A γ-polyglutamate degrading enzyme mutant and its application in preparing small molecule γ-polyglutamate

Abstract

The present invention relates to a γ-polyglutamate degrading enzyme mutant and its application in preparing small molecule γ-polyglutamate. The amino acid sequence of the γ-polyglutamate degrading enzyme mutant is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1. The present invention also provides a method for preparing small molecule γ-polyglutamate using the above-mentioned γ-polyglutamate degrading enzyme mutant. The present invention is based on polyglutamate degrading enzyme pgds, constructs a mutant library, and screens to obtain a γ-polyglutamate degrading enzyme mutant in which the isoleucine (Ile) at position 250 is mutated to serine (Ser) and the proline (Pro) at position 180 is mutated to cysteine ​​(Cys). Compared with polyglutamate degrading enzyme pgds, the enzyme activity of the γ-polyglutamate degrading enzyme mutant provided by the present invention is significantly improved, reaching 579.4U / mL, effectively increasing the activity of degrading γ-polyglutamate, and greatly improving the efficiency of degrading γ-polyglutamate.

Description

Technical Field

[0001] The invention relates to a gamma-polyglutamate degrading enzyme mutant and application thereof in preparing small molecule gamma-polyglutamate, belonging to the technical field of genetic engineering. Background Art

[0002] Polyglutamic acid is formed by the polymerization of two configurations of glutamic acid through γ-glutamine bonds. The molecular weight is generally distributed between 10 and 2000 kDa. The most common production method is fermentation synthesis by Bacillus subtilis. The large number of hydroxyl groups in its long peptides make it extremely hydrophilic and moisturizing. It is also a non-toxic, safe and edible biomaterial with good biocompatibility, biodegradability and non-immunogenicity. It is widely used in agriculture, food, medicine and cosmetics. However, the polyglutamic acid of chain long peptides has the following problems in practical applications: ① The molecular weight of polyglutamic acid from natural sources is usually in the millions, and its high viscosity greatly reduces its rheological properties; ② Chain polyglutamic acid is easily degraded by amide bond degrading enzymes; ③ The structure of long peptides is single and easily affected in application. Compared with chain long peptides, small molecule γ-polyglutamic acid has a smaller molecular weight. Small molecule γ-polyglutamic acid with the same mass has more active groups, more stable properties, is not easy to degrade during the compounding process, and has better activity performance in practical applications.

[0003] At present, the degradation of γ-PGA mainly includes three aspects: physical degradation, chemical degradation, and biological degradation. The main physical degradation methods include high temperature, mechanical shear force, ultrasound, ultraviolet radiation, etc. For example, in 1992, Atsuo degraded the γ-PGA solution through an oil bath at 80°C, 100°C, and 120°C, and the long peptides broke randomly at high temperature. In 1999, Perez degraded the million-molecular-weight γ-PGA to less than 100kDA by ultrasonic treatment at 20,000Hz for 2h. Although physical degradation is easy to operate, it has high equipment requirements and reaction conditions, and its efficiency is relatively low. The chemical degradation method is mainly to hydrolyze γ-PGA by adding acid (HCl) or alkali (NaOH). For example, Chen Guoguang adjusted the γ-PGA solution to pH 3.0 and the temperature to 80°C, and filtered it with an ultrafiltration membrane after 8h to obtain a small molecule γ-PGA of about 30kDa. Although chemical degradation has high hydrolysis efficiency, the introduction of exogenous chemical reagents affects the properties of γ-PGA, increases the difficulty of product purification, and is prone to environmental pollution. The bioenzymatic method uses polyglutamic acid degrading enzymes to specifically act on the γ-glutamine bonds of γ-PGA, causing the bonds to break and degrade. The reaction conditions are relatively mild. For example, Wang Huan et al. compared the genes related to γ-PGA degradation in the genome (ggt, cwl0, pgds, etc.) based on Bacillus licheniformis WX-02 through overexpression screening, and determined that polyglutamic acid degrading enzyme pgds is the key gene for degrading polyglutamic acid. Finally, by optimizing the promoter and signal peptide, the one-step production of 70kDA γ-PGA was successfully achieved. Although this method can produce low molecular weight γ-PGA, it still has the problems of low enzyme activity, poor degradation efficiency, and limited degradation ability. In the literature, the degradation limit of polyglutamic acid degrading enzymes is about 70kDa, and the free plasmid is easily lost when antibiotics are not added for screening. After the bacterial chromosome is integrated into the expression system, the degradation effect of γ-PGA is lost.

[0004] Therefore, there is an urgent need to develop a γ-polyglutamate degrading enzyme with high enzymatic activity and high degradation efficiency, which is of great value for preparing small molecule γ-polyglutamate by biodegradation method. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a γ-polyglutamate degrading enzyme mutant and its application in preparing small molecule γ-polyglutamate. The present invention can effectively improve the enzyme activity and degradation efficiency by mutating the γ-polyglutamate degrading enzyme.

[0006] The technical solution of the present invention is as follows:

[0007] A gamma-polyglutamate degrading enzyme mutant (I250S / P180C), the amino acid sequence of which is shown in SEQ ID NO.2, and the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0008] The γ-polyglutamate degrading enzyme mutant I250S / P180C provided by the present invention is based on the polyglutamate degrading enzyme pgds derived from Bacillus subtilis subsp.subtilis str.168, and the isoleucine (Ile) at position 250 is mutated to serine (Ser), and the proline (Pro) at position 180 is mutated to cysteine ​​(Cys). The amino acid sequence NCBI accession number of the polyglutamate degrading enzyme pgds is NP_391467.1, and the NCBI accession number of the nucleotide sequence of the encoding gene is QJF42721.1.

[0009] A recombinant vector is a plasmid vector into which the coding gene of the above-mentioned gamma-polyglutamate degradation enzyme mutant is inserted.

[0010] Preferably according to the present invention, the plasmid vector is pET-28a(+).

[0011] A recombinant strain is obtained by transforming the above recombinant vector into a host cell.

[0012] Preferably according to the present invention, the host cell is Escherichia coli; further preferably, the host cell is Escherichia coli BL21 (DE3).

[0013] Application of the above-mentioned γ-polyglutamate degrading enzyme mutant in the preparation of small molecule γ-polyglutamate.

[0014] A method for preparing small molecule γ-polyglutamate using the above-mentioned γ-polyglutamate degrading enzyme mutant, characterized in that the specific steps are as follows:

[0015] (1) cloning the mutant gene of the γ-polyglutamate degrading enzyme into an expression vector to obtain a recombinant expression vector; then transforming the recombinant expression vector into a host cell, selecting a positive clone for fermentation culture, and collecting wet cells;

[0016] (2) adding the wet cells obtained in step (1) to a Tris-HCl buffer, ultrasonically disrupting the cells for 35 to 45 minutes, and then centrifuging the cells at 8000 to 12000 rpm for 5 to 15 minutes to obtain a supernatant containing the γ-polyglutamate degrading enzyme mutant;

[0017] (3) adding the supernatant containing the mutant of γ-polyglutamate degrading enzyme obtained in step (2) to the γ-polyglutamate solution, and then reacting for 5 to 10 hours at 35 to 40° C., pH 6.5 to 7.5, and a rotation speed of 200 to 240 rpm, and obtaining a small molecule γ-polyglutamate solution after inactivation of the enzyme, filtration and dialysis;

[0018] (4) The small molecule γ-polyglutamic acid solution obtained in step (3) is filtered under positive pressure, and then activated carbon is added to the filtrate, and stirred at 50-60° C. for 1.5-2.5 hours, and then filtered, ultrafiltered, vacuum rotary evaporated and freeze-dried to obtain small molecule γ-polyglutamic acid.

[0019] Preferably, in step (2), the concentration of the Tris-HCl solution is 50 mM; the mass volume ratio of the Tris-HCl solution to the wet bacteria is 10:1, unit: mL / g; the ultrasonic disruption is performed by using an ultrasonic cell disruptor at a power of 150 to 300 kW for 40 minutes, working for 3 seconds and stopping for 3 seconds.

[0020] Preferably, according to the present invention, in step (2), the concentration of the γ-polyglutamate degrading enzyme mutant in the supernatant containing the γ-polyglutamate degrading enzyme mutant is 0.05-0.15 g / ml.

[0021] Preferably according to the present invention, in step (3), the volume ratio of the γ-polyglutamate solution to the supernatant containing the γ-polyglutamate degrading enzyme mutant is 9:1.

[0022] Preferably according to the present invention, in step (3), the concentration of the small molecule γ-polyglutamic acid solution is 0.1 to 0.2 g / mL.

[0023] Preferably, according to the present invention, in step (4), the positive pressure filtration is performed using a PES material, 0.5 μm microporous filter membrane; the mass volume ratio of the added amount of activated carbon to the filtrate is 1: (30-35), unit: g / mL.

[0024] Preferably, in step (4), the filtration separation is: pouring the solution decolorized by activated carbon onto diatomaceous earth to separate the activated carbon and small molecule γ-polyglutamic acid in the solution; the diatomaceous earth is mainly composed of SiO2 and has a specification of 100 mesh.

[0025] The beneficial effects of the present invention are:

[0026] The present invention is based on polyglutamate degrading enzyme pgds, and the enzyme is subjected to 3D modeling, structural analysis and full plasmid saturation mutation, and a mutant library is constructed. Finally, the γ-polyglutamate degrading enzyme mutant whose isoleucine (Ile) at position 250 is mutated to serine (Ser) and proline (Pro) at position 180 is mutated to cysteine ​​(Cys) is obtained by screening. Compared with polyglutamate degrading enzyme pgds, the enzyme activity of the γ-polyglutamate degrading enzyme mutant provided by the present invention is significantly improved, reaching 579.4U / mL, effectively increasing the activity of degrading γ-polyglutamate, greatly improving the efficiency of degrading γ-polyglutamate, and can hydrolyze polyglutamate in one step, without equipment requirements, and with mild reaction conditions. Compared with the final molecular weight of about 100kDa after the existing polyglutamate degrading enzyme treats polyglutamate of a molecular weight of a million from natural origin, the γ-polyglutamate degrading enzyme mutant provided by the present invention can hydrolyze polyglutamate of a molecular weight of a million from natural origin to less than 2kDa in one step. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is a 3D model diagram of polyglutamate degrading enzyme PGDS.

[0028] Figure 2 This is the docking result diagram of the three-dimensional structural model of γ-polyglutamate and polyglutamate degrading enzyme pgds.

[0029] Figure 3 The figure is a selection diagram of the mutation site of the γ-polyglutamate degrading enzyme mutant of the present invention.

[0030] Figure 4 This is the electrophoresis diagram of the linearized plasmid containing the γ-polyglutamate degrading enzyme mutant I250S / P180C.

[0031] Figure 5 This is the electrophoresis diagram of the engineered bacteria containing the γ-polyglutamate degrading enzyme mutant I250S / P180C.

[0032] Figure 6 This is the GPC test result of the molecular weight of the small molecule γ-polyglutamic acid prepared in the present invention. DETAILED DESCRIPTION

[0033] The following examples and drawings of the present invention only illustrate specific implementation schemes for implementing the present invention. These schemes and drawings should not be understood as limiting the present invention. Any changes made without departing from the principle and essence of the present invention fall within the protection scope of the present invention.

[0034] The experimental techniques and methods used in this example are all conventional technical methods unless otherwise specified. The materials, reagents, etc. used in this example can be obtained through regular commercial channels unless otherwise specified.

[0035] In order to better explain the present invention, the present invention is further described below in conjunction with the examples. The experimental methods in the present invention are conventional methods unless otherwise specified. The molecular biology experimental methods not specifically specified can be specifically referred to the "Molecular Cloning Experiment Guide" compiled by J. Sambrook et al., or carried out according to the kit method. The reagents and biological materials used in the specific examples can be obtained from commercial channels unless otherwise specified.

[0036] Example 1: Selection of mutation sites

[0037] 1. Homology modeling based on the amino acid sequence of polyglutamate degrading enzyme pgds

[0038] The polyglutamate degrading enzyme pgds is derived from Bacillus subtilis subsp. subtilis str.168, the NCBI accession number of its amino acid sequence is NP_391467.1, and the NCBI accession number of the nucleotide sequence of the encoding gene is QJF42721.1.

[0039] According to the amino acid sequence of polyglutamate degrading enzyme pgds, homology modeling was performed on SWISS-MODEL to obtain a 3D model of polyglutamate degrading enzyme pgds, as shown in the following figure: Figure 1 As shown. Then, the molecular docking software Autodock 4 was used to perform molecular simulation docking experiments on the 3D model data of polyglutamate degrading enzyme pgds and substrate polyglutamic acid (γ-PGA). The ligand molecule was downloaded from ZINC and then dehydrated and hydrogenated in AutoDockTools as a docking receptor. The computer simulation docking experiment was performed using docking software, with the chiral carbon of polyglutamic acid as the docking center and the length, width and height as The cube region is set as the substrate binding position. After the substrate polyglutamic acid is treated with water removal and hydrogenation in AutoDockTools, it is set as a ligand and tailed. Open the receptor and ligand files in AutoDockTools, set the docking region, generate a Grid file, select full flexible docking for the ligand in Docking, and select Genetic Algorithm (GA) for analysis. The algorithm is run 100 times, and the other parameters are default. The docking results are grouped by clustering algorithm in AutoDockTools and sorted from small to large by binding energy. Each docking experiment is run 100 times to improve accuracy. The docking structure is displayed on the PyMol software, as shown in the following figure. Figure 2 shown.

[0040] Structural analysis of the polyglutamate degrading enzyme pgds revealed that the three-dimensional structure of the polyglutamate degrading enzyme pgds is composed of three similar spherical structures connected in series, and the catalytic center is composed of a triplet, namely Asp139, His247, and Ser261, which are connected to γ-PGA through chemical bonds. The hydroxyl O atom of Ser261 attacks the C atom on the γ-amide bond of the substrate, breaking the substrate C=O bond and forming a tetrahedral transition state. Subsequently, the proton of His247 is transferred to the amino group of the substrate to release the broken polyglutamate.

[0041] 2. Select mutation sites

[0042] After fixing γ-PGA in the active pocket, the modification strategy is to reshape the substrate binding pocket and enlarge the contact channel to facilitate the contact between the substrate polyglutamate and the catalytic center. When selecting mutation sites, the active site and conserved sequence amino acids should be avoided, and amino acid residues near the substrate active pocket should be selected for mutation. Finally, the selected mutation sites are E174, P180, C194, R212, D233, I250, Q259, and Y275.

[0043] Example 2: Construction and screening of γ-polyglutamate degrading enzyme mutant library

[0044] 1. Entrust Jinweizhi Biotechnology Co., Ltd. to optimize the codons of polyglutamate degrading enzyme pgds and artificially synthesize its coding gene sequence, and then clone its coding gene sequence into pET-28a plasmid to obtain a plasmid vector expressing polyglutamate degrading enzyme pgds, and then transfer the plasmid vector into Escherichia coli BL21 (DE3) competent cells through the calcium chloride method to obtain Escherichia coli expressing polyglutamate degrading enzyme pgds, and store it at -80°C.

[0045] 2. Construction of mutation library

[0046] The plasmid vector expressing polyglutamate degrading enzyme pgds is used as a template, and primers are designed according to the mutation site selected in Example 1. The full plasmid over-lap PCR one-step synthesis of mutant plasmids is performed with a high-fidelity enzyme, and some primers are as shown in Table 1 (a pair of primers for each of the 8 mutation sites). The PCR product is the mutant plasmid we need to construct after methylation template elimination and purification. The mutant plasmid is transformed into Escherichia coli BL21 (DE3) competent cells to complete the construction of mutant engineering bacteria. Then the positive mutant is picked for fermentation culture, and the plasmid is extracted and sent for sequencing. The correct engineering bacteria for sequencing are induced to ferment, and the mutant library is screened using polyglutamate as a substrate to detect enzyme activity.

[0047] Table 1. PCR primers for some mutants

[0048]

[0049]

[0050] Note: The bold part is the mutation site

[0051] The mutant library constructed by the present invention contains 35 mutants, including I250E, I250S, I250K, R212M, R212H, R212W, R212D, R212A, R212P, R212C, R212Q, R212E, Q259S, Q259P, Y275K, Y275W, Y275V, Q259E, Q259A, Q259K, Q259N, Q259R, Q259F, E174N, E174P, E174E, E174S, P180C, P180W, P180N, P180D, P180L, D233E, D233N, and I250S / P180C.

[0052] The plasmids of the above mutants were verified by 1.0% agarose gel electrophoresis, and the electrophoresis diagram of the plasmid containing the γ-polyglutamate degrading enzyme mutant I250S / P180C is shown in Figure 4. At the same time, colony PCR was performed on the engineering bacteria containing the above mutants, and the electrophoresis diagram of the engineering bacteria containing the γ-polyglutamate degrading enzyme mutant I250S / P180C is shown in Figure 4. Figure 5 shown.

[0053] Depend on Figure 4 , 5 It can be seen that the γ-polyglutamate degrading enzyme mutant I250S / P180C was mutated and expressed successfully.

[0054] 3. Mutant screening

[0055] The engineered bacteria with correct sequencing and containing the above mutants were streaked onto solid culture medium and cultured at 37°C for 12 hours; solid flat bacteria were inoculated into seed culture medium and cultured at 37°C and 220r / min for 12 hours; under sterile conditions, the seed solution was inoculated into the fermentation medium at an inoculum concentration of 2% by volume, and cultured at 37°C and 220r / min for 1-3 hours, and the OD 600 When the concentration of IPTG was 0.6-0.8, a final concentration of 0.2 mM IPTG was added, and the shaking flask was transferred to 18°C ​​and 220 r / min conditions for 20 hours of culture to obtain a fermentation broth, and the fermentation broth was centrifuged at 12000 rpm for 10 minutes, the supernatant was discarded, and the wet cells were collected; the wet cells were ultrasonically broken, and the broken supernatant containing the enzyme mutant was taken, and then the γ-PGA solution was used as a substrate for degradation reaction. The reaction system is: the volume ratio of the broken supernatant: the substrate is 1:9; the reaction conditions are: the solution pH is 7.0, the temperature is 37°C, the degradation reaction time is 5 hours, and the enzyme activity of the enzyme mutant is detected after the reaction is completed. The results are shown in Table 2.

[0056] At the same time, wild-type polyglutamate degrading enzyme pgds was used as a control.

[0057] The enzyme activity detection method is as follows: prepare 50mM, pH7.0 phosphate buffer; 2M, pH5.4 acetate buffer and 1g / L polyglutamic acid solution; take 900μL of 1g / L γ-PGA solution, add 100μL crude enzyme solution, hydrolyze for 1h at 37°C and 220rpm, boil in a boiling water bath for 5min to inactivate the enzyme activity, and set up a control; take 0.5mL of the hydrolyzed reaction solution, add 1mL of acetate buffer and 0.5mL of ninhydrin colorimetric solution, mix well and boil in a boiling water bath for 15min, cool to room temperature and detect the absorbance value at an absorbance of 570nm, use acetate buffer as a diluent to match the optimal measurement range of a spectrophotometer, use L-glutamic acid as a substrate, draw a glutamic acid concentration standard curve to calculate the enzyme activity.

[0058] Enzyme activity definition: The amount of enzyme required to hydrolyze 1 g / L γ-PGA solution to generate 1 μmol / L of free amino acids for 1 h at 37°C, expressed in U / mL.

[0059] The solid culture medium is composed of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, 2% agar by mass concentration, deionized water as solvent, pH 7.0. The seed culture medium is composed of: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, deionized water as solvent, pH 7.0, sterilized at 115°C for 30 min. The fermentation culture medium is composed of: 4 g / L glucose, 20 g / L peptone, 15 g / L yeast extract, 2.2 g / L Na2HPO4, 1.0 g / L KH2PO4, 8 g / L NaCl, deionized water as solvent, pH 7.0, sterilized at 115°C for 30 min.

[0060] Table 2. Enzyme activities of various mutants

[0061]

[0062] As shown in Table 2, the degradation activity of the γ-polyglutamate degrading enzyme mutant I250S / P180C reached 579.4 U / mL, which is more than 20 times that of the wild-type polyglutamate degrading enzyme, effectively increasing the activity of degrading γ-polyglutamate and greatly improving the efficiency of degrading γ-polyglutamate.

[0063] Example 3. Preparation of small molecule γ-polyglutamic acid

[0064] A method for preparing small molecule γ-polyglutamic acid using a γ-polyglutamate degrading enzyme mutant (I250S / P180C) comprises the following steps:

[0065] (1) According to the method described in Examples 1 and 2, the mutant I250S / P180C of γ-polyglutamate degrading enzyme was cloned into an Escherichia coli expression vector to obtain an Escherichia coli recombinant expression vector; then the Escherichia coli recombinant expression vector was transformed into a host cell, positive clones were selected for fermentation culture, and wet cells were collected and weighed to 12.56 g;

[0066] (2) dissolving the wet cells in 125.6 mL of 50 mM Tris-HCl solution, treating the cells with an ultrasonic cell disruptor at a power of 200 kW for 40 min, working for 3 s, stopping for 3 s, and centrifuging at 10,000 rpm for 10 min after disruption to obtain a supernatant containing the γ-polyglutamate degrading enzyme mutant, wherein the concentration of the γ-polyglutamate degrading enzyme mutant in the supernatant is 0.1 g / mL;

[0067] (3) Weighing 151.2 g of sodium polyglutamate with a molecular weight of 1200 kDa and dissolving it in 1512 mL of water, adding the supernatant containing the γ-polyglutamate degrading enzyme mutant obtained in step (2) to the γ-polyglutamate solution at a volume ratio of 9:1, and then reacting for 5 h at 37° C., pH 7.0, and a rotation speed of 220 rpm, and incubating in a boiling water bath for 10 min to inactivate the enzyme, thereby obtaining a small molecule γ-polyglutamate solution;

[0068] (4) The small molecule γ-polyglutamic acid solution obtained in step (3) was filtered with a 0.5 μm PES microporous filter membrane under positive pressure to remove macromolecular impurities in the culture medium, and about 1566 mL of filtrate was collected; then 47 g of activated carbon was slowly added to the filtrate, and after it was fully dissolved, it was placed in a magnetic stirrer and stirred at 55° C. for 2 h to obtain a solution decolorized by activated carbon;

[0069] Then, a 2L Buchner funnel is taken, and qualitative filter paper is laid on the bottom layer of the funnel, and a diatomaceous earth solution is prepared and slowly poured into the filter paper, and the liquid is filtered until a diatomaceous earth layer of about 1 cm thick is formed on the filter paper, and the solution decolorized by activated carbon is slowly poured onto the diatomaceous earth to separate the dissolved activated carbon and the small molecule γ-polyglutamic acid, and a filtered separation solution is obtained; the diatomaceous earth is mainly composed of SiO2, and the specification is 100 mesh;

[0070] The filtered separated solution was further ultrafiltered using a 10 kDa nanomembrane to collect about 1513 mL of a small molecule γ-polyglutamic acid solution;

[0071] After removing 776 mL of water from the small molecule γ-polyglutamic acid solution using a vacuum rotary evaporator, the concentrated sample was divided into 3 glass containers with a volume not exceeding half the height of the container, covered with plastic wrap, and holes were made in the plastic wrap with a needle for ventilation. The sample was then placed in a freeze dryer for 48 hours to obtain freeze-dried small molecule γ-polyglutamic acid.

[0072] The freeze-dried small molecule γ-polyglutamic acid sample was ground into a white fine powder, weighing 131.4 g, with a yield of 86.9%.

[0073] Example 4: GPC Detection of Small Molecule γ-Polyglutamic Acid

[0074] 1 g of the freeze-dried small molecule γ-polyglutamic acid powder prepared in Example 3 was taken out for GPC detection. The specific method is as follows:

[0075] The freeze-dried small molecule γ-polyglutamic acid powder was prepared into a solution with a concentration of about 5 mg / mL using a mobile phase, the pH was adjusted to neutral with a NaOH aqueous solution, and the solution was sterilized by filtration with a 0.22 μm microporous filter membrane. The molecular weight was determined using a Malvern gel permeation chromatograph. The results are as follows: Figure 6 As shown, the larger the molecular weight, the earlier the peak time. The control group on the left is the undegraded PGA with a molecular weight of one million, and the experimental group on the right is the degraded PGA of 1.3 kDa.

[0076] Wherein, the chromatographic conditions of the GPC detection are as follows:

[0077] Chromatographic column: TSK-GEL G4000WPXL;

[0078] Detector: laser detector and differential detector;

[0079] Mobile phase: 0.1 M sodium nitrite-0.05% sodium nitrite (natural pH);

[0080] Column temperature: 35 °C;

[0081] Flow rate: 0.8 ml / min;

[0082] The injection volume was 50 μl.

[0083] Depend on Figure 6 It can be seen that the molecular weight of the small molecule γ-polyglutamic acid prepared by the present invention is 1.6 kDa, indicating that the present invention can fully hydrolyze the natural source million-level molecular weight polyglutamic acid to less than 2 kDa through the heterologously expressed γ-polyglutamate degrading enzyme mutant I250S / P180C.

Claims

1. A γ-polyglutamate degrading enzyme mutant, characterized in that: The amino acid sequence is shown in SEQ ID NO.2, and the nucleotide sequence of the coding gene is shown in SEQ ID NO.

1.

2. A recombinant vector, characterized in that: The gene encoding the γ-polyglutamate degrading enzyme mutant according to claim 1 is inserted into a plasmid vector.

3. A recombinant strain, characterized in that: The method is obtained by transforming the recombinant vector according to claim 2 into a host cell.

4. Use of the γ-polyglutamate degrading enzyme mutant according to claim 1 in the preparation of small molecule γ-polyglutamate.

5. A method for preparing small molecule γ-polyglutamate using the γ-polyglutamate degrading enzyme mutant according to claim 1, characterized in that: The specific steps are as follows: (1) Cloning the mutant gene of the γ-polyglutamate degrading enzyme described in claim 1 into an expression vector to obtain a recombinant expression vector; then transforming the recombinant expression vector into a host cell, selecting positive clones for fermentation culture, and collecting wet cells; (2) adding the wet cells obtained in step (1) to Tris-HCl buffer, ultrasonically disrupting for 35 to 45 minutes, and then centrifuging at 8000 to 12000 rpm for 5 to 15 minutes to obtain a supernatant containing the γ-polyglutamate degrading enzyme mutant; (3) adding the supernatant containing the mutant of γ-polyglutamate degrading enzyme obtained in step (2) to the γ-polyglutamate solution, and then reacting at 35-40° C., pH 6.5-7.5, and rotation speed 200-240 rpm for 5-10 hours, and obtaining a small molecule γ-polyglutamate solution after enzyme inactivation, filtration and dialysis; (4) The small molecule γ-polyglutamic acid solution obtained in step (3) is filtered under positive pressure, and then activated carbon is added to the filtrate, and stirred at 50-60° C. for 1.5-2.5 hours. The small molecule γ-polyglutamic acid is obtained after separation by filtration, ultrafiltration, vacuum rotary evaporation and freeze drying.

6. The method for preparing small molecule γ-polyglutamic acid according to claim 5, characterized in that: In step (2), the concentration of the Tris-HCl solution is 50 mM; the mass volume ratio of the Tris-HCl solution to the wet bacteria is 10:1, unit: mL / g; the ultrasonic disruption is carried out by using an ultrasonic cell disruptor at a power of 150-300 kW for 40 min, working for 3 s and stopping for 3 s.

7. The method for preparing small molecule γ-polyglutamic acid according to claim 5, characterized in that: In step (2), the concentration of the γ-polyglutamate degrading enzyme mutant in the supernatant containing the γ-polyglutamate degrading enzyme mutant is 0.05-0.15 g / ml.

8. The method for preparing small molecule γ-polyglutamic acid according to claim 5, characterized in that: In step (3), the volume ratio of the γ-polyglutamate solution to the supernatant containing the γ-polyglutamate degrading enzyme mutant is 9:

1.

9. The method for preparing small molecule γ-polyglutamic acid according to claim 5, characterized in that: The concentration of the small molecule γ-polyglutamic acid solution is 0.1-0.2 g / mL.

10. The method for preparing small molecule γ-polyglutamic acid according to claim 5, characterized in that: In step (4), the positive pressure filtration is performed using a PES material and a 0.5 μm microporous filter membrane; the mass volume ratio of the added amount of activated carbon to the filtrate is 1: (30-35), unit: g / mL; The filtering separation is as follows: pouring the solution decolorized by activated carbon onto diatomaceous earth to separate the activated carbon and small molecule gamma-polyglutamic acid in the solution; the diatomaceous earth is mainly composed of SiO2 and has a specification of 100 meshes.

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