method is preferably to culture the engineered strain in a medium containing antibiotics, induce expression under 0.1 mmol / L IPTG conditions, lyse the bacteria, and collect the supernatant. The medium containing antibiotics is preferably LB medium containing 50 μg / mL kanamycin. The culture conditions are preferably 37°C, 200 rpm in a shaker for 4 to 5 hours. Cultivate until OD 600 The value is 0.5 to 0.7, and more preferably 0.6. The induction expression condition is preferably induced in a shaker at 30°C and 200 rpm for 9 hours.

[0056] After obtaining the solution containing the heparinase mutant recombinant protein, the present invention mixes the solution containing the heparinase mutant recombinant protein with a sodium alginate solution, drips the obtained mixed solution into a CaCl2 solution, cools and solidifies, and obtains an immobilized enzyme.

[0057] In the present invention, the volume ratio of the solution containing the heparinase mutant recombinant protein to the sodium alginate solution is preferably 0.8 to 1.2:1, more preferably 1:1. The mass percentage of the sodium alginate solution is 8%. The solvent of the sodium alginate solution is preferably a 0.0025M calcium acetate buffer containing 0.25M sodium acetate. The concentration of the CaCl2 solution is 0.1mol / L. The method of dripping is preferably to transfer the mixed solution to a syringe, squeeze the syringe pusher, and drip it drop by drop into the CaCl2 solution to form a small ball of a certain diameter.

[0058] In the present invention, the immobilized enzyme is beneficial to improving the temperature, pH value and storage stability of the heparinase mutant H1-6 compared with the free enzyme.

[0059] In view of the fact that the heparinase mutant H1-6, the engineered strain or the immobilized enzyme all have high catalytic activity, the present invention provides the use of the heparinase mutant H1-6, the engineered strain or the immobilized enzyme in the catalytic production of heparin.

[0060] The following is a detailed description of a heparinase mutant H1-6 and its engineered strain and application provided by the present invention in conjunction with the examples, but they should not be construed as limiting the scope of protection of the present invention.

[0061] Example 1

[0062] A method for obtaining heparinase mutant H1-6

[0063] Materials and instruments

[0064] 1. Experimental strains and plasmids

[0065] The laboratory has successfully constructed plasmid pEAS-1b-H1, transferred it into E. coli BL21 and successfully induced the expression of a small amount of heparinase protein. E. coli BL21 competent cells were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd., and E. coli Top10 heat shock competent cells were purchased from Shanghai Weidi Biotechnology Co., Ltd.

[0066] 2. Primers and DNA Sequencing

[0067] The synthesis of primers and DNA sequencing results used in this experiment were provided by Guangzhou Aigene Biotechnology Co., Ltd.

[0068] 3. Experimental reagents and experimental instruments

[0069] The experimental reagents used in this experiment are shown in Table 1, and the experimental instruments involved are shown in Table 2.

[0070] Table 1 Experimental reagents

[0071]

[0072]

[0073] Table 2 Experimental instruments

[0074]

[0075]

[0076] 4 Solution preparation

[0077] (1) LB liquid medium (w / v): 1% tryptone, 0.5% yeast powder, 1% sodium chloride, pH 7.0. Sterilize at 121°C under high pressure for 20 min, cool and store in a refrigerator at 4°C until use.

[0078] (2) Ampicillin-resistant LB liquid medium (containing 100 μg / mL ampicillin): Take out the reserved LB liquid medium, add ampicillin to a final concentration of 100 μg / mL, and mix well.

[0079] (3) LB solid medium (containing 100 μg / mL ampicillin): 1% tryptone, 0.5% yeast powder, 1% sodium chloride, 2% agar powder, pH 7.0, sterilize at 121°C for 20 min, shake the medium to mix the medium. The following operations are performed in a clean workbench: Cool the room temperature to about 50°C, add ampicillin to a final concentration of 100 μg / mL, shake evenly and pour into a sterile culture dish, wait for it to cool and fully solidify, and seal it with a sealing film. If it is not used immediately, it can be placed in a 4°C refrigerator upside down for later use.

[0080] (4) Heparin sodium solution: Add 0.040 g of heparin sodium to every 1 mL of ultrapure water to prepare a 4.0% (w / v) heparin sodium solution. Subsequent dilution operations are all diluted with ultrapure water. Before taking the heparin sodium solution, the solution is placed on a vortex machine for shaking to ensure uniform dissolution of the solute.

[0081] (5) Azure A solution: Accurately weigh 0.50 g of Azure A and dissolve it in 10 mL of ultrapure water. Shake thoroughly to dissolve it completely. Then take 2 mL of the above solution to make it 500 mL. Filter it through filter paper and store it in a refrigerator at 4°C. Dilute it 10 times before use to prepare a 0.002% Azure A solution.

[0082] (6) 0.25 M sodium acetate-0.0025 M calcium acetate (pH 7.0) buffer: 20.5 g sodium acetate and 0.4405 g calcium acetate, adjust pH to 7.0, and make up to 1 L.

[0083] 5. Random Mutation Method

[0084] 5.1 Cultivation of wild strains

[0085] Using the genome of Raoultella sp. NX-TZ-3-15 as a template, the primer pair H1-SF-Sac I: TCGACGAGCTCGATGCGGATGAAGCAGTATTTG (as shown in SEQ ID NO: 15), H1-SR-Xba I: GCTGCTCTAGATTATCCATGACTAACCTTTGCT (as shown in SEQ ID NO: 16) was designed for PCR amplification, and restriction site sequences of Sac I and Xba I were introduced at both ends of the target gene H1 (as shown in SEQ ID NO: 4). The obtained gene H1 was inserted between the two restriction sites of Sac I and Xba I of the pEAS-1b plasmid, and the obtained recombinant plasmid pEAS-1b-H1 was transformed into the competent E. coli BL21, and after resistance screening and culture, the strain E. coli BL21-pEAS-1b-H1 was obtained.

[0086] Shake flask culture: Use a pipette to inoculate 1% of the constructed strain E. coli BL21-pEAS-1b-H1 into 10 mL of LB medium (containing 100 μg / mL of ampicillin), and shake culture in a constant temperature shaker at 30°C and 200 rpm for 12 h.

[0087] 5.2 Determination of high-throughput screening system

[0088] The commonly used method for detecting heparin content is the Azure A detection method. The conventional detection dosage is: add 200μL crude enzyme solution and 50μL heparin solution (0.025g heparin sodium standard is dissolved with 1mL 0.25mmol / L sodium acetate-0.0025M calcium acetate buffer solution) into the EP tube, mix thoroughly, and immediately take 5μL solution into 5mL 0.002% Azure A solution. In the high-throughput screening system, the amount of reagents is reduced, so it is convenient for a large number of mutant screening. First, use a 96-deep-well culture plate to induce the culture of Escherichia coli containing heparinase, and place it in a 37°C water bath to rupture the cells and release the heparinase. Use an 8-channel pipette to add 100 μL crude enzyme solution and 25 μL heparin solution (0.025 g heparin sodium standard is dissolved in 1 mL 0.25 mmol / L sodium acetate-0.0025 M calcium acetate buffer solution) into a 96-well ELISA plate, cover with a sealing film, and place on a metal heat preservation instrument at 30°C and 100 rpm for heating reaction. Immediately take 1 μL of the solution and color it in 1 mL 0.002% Azure A solution. Measure the absorbance of the coloring solution at 620 nm using an ELISA reader.

[0089] In the entire high-throughput system, in order to ensure the reliability of the screening results, the same wild-type heparinase strain was first used to perform high-throughput detection of enzyme activity to observe the relative deviation of the test results.

[0090] 5.3 Plasmid extraction

[0091] The plasmid of the above strains was extracted using Omega's Plasmid Mini Kit ID6943. For specific experimental procedures, see the instructions of Omega's Plasmid Mini Kit ID6943.

[0092] 5.4 Exploration of CepPCR amplification and its optimal manganese ion concentration

[0093] One round of CepPCR mutation: According to the 684bp base number of the target heparinase gene sequence of plasmid pEAS-1b-H1, the target gene H1 was divided into three fragments of about 200bp in length, named P1, P2 and P3 (where P1 fragment length is 220bp, P2 fragment length is 220bp, and P3 fragment length is 244bp), and primer pairs were designed at both ends of the three gene fragments, and CepPCR amplification was performed to obtain mutant gene fragments. The sequence of the primer pair is shown in Table 3. The CepPCR amplification reaction system (50μL) is shown in Table 4. The reaction procedure of CepPCR is shown in Table 5.

[0094] Table 3 Primer sequences

[0095]

[0096] Table 4 CepPCR system

[0097]

[0098]

[0099] Table 5 CepPCR program

[0100]

[0101] Second round of CepPCR mutation: The template selected in the error-prone amplification system is the plasmid of the mutant with the highest enzyme activity screened out after the previous round of mutation. The error-prone segmented PCR is divided into two fragments of about 400 bp in length, named P4 and P5 (wherein the P4 fragment is 440 bp in length and the P5 fragment is 460 bp in length, with about 200 bp of base repeats between the two), and primer pairs are designed at both ends of the two gene fragments, and CepPCR amplification is performed to obtain the mutant heparinase gene fragment. The sequence of the primer pair is shown in Table 6.

[0102] Table 6 Primer sequences

[0103]

[0104] Table 7 CepPCR system

[0105]

[0106] CepPCR reaction procedure: same as Table 5.

[0107] 5.5 Agarose gel electrophoresis of CepPCR products

[0108] The above CepPCR products were analyzed by agarose gel electrophoresis, and the specific operation was as follows:

[0109] Weigh 0.5g agarose and add 50mL 1×TAE solution, heat in a microwave oven for 1min to ensure that the agarose is fully dissolved and a transparent and clear solution is obtained; after cooling to 50℃-60℃ at room temperature, add 5μL 10000×Gel Red nucleic acid stain, shake well, pour into the gelatin plate, insert a tooth comb, and remove any bubbles in time. Let it stand at room temperature until the gelatin solution is completely solidified (about 20-30min);

[0110] After the gel solidifies, gently pull out the tooth comb and place the colloid in an electrophoresis tank filled with 1×TAE solution, with the sample loading hole facing the negative electrode.

[0111] Mix 5 μL of sample with 1 μL of 6× Loading Buffer by pipetting, and add 5 μL of the mixed sample to the loading well. Adjust the voltage to 120V, and perform electrophoresis at room temperature for 20 minutes; after the electrophoresis is completed, place the gel block in the gel imaging system for observation and photography.

[0112] 6. Purification and recovery of CepPCR products

[0113] CepPCR products were recovered using TaKaRa MiniBEST DNA Fragment Purification Kit Ver.4.0. For specific experimental procedures, see the kit instructions.

[0114] 7Megawhop PCR

[0115] Select Mn in CepPCR system 2+ The CepPCR purified product with a concentration of 0.3 mmol / L was used as the large primer for Mega-whopPCR, and the reaction system (50 μL) was as shown in Table 8.

[0116] Table 8 Megawhop PCR system

[0117]

[0118] The reaction procedure of Megawhop PCR is shown in Table 9.

[0119] Table 9 Megawhop PCR program

[0120]

[0121] 8DpnI digestion and purification

[0122] DpnI enzyme is a nucleic acid restriction enzyme that recognizes methylated GATC and is often used to eliminate parental DNA. The template plasmid is derived from conventional Escherichia coli and is modified by dam methylation, while the plasmid with a mutant sequence synthesized in vitro is not methylated and is not cut, and can be successfully transformed in the subsequent transformation competent cells.

[0123] The successfully verified Megawhop PCR product was digested with DpnI, and the reaction system (50 μL) was as shown in Table 10.

[0124] Table 10DpnI restriction enzyme system

[0125]

[0126] The DpnI digestion procedure is:

[0127] Table 11Dpn I restriction enzyme digestion program

[0128]

[0129] 9 Transformation of E.coli Top10 competent cells

[0130] Take 1 μL of the digested product in step 8 and transform it into E. coli Top10 heat shock competent cells. For specific experimental operations, refer to the instructions of E. coli Top10 competent cells. Spread each 100 μL of the transformed bacterial solution evenly on the solid LB medium containing ampicillin resistance, and culture it in a 30°C constant temperature biochemical incubator for 12-17 hours, not more than 20 hours, and observe the growth of the colonies. In this way, a heparinase gene mutation library can be obtained.

[0131] 10. Positive conversion rate verification

[0132] Randomly select more than ten single colonies from the solid LB medium in step 9 and place them in 20 μL ultrapure water, heat at 95°C for 5 minutes, and centrifuge for 1 minute. The supernatant contains the plasmid released by the high temperature cell disruption, which can be used as a template for colony PCR. The positive control is the pEAS-1b-H1 plasmid.

[0133] The colony PCR system (10 μL) is as shown in Table 12:

[0134] Table 12 PCR system

[0135]

[0136] The colony PCR program is shown in Table 13.

[0137] Table 13 PCR program

[0138]

[0139] The above products were subjected to agarose gel electrophoresis, and the experimental method was the same as 5.

[0140] 11 Transformation into E.coliBL21 competent cells

[0141] In an ultra-clean workbench, the solid culture medium plate on which the colonies grew in step 9 was eluted with LB liquid culture medium (containing ampicillin resistance), the eluate was collected in a sterilized centrifuge tube, and the plasmid was extracted according to the method of the plasmid extraction kit.

[0142] All plasmids containing the mutant library were transformed into E. coli BL21 competent cells. The transformation method was described in the instruction manual of E. coli BL21 competent cells. Then the bacterial solution was spread on a solid LB medium containing ampicillin resistance and cultured in a constant temperature incubator at 30°C for 17 hours. After a large number of E. coli BL21-pEAS-1b-H1 mutant single colonies were grown, the solid culture medium was stored in a 4°C refrigerator and waited for subsequent bacterial selection and activation culture.

[0143] 12 Mutant culture and induced expression

[0144] (1) Open the sterilized sealed 96-well plate on a sterile workbench, add 1 mL of 100 μg / mL Amp-resistant liquid LB medium to each well, pick about 1000 monoclonal mutants obtained in step 11 into the liquid medium with a sterilized toothpick, and seal with a sterile sealing film. The culture temperature is 30°C, the rotation speed is 700 rpm, and the activation culture is carried out overnight.

[0145] (2) Induced expression: Add 1 mL of 100 μg / mL Amp resistant liquid LB medium to each 96-well plate, and inoculate the bacterial solution in the same order into a new 96-well plate at a 2% inoculum. Place the plate in a constant temperature shaker at 30°C and 700 rpm. When the OD 600 When the value is 0.70-0.80, IPTG is added to make the final concentration of IPTG in the system 0.25mmol / L, and the induction culture is continued for 9h.

[0146] (3) Preparation of crude enzyme solution: After IPTG-induced expression is completed, the 96-well plate is placed in a 37°C water bath for cell autolysis reaction. After 0.5 h of reaction, the crude enzyme solution is obtained.

[0147] 13 High Throughput Screening

[0148] Determination of enzyme activity in high-throughput screening: 100 μL crude enzyme solution + 25 μL heparin solution (0.025 g heparin sodium standard is dissolved in 1 mL 0.25 M sodium acetate-0.0025 M calcium acetate buffer solution), mix thoroughly and keep warm at 30°C for 15 min. Add 1 mL of 0.002% Azure A to each well of a 96-well deep-well plate for later use. Immediately after the crude enzyme solution and heparin are kept warm for 15 min, take 1 μL of the crude enzyme solution-heparin mixture and add it to 1 mL 0.002% Azure A solution. After fully blowing and mixing, take 200 μL / well and add it to a 96-well ELISA plate, and measure the absorbance value at 620 nm-15 min respectively.

[0149] 14 Enzyme activity detection of mutant strains with excellent enzyme activity

[0150] (1) Cultivation of mutant strains and induced expression: In a clean bench, add 10 mL of LB medium containing ampicillin resistance (100 μg / mL Amp resistance) to each sterilized conical flask, inoculate the selected mutant strains with excellent enzyme activity at a 2% inoculum, place in a constant temperature shaker, and culture at 30°C, 200 rpm until OD 600 When the value was 0.70-0.80, IPTG was added to make the final concentration of IPTG 0.25 mmol / L. After returning to the constant temperature incubator, the cells were induced to culture at the same temperature and rotation speed for 9 hours.

[0151] After the induction culture is completed, the conical flask is placed in a preheated 37°C water bath for lysis reaction. After 0.5 hours of reaction, the crude enzyme solution is obtained by thermal self-lysis. The 37°C thermal reaction initiates SRRz, a phage lysis gene, which is combined with a promoter induced by temperature signals to develop an efficient and simple lysis method. Compared with the traditional ultrasonic lysis method, this new method shortens the time to 30 minutes and has high efficiency, with a lysis rate of 60% to 90%.

[0152] (2) Heparin standard curve determination: Dilute 4.0% heparin sodium solution with ultrapure water to prepare 1 mL of 0.10%, 0.20%, 0.30%, 0.40%, and 0.50% (w / v) heparin sodium standard solution. Take 6 test tubes and add 5 mL of 0.002% Azure A solution to each test tube. Add 5 μL of ultrapure water to one of the test tubes as a blank control. Add 5 μL of 0.10% to 0.50% heparin sodium standard solution to the other test tubes in sequence. After being thoroughly pipetted and mixed, take 200 μL and add it to a 96-well ELISA plate. Measure the absorbance at 620 nm in an ELISA instrument (make 3 sets of parallel samples and take the average value).

[0153] (3) Determination of quasi-enzyme activity: 200 μL crude enzyme solution + 50 μL heparin solution (0.025 g heparin sodium standard is dissolved in 1 mL 0.0025 M calcium acetate buffer solution containing 0.25 M sodium acetate), mix thoroughly and keep warm at 30°C for 15 min, then immediately take 5 μL crude enzyme solution and add it to 5 mL 0.002% azure A solution, mix thoroughly and take 200 μL / well and add it to a 96-well plate (make 3 sets of parallel samples and take the average value), measure the absorbance at 620 nm-0 min and 620 nm-15 min.

[0154] (4) Definition of enzyme activity: Under the conditions of 30°C and pH=7.0, the amount of enzyme required to degrade 1 mg of heparin per hour is one unit (U). Enzyme activity calculation formula I: The heparin content (μg) in the whole system at different times can be obtained from the standard curve. The difference between the heparin content at two different times is the amount of heparin degradation during this period.

[0155]

[0156] Amino acid comparison of 15 mutants

[0157] The mutant strain with good enzyme activity was sent to Ebi Bio-Sequencing Company for sequencing to obtain the gene sequence of the mutant strain. The gene sequence was converted into an amino acid sequence using Snapgene software, and then compared with the original heparanase amino acid sequence using Clustal Omega software (http: / / www.ebi.ac.uk / Tools / msa / clustalo / ).

[0158] 16 Homology modeling and molecular docking

[0159] After sequencing, the target gene sequence of the mutant strain was obtained, and the Alphafold (https: / / alphafold.com / ) online software was used to simulate the wild-type heparinase and the mutant heparinase protein structure model. The binding energy between the heparinase protein and the heparin molecule was calculated using the AutoDock software (https: / / autodock.scripps.edu / ). Then, the hydrogen bonds between the heparinase protein and the heparin molecule were drawn using the Pymol software (https: / / pymol.org / 2 / ).

[0160] result

[0161] 1. Relative deviation of high-throughput screening system

[0162] Table 14 shows the OD of the same strain cultured on a 96-well plate, the crude enzyme solution obtained after thermal lysis and fragmentation, and the enzyme activity measured by the Azure A method 620 The relative deviation of 96 absorbance values ​​was calculated to be 6.34%, which was less than the set value of 12%, indicating that this method was suitable for high-throughput screening of heparinase.

[0163] Table 14 High throughput screening values

[0164]

[0165]

[0166] 2 Optimization of manganese ion concentration in CepPCR system

[0167] A round of error-prone PCR mutation: Figure 2 As shown, the CepPCR products with different manganese ion concentrations were imaged after agarose gel electrophoresis. Lanes 1 to 5 are the electrophoresis results of CepPCR products with template P1 added, lanes 6 to 10 are the results of CepPCR products with template P2 added, and lanes 11-15 are the results of CepPCR products with template P3 added. The CepPCR system is divided into three groups according to the addition of templates. The lanes in each group from left to right are respectively the CepPCR system with Mn added 2+ The concentrations are 0.10, 0.30, 0.50, 0.70, and 0.90 (all in mmol / L). The leftmost lane is the DNA marker.

[0168] Depend on Figure 2 It can be seen that the addition of Mn 2+ The concentration range should be 0.10-0.30mmol / L. The addition of Mn 2+ The concentration range should be 0.10~0.50mmol / L. The addition of Mn 2+The concentration range should be 0.10-0.30 mmol / L. In summary, 0.30 mmol / L was selected as the final concentration of the CepPCR system.

[0169] Second round of mutation: Figure 3 As shown, CepPCR products were imaged after agarose gel electrophoresis. 2+ The CepPCR system can synthesize two gene fragments P4 and P5 of about 400 bp with different primers.

[0170] 3. Megawhop PCR product verification

[0171] A round of error-prone PCR mutation: Figure 4 As shown, the product of the CepPCR system with a manganese ion concentration of 0.30mmol / L was used as the primer in the Megawhop PCR system to amplify the whole plasmid to obtain the mutated plasmid. The annealing temperature of the P1 fragment was 72°C, the annealing temperature of the P2 fragment was 57.5°C, and the annealing temperature of the P3 fragment was 61.1°C. It can be seen from the figure that the DNA length obtained by the amplification product is about 6000-7500bp. The size of pEAS-1b-H1 is known to be about 6000bp, which is consistent with the band in the figure. It is preliminarily judged that the amplification was successful.

[0172] Second round of error-prone PCR mutations: Figure 5 As shown, the product of CepPCR was used as the primer in the MegawhopPCR system to amplify the whole plasmid with high fidelity to obtain the complete plasmid after mutation. The annealing temperature of P4 and P5 fragments was 58°C. It can be seen from the figure that the DNA length of the amplified product is greater than 5000bp. It is known that the size of pEAS-1b-H1 is about 6000bp, which is consistent with the band in the figure, so it is preliminarily judged that the amplification is successful. After gel excision, recovery and purification of the plasmid DNA, it can be transferred into competent cells after demethylation treatment.

[0173] 4. Conversion rate verification

[0174] One round of mutation: Pick 13 monoclonal colonies from each plate and perform colony PCR verification, such as Figure 6 The length of the positive control H1 gene fragment is 684 bp. In lanes 1-13 of the experimental group, there are 12 bands of the same length as the positive control lane 14, indicating that the plasmid pEAS-1b-Hmutant was successfully constructed, with a positive rate of about 92.3%, which meets the requirements for library construction.

[0175] 5. Transformation of competent cells

[0176] Figure 8This is the colony growth after transformation into E.coli BL21 after one round of mutation. The plasmid was extracted from the above transformed E.coli TOP10 competent cells and then transferred into E.coli BL21 competent cells, and the mutant monoclonal colonies with ampicillin resistance were grown.

[0177] 6 High-throughput screening

[0178] The activity of heparinase was detected by the Azure A method. After the reaction, the OD 620nm The absorbance was measured and the data with larger absorbance values ​​were selected. After the initial screening, there were about 20 mutant bacteria in each round that might have better enzyme activity than the wild-type strain, but further enzyme activity detection and verification were needed. These 23 strains were initially selected for the first round of rescreening, and 15 were selected for the second round of rescreening.

[0179] 7 Enzyme activity detection

[0180] Fig. 9 This is the standard curve for detecting heparin content by Azure A. Fig.10 It can be obtained that it is the enzyme activity data of excellent mutants selected after preliminary screening after a round of error-prone PCR mutations. 1-1 is the wild-type bacteria of heparinase, and the crude enzyme liquids obtained by lysing the random mutant strains of 1-6 strains all have significantly higher enzyme activities than the original strains. Among them, the enzyme activity of 1-6 strains is 3726U / L, which is 97.6% higher than the enzyme activity of the wild-type strain 1886U / L. It also has obvious advantages over the enzyme activity of the crude enzyme liquids produced by other mutant strains.

[0181] Amino acid comparison of 8 mutants

[0182] The mutation sites of strains 1-6 are as follows Fig.11 The detailed base sequence changes and analysis are as follows: the amino acid sequence of wild-type heparinase and the amino acid sequence of heparinase expressed by strains 1-6 are shown in Fig.11 The amino acid sequence mutation sites of heparin expressed by the mutant heparinase of strains 1-6 are: P10R, V45A and L160P.

[0183] 9 Homology modeling and molecular docking results

[0184] Fig.12This is the conformation diagram with the minimum docking binding energy between the heparinase molecule and the substrate. The protein structure pdb simulated by AlphaF old software, and then the molecular structure diagram pdb of heparin was drawn by Chemdraw. The protein structure and small molecules were imported into Auto Dock software. Auto Dock molecular docking software was used to dock the heparinase molecule with the heparin substrate small molecule, because small molecules can have a variety of different conformations. The minimum docking binding energy of wild-type heparinase is -5.7kcal / mol, and its root mean square deviation is 0.83; the minimum docking binding energy of heparinase No. 1-6 is -3.4kcal / mol, and its root mean square deviation is 0.73. The Auto dock results show that the potential binding sites of all configurations are wild-type H1-1 heparinase: SER14, ASP179, VAL182, SER202, ALA203. Heparinase mutant H1-6: SER11, ASP38, GLY146 GLY149, ASP179, GLY181, VAL182, VAL199, GLY201, SER202.

[0185] Example 2

[0186] Method for recombinantly expressing free heparinase mutants using strains 1-6 obtained in Example 1 and preparing immobilized enzymes

[0187] 1. Materials and Methods

[0188] 1.1 Experimental strains

[0189] The strain E. coli BL21(DE3)pET-28a-MBP-H1-6 was constructed and deposited by our laboratory.

[0190] 1.2 Experimental Materials and Instruments

[0191] The experimental reagents used in this experiment are shown in Table 15. Some instruments and materials are repeated in Table 1 and Table 2 and are no longer listed.

[0192] Table 15 Reagents used in the experiment

[0193]

[0194]

[0195] 2. Solution Preparation

[0196] The preparation of some solutions refers to step 4 in Example 1, the preparation of the buffer solution:

[0197] By mixing substances A and B in Table 16 below, a buffer solution of the corresponding pH can be prepared.

[0198] Table 16 Buffer solution preparation method

[0199]

[0200] 3 Experimental methods

[0201] 3.1 Strain culture and induced expression

[0202] (1) Take 100 μL of bacterial solution from the glycerol tube containing E. coli BL21 (DE3) pET-28a-MBP-H1-6 and add it to 10 mL of LB medium (containing 50 μg / mL kanamycin) and activate and culture it in a shaker at 37°C and 200 rpm for 12 h.

[0203] (2) Take 1% of the activated bacterial solution in LB medium (containing 50 μg / mL kanamycin), culture it in a shaker at 37°C and 200 rpm for 4-5 hours, and take 200uL of the bacterial solution to measure its OD 600 The value is 0.5~0.7.

[0204] (3) Add 0.1 mmol / L IPTG to the bacterial solution in (2) above and induce in a shaker at 30°C and 200 rpm for 9 h.

[0205] 3.2 Preparation of crude enzyme solution

[0206] (1) The induced bacterial solution was centrifuged at 4°C and 6000 rpm for 10 min, the supernatant was discarded, and 0.25 mmol / L sodium acetate-0.0025 mmol / L calcium acetate (pH 7.0) buffer with a volume half that of the bacterial solution was added to resuspend the cells.

[0207] (2) The resuspended bacterial solution was transferred to a beaker, placed on an ice-water mixture, and the cells were disrupted using an ultrasonic disruptor (power 20%, on for 3 seconds, off for 3 seconds, time for 10 minutes). The disrupted cells were centrifuged at 4°C and 7800 rpm for 10 minutes. The supernatant obtained was the crude enzyme solution.

[0208] 3.3 Determination of heparinase activity

[0209] (1) Draw a heparin standard curve

[0210] 0.04 g of heparin sodium was added to 1 mL of ultrapure water, and then diluted with ultrapure water to different degrees to obtain 1 mL of heparin sodium solution with concentrations of 0.1%, 0.2%, 0.3%, 0.4%, and 0.5%, respectively.

[0211] Take 6 test tubes and add 5mL of 0.002% Azure A solution, take 5μL of the above 0.5% heparin sodium solution and add it to Azure A solution, and take 5μL ddH2O as blank control. After fully mixing, take 200μL of each on the ELISA plate and measure its absorbance at 620nm (each group is tested three times in parallel).

[0212] (2) Determination of heparinase activity

[0213] Add 200 μL crude enzyme solution and 50 μL heparin solution (0.025 g heparin sodium standard is dissolved in 1 mL 0.25 M sodium acetate-0.0025 M calcium acetate buffer solution) into the EP tube, mix thoroughly, immediately take 5 μL of the solution into 5 mL 0.002% azure A solution, shake thoroughly and take 200 μL into the ELISA plate (make 3 sets of parallel samples and take the average value), and use an ELISA reader to measure the absorbance at a wavelength of 620 nm. After reacting in a 35°C water bath for 15 min, take 5 μL and measure the absorbance at a wavelength of 620 nm.

[0214] (3) Definition of heparinase activity

[0215] The amount of enzyme required to degrade 1 mg of heparin within 1 hour at 35°C and pH 7.0 is one enzyme activity unit (U).

[0216] (4) The calculation formula of heparinase activity is shown in Formula I

[0217] The heparin content (mg) in the whole system at different times can be calculated from the standard curve. The amount of heparin degraded during this period can be obtained by calculating the difference between the heparin contents at two different times, and the heparinase activity can be calculated from the definition of heparinase activity.

[0218]

[0219] 3.4 Calcium alginate immobilized heparinase

[0220] Weigh 4g of sodium alginate and dissolve it in 50mL of 0.25M sodium acetate-0.0025M calcium acetate buffer. Heat in a water bath to dissolve it. When cooled to room temperature, add an equal amount of the crude enzyme solution prepared above and stir to mix (sodium alginate concentration is 4%). Use a syringe to squeeze the mixture drop by drop into a 0.1mol / L CaCl2 solution to form small balls of a certain diameter. Place it in a 4℃ refrigerator to solidify for 5h and then take it out. Wash the small balls twice with deionized water and filter them for later use.

[0221] 3.5 Study on the optimal reaction conditions of free enzyme and immobilized enzyme

[0222] (1) Take 200 μL of crude heparinase solution in EP tubes, add 50 μL of 0.25% heparin solution (dissolve 0.025 g of sodium heparin standard in 1 mL of 0.25 mmol / L sodium acetate-0.0025 mmol / L calcium acetate buffer) to each EP tube, shake well, and react in a water bath at 20°C, 25°C, 30°C, 35°C, 40°C, and 45°C for 15 min, and measure the absorption value at 620 nm using the method in 3.3 after the reaction. Take equal amounts of sodium alginate beads of immobilized heparinase in EP tubes, add 200 μL of sodium calcium acetate buffer and 50 μL of 0.25% heparin solution to each EP tube, shake well, and react in a water bath at the above temperature for 15 min, and measure and calculate the enzyme activity.

[0223] (2) Take 200 μL of free heparinase in buffer solutions of different pH values ​​(5, 6, 7, 7.5, 8, 9), add to 50 μL of 0.25% heparin solution of the corresponding pH value, shake well, react in a 35°C water bath for 15 min, measure and calculate the enzyme activity. Take equal amounts of immobilized enzyme in EP tubes, add 200 μL of buffer solutions of different pH values ​​and 50 μL of 0.25% heparin solution of the corresponding pH value to each EP tube, shake well, react in a 35°C water bath for 15 min, measure and calculate the enzyme activity of the immobilized enzyme.

[0224] (3) The maximum enzyme activity between the free enzyme and the immobilized enzyme was taken as 100% to calculate the relative enzyme activity at different reaction pH.

[0225] 3.6 Stability experiments of free enzyme and immobilized enzyme

[0226] (1) Take 200 μL of free enzyme in EP tubes, and store each EP tube at 25℃, 35℃, 45℃, 55℃, 65℃, and 75℃ for 30 min. Take out the EP tubes, add 50 μL of 0.25% heparin solution to each EP tube, shake well, and react in a 35℃ water bath for 15 min. Measure and calculate the enzyme activity. Take equal amounts of immobilized enzyme in EP tubes, and store each EP tube at different temperatures for 30 min. Take out the EP tubes, add 200 μL of sodium calcium acetate buffer and 50 μL of 0.25% heparin solution to each EP tube, shake well, and react in a 35℃ water bath for 15 min. Measure and calculate the enzyme activity of the immobilized enzyme.

[0227] (2) Take 200 μL of free heparinase in buffer solutions of different pH values ​​(5, 6, 7, 7.5, 8, 9) and store them in a 4℃ refrigerator for 1 hour. Take them out and add 50 μL of 0.25% heparin solution of pH value 7. After shaking well, react in a 35℃ water bath for 15 minutes. Measure and calculate the enzyme activity. Take equal amounts of immobilized enzymes and soak them in buffer solutions of different pH values. Store them in a 4℃ refrigerator for 1 hour. Take them out and rinse them 3 times with sodium calcium acetate buffer. Dry them. Add the washed immobilized enzymes to EP tubes. Add 200 μL of 0.25mmol / L sodium acetate-0.0025mmol / L calcium acetate buffer and 50 μL of 0.25% heparin solution to each EP tube. After shaking well, react in a 35℃ water bath for 15 minutes. Measure and calculate the enzyme activity.

[0228] (3) The maximum enzyme activity between the free enzyme and the immobilized enzyme was taken as 100% to calculate the relative enzyme activity of the enzyme after being treated with different pH values.

[0229] 3.7 Storage stability of free and immobilized enzymes

[0230] (1) Take 200 μL of free enzyme in an EP tube, add 50 μL of 0.25% heparin solution, shake well, react in a 35°C water bath for 15 min, measure and calculate the enzyme activity. Store the remaining free enzyme in a 4°C refrigerator, and repeat this step for 15 days.

[0231] (2) Take an equal amount of immobilized enzyme in an EP tube, add 200 μL of sodium calcium acetate buffer and 50 μL of 0.25% heparin solution, shake well, react in a 35°C water bath for 15 min, measure and calculate its total enzyme activity. Repeat this step and measure the enzyme activity for 15 days.

[0232] (3) Taking the maximum enzyme activity between the free enzyme and the immobilized enzyme as 100%, the relative enzyme activity after different storage times was calculated.

[0233] result

[0234] Heparin standard curve Fig.13 .

[0235] Morphological observation results of calcium alginate beads and immobilized enzymes

[0236] Calcium alginate beads provide certain attachment conditions for heparinase. The enzyme can attach to the calcium alginate beads through hydrogen bonds, electrostatic forces or van der Waals forces to achieve the purpose of immobilizing the enzyme. The morphology of calcium alginate immobilized heparinase is a transparent sphere with uniform particle size. The spherical shape has a larger specific surface area, allowing the enzyme to contact the substrate more fully.

[0237] The enzyme activity of both free enzyme and immobilized enzyme first increases with the increase of reaction temperature. When the temperature reaches 35°C, the enzyme activity reaches the highest, reaching 3880U / L (free enzyme) and 3189U / L (immobilized enzyme); then the enzyme activity decreases with the increase of reaction temperature. Whether it is free enzyme or immobilized enzyme, the optimal reaction temperature is 35°C, and the relative enzyme activity of both is the highest. After the enzyme is combined with the carrier, the sites for the enzyme to react with the substrate are reduced, so the enzyme activity of the immobilized enzyme is low at 25°C. As the temperature increases, the diffusion rate of the substrate increases, the reaction rate of the sites with the substrate accelerates, and the enzyme activity increases relatively.

[0238] As the reaction pH value increases, the relative enzyme activities of the free enzyme and the immobilized enzyme both show a trend of first increasing and then decreasing, and the enzyme activities of both enzymes reach the highest when pH = 7, so the optimal reaction pH of the free enzyme and the immobilized enzyme is 7. In addition, it can be seen from the distribution of the curve that the enzyme activity of the free enzyme fluctuates greatly with the change of pH, while the immobilized enzyme is relatively stable, and the relative enzyme activity decreases less. The reason may be that the calcium alginate beads provide a protective or buffering environment for the enzyme.

[0239] Results of thermal stability studies on free and immobilized enzymes

[0240] The enzyme was stored at different temperatures for 30 minutes before reacting. In the low temperature range, the activity of the free enzyme first increased with the increase of storage temperature, reaching a maximum of 3756U / L; after reaching 35°C, the enzyme activity began to decrease, and when it reached above 65°C, the relative enzyme activity was close to 0. After the free enzyme was treated at 55°C for 1 hour, white flocs appeared, indicating that the protein was denatured and inactivated, so the enzyme activity decreased rapidly. When it reached 65-75°C, the flocs became significantly more, and the free enzyme was almost completely denatured, so the enzyme activity was close to 0.

[0241] The relative enzyme activity of the immobilized enzyme first increased with the increase of temperature, reaching a maximum of 3142U / L. The enzyme activity remained stable between 35-45℃, and began to decline at 65℃, but the enzyme activity remained above 60%. Compared with the free enzyme, the immobilized enzyme is more stable and has significantly improved heat resistance. The reason for this is that the structure of the calcium alginate spheres protects the enzyme to a certain extent, reducing the degree of damage to the protein structure.

[0242] Results of pH stability studies of free and immobilized enzymes

[0243] Before the reaction, the enzyme was placed under different pH conditions and stored for 1 hour before the reaction. The activity of the free enzyme first increased with the increase of pH. When the pH reached 6, the enzyme activity reached the highest, and then the enzyme activity began to decrease with the increase of pH. The activity of the immobilized enzyme fluctuated slightly in the pH range of 5-9, showing good stability and good acid and alkali resistance. The optimal storage pH of both the free enzyme and the immobilized enzyme is 6. The good stability of the immobilized enzyme is mainly due to the fact that calcium alginate contains a certain amount of non-free water. Changes in external ion concentration are not easy to change the microenvironment of the enzyme in the carrier, and the enzyme activity is not significantly affected by the external pH.

[0244] Results of storage stability study of free enzyme and immobilized enzyme

[0245] The results showed that compared with the free enzyme, the storage stability of immobilized heparinase was better. The half-life of the free enzyme at 4°C was about 3 days, and the enzyme activity was close to 0 after the 9th day, and the enzyme activity decreased sharply. The storage stability of the immobilized enzyme was improved, and its half-life was on the 7th day. The enzyme activity on the 9th day could still maintain 40% of the original. The immobilized enzyme activity decreased in a buffer period of 3-7 days, which delayed the time of enzyme activity decline. The decrease in the activity of the immobilized enzyme is not only related to the loss of the enzyme's own activity, but also the weakening of the hydrogen bond, electrostatic and van der Waals force between the carrier and the enzyme, which is also an important reason for the inactivation of the enzyme.

[0246] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A heparinase mutant H1-6, characterized in that The amino acid sequence of the heparinase mutant is shown in SEQ ID NO: 1, which is obtained by performing the following mutations on the amino acid sequence of the wild-type heparinase: P10R, V45A and L160P.

2. A gene encoding the heparinase mutant H1-6 according to claim 1, characterized in that: The nucleotide sequence of the encoding gene is shown in SEQ ID NO:

3.

3. A recombinant vector, characterized in that: The nucleotide sequence encoding the heparinase mutant H1-6 according to claim 1 is inserted into the backbone vector.

4. An engineered strain for recombinantly expressing the heparinase mutant according to claim 1, characterized in that: It comprises a nucleotide sequence encoding the heparinase mutant H1-6 according to claim 1 or a recombinant vector according to claim 3.

5. An immobilized enzyme, characterized in that: The heparinase mutant H1-6 described in claim 1 is fixed using calcium alginate gel as a carrier.

6. A method for preparing the immobilized enzyme according to claim 5, characterized in that: The following steps are involved: 1) recombinantly expressing the heparinase mutant H1-6 according to claim 1 to obtain a solution containing the heparinase mutant recombinant protein; 2) The solution containing the heparinase mutant recombinant protein is mixed with a sodium alginate solution, and the obtained mixed solution is dripped into a CaCl2 solution, cooled and solidified to obtain an immobilized enzyme.

7. The preparation method according to claim 6, characterized in that: The method for recombinant expression in step 1) is to culture the engineered strain according to claim 4 in a medium containing antibiotics, induce expression under 0.1 mmol / L IPTG conditions, lyse the bacteria, and collect the supernatant.

8. The preparation method according to claim 6 or 7, characterized in that: The volume ratio of the solution containing the heparinase mutant recombinant protein to the sodium alginate solution in step 2) is 0.8-1.2:1; The mass percentage of the sodium alginate solution is 8%; The concentration of the CaCl2 solution is 0.1 mol / L.

9. Use of the heparinase mutant H1-6 according to claim 1, the engineered strain according to claim 4 or the immobilized enzyme according to claim 5 in catalytic production of heparin.

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