D-tagatose-4-epimerase mutants, engineered bacteria and use of synthetic d-tagatose

Abstract

The application discloses a D-tagatose 4-epimerase mutant, an engineering bacterium and application of the engineering bacterium for synthesizing D-tagatose. The application screens a high-activity D-tagatose 4-epimerase mutant, improves the tolerance of the enzyme to a substrate D-fructose, and enables the enzyme to catalyze at a higher substrate concentration, and meanwhile improves the conversion efficiency. The conversion rate of the superimposed mutant enzyme N139T / Q334A reaches 1.55 and 1.52 times of that of a wild enzyme at substrate concentrations of 10 g / L and 50 g / L, and the mutant enzyme can reach a conversion rate close to that of the wild enzyme at a substrate concentration of 10 g / L at a substrate concentration of 50 g / L. The application improves the problems of poor substrate tolerance and poor catalytic activity at high temperature of the wild enzyme, overcomes the problem of easy generation of three wastes in a chemical synthesis method, and has an important industrial application prospect.

Description

(I) Technical Field

[0002] This invention relates to a highly active D-tagatose-4-epimerase mutant, an engineered bacterium, and the application of synthesized D-tagatose. (II) Background Technology

[0004] In recent years, the incidence of metabolic diseases such as obesity and diabetes has been gradually increasing. Excessive sugar intake is widely considered a significant contributing factor. Therefore, finding sweeteners that combine physiological functions with low-calorie properties is a major research focus in the field of health and wellness. D-Tagatose, as the C-4 epimer of D-fructose, combines excellent taste with low calories. It also possesses various physiological activities, including regulating blood sugar levels, inhibiting fat accumulation, improving gut microbiota structure, and enhancing metabolism, making it a promising candidate as a sucrose substitute.

[0005] Currently, industrial methods for synthesizing D-tagatose are mainly divided into two categories: chemical synthesis and biosynthesis. Chemical synthesis uses soluble alkali metal salts as catalysts to catalyze the isomerization reaction of D-galactose with metal hydroxides to produce D-tagatose. However, it suffers from drawbacks such as numerous reaction byproducts, low purity of the target product, heavy metal residues, and acidic wastewater. Biosynthesis, with its advantages of mild reaction conditions, fewer byproducts, and environmental friendliness, is considered the mainstream direction for the future industrial production of D-tagatose. Currently reported biosynthetic methods for D-tagatose mainly rely on the synergistic catalysis of β-galactosidase and L-arabinose isomerase, using lactose as a substrate and undergoing a two-step enzymatic reaction to generate the target product. However, this pathway faces technical bottlenecks such as poor thermostability and low enzyme activity of L-arabinose isomerase.

[0006] Against this backdrop, many scholars have dedicated themselves to discovering and developing D-tagatose 4-epimerases (T4E) that synthesize D-tagatose using the less expensive D-fructose as a substrate. Currently, publicly reported T4E enzyme resources are scarce, and the catalytic activity of both natural and modified enzymes is generally low, failing to meet the demands for efficient industrial production of D-tagatose. Therefore, molecular modification of T4E to enhance its catalytic activity has become a key research focus in this field. (III) Summary of the Invention

[0008] The purpose of this invention is to provide a highly active D-tagatose-4-epimerase mutant, engineered bacteria, and its application in the synthesis of D-tagatose, thereby solving the problem that the current D-tagatose-4-epimerase has low catalytic activity and cannot meet the requirements for efficient industrial production of D-tagatose.

[0009] The technical solution adopted in this invention is:

[0010] This invention provides a highly active D-tagatose-4-epimerase mutant, which is obtained by single-point or superimposed mutations at positions 139, 31, 92, 95, 172, 192, 220, 258, 294, 320, 334, and 358 of the amino acid sequence shown in SEQ ID NO.2.

[0011] Preferably, the highly active D-tagatose-4-epimerase mutant is the one described in SEQ ID NO. The amino acid sequence shown in NO.2 is mutated to one of the following: (1) N139T, i.e., asparagine at position 139 is mutated to threonine; (2) N139T / E31G, i.e., asparagine at position 139 is mutated to threonine, and glutamic acid at position 31 is mutated to glycine; (3) N139T / P95D, i.e., asparagine at position 139 is mutated to threonine, and proline at position 95 is mutated to aspartic acid; (4) N139T / K172P, i.e., asparagine at position 139 is mutated to threonine, and lysine at position 172 is mutated to proline; (5) N139T / L220H, i.e., asparagine at position 139 is mutated to threonine, and leucine at position 220 is mutated to histidine; (6) N139T / C258I, i.e., asparagine at position 139 is mutated to threonine. (7) N139T / S294E, i.e., asparagine at position 139 is mutated to threonine, and serine at position 294 is mutated to glutamic acid; (8) N139T / A320I, i.e., asparagine at position 139 is mutated to threonine, and alanine at position 320 is mutated to isoleucine; (9) N139T / Q334A, i.e., asparagine at position 139 is mutated to threonine, and glutamine at position 334 is mutated to alanine; (10) N139T / T358I, i.e., asparagine at position 139 is mutated to threonine, and threonine at position 358 is mutated to isoleucine; (11) K92S, i.e., lysine at position 92 is mutated to serine; (12) E192D, i.e., glutamic acid at position 192 is mutated to aspartic acid.

[0012] Preferably, the highly active D-tagatose-4-epimerase mutant is N139T / Q334A, with the amino acid sequence shown in SEQ ID NO.4.

[0013] Due to the specificity of amino acid sequences, any variant of the polypeptide with the aforementioned mutant amino acid sequence, such as its conserved variants, bioactive fragments, or derivatives, is within the scope of protection of this invention, provided that the polypeptide fragment or polypeptide variant shares more than 95% homology with the aforementioned amino acid sequence. The alterations may include the deletion, insertion, or substitution of amino acids in the amino acid sequence; for conserved alterations in variants, the substituted amino acid has a similar structure or chemical properties to the original amino acid, such as replacing isoleucine with leucine; variants may also have non-conserved alterations, such as replacing glycine with tryptophan.

[0014] The present invention also relates to a gene encoding the D-tagatose-4-epimerase mutant, preferably the nucleotide sequence of the encoding gene as shown in SEQ ID NO.3.

[0015] Due to the specific nature of nucleotide sequences, any polynucleotide variant of the aforementioned mutant, provided it shares more than 95% homology with the original polynucleotide, falls within the scope of protection of this invention. The polynucleotide variant refers to a polynucleotide sequence with one or more nucleotide alterations. This polynucleotide variant can be a live or non-live allelic variant, including substitution variants, deletion variants, and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may involve the substitution, deletion, or insertion of one or more nucleotides, but does not substantially alter the function encoding the amino acid.

[0016] This invention also relates to a recombinant vector containing the coding gene and a recombinant genetically engineered bacterium; the recombinant vector uses pET-28a as a basic plasmid, and the recombinant genetically engineered bacterium uses... E. coli BL21(DE3) is the host bacterium.

[0017] The present invention also relates to the application of the D-tagatose-4-epistomerase mutant in the synthesis of D-tagatose. The application is as follows: using wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the encoding gene of the D-tagatose-4-epistomerase mutant as the enzyme source, D-fructose as the substrate, cobalt ions as the co-catalyst, and a buffer solution with pH 6.0-9.0 (preferably pH 8.0) as the reaction medium, the reaction is carried out at 50-80℃ and 500-1200 r / min (preferably at 70℃ and 1000 r / min for 6 h) until the reaction is complete, and a mixture of D-fructose and D-tagatose is obtained.

[0018] Preferably, in the reaction system, the initial concentration of the substrate is 10-100 g / L (preferably 50 g / L), the final concentration of the wet bacterial cells is 10-150 g / L (preferably 40 g / L), and the final concentration of the cobalt ions is 0.1-10 mM (preferably 1 mM).

[0019] Preferably, the reaction medium is a pH 8.0, 50 mM sodium phosphate buffer solution.

[0020] Preferably, the wet bacterial cells are prepared as follows: recombinant genetically engineered bacteria containing the D-tagatose-4-epimerase mutant gene are inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180-200 r / min until OD. 600 =0.8–1.0, to obtain seed culture; the seed culture is transferred at an inoculum concentration of 1–5% to LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37℃ and 180 r / min until OD. 600 =0.6~0.8, add IPTG to a final concentration of 0.1 mM, and induce culture at 28℃ and 180 r / min for 12~14 h to obtain the induced culture suspension. Centrifuge the induced culture suspension at 4℃ and 8000 rpm for 10 min to collect the wet cells.

[0021] LB liquid medium composition: 10 g / L tryptone, 5 g / L yeast extract, 10 g / L NaCl, solvent is ultrapure water; LB solid medium is supplemented with 20 g / L agar; autoclave; add kanamycin to a final concentration of 50 μg / mL before use.

[0022] The D-tagatose-4-epimerase and mutant described in this invention can withstand high temperatures of 40–75°C. Furthermore, at a substrate concentration of 10 g / L and a reaction temperature of 70°C, the conversion rate of the D-tagatose-4-epimerase mutant N139T / Q334A reaches 1.55 times that of the wild type.

[0023] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in the following: the present invention screens and obtains a highly active D-tagatose-4-epimerase mutant, which improves the enzyme's tolerance to the substrate D-fructose, enabling it to catalyze at higher substrate concentrations and improve conversion efficiency.

[0024] The superimposed mutant enzyme N139T / Q334A screened in this invention achieved conversion rates of 13.49% and 7.98% at substrate concentrations of 10 g / L and 50 g / L, respectively, with yields of 1.35 g / L and 3.99 g / L. In contrast, the wild-type enzyme EbT4E achieved a conversion rate of 8.69% at a substrate concentration of 10 g / L, but this decreased to 5.25% at 50 g / L. The mutant enzyme of this invention achieves a conversion rate close to that of the wild-type enzyme at 50 g / L, and its conversion rates at substrate concentrations of 10 g / L and 50 g / L are 1.55 and 1.52 times higher than those of the wild-type enzyme, respectively.

[0025] This invention improves the poor substrate tolerance and low catalytic activity at high temperatures of wild-type enzymes through mutation, expands the relatively scarce D-tagatose-4-epimerase library and mutant library, and demonstrates the technical advantages of D-tagatose-4-epimerase in catalyzing the synthesis of D-tagatose from D-fructose, which is green, environmentally friendly, low in toxicity, and produces few by-products. It overcomes the problem of waste generation in chemical synthesis methods and has important industrial application prospects. (iv) Description of the attached drawings

[0027] Figure 1 This is a schematic diagram of the enzymatic synthesis of D-tagatose from D-fructose.

[0028] Figure 2 High-performance liquid chromatography (HPLC) chromatograms of D-fructose and D-tagatose standards.

[0029] Figure 3 The bar chart shows the relative conversion rates of the original enzyme and the single point mutant.

[0030] Figure 4 Bar charts showing the relative transformation rates of single-site and multi-site mutants. (V) Detailed Implementation Methods

[0032] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0033] Example 1: Wild-type D-tagatose-4-epimerase genetically engineered bacteria E. coli Construction of BL21(DE3) / T4E

[0034] Sourced from NCBI Thermotoga petrophila Using D-tagaturonate epimerase UxaE (GenBank accession number WP_011943119.1) as a probe, potential T4E was screened from the NCBI database. Based on screening principles such as sequence consistency and conservation of active sites, the probe was ultimately selected from... Eubacteriales-bacterium Tagitaturonateepimerase family protein (referred to as wild-type enzyme) Eb Further research will be conducted on T4E (GenBank ID MDD7397597.1).

[0035] The above filtered Eb After codon optimization and the addition of a histidine tag (nucleotide sequence as shown in SEQ ID NO.1, amino acid sequence as shown in SEQ ID NO.2), the T4E gene sequence was synthesized and inserted into the plasmid pET28a(+) to obtain pET28a(+)-. Eb T4E plasmid, transform the plasmid into E. coli Wild-type recombinant genetically engineered bacteria were obtained from BL21(DE3). E. coli BL21(DE3) / Eb T4E, denoted as engineered bacteria Eb T4E.

[0036] SEQ ID NO.2

[0037] MKSIMQLQDFLDKLHQDGSLYVYPRSAEEAEGTCCAIILAGEEKEKLLAVAGEKADTFAGEEVDGIKLCGLTAENAKALMALFPYTRPVSHKDHPFTMGLGDRLGLATPGHVRAMEAYAKFGVF PVLAQQSIRELTLTNRTFEEVIAAAAFGVFQEGYKKGYGADGDHLKTKEEIKYAIESGCTMITLDCSEHIDIHAAAFTQAQVDEAYAALPQEVRSLYEERYLHKELPVIGTLEAEELRRIVLVFW RAIDHARECYAYIESIKPYPVDFELSIDETRTITTPAEHFVVADSLYQAGVRPVSVAPHFSGEFEKGIEYAGNLHDFARDFDIHQKIADHFGYKLSVHSGSDKFSVFSTVGRVTHGHVHVKTAG TNWLVAMEVVAEHDPALYRKAHTFAIAQRHEAEKYYHVSTKVEEIPPIELESDAYLPEYLHLPASRQTIHIAYGLLLQQEWFQEEFFAFMAAHEEEYYEALVRHIGRHLKYLTAQMLEHHHHHH.

[0038] Example 2: Construction of a unit point mutant of D-tagatose-4-epimerase

[0039] 1. Design of mutation sites

[0040] Wild-type enzymes were constructed using Alphafold3. Eb A protein structure model of T4E was developed, and molecular docking with the substrate D-fructose was performed using Maestro software to predict the binding mode and affinity between the substrate and the enzyme active site. To enhance... EbT4E's affinity for the substrate D-fructose allows the substrate to enter the active pocket more smoothly. Amino acids near the substrate channel were selected for modification, specifically at sites: 92, 135, 139, 145, 146, 147, 148, 187, 192, and 368. Following the principle of regression consensus mutation, previously reported dominant amino acid sites were modified... Eb The T4E assay was applied, and the predicted mutants were K92S, L135T, N139T, V145P, I146P, A147P, A148P, T187A, E192D, and H368L.

[0041] Based on the remote modification strategy, some amino acids far from the active pocket were also modified at specific sites: 31, 32, 70, 73, 95, 166, 172, 181, 220, 258, 294, 320, 334, 358, 403, and 463. The mutants were predicted using the online website PROSS, and the mutants were E31G, G32D, G70P, A73H, P95D, G166A, K172P, S181A, L220H, C258I, S294E, A320I, Q334A, T358I, H403E, and M463L.

[0042] 2. Unit point saturation mutation

[0043] Wild-type enzymes screened according to Example 1 Eb Primers for site-directed mutagenesis were designed based on the T4E gene sequence (SEQ ID NO.1) and the mutation site in step 1 (Table 1). Using rapid PCR technology, single point mutations were introduced at positions 92, 135, 139, 145, 146, 147, 148, 187, 192, and 368, respectively, using the recombinant plasmid pET28a / EbT4E as a template.

[0044] Table 1 Mutant Primers

[0045]

[0046] The PCR reaction system (50 μL) consisted of: 25 μL of 2×Phanta Max Buffer, 1 μL of dNTPs, 2 μL of forward primer (5 pmol / μL), 2 μL of reverse primer (5 pmol / μL), 1 μL of template DNA (20 ng / μL), 1 μL of Phanta Max SuperFidelity DNA Polymerase, and ddH2O added to a final volume of 50 μL.

[0047] The PCR reaction program was as follows: 95℃ pre-denaturation for 5 min; 30 cycles (95℃ denaturation for 10 s, 55-60℃ annealing for 15 s, 72℃ extension for 3.5 min); 72℃ extension for 10 min; and finally, incubation at 16℃. The PCR products were verified by 0.9% agarose gel electrophoresis. Once the amplified gene fragment matched the size of the target vector, the next step of the experiment could be carried out.

[0048] After digesting the template with DpnI at 37℃ for 3 hours, 3 μL of the PCR product was added to 100 μL of ice bath. E. coli In a suspension of BL21(DE3) competent cells, the cells were incubated on ice for 30 min. The transformation product was then heat-shocked at 42°C for 90 s, followed by rapid cooling on ice for 5 min. 600 μL of LB liquid medium was added to the tube, and the cells were incubated at 37°C, 150 rpm for 50 min, and centrifuged at 12000 rpm for 1 min. 400 μL of supernatant was discarded, and the bacterial suspension was resuspended. 200 μL of the resuspended suspension was spread onto an LB agar plate containing 50 μg / mL kanamycin-resistant material. After complete absorption of the culture medium, the plates were incubated upside down at 37°C for 12 h. Colonies were then picked and inoculated into 10 mL of LB liquid medium containing 50 μg / mL kanamycin-resistant material and incubated at 37°C for 12 h. The resulting bacterial suspension was sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the sequencing results matched correctly, identifying the mutant recombinant strain as *E. coli* BL21(DE3) / pET28a / . Eb T4E / muts.

[0049] The above-mentioned mutant recombinant bacteria were prepared into wet cells according to the method in Example 3, and the transformation rate was tested according to the method in Example 4. With the wild-type transformation rate set at 100%, the relative transformation rates of other mutants were calculated. The results are shown in […]. Figure 3 As shown, the dominant mutant was obtained. E. coli BL21(DE3) / pET28a / Eb T4E / N139T, E. coli BL21(DE3) / pET28a / Eb T4E / K92S, E. coli BL21(DE3) / pET28a / Eb T4E / E192D, the optimal mutant E. coli BL21(DE3) / pET28a / Eb T4E / N139T is denoted as M1.

[0050] 3. Superposition mutation

[0051] The dominant mutant from step 2 was superimposed with a distal site using the methods in steps 1 and 2. The recombinant mutant bacteria were then prepared into wet cells according to the method in Example 3, and the transformation rate was tested according to the method in Example 4. With M1 transformation rate set at 100%, the relative transformation rates of other mutants were calculated. The results are shown in […]. Figure 4 As shown, the dominant mutants obtained through screening were M1-Q334A, M1-E31G, M1-P95D, M1-G172P, M1-L220H, M1-C258I, M1-S294E, M1-A320I, and M1-T358I. Among them, the mutants... E. coli BL21(DE3) / pET28a / Eb The transformation rate of T4E / N139T / Q334A (amino acid sequence as shown in SEQ ID NO.4, nucleotide sequence as shown in SEQ ID NO.3) was 1.55 times higher than that of the original strain.

[0052] Example 3: Preparation of D-tagatose-4-epomerase and mutant wet cells

[0053] The wild-type recombinant genetically engineered bacteria from Example 1 and the mutant recombinant genetically engineered bacteria constructed in Example 2 were streaked onto LB solid medium plates containing a final concentration of 50 μg / mL kanamycin resistance for activation. After incubation at 37°C for 12 h, single colonies were picked and inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, and cultured at 37°C and 180 rpm for 6-8 h until OD. 600 Once the bacterial OD reaches 0.6-0.8, inoculate 1% (v / v) into 100 mL of fresh LB liquid medium containing a final concentration of 50 μg / mL kanamycin resistance, and incubate at 37°C and 180 r / min until the bacterial OD reaches 0.6-0.8. 600 The concentration of the culture medium was increased to 0.6-0.8. IPTG was added to a final concentration of 0.1 mM, and the mixture was induced and cultured at 28°C and 180 r / min for 12-14 h. After centrifugation at 4°C and 8000 r / min for 10 min, the supernatant was discarded, and the precipitate was collected to obtain the wet bacterial cells. These wet bacterial cells can be used directly as a biocatalyst or for protein purification.

[0054] Example 4: Conversion rate determination of D-tagatose-4-epimerase and mutants

[0055] The D-tagatose-4-epimerase and mutant wet cells obtained in Example 3 were used as enzyme sources to catalyze the substrate D-fructose.

[0056] The wet bacterial cells were thoroughly mixed and suspended in 50 mM sodium phosphate buffer (pH 8.0), and then 1 mM Co was added to the final concentration. 2+A 1 mL reaction system was prepared with D-fructose at a final concentration of 10 g / L, and wet bacterial cells were added to the system to a final concentration of 40 g / L. The reaction was carried out at 70℃ and 1000 r / min for 6 h. Samples were taken, the reaction was terminated by incubating on ice for 10 min, and centrifuged at 12000 r / min for 10 min. The supernatant was then diluted with deionized water to a total sugar concentration (D-fructose + D-tagatose) of less than 2 g / L for later use.

[0057] HPLC detection conditions: A Thermo Fisher Scientific high-performance liquid chromatography system, a Thermo Fisher Scientific differential detector, and a Thermo Fisher Scientific autosampler were used to detect the product D-tagatose and the substrate D-fructose. A Sugar-Park column was used, with 0.5 g / L EDTA-disodium calcium as the mobile phase and ultrapure water as the solvent. Parameters were set at a flow rate of 0.4 mL / min, a column temperature of 80 ℃, and an injection volume of 10 μL. The conversion rate of D-fructose to D-tagatose was determined based on the peak retention time and peak area. The conversion rate (%) was defined as the ratio of the D-tagatose concentration at 6 h of reaction time to the initial D-fructose concentration. A schematic diagram of the HPLC detection of the substrate D-fructose and the product D-tagatose standard is shown below. Figure 2 As shown.

[0058] Example 5

[0059] To verify the improved substrate tolerance of the mutant, D-tagatose-4-epimerase obtained in Example 3 and mutant wet cells were used as enzyme sources to catalyze the substrate D-fructose. The final concentration of D-fructose was changed to 50 g / L, and the remaining catalytic system and measurement methods were the same as in Example 4. The wild-type enzyme EbT4E achieved a conversion rate of 8.69% at a substrate concentration of 10 g / L, while the conversion rate decreased to 5.25% at a substrate concentration of 50 g / L. The mutant N139T / Q334A achieved conversion rates of 13.49% and 7.98% at substrate concentrations of 10 g / L and 50 g / L, respectively, with yields of 1.35 g / L and 3.99 g / L, respectively. This demonstrates that the mutant N139T / Q334A can maintain almost the same high conversion rate as the wild-type enzyme at low substrate concentrations while increasing the substrate concentration, thus improving the catalytic efficiency of the enzyme and reducing economic costs.

Claims

1. A highly active D-tagatose-4-epimerase mutant, characterized in that, The mutant was obtained by single-point or superimposed mutations at positions 139, 31, 92, 95, 172, 192, 220, 258, 294, 320, 334, and 358 of the amino acid sequence shown in SEQ ID NO.

2.

2. The D-tagatose-4-epimerase mutant as described in claim 1, characterized in that, The mutant is the amino acid sequence shown in SEQ ID NO.2 that has been mutated to one of the following: (1) N139T; (2) N139T / E31G; (3) N139T / P95D; (4) N139T / K172P; (5) N139T / L220H; (6) N139T / C258I; (7) N139T / S294E; (8) N139T / A320I; (9) N139T / Q334A; (10) N139T / T358I; (11) K92S; (12) E192D.

3. A recombinant genetically engineered bacterium containing the gene encoding the D-tagatose-4-epimerase mutant as described in claim 1.

4. The use of the D-tagatose-4-epimerase mutant of claim 1 in the synthesis of D-tagatose.

5. The application as described in claim 4, characterized in that, The application is as follows: using wet bacterial cells obtained by fermentation culture of recombinant genetically engineered bacteria containing the encoding gene of the D-tagatose-4-epistomerase mutant as the enzyme source, D-fructose as the substrate, cobalt ions as the co-catalyst, and a buffer solution with pH 6.0-9.0 as the reaction medium to form a reaction system, and after the reaction is completed at 50-80℃ and 500-1200r / min, a mixture of D-fructose and D-tagatose is obtained.

6. The application as described in claim 5, characterized in that, In the reaction system, the initial concentration of the substrate is 10–100 g / L, the final concentration of the wet bacterial cells is 10–150 g / L, and the final concentration of the cobalt ions is 0.1–10 mM.

7. The application as described in claim 5, characterized in that, The reaction medium was a pH 8.0, 50 mM sodium phosphate buffer solution.

8. The application as described in claim 5, characterized in that, The wet bacterial cells were prepared as follows: recombinant genetically engineered bacteria containing the D-tagatose-4-epimerase mutant gene were inoculated into LB liquid medium containing a final concentration of 50 μg / mL kanamycin and cultured at 37°C and 180-200 r / min until OD. 600 =0.8–1.0, to obtain seed culture; the seed culture is transferred at an inoculum concentration of 1–5% to LB liquid medium containing a final concentration of 50 μg / mL kanamycin, and cultured at 37℃ and 180 r / min until OD. 600 =0.6~0.8, add IPTG to a final concentration of 0.1 mM, and induce culture at 28℃ and 180 r / min for 12~14 h to obtain the induced culture suspension. Centrifuge the induced culture suspension at 4℃ and 8000 rpm for 10 min to collect the wet cells.

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