Application of a β-agarase mutant and its immobilized enzyme

Abstract

The present invention discloses an application of a β-agarase mutant and its immobilized enzyme, belonging to the fields of genetic engineering technology and enzyme engineering. After characterizing and immobilizing the agarase from wild-type Saccharophagus degradans 2-40 (NCBI accession number: ABD81904) and the mutant of the present invention, an immobilized β-agarase mutant with enhanced thermal stability and reusability is obtained, endowing the β-agarase with greater industrial application value. After characterization and immobilization, the present invention obtains a mutant immobilized enzyme with higher heat resistance compared to the free enzyme, greatly enhancing the industrial application prospect of agarase.

Description

Technical Field

[0001] The present invention relates to a β-agarase mutant and its application in immobilized enzyme, belonging to the fields of genetic engineering technology and enzyme engineering. Background Art

[0002] Agar is the main component of the cell walls of red algae in the Gelidiaceae and Gracilariaceae families. It is one of the most widely used marine polysaccharides in the world, mainly composed of agarose and agaropectin. Among them, agarose is a linear chain molecule composed of alternating (1-3)-O-β-D-galactose and (1-4)-O-3,6-anhydro-α-L-galactose. Agar is composed of polysaccharide chains with complex galactose residues of different lengths. Research shows that agarase can degrade agar polysaccharide into agarooligosaccharides (NAOS) with a degree of polymerization of 2-10. Agarooligosaccharides have various physiological activities such as antioxidant, lipid-lowering, immunomodulatory, anti-allergic, and glycosidase inhibitory activities. At the same time, they are also widely used in single-cell isolation of seaweeds and protoplast preparation. They are a kind of functional oligosaccharide with great development potential.

[0003] Agarase is considered to be the main method for sustainable commercial production of agar oligosaccharides due to its advantages of specifically hydrolyzing agar and not forming harmful compounds. However, free enzymes have some disadvantages (low thermal stability, narrow pH range, effective activity in an aqueous environment, and loss of catalytic activity after recycling), resulting in limited applications in industry.

[0004] Immobilized enzyme technology refers to the method of restricting free enzymes in a certain space or completely attaching them to a certain carrier through certain methods, so that they cannot move freely, but still maintain the integrity of the spatial structure and active center of the enzyme. It is a mainstream enzyme catalysis technology in enzyme engineering applications. Compared with free enzymes, immobilized enzymes have more stable performance, are less affected by external factors such as temperature, organic solvents, and pH, can be reused, and are easy to separate from reaction products. They greatly improve catalytic activity and operational stability, providing the possibility for wide applications in industrial production.

[0005] There are many methods for preparing immobilized enzymes, and the main methods include: adsorption, entrapment, cross-linking, and conjugation. Adsorption is also called the non-covalent method, mainly through the interaction between some non-covalent bonds. However, this binding is quite weak and unstable, usually does not destroy the natural structure of the enzyme, and allows the enzyme to maintain its activity. Entrapment is to embed the enzyme in a semi-permeable carrier so that the enzyme can be separated. Immobilized enzymes have the advantages of simple preparation, mild conditions, and high enzyme activity. However, the effect of single entrapment is affected by many factors and is unstable. Cross-linking uses bifunctional or multifunctional cross-linking reagents for enzyme molecules and carriers to form relatively strong covalent connections between the cross-linking reagents and the enzymes. However, this method may greatly affect the enzymatic properties of the enzymes. Covalent bonds can be divided into random covalent bonds and directed covalent bonds. Since intermolecular covalent bonds are formed between the carrier and the enzyme, it may endow the enzyme with structural rigidity, thereby improving its stability. The covalent bond method is the most deeply studied and widely used method in the research of immobilized enzymes. However, random covalent immobilization is usually carried out by randomly immobilizing amino acid residues (such as lysine residues) on the enzyme molecule, which may lead to the binding of multiple sites of the enzyme to the carrier, destroying the natural conformation of the enzyme, or causing steric hindrance to prevent the substrate from entering the active site of the enzyme, and finally greatly reducing the activity of the immobilized enzyme. Moreover, random immobilization can lead to the blocking of the active sites of the immobilized enzyme, making it difficult for the substrate to bind and the product to be released. On the contrary, directed covalent immobilization can immobilize the enzyme on a suitable carrier in a certain direction (away from the active center), so that the enzyme can better bind to the substrate. Therefore, site-specific immobilization is crucial for improving enzyme activity and productivity.

[0006] With the rapid development of structural biology, bioinformatics, and computer technology, protein computational design has become a reliable means to modify protein properties. By using computer means to determine the mutation sites and then carrying out directed covalent immobilization of the enzyme, finally, β-agarase with great industrial application potential can be obtained. Summary of the Invention

[0007] After characterizing and immobilizing the agarase from wild-type Saccharophagus degradans 2-40 (NCBI accession number: ABD81904) and the mutants of the present invention, an immobilized β-agarase mutant with enhanced thermal stability and reusability is obtained, endowing β-agarase with greater industrial application value.

[0008] The present invention provides an agarase mutant, which is obtained by mutating the arginine at the 66th position of the agarase parental enzyme with the amino acid sequence shown in SEQ ID NO.2 to cysteine, and is named R66C;

[0009] Or it is obtained by mutating the lysine at position 211 of the agarase parental enzyme with the amino acid sequence shown in SEQ ID NO.2 into cysteine, and is named K211C;

[0010] Or it is obtained by mutating the asparagine at position 452 of the agarase parental enzyme with the amino acid sequence shown in SEQ ID NO.2 into cysteine, and is named N452C.

[0011] In one embodiment of the present invention, the nucleotide sequence of the agarase parental enzyme is as shown in SEQ ID NO.1.

[0012] The present invention also provides a gene encoding the above mutant.

[0013] The present invention also provides a recombinant vector carrying the above gene.

[0014] In one embodiment of the present invention, the recombinant vector uses pET-28a as the expression vector.

[0015] The present invention also provides a recombinant cell containing the above mutant, or containing the above gene, or containing the above recombinant vector.

[0016] In one embodiment of the present invention, the recombinant cell uses a prokaryotic cell or a eukaryotic cell as the expression host.

[0017] In one embodiment of the present invention, the expression host is Escherichia coli.

[0018] The present invention also provides a recombinant Escherichia coli, which is characterized in that the recombinant Escherichia coli expresses the above agarase mutant.

[0019] The present invention also provides an immobilized agarase, which is prepared by mixing and incubating the above agarase mutant with maleimide-modified magnetic nanoparticles.

[0020] The present invention provides a method for selectively immobilizing a β-agarase mutant, and the method includes the following steps:

[0021] (1) Preparation of an amino-functionalized magnetic nanoparticle carrier;

[0022] (2) Preparation of maleimide-modified magnetic nanoparticles;

[0023] (3) Fixing β-agarase on magnetic nanoparticles by means of a Michael addition reaction between maleimide and the CYS group, that is: the immobilized β-agarase mutant is prepared by incubating the magnetic nanoparticles prepared in step (2) with the above β-agarase mutant.

[0024] In one embodiment of the present invention, the specific steps of the method are as follows:

[0025] (1) Preparation of amino-functionalized magnetic nanoparticle carriers:

[0026] Preparation of magnetic nanoparticles by solvothermal method: Dissolve FeCl3·6H2O in ethylene glycol, then add sodium acetate and chitosan, stir the mixture, and then seal it in an autoclave; heat at 200 °C for 8 h, wash it with ethanol several times after cooling to room temperature, and dry it at 60 °C for 6 h;

[0027] (2) Preparation of maleimide-modified magnetic nanoparticles:

[0028] Add sulfo-SMCC to the amino-functionalized magnetic nanoparticles, and shake the mixture in a water bath at 25 °C for 6 h to obtain maleimide-modified magnetic nanoparticles;

[0029] (3) Immobilize β-agarase on magnetic nanoparticles by Michael addition reaction between maleimide and CYS groups:

[0030] Add the magnetic nanoparticles prepared in step (2) to the agarase mutant solution, shake the mixture at 25 °C for 12 h, and wash it repeatedly to obtain the immobilized enzyme β-agarase.

[0031] The present invention also provides a method for improving the thermal stability of the immobilized enzyme. The method is as follows:

[0032] (1) Preparation of β-agarase mutants:

[0033] Using the plasmid containing the original enzyme as a template, perform site-directed mutagenesis with primer sequences to obtain R66C, K211C, and N452C mutants respectively;

[0034] (2) Mix and incubate the agarase mutants obtained in step (1) with amino-functionalized and maleimide-modified magnetic nanoparticles to prepare immobilized agarase.

[0035] The present invention also provides the use of the above-mentioned agarase mutants, or the above-mentioned genes, or the above-mentioned recombinant vectors, or the above-mentioned recombinant cells, or the above-mentioned immobilized agarase in the preparation of agarooligosaccharides or products containing agarooligosaccharides.

[0036] In one embodiment of the present invention, the application is to add the agarase mutant or recombinant cell or immobilized agarase to a reaction system containing agar to prepare.

[0037] Beneficial effects

[0038] (1) After characterizing and immobilizing the wild-type agarase derived from Saccharophagus degradans 2-40 (NCBI accession number: ABD81904) and the mutants of the present invention, an immobilized β-agarase mutant with enhanced thermal stability and reusability was obtained, endowing β-agarase with greater industrial application value. After the present invention was characterized and immobilized, a mutant immobilized enzyme with higher heat resistance than the free enzyme was obtained, greatly improving the industrial application prospect of agarase.

[0039] (2) For the R66C, K211C, and N452C mutant agarases, at 40 °C, after a 30-min water bath, the immobilized enzymes and relative enzyme activities were 94.62%, 92.00%, and 98.17% respectively, while that of the free enzyme was 79.84%;

[0040] After a 60-min water bath, the relative enzyme activity of the free enzyme was further reduced to 37.77%, and the immobilized enzymes R66C, K211C, and N452C retained 93.55%, 88.00%, and 98.17% of the relative enzyme activity respectively;

[0041] After a 6-h water bath, the immobilized enzymes R66C, K211C, and N452C still retained 59.25%, 45.10%, and 46.88% of the initial enzyme activity respectively, while the free enzyme almost lost all its enzyme activity; it can be seen that the stability of the immobilized enzyme of the present invention was significantly improved. Description of the Drawings

[0042] Figure 1 : SDS-PAGE diagram of the purified wild enzyme Aga50D and mutant enzymes; where, in the figure, M: protein molecular weight standard, 1: purified wild-type Aga50D, 2-4: purified mutants, which are R66C, K211C, and N452C in sequence.

[0043] Figure 2: Enzyme activity characterization of the wild enzyme Aga50D and its mutants; where, Figure 2a : Relative enzyme activity of the mutant relative to the wild type, Figure 2b : Tm values of the wild type and the mutant, Figure 2c : Optimal temperature characterization of the wild type and its mutants, Figure 2d : Optimal pH characterization of the wild type and its mutants, Figure 2e : Thermal stability characterization of the wild type and its mutants at 40 °C, Figure 2f : Thermal stability characterization of the wild type and its mutants at 45 °C.

[0044] Figure 3 : Schematic diagram of the preparation process of maleimide-modified magnetic nanoparticles.

[0045] Figure 4: Fourier transform infrared spectroscopy (FTIR) spectrum; among them Figure 4a : FTIR spectra of different mutant enzymes immobilized on maleimide-modified magnetic nanoparticles, maleimide-modified magnetic nanoparticles, and amino-modified magnetic nanoparticles, Figure 4b : FTIR spectra of amino-modified magnetic nanoparticles, maleimide-modified magnetic nanoparticles, and the N452C mutant enzyme immobilized on maleimide-modified magnetic nanoparticles.

[0046] Figure 5: Transmission electron microscopy (TEM) images of amino-modified magnetic nanoparticles and enzymes immobilized on maleimide-modified magnetic nanoparticles, among which, Figure 5a : TEM image of amino-modified magnetic nanoparticles, Figure 5b : TEM image of the N452C mutant enzyme immobilized on maleimide-modified magnetic nanoparticles.

[0047] Figure 6: Enzymatic property characterization of different mutant immobilized agarases; Figure 6a : Immobilization efficiency of different mutant immobilized enzymes, Figure 6b : Enzyme activity retention rate of different mutant immobilized enzymes, Figure 6c : Thermal stability characterization of different mutant immobilized enzymes at 40 °C, Figure 6d : Thermal stability characterization of different mutant immobilized enzymes at 45 °C, Figure 6e : Reusability of different mutant immobilized enzymes. Specific implementation methods

[0048] pET28a-Aga50D (the construction method is described in the Chinese invention patent with the publication number CN114836405A) and its mutants involved in the following examples were synthesized by Suzhou Anshengda Biotechnology Co., Ltd. The main reagents involved in the following examples: BCA concentration assay kit was purchased from Beyotime Biotechnology Co., Ltd., gene synthesis was completed by Suzhou Anshengda Biotechnology Co., Ltd., Sulfo-SMCC was purchased from Shanghai Leyan Co., Ltd., and other common reagents were of domestic analytical grade.

[0049] The media involved in the following examples are as follows:

[0050] LB liquid medium: Weigh 1 g of peptone, 0.5 g of yeast extract, and 1 g of NaCl. Dissolve with deionized water and make up the volume to 100 mL. Autoclave at 121 °C for 20 min.

[0051] LB solid medium: Add 1.8% agar powder to the LB liquid medium.

[0052] LB liquid resistant medium: Kanamycin was added to LB liquid medium at a final concentration of 50 μg / mL. LB solid medium: Kanamycin was added to LB solid medium at a final concentration of 50 μg / mL.

[0053] The purification method of the enzyme involved in the following examples is as follows:

[0054] (1) Equilibration: Equilibrate the nickel column with a buffer of 50 mM Tris-HCl, 500 mM NaCl, pH 7.5.

[0055] (2) Loading: Load the pretreated sample at a flow rate of 1 mL / min.

[0056] (3) Washing: Wash with a buffer of 50 mM Tris-HCl, 500 mM NaCl, pH 7.5, 20 mM imidazole.

[0057] (4) Elution: Elute and collect the target protein with a buffer of 50 mM Tris-HCl, 500 mM NaCl, 300 mM imidazole, pH 7.5 to obtain the purified enzyme.

[0058] The detection method involved in the following examples is as follows:

[0059] Determination of mutant enzyme activity:

[0060] The enzyme activity was determined by the 3,5-dinitrosalicylic acid method (DNS). Agarase catalyzes the hydrolysis of agarose to produce reducing sugars under certain conditions. 3,5-Dinitrosalicylic acid reacts with reducing sugars to produce a brown-red amino complex under heating conditions. The color depth is proportional to the amount of reducing sugars within a certain range and can be measured at a wavelength of 520 nm to calculate the enzyme activity.

[0061] Definition of enzyme activity unit:

[0062] Under the conditions of 30 °C and pH 7.5, the amount of enzyme required to catalyze the production of 1 μmol of D-galactose per minute is defined as one activity unit.

[0063] Steps for enzyme activity determination:

[0064] (1) Preheating: Take 2 mL of 1 mg / mL agarose solution (pH 7.5) in a colorimetric tube.

[0065] (2) Reaction: Add 0.1 mL of enzyme solution, mix well by shaking, react for 5 min, add 1.5 mL of DNS to terminate the reaction, heat in a boiling water bath for 5 min, and cool immediately.

[0066] (3) Measurement: Measure the absorbance value at 520 nm and calculate the enzyme activity.

[0067] Tm value determination:

[0068] Differential scanning fluorimetry (DSF) was used. Native proteins are in a folded state with hydrophobic parts hidden inside. As the temperature rises, the protein structure gradually disintegrates, exposing the hydrophobic parts. At this time, a dye that has an affinity for the hydrophobic parts binds to the protein, showing an increase in the fluorescence signal intensity of the system. When the temperature reaches a certain point, the unfolded protein chains aggregate, the fluorescent dye cannot bind and returns to the environment or undergoes fluorescence quenching at high temperatures, resulting in a decrease in the fluorescence signal. By tracking the changes in the fluorescence signal, the Tm of the protein can be determined. The SYPRO Orange dye was diluted 100 times, and 5 μL of the dye was mixed with 20 μL of the protein and placed in a 96-well thin-walled PCR plate. It was then heated from 25 °C to 99 °C in an ABI StepOnePlus real-time fluorescence quantitative PCR instrument system to monitor the fluorescence changes.

[0069] Steps for determining the thermal stability of the enzyme at 40 and 45 °C:

[0070] (1) Preheating: Take 2 mL of agarose solution (1 mg / mL, pH 7.5) in a colorimetric tube and incubate it in a water bath at 40 °C or 45 °C for 30, 60, 90, 120, 180 min and 6 h respectively;

[0071] (2) Reaction: Add 0.1 mL of the enzyme solution, mix well by shaking, react for 5 min, add 1.5 mL of DNS to terminate the reaction, and incubate in a boiling water bath for 5 min, then cool immediately.

[0072] (3) Measurement: Measure the absorbance at 520 nm and calculate the enzyme activity.

[0073] Calculation of the enzyme activity retention rate:

[0074] Enzyme activity retention rate (%) = free enzyme activity / immobilized enzyme activity * 100.

[0075] Calculation of the immobilization rate:

[0076] Immobilization rate (%) = (mass of enzyme added - mass of enzyme in the first wash supernatant - mass of enzyme in the second wash supernatant) / mass of enzyme added * 100.

[0077] The present invention will be specifically described below in conjunction with the accompanying drawings and embodiments.

[0078] Example 1: Construction of mutants

[0079] The specific steps are as follows:

[0080] Construction of a recombinant vector containing mutants:

[0081] Design site-directed mutagenesis primers and perform site-directed mutagenesis using the recombinant plasmid pET28a-Aga50D as a template to obtain mutants R66C, K211C, and N452C, respectively.

[0082] The primer sequences involved are as follows:

[0083] The site-directed mutagenesis primer for introducing the R66C mutation is:

[0084] R66C-F:5'-atcaagtcccttcaaatattcattttttaaatgcgtgcgcaagtatagaaacctata-3';

[0085] The site-directed mutagenesis primer for introducing the K211C mutation is:

[0086] K211C-F:5'-tgaaaaatttagatttgtcgggcattgcttgcatatcgctaagtgtgcaaagcgcaa-3';

[0087] K211C-R:5'-ttgcgctttgcacacttagcgatatgcaagcaatgcccgacaaatctaaatttttca-3';

[0088] The site-directed mutagenesis primer for introducing the N452C mutation is:

[0089] N452C-F:5'-tggatgctagtagacccagaagggtacccttactttgctacgggtttagac-3';

[0090] N452C-R:5'-tcagtccagttgcctagcgaggtaaagccccagcatagcattctgtctaccgttactt-3';

[0091] The PCR reaction system is shown in Table 1:

[0092] Table 1 Mutation PCR reaction system

[0093]

[0094] The PCR reaction conditions are: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 3 min 48 s, for a total of 30 cycles.

[0095] After gel extraction of the target fragment, it was transformed into E. coli BL21(DE3). The transformants were spread on LB plates containing kanamycin (50 μg / mL) and cultured statically overnight at 37°C. After colonies grew, single colonies were picked and inoculated into liquid LB medium containing kanamycin (50 μg / mL), and cultured overnight at 37°C with a shaking speed of 200 rpm. The bacterial solution was sent to Suzhou Anshengda Biotechnology Co., Ltd. for determination.

[0096] Mutant engineering bacteria containing the correct mutants were obtained respectively: E. coli BL21(DE3) / pET28a-R66C, E. coli BL21(DE3) / pET28a-K211C, E. coli BL21(DE3) / pET28a-N452C.

[0097] As a control: pET28a-Aga50D was transformed into E. coli BL21(DE3). According to the above method, wild-type agarase engineering bacteria: E. coli BL21(DE3) / pET28a-Aga50D were prepared.

[0098] Example 2: Purification of the enzyme and determination of its enzymatic properties

[0099] (1) Shake flask fermentation for enzyme production:

[0100] The genetic engineering bacteria obtained in Example 1 were streaked on LB plates containing kanamycin (50 μg / mL). After culturing at 37°C for 12 h, single colonies were picked and inoculated into LB liquid medium containing kanamycin (50 μg / mL) for shake flask fermentation, and cultured at 37°C with a shaking speed of 200 rpm for 12 h to obtain seed liquid.

[0101] 1 mL of the seed liquid was transferred to 100 mL of LB liquid medium containing kanamycin (50 μg / mL) for shake flask fermentation. When the OD600 reached 0.6 - 0.8 at 37°C, IPTG with a final concentration of 0.5 mM was added, and the temperature was reduced to 16°C to induce enzyme production. After culturing for 12 h, the cells were collected by centrifugation.

[0102] (2) Purification of the enzyme:

[0103] The centrifuged cells were resuspended in an appropriate amount (2 - 3 volumes) of lysis buffer (50 mmol / L Tris-HCl, 100 mmol / L NaCl, pH = 7.5), sonicated on ice for 15 - 20 min at 30% power, sonicated for 2 s and stopped for 3 s. After sonication, the supernatant was collected by centrifugation at 4°C and 8000 rpm for 10 min, and filtered through a 0.22 μm filter membrane. Then, Ni 2+ column affinity chromatography was performed to purify the mutant enzyme.

[0104] Pure enzyme solutions containing wild-type agarase and mutant enzymes R66C, K211C, and N452C were separately prepared. After SDS-PAGE electrophoresis detection, the results are as Figure 1 shown.

[0105] From Figure 1 the electrophoresis pattern, it can be seen that the wild-type WT is a single band without other heteroprotein bands, and the molecular weight is 84 kDa. The other mutants R66C, K211C, and N452C are also single bands, and the molecular weights are the same as that of the wild-type; it is proved that both the wild-type enzyme and the mutants of the present invention have been expressed.

[0106] (3) Enzyme activity assay:

[0107] The enzyme activities of the above wild-type agarase and mutant enzymes R66C, K211C, and N452C were measured. The wild-type enzyme activity was set as 100%, and the enzyme activity properties of the wild-type and mutant enzymes were detected. The results are shown in Figure 2.

[0108] From Figure 2a it can be seen that: the relative enzyme activities of the mutant enzymes relative to the wild-type enzyme are mostly around 110%, and only the relative enzyme activity of the K211C mutant compared with the wild-type enzyme is 98.08%;

[0109] From Figure 2b it can be seen that: among the three mutants, the Tm value of R66C is higher than that of the wild-type, and the Tm values of the other mutants are lower than that of the wild-type.

[0110] From Figure 2c the curves of d, f, e, it can be seen that the optimal temperature of the wild-type and mutant types: 30 °C, the optimal pH = 7, and at 40, 45 °C, the inactivation rates of the mutants and the wild-type are almost the same.

[0111] The above charts can prove that there are almost no differences in the enzymatic properties between the mutants and the wild-type.

[0112] Example 3: Immobilization of mutant enzyme:

[0113] (1) Selection of reaction groups:

[0114] The mutant enzyme surface contains CYS residues. Taking advantage of the characteristics that maleimide undergoes conjugate addition with thiol at physiological pH values (6.5 - 7.5) quickly and without any other reagents or catalysts, the mutant enzyme is directionally immobilized.

[0115] (2) Preparation of amino-functionalized magnetic nanoparticle carrier (NH2-MNPS):

[0116] Preparation of magnetic nanoparticles by solvothermal method:

[0117] Dissolve 6.25 mM FeCl3·6H2O in 30 mL of ethylene glycol, then add 50 mM sodium acetate and 0.02 g of chitosan. Vigorously stir the resulting mixture for 30 min, and then seal it in an autoclave (with a capacity of 50 mL). Heat at 200 °C for 8 h, wash it several times with ethanol after cooling to room temperature, and dry it at 60 °C for 6 h to prepare amino-functionalized magnetic nanoparticles (NH2-MNPS).

[0118] (3) Preparation of maleimide-modified magnetic nanoparticles (MAL-NH2-MNPS):

[0119] Add 1 mg of sulfo-SMCC to 50 mg of the amino-functionalized magnetic nanoparticles NH2-MNPS prepared in step (2), and shake the resulting mixture in a water bath at 25 °C for 6 h to obtain maleimide-modified magnetic nanoparticles MAL-NH2-MNPS. The reaction process is as Figure 3 shown.

[0120] (4) Immobilization of β-agarase:

[0121] Add 50 mg of the maleimide-modified magnetic nanoparticles prepared in step (3) to 1 mg of wild-type and mutant agarase solutions with the mass of the enzyme respectively, so that the final content of the carrier and the enzyme is 50:1 (w / w). Shake the mixture at 25 °C for 12 h, and wash it twice with PBS buffer to obtain immobilized enzyme wild-type β-agarase and immobilized enzyme mutant β-agarase.

[0122] (5) FT-IR test:

[0123] Adopt the KBr tablet method to analyze the structure of the sample using a Fourier transform infrared spectrometer. The wave number range is 4000~400 cm -1 , and the resolution is 0.01 cm -1 . The results are as Figure 4a ~b shown.

[0124] It can be seen from the FT-IR spectrum that: the stretching vibration peak of the primary amine in NH2-MNPS is at 1634 cm -1 ; the stretching vibration peaks of C=0 and amide N-H in the maleimide unit of MAL-NH2-MNPS are at 1614 cm -1 and 1407 cm -1 respectively, which proves that NH2-MNPS has been successfully maleimidized; 1699 cm -1The absorption peak at this position is the absorption peak of C=C in the maleimide unit of MAL-NH2-MNPS; while there is no absorption peak of C=C in N452C-MAL-NH2-MNPS, which proves that the enzyme has successfully undergone a Michael addition reaction with MAL-NH2-MNPS.

[0125] (6) Transmission electron microscope test:

[0126] Take 1 mg of the sample (the sample is the maleimide-modified magnetic nanoparticles prepared in step (3), the immobilized enzyme wild-type β-agarase and the immobilized enzyme mutant β-agarase prepared in step (4)) and disperse it ultrasonically in 2 mL of distilled water. Use a capillary pipette to suck the sample and drop it on the copper grid placed on the round filter paper, and drop it repeatedly several times. Place the filter paper with the copper grid under the sunlight to dry; place the dried copper grid in the sample holder for detection, and the detection instrument is a JEM-2100 high-resolution transmission electron microscope; the results are as Figure 5a ~b shown.

[0127] Transmission electron microscope analysis shows that the particle size of the amino-functionalized magnetic nanoparticles is uniform and there are few gaps between the nanoparticles; after the agarase is immobilized, the nanoparticles become dispersed, the gaps become larger, and there is a thin film on the surface of the particles. These results indicate that the enzyme is immobilized on the maleimide-modified magnetic nanoparticles.

[0128] Example 4: Determination of the enzymatic properties of immobilized wild-type and mutant β-agarases

[0129] 1. Immobilization efficiency of the immobilized enzyme:

[0130] (1) Take 2 mL of agarose solution (1 mg / mL, pH 7.5), and add 100 μL of the immobilized wild-type (initial enzyme activity is 13 ± 0.82 U / mg) and the immobilized mutant β-agarase prepared in Example 3 (the initial enzyme activities are R66C: 13.10 ± 0.54 U / mg; K211C: 11.2 ± 0.76 U / mg; N452C: 15.4 ± 0.94 U / mg) respectively;

[0131] React the above reaction system at 30 °C for 5 min and measure its enzyme activity.

[0132] (2) After the reaction is completed, measure the enzyme activities of the above immobilized wild-type agarase and the immobilized mutant enzymes R66C, K211C, and N452C. Set the enzyme activities of the free wild-type agarase and the free mutant enzymes prepared in Example 2 as 100%, and calculate the immobilization efficiencies of the wild-type and mutant enzymes.

[0133] The results show that the immobilization efficiency of the immobilized wild-type agarase is 0.

[0134] The immobilization yields of the immobilized mutant enzymes R66C, K211C, and N452C were 51.91%, 42.25%, and 37.71%, respectively.

[0135] From Figure 6a It can be seen that: Since the wild-type agarase has no CYS group, it cannot be immobilized on the MAL-MH2-MNPS carrier, and other mutant agarases were all normally immobilized on the MAL-MH2-MNPS carrier as expected.

[0136] The immobilization yields of the mutant agarases were mostly between 30% and 50%. It may be because there is only one CYS group on the mutant agarase molecule and only one immobilization site, resulting in a relatively low immobilization yield.

[0137] 2. Enzyme activity retention rate of the immobilized enzyme:

[0138] Take 2 mL of agarose solution (1 mg / mL, pH 7.5), add 100 μL of the immobilized wild-type (initial enzyme activity of 13 ± 0.82 U / mg) and immobilized mutant β-agarases (initial enzyme activities were R66C: 13.10 ± 0.54 U / mg; K211C: 11.2 ± 0.76 U / mg; N452C: 15.4 ± 0.94 U / mg) prepared in Example 3, and react at 30 °C for 5 min, and measure their enzyme activities.

[0139] Measure the enzyme activities of the above immobilized mutant enzymes R66C, K211C, and N452C. Set the free mutant enzyme activity as 100% respectively, and calculate the enzyme activity retention rate of the immobilized mutant enzymes.

[0140] The results show that from Figure 6b It can be seen that: The enzyme activity retention rates of the mutant agarases were all above 90%. The enzyme activity retention rates of R66C, K211C, and N452C reached 96.72%, 87.91%, and 94.93% respectively. It proves that the strategy of directional immobilization of agarase is successful.

[0141] (3) Thermal stability of the immobilized enzyme and free enzyme at 40 °C and 45 °C

[0142] Take 2 mL of agarose solution (1 mg / mL, pH 7.5), add 100 μL of the free wild enzyme (initial enzyme activity of 13 ± 0.82 U / mg) and immobilized mutant β-agarases (initial enzyme activities were R66C: 13.10 ± 0.54 U / mg; K211C: 11.2 ± 0.76 U / mg; N452C: 15.4 ± 0.94 U / mg) prepared in Example 3, place them in a water bath at 40 °C and incubate for 30, 60, 90, 120, 180 min and 6 h respectively, then react at 30 °C for 5 min, and measure their enzyme activities;

[0143] Meanwhile, according to the above method, the free wild enzyme and the immobilized mutant β-agarase were respectively placed in a water bath at 45°C, incubated for 30, 60, 90, 150, 270 min and 6 h respectively, and then reacted at 30°C for 5 min, and their enzyme activities were measured. The results are shown in Figure 6 and Tables 2 - 3:

[0144] Table 2: Relative enzyme activities of the immobilized enzymes and their free enzymes of different mutants at 40°C

[0145]

[0146]

[0147] Table 3: Relative enzyme activities of the immobilized enzymes and their free enzymes of different mutants at 45°C

[0148]

[0149] The results show that from Figure 6c and Figure 6d it can be seen that with the increase of the water bath time, the enzyme activities of the immobilized enzymes all gradually decrease, but the enzyme activity loss rate of the immobilized β-agarase is significantly less than that of the free enzyme.

[0150] For the R66C, K211C, and N452C mutant agarases, at 40°C, after a 30-min water bath, the immobilized enzymes and their relative enzyme activities are 94.62%, 92.00%, and 98.17% respectively, while the free enzyme is 79.84%;

[0151] After a 60-min water bath, the relative enzyme activity of the free enzyme further decreases to 37.77%, and the immobilized enzymes R66C, K211C, and N452C respectively retain 93.55%, 88.00%, and 98.17% of their relative enzyme activities;

[0152] After a 6-h water bath, the immobilized enzymes R66C, K211C, and N452C still respectively retain 59.25%, 45.10%, and 46.88% of their initial enzyme activities, while the free enzyme almost loses all its enzyme activity; it can be seen that the stability of the immobilized enzyme of the present invention is significantly improved.

[0153] (4) Reusability of the immobilized enzyme:

[0154] Take 2 mL of agarose solution (1 mg / mL, pH 7.5), add 100 μL of the immobilized mutant β-agarases R66C, N452C, and K211C prepared in Example 3, react at 30°C for 5 min, measure their enzyme activities, and take the first enzyme activity of each immobilized mutant β-agarase as 100%, and observe the reusability of the immobilized enzyme after 5 repeated uses.

[0155] The results show that from Figure 6e it can be seen that about 20-50% of the enzyme activity still remains after the immobilized enzyme is reused 5 times, while the free enzyme cannot be recovered and reused.

[0156] The decrease in the enzyme activity of the immobilized enzyme after repeated use is due to the fact that under laboratory conditions, the repeatability is measured, and the solid and liquid cannot be completely separated by 100%. After each use, a part of the immobilized enzyme will be lost due to reasons such as agar solidification. Therefore, it will cause a partial decrease in the enzyme activity. Therefore, an appropriate amount of immobilized enzyme can be supplemented after each use, which has a more obvious effect on maintaining the enzyme activity.

[0157] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. An agarase mutant, characterized in that, The mutant is obtained by mutating asparagine at position 452 of the agarase with the amino acid sequence shown in SEQ ID NO.2 into cysteine.

2. A gene encoding the mutant according to claim 1.

3. A recombinant vector carrying the gene according to claim 2.

4. The recombinant vector according to claim 3, characterized in that, The recombinant vector uses pET-28a as the expression vector.

5. A recombinant cell expressing the mutant according to claim 1, or carrying the gene according to claim 2, or carrying the recombinant vector according to claim 3 or 4.

6. The recombinant cell according to claim 5, wherein The recombinant cell uses a prokaryotic cell or a eukaryotic cell as the expression host.

7. The recombinant cell according to claim 6, wherein The expression host is Escherichia coli.

8. An immobilized agarase, characterized in that, The immobilized agarase is prepared by mixing and incubating the agarase mutant according to claim 1 with maleimide-modified magnetic nanoparticles.

9. A method for improving the thermal stability of immobilized enzymes, characterized in that, The method is as follows: (1) Preparation of β-agarase mutant: Mutate asparagine at position 452 of the agarase with the amino acid sequence shown in SEQ ID NO.2 into cysteine; (2) Mix and incubate the agarase mutant obtained in step (1) with amino-functionalized and maleimide-modified magnetic nanoparticles to prepare the immobilized agarase.

10. Use of the agarase mutant according to claim 1, or the gene according to claim 2, or the recombinant vector according to claim 3 or 4, or the recombinant cell according to any one of claims 5 to 7, or the immobilized agarase according to claim 8 in the preparation of agarooligosaccharides or products containing agarooligosaccharides.

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