A mutant of alginate lyase Alyw203 with improved thermal stability

Abstract

The present invention belongs to the field of enzymes, and specifically relates to a mutant of alginate lyase Alyw203 with improved thermal stability. The present invention performs mutations on the basis of alginate lyase Alyw203 to obtain mutants with mutation sites of D40W, S136I, L172V, T206W and D248L. The thermal stability of the mutants is improved, and the mutant L172V is improved most significantly. The residual activity is increased from 30.46% to 76.59% after incubation at 40°C for 1 hour; the half-life at 40°C is increased to 388.78min, which is 10.91 times that of the original enzyme, and there is still 37.80% residual enzyme activity after incubation for 12h. The present invention also provides a coding gene for the mutant, a recombinant expression vector for producing the mutant, and a genetically engineered bacterium.

Description

Technical Field

[0001] The invention belongs to the field of enzymes, and in particular relates to an alginate lyase Alyw203 mutant with improved thermal stability. Background Art

[0002] Alginate is difficult to develop and utilize due to its complex structure, large molecular weight, high viscosity and high degree of polymerization. The brown algae oligosaccharides obtained by degradation of alginate have multiple biological activities such as anti-inflammatory, antioxidant, immunomodulatory, anti-tumor, and promotion of plant root growth. Since physical and chemical methods are costly and cause environmental pollution, enzyme catalysis has become a green and efficient method for preparing brown algae oligosaccharides.

[0003] Alginate lyase produces an unsaturated C=C double bond between the non-reducing end C4 and C5 through a β-elimination reaction. Alginate lyase is widely found in animals, plants, bacteria and fungi, among which marine bacteria (Vibrio, Pseudomonas, Alteromonas, Bacillus, Flavobacterium, etc.) are the main source of alginate lyase. The inventor's prior study (Liu, Lu, et al. Frontiers in Microbiology, 2021) cloned an alginate lyase (abbreviated as Alyw203) from Vibrio W2 and expressed it in food-grade Yarrowia lipolytica. Alyw203, as an alginate lyase that does not depend on NaCl, is resistant to metal ions and has high activity in various ionic environments.

[0004] Since most marine bacteria are mesophilic or psychrophilic, the corresponding alginate lyase has inherent limitations in thermal stability. Most alginate lyases have poor thermal stability in environments above 40°C, with residual enzyme activity less than 50% after 0.5 hours of treatment. Poor thermal stability greatly limits the industrial application potential of alginate lyase Alyw203. Using protein engineering methods such as directed evolution and rational design is an effective way to improve the thermal stability of alginate lyase. Summary of the invention

[0005] In order to solve the technical problem of poor thermal stability of alginate lyase, the present invention, based on Alyw203, obtains a mutant of Alyw203 with improved thermal stability by using genetic engineering technology, and performs partial amino acid substitution on the basis of Alyw203. The half-life of the mutant at 40°C can be as high as 10 times that of the original enzyme, and the mutant can be used for alginate degradation in a higher temperature environment.

[0006] The present invention provides a mutant of alginate lyase with improved thermal stability. Compared with the alginate lyase Alyw203 whose amino acid sequence is shown in SEQ ID NO: 1, the mutant of alginate lyase comprises one or more of the following amino acid substitution mutations: D40W, S136I, L172V, T206W and D248L; and the alginate lyase mutant has improved thermal stability compared with the alginate lyase Alyw203. The mutant may also comprise other mutations, for example, further mutations are made on the basis of the mutations, but the thermal stability thereof is not significantly reduced. D40W represents that the aspartic acid at the 40th position of SEQ ID NO: 1 is replaced by tryptophan, S136I represents that the serine at the 136th position is replaced by isoleucine, L172V represents that the leucine at the 172th position is replaced by valine, T206W represents that the threonine at the 206th position is replaced by tryptophan, and D248L represents that the aspartic acid at the 248th position is replaced by leucine.

[0007] The improved thermal stability described in the present invention refers to: the time required for the enzyme activity to decrease to the initial activity at a certain temperature (eg, 40° C.) is increased (half-life is increased).

[0008] In some embodiments, the alginate lyase mutant comprises any one of the following amino acid substitution mutations compared to alginate lyase Alyw203: D40W, S136I, L172V, T206W, and D248L.

[0009] In some embodiments, the amino acid sequence of the alginate lyase mutant is shown in any one of SEQ ID NOs: 2-6.

[0010] The present invention also provides a coding gene of the alginate lyase mutant. In the case of a known amino acid sequence, the coding gene can be modified on the basis of the Alyw203 wild-type coding gene, or a new coding gene can be obtained by codon optimization.

[0011] The present invention also provides a recombinant expression vector of the coding gene, which contains an expression frame of the coding gene and can be used to express and produce the alginate lyase mutant. The recombinant vector can be constructed by connecting the nucleotide sequence of the coding gene of the alginate lyase to various vectors by conventional methods in the art. These vectors can be selected from various conventional vectors in the art, such as various plasmids, phages or virus vectors, etc., preferably pET-22b (+) vector.

[0012] The present invention also provides a genetically engineered bacterium expressing the alginate lyase mutant, wherein the genetically engineered bacterium contains the coding gene of the alginate lyase mutant, or contains the above-mentioned recombinant expression vector. It can be obtained by transforming the recombinant expression vector of the present invention into a host microorganism. The host microorganism can be various conventional host microorganisms in the art, as long as the recombinant expression vector can stably replicate itself and the alginate lyase coding gene carried by it is effectively expressed, preferably Escherichia coli, more preferably Escherichia coli BL21.

[0013] The present invention also provides a method for preparing alginate lyase mutant with improved thermal stability, comprising: inoculating the genetically engineered bacteria into a fermentation medium, culturing at 37°C and 180 rpm until OD 600 IPTG was added to a final concentration of 0.2 mM for induction, and cultured overnight at 16°C and 180 rpm. Preferably, the inoculation is 3-5% of the seed solution by volume.

[0014] Compared with the prior art, the beneficial effects of the present invention include at least:

[0015] The present invention obtains 6 single mutants with reduced free energy through virtual mutation, and the relative enzyme activities of 5 mutants are improved; the optimum pH of 2 mutants is increased to 10.0. The thermal stability of the mutants is improved, and the mutant L172V is improved most significantly, with the residual activity increased from 30.46% to 76.59% after incubation at 40°C for 1 hour; the half-life of L172V at 40°C increases to 388.78min, which is 10.91 times that of the original enzyme, and there is still 37.80% residual enzyme activity after incubation for 12h. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The three-dimensional structure of alginate lyase Alyw203;

[0017] Figure 2 SDS-PAGE protein electrophoresis analysis of mutant enzymes, wherein lane M: protein marker, lanes 1-6: D40W, D44I, S136I, L172V, T206W, D248L;

[0018] Figure 3 This is a comparison of the relative enzyme activities of Alyw203 and mutant enzymes;

[0019] Figure 4 The optimum temperature diagram of Alyw203 and mutant enzymes;

[0020] Figure 5The optimal pH diagram of the recombinant enzyme Alyw203 and the mutant enzyme; AG are: mutant enzymes D40W, D44I, S136I, L172V, T206W, D248L and Alyw203;

[0021] Figure 6 The results of thermal stability test of Alyw203 and mutant enzymes; Figure 6 Middle A: Residual activity of Alyw203 and mutant enzymes at different temperatures; Figure 6 Middle B: Half-life of Alyw203 and mutant enzymes at 40°C. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical scheme and beneficial effects of the present invention clearer, the present invention is further described in detail below in conjunction with the accompanying drawings and specific embodiments. Examples of the embodiments are shown in the accompanying drawings. It should be understood that the specific embodiments described in the following embodiments of the present invention are only exemplary descriptions of the specific embodiments of the present invention, and are intended to be used to explain the present invention, but do not constitute a limitation of the present invention.

[0023] The endpoints of ranges and any values ​​disclosed herein are not limited to the exact range or value, and these ranges or values ​​should be understood to include approximations to these ranges.

[0024] Example 1, selection of mutation sites

[0025] The alginate lyase Alyw203 contains typical conserved regions of the PL7 family, namely "QIH", "RTELREMLR" and "MYFKAG". A0A853RBM7.1.A (sequence homology 73.05%) was used as a template for homology modeling of Alyw203. Like most PL7 family alginate lyases, Alyw203 has a small amount of α-helical structure and is mainly composed of β-folded structures (such as Figure 1 ). Virtual mutagenesis was performed by Discovery Studio to obtain 6 single mutants with reduced free energy, which were named D40W, D44I, S136I, L172V, T206W and D248L (as shown in Table 1) based on the mutation sites.

[0026] Table 1: Six mutation sites with reduced free energy

[0027]

[0028] Example 2, Recombinant preparation and purification of Alyw203 and mutants

[0029] Connect the confirmed sequence mutant to the pET-22b(+) vector and transform it into Escherichia coli BL21(DE3) competent cells, culture at 37°C overnight, and pick a single colony for further culture. Inoculate the seed culture into 100mL LB medium supplemented with kanamycin at a ratio of 3-5% (w / v) seed liquid, and culture at 37°C and 180rpm until OD 600 is 0.6-0.8. Add IPTG to a final concentration of 0.2mM for induction, and culture at 16°C and 180rpm overnight. After induction, centrifuge at 8000rpm for 10min, and resuspend the cell pellet in 10mL pH 7.0PB buffer. Ultrasonic treatment for 10min, and then centrifuge at 8000rpm for 10min to obtain a supernatant containing crude enzyme. The crude enzyme is purified by nickel column to obtain pure enzyme solution, which is verified by SDS-PAGE protein electrophoresis. Figure 2 As shown, the mutants all showed a clear band near 55.16 kDa, which was consistent with the band of Alyw203, indicating that all single mutants were successfully induced. The activity of alginate lyase was measured. Compared with Alyw203, the relative enzyme activities of single mutants S136I, L172V, T206W, and D248L increased by 130%, 103%, 113%, and 102%, respectively (Figure 2). Figure 3 The relative enzyme activities of the other two mutants were slightly lower than those of Alyw203. The above results prove that replacing the 136th serine (Ser), 172th leucine (Leu), 206th threonine (Thr), and 248th aspartic acid (Asp) in the original Alyw203 enzyme gene with isoleucine (Ile), valine (Val), tryptophan (Trp), and leucine (Leu), respectively, can enhance the specific activity of the original Alyw203 enzyme.

[0030] Example 3, Determination of the Optimal Reaction Temperature and pH of Alyw203 and Mutants

[0031] The activity of alginate lyase was determined by DNS method. 1.0 mL of 10 mmol / L pH 7.0 PB buffer containing 1% (w / v) sodium alginate was mixed with 1.0 mL of enzyme solution and reacted at 30°C for 30 min. After the reaction, 1 mL of DNS reagent was added, boiled for 5 min, cooled on ice and diluted to 25 mL with distilled water. The absorbance was measured at 520 nm, and the amount of reducing sugar produced was calculated based on the glucuronic acid standard curve. Enzyme activity is expressed in units (U), which is defined as the amount of enzyme required to produce 1 μg of reducing sugar per minute. 0.2 mL of 10 mmol / L pH 7.0 PB buffer (containing 1% (w / v) sodium alginate) was taken and reacted at different temperatures (10-60°C, interval 5°C) for 10 minutes, and then 0.2 mL of enzyme solution was added and reacted for 30 minutes. The optimal enzyme activity was defined as 100%, and the relative activity was calculated based on this. pH optimization: PB buffer with pH 3.0 to 12.0 was prepared and measured at the optimal temperature according to the above method.

[0032] like Figure 4 As shown, the optimum temperature of Alyw203 is 35°C, and the optimum temperatures of mutant enzymes D44I and S136I are the same as those of Alyw203. Figure 5 As shown, the optimum temperatures of mutant enzymes D40W, L172V, T206W and D248L are 20℃, 30℃, 25℃ and 15℃, respectively, which are all lower than 35℃ of the original enzyme. The optimum pH of Alyw203 is 8.0, while the optimum pH of mutant enzymes T206W and D248L is 10.0, and the optimum pH of the remaining mutant enzymes is 9.0. Compared with Alyw203, mutant enzymes T206W and D248L show higher optimum pH values. Alginate dissolves better in a weakly alkaline environment, so mutant enzymes with a preference for alkalinity can be more effectively used in industrial production.

[0033] Example 4, Determination of thermal stability of Alyw203 and mutant enzymes

[0034] Alyw203 and mutant enzymes were incubated at different temperatures (10-50°C, interval 5°C) for 1 h, immediately cooled in an ice bath, and the residual enzyme activity was determined under the optimal conditions; the enzyme was incubated at 40°C for different time periods (0, 0.5, 1, 2, 3, 4, 6, 8, 12, 16 h), and the residual enzyme activity was determined. The time required for the enzyme activity to drop to 50% of the initial activity was taken as the half-life, reflecting the thermal stability of the enzyme.

[0035] like Figure 6As shown in Figure A, the thermal stability of mutant enzymes S136I, L172V, and T206W at different temperatures was higher than that of Alyw203. Alyw203 showed good thermal stability after being cultured at 10-35°C for 1 hour, and the relative enzyme activity remained above 60%, but after being incubated at 40°C for 1 hour, the relative enzyme activity dropped to 30.46%, and it was completely inactivated after 45°C. After being cultured at 35°C for 1 hour, the relative enzyme activity of mutant enzymes D40W, D44I, and D248L was significantly lower than that of Alyw203, and the thermal stability decreased significantly. The mutant enzymes S136I, L172V, and T206W can all maintain a relative enzyme activity of more than 75% before 35°C, which is comparable to the thermal stability of Alyw203. After culturing at 40°C for 1 hour, the relative enzyme activities of S136I and T206W were 32.71% and 40.43%, respectively, and the thermal stability was improved compared with Alyw203, but not significantly. The relative enzyme activity of L172V increased from 30.46% of Alyw203 to 76.59%, and the thermal stability was significantly improved.

[0036] like Figure 6 As shown in Figure B, except for the mutant enzyme S136I, the thermal stability of the other mutant enzymes at 40°C was significantly higher than that of Alyw203 (P<0.05). The half-lives of D40W, D44I, S136I, L172V, T206W, and D248L at 40°C were 45.35min, 45.21min, 35.71min, 388.78min, 41.81min, and 41.37min, respectively. The mutant enzyme S136I had a shorter half-life, mainly because the enzyme activity after S136I mutation was the highest, and 40°C was not the optimal temperature for S136I. The half-life of L172V at 40°C increased to 388.78min, which was 10.91 times that of Alyw203 at 40°C, and L172V still had 37.80% residual enzyme activity after incubation at 40°C for 12h.

[0037] Example 5, Comparison of kinetic parameters of Alyw203 and mutant enzymes

[0038] Sodium alginate solutions of different concentrations were prepared in 10mM pH 7.0PB buffer. After the substrate solution was preheated at the optimum temperature for 10 minutes, the enzyme solution was added, the reaction was carried out for 30 minutes, and then the DNS reagent was added to quantify the reducing sugar. The Lineweaver-Burk plot was drawn with the reciprocal of the substrate concentration (1 / S) as the horizontal axis and the reciprocal of the reaction rate (1 / V) as the vertical axis. The Michaelis constant (Km), maximum reaction rate (Vmax), turnover number (Kcat) and catalytic efficiency (Kcat / Km) were calculated from the figure. As shown in Table 2, compared with Alyw203, the Km values ​​of the forward mutants S136I, L172V, T206W and D248L were reduced, and the Vmax and Kcat / Km values ​​were increased. The Km value of the mutant enzyme S136I was reduced to 86.26 mg / mL, which was 2.47 times lower than that of Alyw203 (212.76 mg / mL). The Km value of mutant L172V was reduced to 129.83 mg / mL, and the Kcat / Km value was increased by 4.92 times compared with Alyw203, indicating that the mutation of leucine (Leu) to valine (Val) at position 172 not only improved the thermal stability of the enzyme, but also had a certain effect on the catalytic efficiency. L172V is a mutant with significantly improved catalytic efficiency and thermal stability.

[0039] Table 2: Kinetic parameters of Alyw203 and mutant enzymes

[0040]

[0041] The amino acid sequence involved in the present invention is as follows:

[0042] Alyw203 (SEQ ID NO: 1):

[0043] VGCTSNGNDTSNLHPQSETGAPLLTPVAIEASSHDGNDPDRLFDQDINTRWSANGDGEWAVLDYGSVHEFDAVRAAFSKGNERKSKFDILVSTDGKTWTPVLQNQESSGGVIGYERFEFSPVQARYVKYVGHGNTSNGWNSVTELAAVKCGVNACPSNQIITPAVIAAEQGLIAQQKEAEKARQAARKDLRKGNFGVPAVYPCQTTVKCAKTALPVPTGLPTTPKAGNKPSQNFDLTSWYLSQPFDHDNNNRPDDVSEWDLANGYEHPDVFYTAKDGGLVFKSFVKGVRTSPNTKYARTEMREMLRRGDTSIPTKGVNKNNWVFSSAPVADQKAAGGVDGVMEATLKIDHTTTTGEAGEVGRFIIGQIHDQDDEPIRLYYRKLPNHEKGTVYFAHENTLKGTDQYFDLVGGMTGEIGDDGIALGEKFSYRIAVKGNTLTVTVMRDGKPDAKQVVDMSQSGYDVGGKYMYFKAGVYNQNITGEMDDYVQATFYKLEKSHGTYQGK

[0044] Mutant D40W (SEQ ID NO: 2):

[0045] VGCTSNGNDTSNLHPQSETGAPLLTPVAIEASSHDGNDPWRLFDQDINTRWSANGDGEWAVLDYGSVHEFDAVRAAFSKGNERKSKFDILVSTDGKTWTPVLQNQESSGGVIGYERFEFSPVQARYVKYVGHGNTSNGWNSVTELAAVKCGVNACPSNQIITPAVIAAEQGLIAQQKEAEKARQAARKDLRKGNFGVPAVYPCQTTVKCAKTALPVPTGLPTTPKAGNKPSQNFDLTSWYLSQPFDHDNNNRPDDVSEWDLANGYEHPDVFYTAKDGGLVFKSFVKGVRTSPNTKYARTEMREMLRRGDTSIPTKGVNKNNWVFSSAPVADQKAAGGVDGVMEATLKIDHTTTTGEAGEVGRFIIGQIHDQDDEPIRLYYRKLPNHEKGTVYFAHENTLKGTDQYFDLVGGMTGEIGDDGIALGEKFSYRIAVKGNTLTVTVMRDGKPDAKQVVDMSQSGYDVGGKYMYFKAGVYNQNITGEMDDYVQATFYKLEKSHGTYQGK

[0046] Mutant S136I (SEQ ID NO: 3):

[0047] VGCTSNGNDTSNLHPQSETGAPLLTPVAIEASSHDGNDPDRLFDQDINTRWSANGDGEWAVLDYGSVHEFDAVRAAFSKGNERKSKFDILVSTDGKTWTPVLQNQESSGGVIGYERFEFSPVQARY VKYVGHGNTINGWNSVTELAAVKCGVNACPSNQIITPAVIAAEQGLIAQQKEAEKARQAARKDLRKGNFGVPAVYPCQTTVKCAKTALPVPTGLPTTPKAGNKPSQNFDLTSWYLSQPFDHDNNNR PDDVSEWDLANGYEHPDVFYTAKDGGLVFKSFVKGVRTSPNTKYARTEMLRRGDTSIPTKGVNKNNWVFSSAPVADQKAAGGVDGVMEATLKIDHTTTTGEAGEVGRFIIGQIHDQDDEPIRL YYRKLPNHEKGTVYFAHENTLKGTDQYFDLVGGMTGEIGDDGIALGEKFSYRIAVKGNTLTVTVMRDGKPDAKQVVDMSQSGYDVGGKYMYFKAGVYNQNITGEMDDYVQATFYKLEKSHGTYQGK

[0048] Mutant L172V(SEQ ID NO:4):

[0049] VGCTSNGNDTSNLHPQSETGAPLLTPVAIEASSHDGNDPDRLFDQDINTRWSANGDGEWAVLDYGSVHEFDAVRAAFSKGNERKSKFDILVSTDGKTWTPVLQNQESSGGVIGYERFEFSPVQARYVKYVGHGNTSNGWNSVTELAAVKCGVNACPSNQIITPAVIAAEQGVIAQQKEAEKARQAARKDLRKGNFGVPAVYPCQTTVKCAKTALPVPTGLPTTPKAGNKPSQNFDLTSWYLSQPFDHDNNNRPDDVSEWDLANGYEHPDVFYTAKDGGLVFKSFVKGVRTSPNTKYARTEMREMLRRGDTSIPTKGVNKNNWVFSSAPVADQKAAGGVDGVMEATLKIDHTTTTGEAGEVGRFIIGQIHDQDDEPIRLYYRKLPNHEKGTVYFAHENTLKGTDQYFDLVGGMTGEIGDDGIALGEKFSYRIAVKGNTLTVTVMRDGKPDAKQVVDMSQSGYDVGGKYMYFKAGVYNQNITGEMDDYVQATFYKLEKSHGTYQGK

[0050] Mutant T206W (SEQ ID NO: 5):

[0051] VGCTSNGNDTSNLHPQSETGAPLLTPVAIEASSHDGNDPDRLFDQDINTRWSANGDGEWAVLDYGSVHEFDAVRAAFSKGNERKSKFDILVSTDGKTWTPVLQNQESSGGVIGYERFEFSPVQARY VKYVGHGNTSNGWNSVTELAAVKCGVNACPSNQIITPAVIAEQGLIAQQKEAEKARQAARKDLRKGNFGVPAVYPCQTWVKCAKTALPVPTGLPTTPKAGNKPSQNFDLTSWYLSQPFDHDNNNR PDDVSEWDLANGYEHPDVFYTAKDGGLVFKSFVKGVRTSPNTKYARTEMLRRGDTSIPTKGVNKNNWVFSSAPVADQKAAGGVDGVMEATLKIDHTTTTGEAGEVGRFIIGQIHDQDDEPIRL YYRKLPNHEKGTVYFAHENTLKGTDQYFDLVGGMTGEIGDDGIALGEKFSYRIAVKGNTLTVTVMRDGKPDAKQVVDMSQSGYDVGGKYMYFKAGVYNQNITGEMDDYVQATFYKLEKSHGTYQGK

[0052] Mutant D248L(SEQ ID NO:6):

[0053] VGCTSNGNDTSNLHPQSETGAPLLTPVAIEASSHDGNDPDRLFDQDINTRWSANGDGEWAVLDYGSVHEFDAVRAAFSKGNERKSKFDILVSTDGKTWTPVLQNQESSGGVIGYERFEFSPVQARY VKYVGHGNTSNGWNSVTELAAVKCGVNACPSNQIITPAVIAAEQGLIAQQKEAEKARQAARKDLRKGNFGVPAVYPCQTTVKCAKTALPVPTGLPTTPKAGNKPSQNFDLTSWYLSQPFDHLNNNR PDDVSEWDLANGYEHPDVFYTAKDGGLVFKSFVKGVRTSPNTKYARTEMREMLRRGDTSIPTKGVNKNNWVFSSAPVADQKAAGGVDGVMEATLKIDHTTTTGEAGEVGRFIIGQIHDQDDEPIRL YYRKLPNHEKGTVYFAHENTLKGTDQYFDLVGGMTGEIGDDGIALGEKFSYRIAVKGNTLTVTVMRDGKPDAKQVVDMSQSGYDVGGKYMYFKAGVYNQNITGEMDDYVQATFYKLEKSHGTYQGK

[0054] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention and do not constitute a limitation on the content of the present invention. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A mutant of alginate lyase with improved thermal stability, characterized in that: The amino acid sequence of the alginate lyase mutant is shown in SEQ ID NO:

4.

2. The gene encoding the alginate lyase mutant according to claim 1.

3. A recombinant expression vector, characterized in that: The recombinant expression vector contains the expression cassette of the encoding gene according to claim 2.

4. The recombinant expression vector according to claim 3, characterized in that The recombinant expression vector is a plasmid vector, a phage or a virus vector.

5. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria contains the recombinant expression vector according to claim 3 or 4.

6. The genetically engineered bacterium according to claim 5, characterized in that The genetically engineered bacteria is Escherichia coli.

7. The method for preparing the alginate lyase mutant according to claim 1, characterized in that: include: The genetically engineered bacteria according to claim 5 or 6 are inoculated into a fermentation medium, and cultured at 37°C and 180 rpm until OD600 is 0.6-0.8; IPTG is added to a final concentration of 0.2 mM for induction, and cultured overnight at 16°C and 180 rpm.

8. The preparation method according to claim 7, characterized in that: The inoculation is 3-5% seed liquid by volume.

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