Type II L-asparaginase and its application

Abstract

The present invention discloses a type II L-asparaginase and its application, and relates to the field of genetic engineering technology. The amino acid sequence of the enzyme is shown in SEQ ID NO.4, and the nucleotide sequence of the gene encoding the enzyme is shown in SEQ ID NO.3. The type II L-asparaginase provided by the present invention has an optimum temperature of 45°C, an optimum pH of 8.0, and has a high specific activity of 598.907U / mg, a half-life of 102h in animal serum at 37°C, and a T of 1.57kcal / cm3 in trypsin. 1 / 2 =33min, the enzyme has no glutaminase activity. The enzyme can induce apoptosis of human liver cancer cell line QGY-7703 and human malignant melanoma cell line A-375, IC 50 The concentrations of 5.7 and 6.5 U / mL respectively, and it will not cause hemolysis. The enzyme has potential application value in the pharmaceutical industry.

Description

Technical Field

[0001] The present invention relates to the technical field of genetic engineering, and in particular to a type II L-asparaginase and an application thereof. Background Art

[0002] Type II L-asparaginase (EC3.5.1.1) can catalyze asparagine (Asn) into aspartic acid (Asp), which is a substance necessary for cells to synthesize glycoproteins and other nutrients. After treatment with type II L-asparaginase, the DNA and protein synthesis of tumor cells is blocked, and they are transformed into a malnutrition state, which eventually leads to apoptosis of cancer cells. However, normal cells are not affected. They can use transaminases to convert oxaloacetate into Asp, and then use asparagine synthetase (ASNS) to synthesize Asn to meet their own nutritional needs. However, some tumor cells have a low expression level of ASNS and lose the ability to synthesize Asn, so they can only rely on Asp in serum. However, one challenge faced by therapeutic type II L-asparaginase is instability, short serum half-life and high trypsin clearance rate. Another challenge is that asparaginase contains a second activity: glutaminase activity, which can cause serious side effects. Therefore, type II L-asparaginases from different sources may have different antigenic, physiological, pharmacological, serological and pharmacokinetic properties, and their tendency to induce side effects and their effectiveness in treating cancer are also different. Exploring new and excellent enzymes from hitherto unexplored niches is of great significance for promoting the development of drugs and treatment regimens based on individual patient needs. Summary of the invention

[0003] The purpose of the present invention is to provide a type II L-asparaginase and its application, wherein the half-life of the enzyme in animal serum at 37°C is the longest in existing studies. The enzyme can induce apoptosis of human liver cancer cell QGY-7703 and human malignant melanoma cell A-375 cell line, and does not contain glutaminase activity, and has potential application value and application potential in the medical field, especially in the development of anticancer drugs and drug preparation.

[0004] In order to achieve the above object, the present invention provides a type II L-asparaginase, the amino acid sequence of which is shown in SEQ ID NO.4, the half-life of which in animal serum at 37°C is 102h, and the half-life of which in trypsin is 102h. 1 / 2 =33min.

[0005] The type II L-asparaginase provided by the present invention can be used to prepare anticancer drugs, wherein the anticancer drugs can be used to inhibit human liver cancer cells QGY-7703 and / or human malignant melanoma cells A-375, and induce apoptosis of QGY-7703 and / or A-375 cells.

[0006] The present invention also provides an anticancer drug prepared by the above type II L-asparaginase, which can be used to inhibit human liver cancer cells QGY-7703 and / or human malignant melanoma cells A-375, and induce apoptosis of QGY-7703 and / or A-375 cells.

[0007] The present invention also provides a gene encoding the above-mentioned type II L-asparaginase, and the nucleotide sequence of the gene encoding is shown in SEQ ID NO.3.

[0008] The present invention also provides a recombinant expression vector comprising the above encoding gene.

[0009] Preferably, the above-mentioned recombinant expression vector is selected from pEASY-E2.

[0010] The present invention also provides a recombinant bacterium comprising the above encoding gene.

[0011] Preferably, the above-mentioned recombinant bacteria is selected from Escherichia coli BL21 (DE3).

[0012] The present invention also provides a method for preparing type II L-asparaginase, comprising the following steps:

[0013] (1) Extraction of metagenomic DNA from fecal microorganisms of slow loris;

[0014] (2) using primers with nucleotide sequences such as SEQ ID NO.1 and SEQ ID NO.2 to perform PCR amplification using the genomic DNA in step (1) as a template;

[0015] (3) connecting the amplified product obtained in step (2) with the recombinant vector to obtain a recombinant expression vector, and transferring the recombinant expression vector into an expression bacterium to obtain a recombinant bacterium;

[0016] (4) Cultivating the recombinant bacteria obtained in step (3), inducing with IPTG, collecting the bacteria and disrupting them by ultrasonication, collecting the supernatant and purifying it with Nickel-NTAAgarose to obtain type II L-asparaginase.

[0017] The type II L-asparaginase of the present invention solves the problems of instability and short serum half-life of the existing type II L-asparaginase and has the following advantages:

[0018] The type II L-asparaginase provided by the present invention has good stability, and its optimum pH is 8.0. After the enzyme is treated in the range of pH=5.0-10.0 for 1 hour, the residual enzyme activity still maintains more than 80%; the optimum temperature of the enzyme is 45°C; after the enzyme is tolerated for 90 minutes under the conditions of 20°C, 30°C, 35°C, 40°C, 45°C, and 50°C, the residual enzyme activity is more than 80%, and the residual activity is still greater than 100% at 20-40°C; the enzyme is at pH=8.0 and 45°C, and the K m , and V max 5.733±0.512mM and 156.2±3.056U / mg respectively; various ions (Li + , Hg 2+ , K + 、Ag + , Ca 2+ , Cu 2+ 、Co 2+ , Mn 2+ 、Na + 、Al 3+ Mg 2+ 、Zn 2+ 、Ni 2 + , Fe 2+ , Fe 3+ ), SDS, EDTA and Tween-80 can inhibit the activity of the recombinant enzyme. Compared with type II L-asparaginase from other sources, the enzyme has the highest specific activity of 598.907U / mg. The half-life of the enzyme in animal serum at 37°C is 102h, and in trypsin T 1 / 2 =33min.

[0019] At present, most asparaginases have glutaminase activity, and Escherichia coli asparaginase and Chrysanthemi asparaginase used for clinical treatment both have glutaminase activity. The type II asparaginase NPASN10 provided by the present invention has no glutaminase activity, and this property may reduce the side effects produced during medication. This study may provide a therapeutic enzyme with better properties for the pharmaceutical field to fill the gap in therapeutic enzymes.

[0020] At present, the great challenge faced by type II asparaginase is that it is unstable and may cause reduced enzyme activity due to the particularity of its administration route (injection), so stability is an important indicator. The NPASN10 provided by the present invention has the longest trypsin and serum half-life tolerance at physiological temperature in existing studies, which can largely solve the problem of asparaginase instability.

[0021] The NPASN10 provided by the present invention can cause apoptosis of QGY-7703 (human liver cancer cells) and A-375 (human malignant melanoma cells) cells and does not contain glutaminase activity and does not cause hemolysis. The enzyme has potential application value in the medical field such as the pharmaceutical industry. Currently, there is no study on the cytotoxicity of asparaginase to these two cells. Therefore, this study can provide a supplement to existing research in this field and provide a reference for future research. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The SDS-PAGE results of the recombinant type II L-asparaginase expressed in Escherichia coli in the present invention are shown in FIG.

[0023] Figure 2 This is the optimum temperature determination result of type II L-asparaginase provided in the present invention.

[0024] Figure 3 The temperature stability test results of the type II L-asparaginase provided in the present invention are shown in FIG.

[0025] Figure 4 This is the optimum pH measurement result of type II L-asparaginase provided in the present invention.

[0026] Figure 5 The pH stability test results of the type II L-asparaginase provided in the present invention are as follows

[0027] Figure 6 The metal ions and chemical reagents for the type II L-asparaginase provided in the present invention affect the assay results.

[0028] Figure 7 The results of the serum half-life determination of type II L-asparaginase provided in the present invention are shown in FIG.

[0029] Figure 8 The half-life measurement result of type II L-asparaginase trypsin provided in the present invention is shown in FIG.

[0030] Fig. 9 The results are shown in Table 1. These are the inhibition results of type II L-asparaginase provided in the present invention on QGY-7703 cells.

[0031] Fig.10 The results are shown in Table 1. These are the inhibition results of type II L-asparaginase on A-375 cells provided by the present invention.

[0032] Fig.11 These are the results of microscopic examination of QGY-7703 cells.

[0033] Fig.12 The figure shows the microscopic examination results of A-375 cells.

[0034] Fig.13 The results of fluorescence microscopy of QGY-7703 cells after DAPI staining.

[0035] Fig.14 The fluorescence microscopy results of A-375 cells after DAPI staining.

[0036] Fig.15 This is the case of hemolysis by type II L-asparaginase provided in the present invention. DETAILED DESCRIPTION

[0037] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0038] Some of the test materials and reagents used in the present invention are as follows:

[0039] 1. Strains and vectors: Vectors pEASY-E2 and BL21 (DE3) were purchased from Beijing Qingke Xinye Biotechnology Co., Ltd.

[0040] 2. Genetic engineering enzymes, kits and other biochemical reagents: restriction endonucleases, DNA polymerases and ligases were purchased from TaKaRa, and plasmid extraction kits and gel recovery and purification kits were from Omega, USA; other reagents were of analytical grade.

[0041] 3. LB medium: Peptone 10g, Yeast extract 5g, NaCl 10g, add distilled water to 1000ML, natural pH (about 7); solid medium plus 2.0% (w / v) agar on the above basis.

[0042] Note: The molecular biology experimental methods not specifically described in the following examples are all carried out with reference to the specific methods listed in the book Molecular Cloning Experiment Guide (3rd Edition) by J. Sambrook, or according to the kits and product instructions.

[0043] Experimental Example 1 Obtaining Type II L-asparaginase Gene NPasn10

[0044] 1. Screening of Type II L-asparaginase Genes from the Metagenomics of Slow Slow Loris Fecal Microorganisms

[0045] In the fecal microbial metagenome of the pygmy slow loris, the gene of type II L-asparaginase was obtained based on gene prediction and functional annotation. A phylogenetic tree was constructed based on sequence homology, and type II L-asparaginase was screened based on its phylogenetic position, tertiary structure and substrate formation BD energe, amino acid composition and blast results. Finally, the NPasn10 gene was screened out for further cloning and expression.

[0046] 2. Cloning of Type II L-asparaginase Gene NPasn10

[0047] The metagenomic DNA of fecal microorganisms of pygmy slow loris was extracted, and PCR amplification was performed using primers NPasn10-F and NPasn10-R (the specific sequences are shown below) with the extracted genomic DNA as a template.

[0048] Among them, the PCR reaction system is 50.0 μL: 1 μL of metagenomic DNA, 10 μL of Ex Taq, 10.5 μL of RBasn10-F, 10.5 μL of RBasn10-R, 1 μL of dNTP, and 50 μL of ddH2O;

[0049] The PCR amplification program was: 98°C for 30s; 55°C for 30s, 72°C for 1min, 30 cycles; 4°C for 20min. The PCR result obtained the target gene NPasn10, and the nucleotide sequence of the gene was shown in SEQ ID NO.3 through sequencing.

[0050] The primer sequences used for amplification are as follows (5'→3'):

[0051] NPasn10-F (SEQ ID NO.1):

[0052] TAAGAAGGAGATATA CATATG GAATTGATGCCGAACCCGCTTTT;

[0053] NPasn10-R (SEQ ID NO.2):

[0054] GTGGTGGTGGTGGTG CTCGAG GGTATGGCCGTGCGTGT.

[0055] The nucleotide sequence of gene NPasn10 is shown in SEQ ID NO.3, and the specific sequence is as follows (5'→3'):

[0056]

[0057] Experimental Example 2 Preparation of Type II L-asparaginase NPASN10

[0058] Through conventional construction methods, the NPasn10 gene sequence obtained in Experimental Example 1 was connected to the plasmid pEASY-E2 to obtain the recombinant expression vector pEASY-E2-NPasn10, and then transformed into Escherichia coli BL21 (DE3) to obtain the recombinant Escherichia coli strain BL21 (DE3) / NPasn10.

[0059] The recombinant bacteria BL21(DE3) / NPasn10 containing the recombinant expression vector pEASY-E2-NPasn10 were inoculated into LB (containing 100 μg / mL Amp) culture medium at 0.1% inoculum and cultured overnight at 37°C and 180 rpm. Then the cultured bacterial solution was inoculated into fresh LB (containing 100 μg / mL Amp) culture medium at 1% inoculum and cultured at 37°C and 180 rpm for 2-3 h (OD 600 After reaching 0.6-0.8), add IPTG with a final concentration of 0.5mmol / L for induction, and culture at 25℃ and 180rpm for about 20h. Centrifuge at 6000rpm for 10min to collect the bacteria. Suspend the bacteria with an appropriate amount of 50mM PBS (pH=7.5) buffer, and then ultrasonically disrupt the bacteria under ice bath conditions. Take the above-mentioned intracellular concentrated primary enzyme solution and centrifuge it at 12,000rpm and 4℃ for 10min, then aspirate the supernatant and purify the target protein with Nickel-NTAAgarose to obtain type II L-asparaginase NPASN10, whose amino acid sequence is shown in SEQ ID NO.4.

[0060] The amino acid sequence of type II L-asparaginase NPASN10 is as follows (SEQ ID NO.4):

[0061] MPNPLLHTPPSPQHRQVLALFATGGTIAGIAQDQHSHVQYQAAALPVTALLDAVPALQHLPAEIRAEQLLAKDSKDLDPEDWLRIAQAVDQALQDPEVAGVVITHGTDTLEETAWFLHRTLRPSPKPVVLVSAMRPATALGADGPQNLLDACCVALSQVAGVVCVAAGEVHRAETVQKL QPYRIPAFGSGECGPMAWVEEGRIAWPAYAAGAMHNAAINATIGATSNDTAGSLVSRGRYHDLLNACVPPPAVAWITSHAGFDARLVKALQAAGFAGVVVAGTGNASLHHALETALDAVAATGMATAITSRCAQGHIVVPPPAQSRHRRWALPAAKARIELLMELWLRDTNQHTHGHT.

[0062] The purified target protein was analyzed by SDS-PAGE. Figure 1 As shown, M is a protein marker; Lane 1 is the initial enzyme induced by E. coli containing only the pEASY-E2 vector; Lane 2 is the unpurified recombinant type II L-asparaginase; Lane 3 is the purified recombinant type II L-asparaginase. Figure 1 It can be seen that the recombinant type II L-asparaginase was expressed in Escherichia coli and was a single band after Nickel-NTAAgarose purification.

[0063] Experimental Example 3 Determination of properties of type II L-asparaginase NPASN10

[0064] The properties of type II L-asparaginase NPASN10 obtained in Experimental Example 2 were determined by using Nessler's reagent to detect the enzyme activity, as follows:

[0065] 500μL of the mixed solution system: 200μL 50mM Tris-Hcl buffer, 200μL 200mM substrate Asn, 50μL appropriately diluted enzyme solution, placed in a water bath at the optimum temperature for the enzyme to react for 10min, then immediately added 50μL 1.5M trichloroacetic acid to terminate the reaction. Centrifuge at 4℃, 12000r / min for 10min, take 200μL of the supernatant and transfer it to a new test tube, add 1.2mLddH2O and 100μL Nessler's reagent, let it stand at room temperature for 10min, then measure the OD 450The blank control was to add trichloroacetic acid to terminate the reaction before the subsequent water bath treatment. Definition of enzyme activity unit: One enzyme activity unit (U) is the amount of enzyme required to hydrolyze Asn and release 1 μmol of ammonia per minute under the optimal reaction conditions of the enzyme.

[0066] 1. Determination of the optimum temperature and temperature stability of type II L-asparaginase

[0067] Determination of the optimum temperature of the enzyme: The type II L-asparaginase NPASN10 purified in Experimental Example 2 was subjected to an enzymatic reaction at pH = 8.0 at 30-60°C, and the relative enzyme activity was determined. The results of the optimum temperature determination are as follows: Figure 2 shown.

[0068] Determination of enzyme temperature stability: The enzyme solution with the same amount of enzyme was placed at a set temperature (20°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C) for 90 minutes, and the enzymatic reaction was carried out at pH = 8.0 and 45°C every 10 minutes. The untreated enzyme solution was used as a control, and the relative enzyme activity was measured. The results of the temperature stability determination are shown in the figure. Figure 3 shown.

[0069] comprehensive Figure 2 and Figure 3 It can be seen that the optimum temperature of L-asparaginase is 45°C, and the relative enzyme activity remains stable at 20, 30, 35, 40, 45, and 50°C. After enduring 90 minutes at 20, 30, 35, 40, 45, and 50°C, the remaining enzyme activity is above 80%; after enduring 90 minutes at 20-40°C, its remaining activity is still greater than 100%, which shows that the enzyme has strong temperature tolerance and stability.

[0070] 2. Determination of the Optimal pH and pH Stability of Type II L-Asparaginase

[0071] Determination of the optimum pH of the enzyme: Carry out the enzymatic reaction in a buffer solution at 45°C and pH = 4.0-12.0, and measure the relative enzyme activity. The results are as follows: Figure 4 shown.

[0072] Determination of enzyme pH stability: Place the purified enzyme solution in a buffer solution with a pH of 4.0-12.0 and treat at 37°C for 1 hour before performing an enzymatic reaction. Use the untreated enzyme solution as a control and determine the relative enzyme activity. The results are as follows: Figure 5 shown.

[0073] The buffer is: 0.5mol / L citric acid-sodium citrate buffer (pH=4.0-6.0); 0.5mol / L Tris-Hcl buffer (pH=7.0-8.0); 0.5mol / L glycine-sodium hydroxide buffer (pH=9.0-12.0). Asn is used as substrate, the reaction is carried out for 10 minutes, and the enzymatic properties of the purified L-asparaginase are determined.

[0074] comprehensive Figure 4 and Figure 5 It can be seen that the optimum pH of L-asparaginase is 8.0; after being treated with a buffer solution of pH = 5.0-10.0 for 1 hour, the relative enzyme activity remains above 80%.

[0075] 3. Determination of the effects of different metal ions and chemical reagents on type II L-asparaginase activity

[0076] Metal ions and chemical reagents with a final concentration of 1mM were added to the enzyme reaction system to study their effects on enzyme activity. The enzyme activity was measured at 45°C and pH=8.0 (the enzyme reaction without metal ions and chemical reagents was used as a control under the same conditions). The results are shown in Figure 6 .

[0077] Depend on Figure 6 It can be seen that all metal ions and chemical reagents have an inhibitory effect on its activity. + , Cu 2+ 、Na + 、Ni 2 + , Fe 2+ and Ca 2+ The activity of the enzyme was slightly inhibited by Mg 2+ , Li + , Fe 3+ 、Ag + , EDTA and Tween-80 almost completely inhibited the activity of the enzyme.

[0078] 4. Determination of Kinetic Parameters of Type II L-Asparaginase

[0079] Kinetic parameters were determined at pH = 8.0, temperature 45 °C and first-order reaction time with different concentrations of Asn as substrate (2-80 mM). K was calculated according to the Lineweaver-Burk method. m and V max The enzyme K value was determined at pH = 8.0 and temperature 45°C. m and V max They are 5.733±0.512mM and 156.2±3.056U / mg respectively.

[0080] 5. In vitro serum tolerance assay for type II L-asparaginase

[0081] In vitro serum tolerance test of enzyme: Purified L-asparaginase (50 IU, 0.1 mL) was mixed with 2.4 mL of animal serum, shaken vigorously, and incubated at 30, 37, and 40 °C. The relative enzyme activity was measured every 12 h. The results were as follows: Figure 7 shown.

[0082] Depend on Figure 7 It can be seen that the initial activity of the purified L-asparaginase decreased by 50% after being incubated at 30°C, 37°C and 40°C for about 50h, 102h and 92h, respectively.

[0083] 6. Determination of the Trypsin Half-Life of Type II L-Asparaginase

[0084] Determination of the half-life of trypsin: Add 0.1 mL of purified asparaginase (50 IU) to 2.4 mL of 50 IU trypsin (pH = 7.5), shake the reaction mixture vigorously, react at 37°C, and measure the relative enzyme activity every 5 minutes. The results are as follows: Figure 8 shown.

[0085] Depend on Figure 8 It can be seen that after the purified L-asparaginase was incubated in 50U / mL trypsin at 37°C for about 33 minutes, its initial activity decreased by 50%.

[0086] 7. Effect of Type II L-asparaginase on the survival rate of QGY-7703 and A-375 cells

[0087] The cytotoxicity of purified asparaginase was studied by MTT assay. 5×10 4 QGY-7703 (human liver cancer cells) and A-375 (human malignant melanoma cells) cells were inoculated into 96-well plates. After the cells adhered to the wall, the cells were treated with different concentrations of asparaginase for 24 hours, the culture medium was discarded, and the cells were washed twice with PBS. 20 μL of MTT dye (5 mg / mL) and 180 μL of culture medium were added to each well and cultured for 4 hours. The liquid in each well was discarded, 100 μL of DMSO was added, and the cells were slowly shaken on a shaker for 10 minutes. After the crystals were fully dissolved, the light absorption value was measured at 490 nm with an enzyme marker, and the cell survival rate was calculated. The results are as follows: Fig. 9 (QGY-7703) and Fig.10 (A-375) shown.

[0088] comprehensive Fig. 9 and Fig.10 It can be seen that the number of cells treated with purified NPASN10 was significantly reduced, and IC 50The concentrations of NPASN10 and A-375 in the samples were 5.7 U / mL and 6.5 U / mL, respectively. Compared with A-375, the enzyme showed better inhibitory effect on QGY-7703 cells. It can be seen that the type II L-asparaginase NPASN10 provided by the present invention can be applied in the medical field, especially in the preparation of anticancer drugs, and in the preparation of drugs for the treatment of liver cancer and malignant melanoma, and has potential application value.

[0089] 8. Effects of Type II L-asparaginase on the morphology of QGY-7703 and A-375 cells

[0090] Approximately 1.5 × 10 cells were cultured per well in a 24-well plate. 5 The cells were treated with 2, 4, 6, 8 and 10 U / mL of asparaginase. The cells were cultured in a 37°C, 5% CO2 incubator for 24 h and photographed under an inverted microscope (magnification 100 times). Fig.11 This is the microscopic examination result of QGY-7703 cells. Fig.12 The results of microscopic examination of A-375 cells. A in the figure is QGY-7703 and A-375 cells that have not been treated in any way, and B in the figure is IC 50 QGY-7703 and A-375 cells were treated with type II L-asparaginase at different concentrations.

[0091] comprehensive Fig.11 and Fig.12 It can be seen that in IC 50 Under the treatment of high concentration of enzyme, the number of cells decreased significantly, and the cells showed blistering, swelling and rupture. However, no morphological changes were observed in the untreated cells, and the cells were highly transparent, with clear outlines and neat arrangement.

[0092] 9. Effects of Type II L-asparaginase on the Nuclei of QGY-7703 and A-375 Cells

[0093] To study the effect of asparaginase on the level of cell apoptosis, approximately 1.5 × 10 5 cells, treated with different concentrations of asparaginase, cultured in a 37°C, 5% CO2 incubator for 24 h, washed three times with PBS (pH 7.0), fixed with 100 μL 4% paraformaldehyde for 10 min, washed three times with 0.1% Triton-X-100, washed three times with PBS, stained with 100 μL, 1.25 μg / ml 4′,6-diamino-2-phenylindole dihydrochloride (DAPI) for 10 min, observed and photographed under an inverted fluorescence microscope (magnification 100 times), where, Fig.13 This is the result of fluorescence microscopy of QGY-7703 cells after DAPI staining. Fig.14Figure 1 is the fluorescence microscopy result of A-375 cells after DAPI staining. Figure A is the DAPI staining result of QGY-7703 and A-375 cells without any treatment, and Figure B is the DAPI staining result of QGY-7703 and A-375 cells after IC 50 DAPI staining results of QGY-7703 and A-375 cells treated with type II L-asparaginase at different concentrations.

[0094] comprehensive Fig.13 and Fig.14 It can be seen that in IC 50 Under the treatment of enzymes with high concentrations, the cells showed obvious apoptotic morphological characteristics such as nuclear condensation, chromatin condensation, DNA exposure, DNA breakage, loss of normal nuclear structure, and nuclear fragmentation into round bodies of varying sizes surrounded by cell membranes. However, in the control group, the cells had intact cell membranes and nuclei, and no morphological changes.

[0095] 10. Type II L-asparaginase hemolytic test

[0096] The hemolytic effect of crude asparaginase and purified asparaginase on mammalian blood was studied by hemolysis test. 25 μL of crude and purified asparaginase were placed in the wells previously punched on the blood agar plate. After incubation at 37°C for 24 hours, the transparent or translucent halo on the plate was checked. The hemolysis of type II L-asparaginase on the plate was as follows: Fig.15 shown.

[0097] Depend on Fig.15 It can be seen that both unpurified and purified asparaginase did not form a transparent circle on the blood agar plate, indicating that the L-ASNase derived from the fecal metagenomics of the slow loris has no glutaminase activity and is non-toxic to red blood cells.

[0098] According to the results of the above research, some existing information related to type II L-asparaginase from different sources was collected, and their enzymatic properties were compared. The results are shown in Table 1 below. Due to insufficient research, relevant information of some enzymes is not listed. It can be seen from Table 1 that the present invention has made a relatively comprehensive study on the properties and functions of type II L-asparaginase, and the type II L-asparaginase NPASN10 provided by the present invention has better comprehensive performance and has a wider application value.

[0099] Table 1 Comparison of properties of type II L-asparaginase from different sources

[0100]

[0101]

[0102] [1]J.K.Arjun,B.P.Aneesh,T.Kavitha,K.Harikrishnan,Characterization ofanovel

[0103] asparaginase from soil metagenomic libraries generated from forestsoil,Biotechnol.Lett.

[0104] 40(2018)343-348.

[0105] [2]V.Patial,V.Kumar,R.Joshi,M.Gupta,D.Singh,Acrylamide mitigation infoods usingrecombinant L-asparaginase:An extremozyme fromHimalayanPseudomonassp.

[0106] PCH182,FoodRes.Int.162(2022)111936.

[0107] [3]M.Sobat,S.Asad,M.Kabiri,M.Mehrshad,Metagenomicdiscoveryandfunctional

[0108] validationofL-asparaginases withanti-leukemic effect fromthe CaspianSea,IScience. 24(2021)101973.

[0110] [4]D.Cappelletti,L.R.Chiarelli,M.V.Pasquetto,S.Stivala,G.Valentini,C.Scotti,

[0111] Helicobacterpylori l-asparaginase:Apromising chemotherapeutic agent,Biochem.

[0112] Biophys.Res.Commun.377(2008)1222-1226.

[0113] [5]I.Husain,A.Sharma,S.Kumar,F.Malik,Purificationandcharacterizationof

[0114] glutaminase free asparaginase from enterobactercloacae:In-vitroevaluationofcytotoxic

[0115] potential against humanmyeloidleukemiaHL-60cells,PLoS One.11(2016)1-27.

[0116] In summary, the present invention uses metagenomic technology to screen the type II L-asparaginase gene from the fecal microbial metagenomics of the pygmy slow loris, and heterologously expresses the type II L-asparaginase in Escherichia coli. By studying its enzymatic properties and anti-cancer properties, a type II L-asparaginase NPASN10 with higher activity, more stability and no glutaminase activity is obtained.

[0117] The type II L-asparaginase without glutaminase activity provided by the present invention may reduce the side effects caused by medication, and will not cause hemolysis, and has great economic value and strong application potential in pharmaceutical industry applications. The type II L-asparaginase provided by the present invention has a high serum and trypsin half-life, has a high enzyme activity at human physiological temperature and pH, and can be applied to the application and development of anticancer drugs. The type II L-asparaginase provided by the present invention comes from a new microbial strain in an ecological niche that has not been explored so far, which is of great significance for promoting the development of drugs and treatment plans based on individual patient needs.

[0118] Although the content of the present invention has been described in detail through the above preferred embodiments, it should be appreciated that the above description should not be considered as a limitation of the present invention. After reading the above content, it will be apparent to those skilled in the art that various modifications and substitutions of the present invention will occur. Therefore, the protection scope of the present invention should be limited by the appended claims.

Claims

1. A type II L-asparaginase, characterized in that The amino acid sequence of the enzyme is shown in SEQ ID NO.

4.

2. Use of the type II L-asparaginase according to claim 1 in the preparation of drugs against liver cancer or human malignant melanoma.

3. The gene encoding type II L-asparaginase according to claim 1, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

3.

4. A recombinant expression vector comprising the coding gene according to claim 3.

5. The recombinant expression vector according to claim 4, characterized in that: The recombinant expression vector is selected from pEASY-E2.

6. A recombinant bacterium comprising the coding gene as claimed in claim 3.

7. The recombinant bacterium according to claim 6, characterized in that The recombinant bacteria are selected from Escherichia coli BL21 (DE3).

8. A method for preparing type II L-asparaginase as claimed in claim 1, characterized in that: The following steps are included: (1) Transforming the recombinant expression vector of claim 4 into an expression bacterium to obtain a recombinant bacterium; (2) Cultivating the recombinant bacteria obtained in step (1), inducing with IPTG, collecting the bacteria and disrupting them by ultrasonication, collecting the supernatant and purifying it with Nickel-NTAAgarose to obtain type II L-asparaginase.

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