Method for producing linear maltotetraose generating enzyme by means of fermentation of bacillus subtilis

Abstract

The present invention relates to the technical field of fermentation engineering. Disclosed is a method for producing a linear maltotetraose generating enzyme by means of fermentation of Bacillus subtilis. The present invention comprises the steps of: activating recombinant Bacillus subtilis to obtain a seed culture liquid; inoculating the seed culture liquid into a secondary seed culture medium to obtain a secondary seed culture liquid; inoculating the secondary seed culture liquid into a fermentation culture medium for fermentation culture, wherein no phosphate is added to the secondary seed culture medium and the fermentation culture medium; and during the fermentation culture, starting nitrogen replenishing at the 20th-36th hours, starting carbon replenishing at the 24th-36th hours, and introducing a carbon starvation phase at the 55th-65th hours, during the phase, the carbon replenishing being stopped. In the fermentation system, the recombinant Bacillus subtilis used in the present invention has an enzyme producing activity of up to 1393.51 U / mL in a 15 L fermentation tank, which is 450% of the enzyme activity during shake flask fermentation.

Description

A method for producing linear maltotetrasaccharide-producing enzyme by fermentation of Bacillus subtilis Technical Field

[0001] This invention relates to a method for producing linear maltotetrasaccharide-producing enzyme by fermentation of Bacillus subtilis, belonging to the field of fermentation engineering technology. Background Technology

[0002] Linear-chain maltotetrasaccharide (LMMT) is one of the most promising maltodextrins and a novel functional sugar source. It not only possesses excellent food processing properties and unique physiological effects but also plays a special role in various biological processes, thus showing broad application prospects in the food, pharmaceutical, and chemical industries. Studies have shown that it may help maintain gut health, improve microbial balance, and even possess antioxidant properties, protecting the human body from oxidative stress damage. Further research has also found that LMT may be beneficial in regulating the immune system, potentially playing a role in the prevention and treatment of immune-related diseases. However, due to its low production efficiency and high price, its functional value and industrial potential have not been fully explored, severely restricting its industrial production and application.

[0003] The production of linear maltotetrasaccharides mainly employs enzymatic processes, with linear maltotetrasaccharide synthase (EC 3.2.1.60) being the key enzyme preparation. As a member of the GH13 family, linear maltotetrasaccharide synthase exhibits significant exonuclease characteristics, specifically acting on the fourth α-1,4 glycosidic bond starting from the non-reducing end of starch, thereby efficiently generating maltooligosaccharides primarily composed of linear maltotetrasaccharides. The performance of this enzyme is one of the core factors determining the industrial value of maltotetrasaccharides, directly affecting the yield, production cost, and other factors of the target product. Most linear maltotetrasaccharide synthases reported domestically and internationally have unsatisfactory protein expression levels, limiting their industrial application. Besides directly screening for new enzymes, optimizing enzyme activity through fermentation with wild-type microorganisms, or expressing and optimizing the fermentation process in a heterologous host, are two common strategies for increasing the expression level of the target protein. The latter has advantages such as higher efficiency and greater versatility.

[0004] The applicant previously successfully synthesized a linear maltodextrose synthase (MFA) derived from Pseudomonas saccharophila STB07. PS The enzyme was recombinantly expressed in Bacillus subtilis, exhibiting superior stability and product specificity. Based on this, a mutant (MFA) with significantly improved substrate specificity and substrate conversion rate was also constructed. PS(ΔCBM enzyme). However, the protein expression level of this enzyme is still at a low level, significantly lower than that of industrial enzymes, limiting its industrial application. Enzyme stability directly affects production efficiency and product quality, meaning a deep understanding of the enzyme's working mechanism is needed for effective improvement and optimization. Secondly, how to achieve large-scale, low-cost enzyme production while ensuring activity and stability is a key problem to be solved. Enzyme production consumes labor costs and significant amounts of water, steam, and energy. Minimizing fermentation time while maintaining enzyme activity, and achieving green and environmentally friendly enzyme production, are also current challenges. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method for producing linear maltodextrin-producing enzyme (MFA) from Bacillus subtilis via fermentation. The method optimizes the fermentation conditions of recombinant Bacillus subtilis by restricting phosphate nutrition in the culture medium and introducing a carbon source starvation phase during fermentation, thereby significantly improving the production of recombinant linear maltodextrin-producing enzyme (MFA) by Bacillus subtilis. PS -ΔCBM enzyme activity level. The steps include activating recombinant Bacillus subtilis to obtain a seed culture; inoculating the seed culture into a secondary seed culture medium to obtain a secondary seed culture; inoculating the secondary seed culture into a fermentation medium for fermentation culture, with nitrogen feeding starting from the 20th to 36th hour of fermentation, carbon feeding starting from the 24th to 36th hour, and introducing a carbon starvation phase at the 55th to 65th hour, during which carbon feeding is stopped.

[0006] The recombinant Bacillus subtilis used in this invention is Bacillus subtilis WB600 / pP43nmk-SP Bgls -MFA PS -ΔCBM, the construction method is described in "Expression, Modification and Application Study of Linear Maltotetrasaccharide Genotype Derived from Pseudomonas saccharophila STB07, Duan Kaiwen, Jiangnan University", specifically:

[0007] Using Bacillus subtilis WB600 as the host and pP43nmk as the vector, the signal peptide SP was selected. Bgls Recombinant maltodextrose synthase MFA with its amino acid sequence truncated (as shown in SEQ ID NO.2) was heterologously expressed. PS -ΔCBM.

[0008] Signal peptide SP Bgls The sequence is shown in SEQ ID NO.3:

[0009] SEQ ID NO.2:

[0010] The first objective of this invention is to provide a Bacillus subtilis fermentation enzyme for the production of linear maltotetrasaccharide MFA. PS The -ΔCBM method includes the following steps:

[0011] Step S1: Activate recombinant Bacillus subtilis to obtain seed culture medium. The recombinant Bacillus subtilis uses pP43nmk as a vector and employs the signal peptide SP. Bgls Heterologous expression of recombinant linear maltotetrasaccharide synthase MFA with starch-binding module truncated PS -ΔCBM;

[0012] Step S2: Inoculate the seed culture medium into the secondary seed culture medium to obtain the secondary seed culture medium;

[0013] Step S3: Inoculate the secondary seed culture medium into the fermentation medium for culture;

[0014] Step S4: Nitrogen source feeding begins 20-36 hours into fermentation, and carbon source feeding begins 24-36 hours into fermentation.

[0015] Furthermore, in step S1, the expression host of the recombinant Bacillus subtilis is Bacillus subtilis WB600.

[0016] Further, in step S1, the recombinant linear maltotetrasaccharide synthase MFA PS The nucleotide sequence of -ΔCBM is shown in SEQ ID NO.1.

[0017] SEQ ID NO.1:

[0018] Further, in step S1, the recombinant linear maltotetrasaccharide synthase MFA PS The amino acid sequence of -ΔCBM is shown in SEQ ID NO.2.

[0019] Further, in step S2, the inoculation amount of the seed culture solution is 3-5% (v / v).

[0020] Furthermore, in step S2, the inoculation amount of the seed culture medium is 3% (v / v), 4% (v / v), or 5% (v / v). Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0021] Furthermore, in step S2, the secondary seed culture medium is composed of a nitrogen source and a carbon source.

[0022] Furthermore, the carbon source includes, but is not limited to, glucose, sucrose, starch, and molasses, and the nitrogen source includes, but is not limited to, protein, amino acids, urea, beef extract, peptone, and yeast extract.

[0023] Furthermore, no phosphates are added to the secondary seed culture medium.

[0024] Furthermore, the phosphates include, but are not limited to, dihydrogen phosphate, hydrogen phosphate, orthophosphate, pyrophosphate, tripolyphosphate, and metaphosphate.

[0025] Furthermore, the carbon source is corn starch.

[0026] Furthermore, in step S3, the inoculation amount of the secondary seed culture medium is 10-15% (v / v).

[0027] Furthermore, in step S3, the inoculation amount of the secondary seed culture medium is 10% (v / v), 11% (v / v), 12% (v / v), 13% (v / v), 14% (v / v), or 15% (v / v). Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0028] Furthermore, in step S3, the fermentation medium is composed of yeast powder, peptone, carbon source, magnesium sulfate and defoamer, wherein the carbon source includes, but is not limited to, glucose, sucrose, starch and molasses.

[0029] Furthermore, no phosphates are added to the fermentation medium, and the phosphates include, but are not limited to, dihydrogen phosphate, hydrogen phosphate, orthophosphate, pyrophosphate, tripolyphosphate, and metaphosphate.

[0030] Furthermore, the carbon source is corn starch.

[0031] Furthermore, in step S3, trace elements are added to the fermentation medium, including iron, calcium, manganese, cobalt, sodium, molybdenum, zinc, aluminum, copper, and boron.

[0032] Specifically, the fermentation medium contains a trace element solution, which contains FeSO4, CaCl2, MnSO4, CoCl2, NaMoO4, ZnSO4, AlCl3, CuCl2 and H3BO4.

[0033] Furthermore, in step S4, the nitrogen source includes yeast powder and peptone, and the carbon source is glucose.

[0034] Furthermore, in step S4, the flow rate of the nitrogen source feed is 24-36 mL / h.

[0035] Furthermore, in step S4, the flow rate of the nitrogen source feed is 24 mL / h, 25 mL / h, 26 mL / h, 27 mL / h, 28 mL / h, 29 mL / h, 30 mL / h, 31 mL / h, 32 mL / h, 33 mL / h, 34 mL / h, 35 mL / h, or 36 mL / h. Other specific values ​​within the above ranges can be selected, and will not be elaborated here.

[0036] Preferably, the flow rate of the nitrogen source is 30 mL / h.

[0037] Furthermore, in step S4, the flow rate of the carbon source feed is 18-22 mL / h.

[0038] Furthermore, in step S4, the flow rate of the nitrogen source feed is 18 mL / h, 19 mL / h, 20 mL / h, 21 mL / h, or 22 mL / h. Other specific values ​​within the above range can be selected, and will not be elaborated here.

[0039] Furthermore, in step S4, a carbon source starvation phase is introduced during the 55th-65th hour of fermentation, during which carbon source feeding is stopped.

[0040] Furthermore, in step S4, the carbon source starvation phase begins at the 55th, 56th, 57th, 58th, 59th, 60th, 61st, 62nd, 63rd, 64th, and 65th hours of fermentation. Other specific point values ​​within the above ranges can be selected, and will not be elaborated here.

[0041] Preferably, the carbon source starvation phase begins at the 60th hour of fermenter culture.

[0042] Furthermore, the carbon source starvation phase lasts for 4-6 hours.

[0043] Furthermore, the carbon source starvation phase lasts for 4 hours, 4.5 hours, 5 hours, 5.5 hours, and 6 hours. Other specific values ​​within the above ranges can also be selected, and will not be elaborated on here.

[0044] Preferably, the glucose starvation phase lasts for 4 hours. Beneficial effects

[0045] This invention improves the expression level of target proteins through fermentation regulation strategies, enabling large-scale, low-cost enzyme production and providing strong support for the industrial production of tetrasaccharides.

[0046] In the fermentation system provided by this invention, the recombinant Bacillus subtilis produced enzymes with a maximum activity of 1393.51 U / mL in a 15L fermenter, which is 450% of the level of shake flask fermentation. The overall fermentation time was shortened, production efficiency was improved, and raw material costs were reduced. Attached Figure Description

[0047] To make the content of this invention easier to understand, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings, wherein:

[0048] Figure 1 shows the growth and enzyme production pattern of recombinant Bacillus subtilis in a 15L fermenter in Example 1 of the present invention.

[0049] Figure 2 is an SDS-PAGE image of the supernatant of recombinant Bacillus subtilis in a 15L fermenter in Example 1 of the present invention, where lane M is the standard protein and lane 1 is the supernatant of the fermenter.

[0050] Figure 3 shows the growth curves of recombinant Bacillus subtilis under different nitrogen source feeding times in Example 2 of the present invention;

[0051] Figure 4 shows the enzyme activity variation curves of recombinant Bacillus subtilis under different nitrogen source feeding times in Example 2 of the present invention.

[0052] Figure 5 shows the growth curves of recombinant Bacillus subtilis under different nitrogen source feed rates in Example 2 of the present invention.

[0053] Figure 6 shows the enzyme activity variation curves of recombinant Bacillus subtilis under different nitrogen source feed flow rates in Example 2 of the present invention.

[0054] Figure 7 illustrates the nitrogen source feeding strategy in Example 2 of this invention.

[0055] Figure 8 shows the growth and enzyme activity curves of recombinant Bacillus subtilis under an optimized nitrogen source feeding strategy in Example 2 of the present invention.

[0056] Figure 9 shows the growth curves of recombinant Bacillus subtilis under different starvation conditions in Example 3 of the present invention.

[0057] Figure 10 shows the enzyme activity curves of recombinant Bacillus subtilis under different starvation conditions in Example 3 of the present invention.

[0058] Figure 11 shows the enzyme production curves of recombinant Bacillus subtilis under different starvation durations (20h starvation) in Example 3 of the present invention;

[0059] Figure 12 shows the enzyme production curves of recombinant Bacillus subtilis under different starvation durations (60h starvation) in Example 3 of the present invention;

[0060] Figure 13 shows the growth and enzyme production curves of recombinant Bacillus subtilis under a glucose starvation strategy in Example 3 of the present invention.

[0061] Figure 14 shows the effect of phosphate addition amount on MFA in Example 4 of the present invention. PS The effect of -ΔCBM enzyme activity, where A is K2HPO4 and B is KH2PO4;

[0062] Figure 15 shows the growth and enzyme production curves of recombinant Bacillus subtilis in a 15L fermenter under different phosphate nutrient environments in Example 5 of the present invention.

[0063] Figure 16 shows the relationship between enzyme production and carbon source of recombinant Bacillus subtilis under different phosphate nutrient environments in Example 6 of the present invention.

[0064] Figure 17 is an SDS-PAGE image of the fermentation supernatant of recombinant Bacillus subtilis in different phosphate nutrient environments and different carbon source types in culture media in Example 6 of the present invention. Lane M is the standard protein.

[0065] Figure 18 shows the relationship between the enzyme production of recombinant Bacillus subtilis under different phosphate nutrient environments and the corn starch concentration in Example 7 of the present invention.

[0066] Figure 19 shows the relationship between enzyme production and glucose concentration of recombinant Bacillus subtilis under different phosphate nutrient environments in Example 7 of the present invention.

[0067] Figure 20 shows the effect of glucose addition timing under different phosphate nutrient environments on the growth and enzyme production of recombinant Bacillus subtilis in Example 8 of the present invention.

[0068] Figure 21 shows the growth and enzyme production curves of recombinant Bacillus subtilis under different phosphate nutrient environments and feeding strategies in Example 9 of the present invention.

[0069] Figure 22 shows the growth and enzyme production curves of recombinant Bacillus subtilis under a feeding strategy during dissolved oxygen rebound (24h) in different phosphate nutrient environments in Example 9 of the present invention.

[0070] Figure 23 shows the growth and enzyme production curves of recombinant Bacillus subtilis under a feeding strategy with stable OD values ​​(36h) in different phosphate nutrient environments in Example 9 of the present invention.

[0071] Figure 24 is an SDS-PAGE image of the fermentation supernatant of recombinant Bacillus subtilis under an OD-stabilized (36h) fed-feeding strategy in a phosphate-limited environment. Lane M is the standard protein and lane Y is the supernatant of the shake flask control group.

[0072] Figure 25 shows the growth and enzyme production of recombinant Bacillus subtilis under phosphate-restricted conditions in a 15L fermenter under both fed-feed and glucose-starved conditions in Example 10 of this invention.

[0073] Figure 26 shows the effects of different nutrient deficiencies on the growth of recombinant Bacillus subtilis and MFA. PS The effect of -ΔCBM enzyme expression. Detailed Implementation

[0074] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, so that those skilled in the art can better understand and implement the present invention. However, the embodiments described are not intended to limit the present invention.

[0075] Technical terms

[0076] culture medium

[0077] The primary function of culture media is to meet the nutritional and energy requirements of microorganisms during their growth and metabolism. Different media components affect the growth capacity of microorganisms, the efficiency of product synthesis, and the yield of metabolic products. Common optimized conditions for culture media include carbon sources, nitrogen sources, and inorganic salts.

[0078] carbon source

[0079] The primary function of carbon sources in culture media is to provide the energy needed for bacterial growth and reproduction, and to provide carbon for the carbon skeleton of microbial cells and metabolic products. Both excessively high and low carbon source content in fermentation media are detrimental to bacterial growth and reproduction; the former inhibits normal bacterial life activities, while the latter leads to bacterial senescence. Common carbon sources in fermentation media include glucose, starch, and glycerol, and a suitable carbon source facilitates absorption and utilization by the bacteria. Starch includes, but is not limited to, corn starch, wheat starch, glutinous rice starch, sorghum starch, cassava starch, and potato starch.

[0080] nitrogen source

[0081] Nitrogen sources are equally important for the growth and metabolism of microorganisms, primarily providing nitrogen for microbial cell matter and nitrogenous metabolites. Nitrogen sources are further divided into inorganic and organic nitrogen sources, with different microbial species having different nitrogen requirements. Organic nitrogen sources include complex organic compounds such as proteins, amino acids, and nucleic acids, such as beef extract, yeast extract, oilseed meal, and silkworm pupa powder. Inorganic nitrogen sources include ammonium salts, nitrates, and urea. In industrial and fermentation industries, commonly used nitrogen sources also include corn steep liquor, soybean flour, peanut flour, and whey powder.

[0082] Linear maltotetrasaccharide synthase:

[0083] The linear maltotetrasaccharide synthase of the present invention is derived from *Pseudomonas saccharophila* STB07; the linear maltotetrasaccharide synthase is a linear maltotetrasaccharide synthase with the amino acid sequence shown in SEQ ID NO.2, or a linear maltotetrasaccharide synthase having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the sequence shown in SEQ ID NO.2.

[0084] The linear maltotetrasaccharide synthase is a linear maltotetrasaccharide synthase encoding the nucleotide sequence shown in SEQ ID NO.1, or a linear maltotetrasaccharide synthase encoding a sequence having at least 60%, at least 70%, at least 80%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less than 100% sequence identity with the sequence shown in SEQ ID NO.1.

[0085] Sequence identity:

[0086] The degree of association between two amino acid sequences or two nucleotide sequences is described by the parameter "sequence identity".

[0087] For the purposes of this invention, the Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol. Biol. [Journal of Molecular Biology] 48:443-453) is used to determine sequence identity between two amino acid sequences. This algorithm is implemented in the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice et al., 2000, Trends Genet. [Trends in Genetics] 16:276-277) (preferably version 5.0.0 or later). The parameters used can be a vacancy opening penalty of 10, a vacancy extension penalty of 0.5, and an EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of "longest identity" marked by Needle (obtained using the -nobrief option) is used as the identity percentage and calculated as follows:

[0088] (identical residues x 100) / (alignment length - total number of vacancies in the alignment)

[0089] Alternatively, the parameters used can be a vacancy open penalty of 10, a vacancy extension penalty of 0.5, and EDNAFULL (the EMBOSS version of NCBI NUC4.4) to replace the matrix. The output of "Longest Identity" marked with Needle (obtained using the -nobrief option) is used as the identity percentage and calculated as follows:

[0090] (identical deoxyribonucleotides × 100) / (alignment length – total number of gaps in alignment).

[0091] Microbial fermentation

[0092] Microbial fermentation refers to the process by which microorganisms (such as bacteria, yeast, and mold) transform raw materials into products needed by humans through specific metabolic pathways under suitable conditions. This process is widely used in food, medicine, energy, chemical industry, and agriculture, for example, in the production of alcohol, antibiotics, enzyme preparations, and food additives.

[0093] Fermentation tank

[0094] Fermentation tanks are primarily used in microbial fermentation processes, providing suitable conditions such as temperature, pH, and oxygen supply to promote microbial growth and metabolism. Fermentation tanks have functions including heating, cooling, stirring, aeration, and sterilization to ensure the high efficiency and stability of the fermentation process.

[0095] Enzyme activity

[0096] Enzyme activity refers to the ability of an enzyme to catalyze a chemical reaction, and its magnitude is usually measured by the rate at which the enzyme catalyzes a particular chemical reaction. The unit of enzyme activity (U) is usually defined as the amount of enzyme that can convert 1 micromolar of substrate per minute under specific conditions (such as temperature, pH value, etc.).

[0097] Feed fermentation

[0098] Feeding is a crucial step in the fermentation process. Its main purpose is to replenish the microbial environment with fresh nutrients during fermentation, maintaining suitable growth conditions and metabolic processes, thereby improving fermentation efficiency and product yield. The primary objective of feeding is to maintain appropriate nutrient concentrations by intermittently or continuously adding fresh nutrients during fermentation, avoiding environmental abrupt changes or inhibitory effects caused by excessive feeding at once. Feeding can: control the concentration of inhibitory substrates, preventing the growth-inhibiting effects of high nutrient concentrations on microorganisms; relieve the repression of catabolites, improving product synthesis efficiency; and maintain suitable pH and nutrient conditions, promoting microbial growth and product synthesis.

[0099] Protein electrophoresis

[0100] Protein electrophoresis is a technique based on the migration of charged molecules under the influence of an electric field. Proteins, as amphoteric electrolytes, carry different charges under different pH conditions, and therefore migrate towards the electrode with the opposite charge in an electric field. The migration rate depends on factors such as the protein's size, shape, charge, and the strength of the electric field.

[0101] Microbial growth period

[0102] The lag phase (also known as the adaptation phase) is the stage in which microorganisms adapt to a new environment or change in culture conditions. During this phase, the number of microbial cells does not increase, but cell metabolism is active, synthesizing new enzymes and metabolites to prepare for subsequent rapid growth.

[0103] The logarithmic growth phase is the fastest growth phase for microorganisms, characterized by a geometric increase in cell number, peak metabolic activity, and the fastest rate of cell division. This phase is characterized by: exponential growth in cell number, a rapid increase in metabolic products, the fastest rate of cell division, and the shortest generation time.

[0104] The stationary phase is the third stage of the microbial growth curve, marking a slowdown in growth, with the cell number reaching its maximum and remaining relatively stable. This stage is characterized by: a slower rate of cell division, and a balance between the number of new and dead cells. Cells begin to accumulate metabolic products, such as secondary metabolites (e.g., antibiotics) and storage substances. Nutrient depletion and the accumulation of harmful metabolites lead to a decrease in growth rate. In some cases, microorganisms may form spores to adapt to adverse conditions.

[0105] In one embodiment of the present invention, after the recombinant Bacillus subtilis of the present invention is fermented in a 15L fermenter, the cell count is in a stagnant phase for 0-4 hours, in a vigorous logarithmic growth phase for 4-60 hours, and enters a stationary phase around 60 hours. During the logarithmic growth phase, enzyme production and cell growth show a consistent trend. When cell growth enters the stationary phase, i.e., around 60 hours of fermentation, enzyme production tends to peak.

[0106] In one embodiment of the present invention, the nitrogen source feeding strategy is as follows:

[0107] Nitrogen was supplemented at a flow rate of 30 mL / h during the early logarithmic growth phase, 24 mL / h during the mid-logarithmic growth phase (20-40 h), and 30 mL / h during the late logarithmic growth phase (after 40 h). The culture was terminated when the activity of linear maltodextrin synthase decreased.

[0108] The early stage of logarithmic growth can be 4h, 5h, 6h, 7h, 8h, 9h, or 10h.

[0109] The logarithmic growth mid-terms can be: 20h, 21h, 22h, 23h, 24h, 25h, 26h, 27h, 28h, 29h, 30h, 31h, 32h, 33h, 34h, 35h, 36h, 37h, 38h, 39h, 40h;

[0110] The logarithmic growth phase can be: 40h, 41h, 42h, 43h, 44h, 45h, 46h, 47h, 48h, 49h, 50h, 51h, 52h, 53h, 54h, 55h, 56h, 57h, 58h, 59h, 60h.

[0111] In one embodiment of the present invention, the glucose starvation strategy of the present invention is as follows: glucose starvation stages of 2, 4 and 6 hours are introduced at the 20th hour of fermentation, and the culture is terminated when the activity of linear maltotetrasaccharide synthase decreases.

[0112] Glucose starvation phases of 2, 4, and 6 hours were introduced at 60 hours, and the culture was terminated when the activity of linear maltodextrin synthase decreased.

[0113] In one embodiment of the present invention, the carbon source feeding strategy of the present invention is as follows:

[0114] Under phosphate-restricted conditions:

[0115] (1) Pre-feeding strategy, that is, from the start of the fermentation process until the end of the fermentation process, glucose is intermittently supplemented to control the glucose concentration in the culture medium at 0.2-0.4 g / L;

[0116] (2) Post-feeding strategy, when OD 600 When the condition is stable, glucose concentration in the culture medium is controlled at 0.2-0.4 g / L by intermittent glucose supplementation;

[0117] The recombinant Bacillus subtilis described in the following examples is disclosed in "Expression, Modification and Application Study of Linear Maltotetrasaccharide Generator Derived from Pseudomonas saccharophila STB07, Duan Kaiwen, Jiangnan University". Bacillus subtilis WB600 was used as the host, pP43nmk as the vector, and the signal peptide SP was selected. Bgls Heterologous expression of linear maltotetrasaccharide synthase MFA derived from Pseudomonas saccharophila STB07 PS The starch-binding module CBM was truncated to obtain the recombinant linear maltotetrasaccharide synthase pP43nmk-SP. Bgls -MFA PS -ΔCBM (hereinafter referred to as MFA) PS -ΔCBM);

[0118] The glucose starvation strategy involved in the following embodiments is that during the glucose starvation stage, no glucose is fed, that is, feeding is stopped, and the residual sugar in the fermenter is consumed.

[0119] The culture medium and reagent formulations involved in the following examples

[0120] LB liquid medium (g / L): 5g analytical grade yeast extract, 10g analytical grade tryptone, 10g NaCl.

[0121] LB solid medium: Add 1.5-2% (w / v) agar powder to LB liquid medium.

[0122] Shake flask fermentation medium (g / L): 24 g of analytical grade yeast extract, 12 g of analytical grade tryptone, 10 g of corn starch, 0-34 mmol / L of KH2PO4, 0-110 mmol / L of K2HPO4·3H2O (total phosphate concentration range 0-144 mmol / L), and 20 μg / mL of kanamycin added before inoculation.

[0123] Secondary culture medium (g / L): industrial grade yeast extract 24, industrial grade soybean peptone 6, corn starch 5, glucose 4, K2HPO4·3H2O 16.43, KH2PO4 2.32, and kanamycin at a final concentration of 20 μg / mL was added before inoculation.

[0124] Phosphate-limited secondary seed culture medium (g / L): 24 g of industrial-grade yeast extract, 6 g of industrial-grade soybean peptone, 10 g of corn starch, and kanamycin at a final concentration of 20 μg / mL added before inoculation.

[0125] Fermentation medium (g / L): 24 g of industrial-grade yeast extract, 6 g of industrial-grade soybean peptone, 5 g of corn starch, 4 g of glucose, 16.43 g of K2HPO4·3H2O, 2.32 g of KH2PO4, 0.06 g of MgSO4, 10 mL / L of trace element solution, with an appropriate amount of antifoaming agent added. Before inoculation, add kanamycin at a final concentration of 20 μg / mL.

[0126] Phosphate-limited fermentation medium (g / L): 24 g of industrial-grade yeast extract, 6 g of industrial-grade soybean peptone, 10 g of corn starch, 0.06 g of MgSO4, 10 mL / L of trace element solution, with an appropriate amount of defoamer added. Before inoculation, add kanamycin at a final concentration of 20 μg / mL.

[0127] Trace element solution (g / L): FeSO4·7H2O 4, CaCl2 4, MnSO4·5H2O 1, CoCl2·6H2O 0.4, NaMoO4·2H2O 0.2, ZnSO4·7H2O 0.2, AlCl3·6H2O 0.1, CuCl2·H2O 0.1, H3BO4 0.05. After aliquoting into 50mL centrifuge tubes and sterilizing, add the solution before inoculation.

[0128] Carbon source supplement or carbon source supplemented medium (g / L): 550g glucose monohydrate.

[0129] Nitrogen-supplemented feed or nitrogen-supplemented culture medium (g / L): 240 g of industrial-grade yeast extract and 60 g of industrial-grade soybean peptone.

[0130] The detection methods involved in the following embodiments are as follows:

[0131] Preparation of DNS solution and plotting of standard curve

[0132] DNS solution preparation (1L): Weigh 182g of potassium sodium tartrate, add 500mL of distilled water, heat in a water bath, and while hot, add 6.3g of DNS (3,5-dinitrosalicylic acid). Then quickly add 21g of NaOH (weighed in a beaker and dissolved in a small amount of water beforehand), along with 5g of phenol. Then add 5g of Na2SO3, stir to dissolve, cool with ice water, and bring the volume to 1L. Store in a brown bottle away from light. It can be used after 7 days and has a shelf life of 45 days.

[0133] Preparation of glucose standard solution: First, dry the glucose reagent (analytical grade), add distilled water to prepare a glucose solution with a concentration of 1 mg / mL, and store it in a refrigerator at -80℃.

[0134] Procedure for constructing a glucose standard curve: Measure 0 mL, 0.05 mL, 0.1 mL, 0.15 mL, 0.2 mL, 0.25 mL, 0.3 mL, 0.4 mL, and 0.5 mL of glucose standard solution, respectively. Add distilled water to a final volume of 1 mL, then add 1.0 mL of DNS and mix well. Boil in water for 5 minutes, immediately cool in ice water, then add 2 mL of distilled water. Vortex to mix, and measure the absorbance at 540 nm. Plot a scatter plot with glucose mass (mg) on ​​the x-axis and absorbance on the y-axis.

[0135] Definition and determination method of enzyme activity

[0136] Enzyme activity is defined as the amount of enzyme required to generate 1 μmol of reducing sugar (calculated as glucose) per minute, which is 1 unit of enzyme activity (U).

[0137] The hydrolytic activity of the enzyme was characterized by changes in reducing sugar content measured by the DNS method. A 1% (w / v) fully gelatinized soluble starch solution was prepared using deionized water or PBS buffer (pH = 7.0) as the substrate. 100 μL of enzyme solution was diluted a certain factor and added to 900 μL of substrate. The reaction was carried out at 50 °C for 15 minutes, and then 1 mL of DNS solution was added to terminate the reaction. The mixture was heated in a boiling water bath for 5 minutes to develop color, and immediately cooled to room temperature in an ice-water bath. Then, 2 mL of deionized water was added, and the mixture was vortexed to mix. The absorbance was measured at 540 nm.

[0138] Determination of bacterial cell concentration

[0139] The bacterial cell concentration was measured by absorbance (OD) at 600 nm using a spectrophotometer. 600 express.

[0140] Example 1: Growth and enzyme production patterns of recombinant Bacillus subtilis in a 15L fermenter

[0141] Seed culture:

[0142] Take a loopful of recombinant Bacillus subtilis from a glycerol tube stored at -80℃, streak it on an LB solid medium plate containing kanamycin resistance, incubate at 37℃ for 12h, pick a single colony and add it to 50mL of LB liquid medium (containing 20μg / mL kanamycin), incubate at 37℃ and 200r / min for 6h to obtain the seed culture.

[0143] Secondary seed culture:

[0144] Wait for the seed culture medium to grow to OD 600 The inoculum was 0.6-1.2, and 3-5% (v / v) was inoculated into the secondary seed culture medium. The culture was then incubated in a water bath at 37°C and 200 r / min for 5 h to obtain the secondary seed culture solution.

[0145] 15L fermenter fermentation culture:

[0146] The fermentation medium volume was 10L. After the fermentation medium was sterilized, the temperature was lowered to 30℃ and the initial pH was adjusted to 7.0. After igniting the flame ring, 10mL / L of trace element solution, 20μg / mL of kanamycin, 0.06g / L of MgSO4 and 4g / L of glucose were added to the fermenter. Then, 10% (v / v) of secondary seed culture medium was added. After inoculation, dissolved oxygen was calibrated to 100%.

[0147] During fermentation, the fermentation temperature was maintained at 30℃, and the pH was maintained at 7.0 with 40% phosphoric acid and 29% ammonia. The dissolved oxygen was maintained at around 30% by adjusting the stirring and air intake.

[0148] The culture was terminated when the activity of linear maltodextrin-producing enzyme decreased. The growth and enzyme production patterns of recombinant Bacillus subtilis under unfeeded conditions are shown in Figure 1. The protein electrophoresis diagram is shown in Figure 2, where lane M represents the standard protein and lane 1 represents the supernatant after fermentation.

[0149] As shown in Figure 1, the period from 0 to 4 hours is a stagnant phase, from 4 to 60 hours the cells are in a vigorous logarithmic growth phase, and around 60 hours the cells enter a stationary phase. During the logarithmic growth phase, enzyme production and cell growth show a consistent trend. When cell growth enters the stationary phase, around 60 hours of fermentation, enzyme production tends to peak. Under conditions of no feeding, the recombinant bacteria fermented in a 15L fermenter for 60 hours showed a high MFA (microenzyme oxidative stress). PS The highest enzyme activity of -ΔCBM was 493.04 U / mL, which is 1.59 times the highest level optimized by shake flask. At this point, the OD... 600 It reached 19.33.

[0150] Example 2: Determination of Nitrogen Source Feeding Strategy

[0151] (1) Effects of nitrogen source feeding timing on the growth and enzyme production of recombinant Bacillus subtilis

[0152] Fermentation was carried out in a 15L fermenter according to the method in Example 1. When the glucose concentration was lower than 0.4 g / L during fermentation, carbon source was added to the culture medium at a flow rate of 20 mL / h to maintain the glucose concentration in the tank at 0.4-0.5 g / L.

[0153] After inoculating 10% (v / v) of the secondary seed culture medium according to the method in Example 1, fermentation culture was started;

[0154] Nitrogen was added at a flow rate of 30 mL / h at the early logarithmic growth stage (4 h of fermentation), mid-logarithmic growth stage (20 h of fermentation), and late logarithmic growth stage (60 h of fermentation). Fermentation was stopped at 72 h. The growth and enzyme production curves of the recombinant bacteria at different feeding times are shown in Figures 3 and 4.

[0155] The results showed that supplementing the nitrogen source at the mid-logarithmic growth stage of the cells (20 hours of fermentation) had the best effect, and the enzyme activity reached 602.48 U / mL after 60 hours of fermentation.

[0156] Supplementing nitrogen sources at the beginning of the logarithmic growth phase (4 hours of fermentation) and at the end of the growth phase (60 hours of fermentation) is not conducive to improving enzyme activity.

[0157] (2) Effect of nitrogen source feed flow rate on growth and enzyme production of recombinant Bacillus subtilis

[0158] Fermentation was carried out in a 15L fermenter according to the method in Example 1. When the glucose concentration was lower than 0.4 g / L during fermentation, carbon source was added to the culture medium at a flow rate of 20 mL / h to maintain the glucose concentration in the tank at 0.4-0.5 g / L.

[0159] After inoculating 10% (v / v) of the secondary seed culture medium according to the method in Example 1, fermentation was started;

[0160] During the mid-logarithmic growth phase of the bacteria (20 h of fermentation), nitrogen was added at rates of 24 mL / h, 30 mL / h, and 36 mL / h, respectively, and fermentation was stopped at 72 h. The growth and enzyme production curves of the recombinant bacteria at different nitrogen feed rates are shown in Figures 5 and 6, respectively.

[0161] As shown in the cell growth curves (Figure 5), during the mid-logarithmic growth phase (20-40 h of fermentation), there was no significant difference in cell growth under the three nitrogen source feed rates. After 40 h of fermentation, the higher the feed rate, the faster the cell growth. The enzyme production curves (Figure 6) show that the enzyme activity levels were comparable in the experimental groups fed with 24 mL / h and 30 mL / h for the first 40 h of fermentation. After 40 h, the differences between different feed rates became more significant, and the final differences in enzyme activity levels were smaller when fed with nitrogen source at 30 mL / h and 36 mL / h.

[0162] (3) Formulation of nitrogen source feeding strategy and its effect on the growth and enzyme production of recombinant Bacillus subtilis

[0163] Fermentation was carried out in a 15L fermenter according to the method in Example 1. When the glucose concentration was lower than 0.4 g / L during fermentation, the carbon source feeding medium was used as the feeding carbon source, and the feeding flow rate was 20 mL / h to maintain the glucose concentration in the tank at 0.4-0.5 g / L.

[0164] To save costs, a nitrogen feeding strategy as shown in Figure 7 was developed based on the optimized nitrogen feed rate described above. This strategy involves supplementing nitrogen at a rate of 24 mL / h during the mid-logarithmic growth phase (20-40 h of fermentation), and at a rate of 30 mL / h during the late-logarithmic growth phase (after 40 h of fermentation). The culture is terminated when the activity of linear maltodextrin-producing enzyme decreases. The growth and enzyme production curves of the recombinant bacteria under this nitrogen feeding strategy are shown in Figure 8.

[0165] Under this fed-batch strategy, the enzyme activity of the recombinant Bacillus subtilis reached a peak of 679.05 U / mL after 62 hours of fermentation, which was 119% higher than that of the original strain. This fed-batch strategy achieved a high enzyme activity level with a relatively low feed volume, and the flow rate switching is easy to control, which is beneficial for industrial production.

[0166] Example 3: Determination of Glucose Starvation Strategy

[0167] (1) Effects of glucose starvation timing on the growth and enzyme production of recombinant Bacillus subtilis

[0168] Fermentation was carried out in a 15L fermenter according to the method in Example 1. When the glucose concentration was lower than 0.4 g / L during fermentation, carbon source was added to the culture medium at a flow rate of 20 mL / h to maintain the glucose concentration in the tank at 0.4-0.5 g / L.

[0169] After inoculating 10% (v / v) of the secondary seed culture medium according to the method in Example 1, fermentation was started;

[0170] During the mid-logarithmic growth phase (20-40 h of fermentation), nitrogen was supplemented at 24 mL / h, and during the late-logarithmic growth phase (after 40 h of fermentation), nitrogen was supplemented at 30 mL / h. A 4-hour glucose starvation phase was introduced at 20, 40, and 60 h of logarithmic growth (20, 40, and 60 h of fermentation). The culture was terminated when the activity of linear maltodextrin synthase decreased. The growth and enzyme production curves of the recombinant bacteria under different starvation conditions are shown in Figures 9 and 10, respectively.

[0171] As shown in Figure 10, the introduction of the starvation stage at 20h and 60h (the 20th and 60th hours of fermentation) was beneficial to the improvement of enzyme activity. The enzyme activity reached 735.05 U / mL and 759.29 U / mL at 60h and 64h of fermentation, respectively, which were 137% and 145% higher than the enzyme activity of the starting strain.

[0172] (2) Effects of glucose starvation duration on the growth and enzyme production of recombinant Bacillus subtilis

[0173] Fermentation was carried out in a 15L fermenter according to the method in Example 1. When the glucose concentration was lower than 0.4 g / L during fermentation, carbon source was added to the culture medium at a flow rate of 20 mL / h to maintain the glucose concentration in the tank at 0.4-0.5 g / L.

[0174] During the mid-logarithmic growth phase (20-40 h of fermentation), nitrogen was supplemented at 24 mL / h, and during the late-logarithmic growth phase (after 40 h of fermentation), nitrogen was supplemented at 30 mL / h.

[0175] Option 1: In the 20th hour of fermentation, glucose starvation stages of 2h, 4h and 6h were introduced respectively. The culture was ended when the activity of linear maltodextrin synergist decreased. The enzyme production of recombinant Bacillus subtilis under different starvation durations is shown in Figure 11.

[0176] Option 2: Introduce glucose starvation phases of 2, 4, and 6 hours at 60 hours. End the culture when the activity of linear maltodextrin-producing enzyme decreases. The growth and enzyme production curves of recombinant Bacillus subtilis under different starvation durations are shown in Figure 12.

[0177] Introducing a 2-hour starvation phase at 20 hours of fermentation was most conducive to increasing enzyme activity. The enzyme activity reached its highest level of 725.05 U / mL at 62 hours after fermentation, which was 134% higher than that of the starting strain.

[0178] Introducing a 4-hour starvation period at 60 hours of fermentation was most conducive to increasing enzyme activity. At 64 hours of fermentation, the enzyme activity reached 762.05 U / mL, which was 146% higher than that of the starting strain.

[0179] (3) The formulation of glucose starvation strategy and its effect on the growth and enzyme production of recombinant Bacillus subtilis

[0180] Fermentation was carried out in a 15L fermenter according to the method in Example 1. When the glucose concentration was lower than 0.4 g / L during fermentation, the carbon source feeding medium was used as the feeding carbon source, and the feeding flow rate was 20 mL / h to maintain the glucose concentration in the tank at 0.4-0.5 g / L.

[0181] During the mid-logarithmic growth phase (20-40 h of fermentation), nitrogen was supplemented at 24 mL / h, and during the late-logarithmic growth phase (after 40 h of fermentation), nitrogen was supplemented at 30 mL / h. Simultaneously, glucose starvation phases of 2 h and 4 h were introduced at 20 h and 60 h of fermentation, respectively. The culture was terminated when the activity of linear maltodextrin synthase decreased. The growth and enzyme production curves of the recombinant bacteria under these fermentation conditions are shown in Figure 13.

[0182] Under these fermentation conditions, the recombinant strain achieved a peak enzyme activity of 850.05 U / mL after 64 hours of fermentation, representing a 174% increase compared to the original strain. This fermentation process not only significantly enhances enzyme activity but also shortens the fermentation cycle, enabling comprehensive and efficient utilization of nutrients in the fermentation system. It addresses the issues of low enzyme activity and high production costs in existing fermentation processes, making it suitable for large-scale industrial production.

[0183] Example 4: Effect of phosphate addition on MFA PS Effect of -ΔCBM enzyme activity

[0184] 1. Before investigating the effects of phosphate, we will first explore the effects of different nutrient deficiencies on the growth of recombinant Bacillus subtilis and MFA. PS Effect of -ΔCBM enzyme expression

[0185] Nutrient restriction, a common stressor in natural and industrial environments, significantly affects the physiological state of microorganisms. Limiting specific nutrients can trigger widespread changes in gene expression, activate stress response pathways, promote resource reallocation, and thereby upregulate specific metabolic pathways, promoting the production of secondary metabolites or enzymes. This embodiment constructs a single-factor nutrient-limited culture medium based on commonly used microbial culture medium (TB medium) to systematically investigate the effects of different nutrient deficiencies on the growth of recombinant Bacillus subtilis and MFA. PS The effect of -ΔCBM enzyme expression;

[0186] Specifically:

[0187] (1) Preparation of culture medium

[0188] Single-factor influence on limiting culture medium: Using TB medium as a control, glycerol, yeast extract, tryptone and phosphate were removed from TB medium respectively;

[0189] (2) Enzyme production by recombinant bacteria fermentation

[0190] Seed activation: Take a loopful of recombinant Bacillus subtilis from a glycerol tube stored at -80℃, streak it on a plate containing kanamycin resistance, incubate at 37℃ for 12h, pick a single colony and add it to 50mL of LB medium (containing 20μg / mL kanamycin), incubate at 37℃ and 200r / min for 6h to obtain seed culture.

[0191] Shake-flask fermentation: Kanamycin at a final concentration of 20 μg / mL was added to a 250 mL Erlenmeyer flask containing 50 mL of single-factor nutrient-limiting medium. Then, 2 mL of the activated seed culture was transferred to the flask and cultured in a shaker at 30 °C and 200 rpm / min for 72 h. After fermentation was stopped, the fermentation broth was centrifuged at 4 °C and 10000 rpm / min for 20 min, and the supernatant was collected to obtain recombinant MFA. PS -ΔCBM crude enzyme solution was used to detect enzyme activity, and the results are shown in Figure 26.

[0192] Experimental results show that:

[0193] (1) The lack of both carbon and nitrogen sources significantly inhibited cell growth and enzyme expression;

[0194] (2) When glycerol, tryptone, or yeast extract were removed from the culture medium, the cell biomass decreased significantly, and MFA... PS -ΔCBM enzyme activity was significantly reduced. This may be because glycerol, as the main carbon source, not only provides cellular structural components but also directly affects energy metabolism. Similarly, nitrogen sources also play an important role in microbial fermentation, participating in the synthesis of purines, pyrimidines, amino acids, and nucleic acids, and are essential components for maintaining enzymatic reactions and the biosynthesis of high-value-added products.

[0195] (3) In this embodiment, phosphate restriction did not inhibit bacterial growth, but rather significantly promoted biomass accumulation and increased MFA. PS -ΔCBM enzyme activity: After 72 h of fermentation, the enzyme activity reached 338.50 U / mL, which was 47.15% higher than the control group and significantly higher than the enzyme activity under other nutrient restriction conditions. This indicates that phosphate restriction may promote the synthesis of specific secondary metabolites by activating stress response pathways, thereby increasing enzyme activity. This finding suggests that phosphate may play a more complex role in metabolic regulation.

[0196] 2. The effect of phosphate addition on MFA PS Effect of -ΔCBM enzyme activity

[0197] To further improve MFA based on the above embodiments PS -ΔCBM enzyme activity was investigated to explore the relationship between the amount of phosphate added in the culture medium and enzyme activity.

[0198] 1. Prepare different shake-flask fermentation media:

[0199] Analytical grade yeast extract 24, analytical grade tryptone 12, corn starch 10, KH2PO4 0-34mmol / L (B), K2HPO4·3H2O 0-110mmol / L (A), kanamycin at a final concentration of 20μg / mL was added before inoculation;

[0200] The amounts of A and B are as follows: 0 mM A + 0 mM B; 7 mM A + 2 mM B; 14 mM A + 4 mM B; 27.5 mM A + 8.5 mM B; 55 mM A + 17 mM B; 83 mM A + 26 mM B; 110 mM A + 34 mM B

[0201] 2. Fermentation experiment

[0202] Seed activation: Take a loopful of recombinant Bacillus subtilis from a glycerol tube stored at -80℃, streak it on a plate containing kanamycin resistance, incubate at 37℃ for 12h, pick a single colony and add it to 50mL of LB medium (containing 20μg / mL kanamycin), incubate at 37℃ and 200r / min for 6h to obtain seed culture.

[0203] Shake-flask fermentation: Kanamycin at a final concentration of 20 μg / mL was added to a 250 mL Erlenmeyer flask containing 50 mL of the different shake-flask fermentation media described above. Then, 2 mL of the activated seed culture was transferred to the flask and cultured in a shaker at 30℃ and 200 rpm for 72 h. After fermentation was stopped, the fermentation broth was centrifuged at 4℃ and 10000 rpm for 20 min, and the supernatant was collected to obtain recombinant MFA. PS -ΔCBM crude enzyme solution.

[0204] The amount of phosphate added in the culture medium and MFA PS The relationship between -ΔCBM enzyme activity is shown in Figure 14.

[0205] Experimental results show that recombinant Bacillus subtilis produces MFA. PS The activity of -ΔCBM enzyme decreased with increasing phosphate addition, reaching its highest level when no phosphate was added. This indicates that phosphate nutrient restriction has a certain promoting effect on the metabolism and protein secretion and expression of recombinant bacteria. A possible reason is that Bacillus subtilis, being a soil bacterium, frequently faces nutrient depletion. When Bacillus subtilis transitions from exponential growth to a plateau phase, previously silenced genes are activated, providing the products necessary for survival under stress conditions.

[0206] Example 5: Scale-up and verification of recombinant bacteria in a 15L fermenter under different phosphorus nutrient conditions

[0207] Based on Example 4, scale-up cultivation and phenomenon verification were carried out in a 15L fermenter. The steps for fermenting and culturing recombinant Bacillus subtilis in a 15L fermenter are as follows:

[0208] Seed culture: Take a loopful of recombinant Bacillus subtilis from a glycerol tube stored at -80℃, streak it on a plate containing kanamycin resistance, incubate at 37℃ for 12h, pick a single colony and add it to 50mL of LB medium (containing 20μg / mL kanamycin), incubate at 37℃ and 200r / min for 6h to obtain seed culture solution.

[0209] Secondary seed culture: wait until the seed culture medium grows to OD 600 The inoculum was 0.6-1.2, and 3-5% (v / v) was inoculated into the phosphate-limited secondary seed medium. The culture was then incubated in a water bath at 37°C and 200 r / min for 5 h to obtain the secondary seed culture solution.

[0210] Fermentation culture in a 15L fermenter: The phosphate-limited fermentation medium was 10L in volume. After sterilization, once the temperature had dropped to 30℃ and the initial pH reached 7.0, the flame ring was ignited, and 10mL / L of trace element solution, 20μg / mL of kanamycin, and 0.06g / L of MgSO4 were added to the fermenter. Then, 10% (v / v) of secondary seed culture was added, and dissolved oxygen was calibrated to 100% after inoculation. During fermentation, the fermentation temperature was maintained at 30℃, and the pH was maintained at 7.0 using 3% sulfuric acid and 10% ammonia. Dissolved oxygen was maintained at approximately 30% by adjusting the stirring and aeration rate.

[0211] In both the secondary culture and fermenter culture described above, the phosphate-containing group served as the control group, while the phosphate-free group served as the experimental group. Figure 15 shows the growth and enzyme production of recombinant Bacillus subtilis under different phosphorus nutrient conditions in a 15L fermenter.

[0212] The experimental results showed that under phosphate-limited nutrient conditions, the enzyme activity of recombinant Bacillus subtilis reached a maximum of 729.15 U / mL after 52 hours of fermentation in a 15L fermenter. This was 47.88% higher than that of the control group (with normal phosphate addition, fermentation for 64 hours, enzyme activity reached a maximum of 493.04 U / mL, see Example 1), and the fermentation time was shortened by 12 hours.

[0213] This indicates that the phenomenon of phosphate restriction promoting enzyme activity still exists in larger fermentation systems. Furthermore, the recombinant bacteria entered the stationary phase more quickly under phosphate restriction conditions, resulting in a shorter fermentation time. This suggests that phosphate restriction can significantly affect the growth, metabolism, and protein secretion of recombinant Bacillus subtilis, thus promoting the increased activity of heterologous expressed enzymes.

[0214] Example 6: Relationship between enzyme production and carbon source type of recombinant bacteria under phosphate-restricted conditions

[0215] Since phosphates are often closely related to the transport and utilization of carbon sources, the relationship between phosphate nutrient limitation and the types of carbon sources was investigated.

[0216] Recombinant Bacillus subtilis was cultured in a fermentation system under phosphate-containing and phosphate-limited conditions, using 5 g / L glycerol, 5 g / L corn starch, 5 g / L glucose, and a mixture of glucose and corn starch (2.5 g / L glucose and 2.5 g / L corn starch) as the carbon source. The culture steps are as follows.

[0217] (1) Preparation of fermentation culture medium:

[0218] The phosphate-containing medium formula is as follows: yeast extract 24 g / L, soybean peptone 6 g / L, K2HPO4·3H2O 16.43 g / L, KH2PO4 2.32 g / L, carbon source, and kanamycin at a final concentration of 20 μg / mL added before inoculation;

[0219] The phosphate-limited medium formula is: 24 g / L yeast extract, 6 g / L soybean peptone, carbon source, and kanamycin at a final concentration of 20 μg / mL added before inoculation.

[0220] The carbon source is 5 g / L glycerol, 5 g / L corn starch, 5 g / L glucose, and a mixture of glucose and corn starch (2.5 g / L glucose and 2.5 g / L corn starch).

[0221] (2) Fermentation culture

[0222] Seed activation: Take a loopful of recombinant Bacillus subtilis from a glycerol tube stored at -80℃, streak it on a plate containing kanamycin resistance, incubate at 37℃ for 12h, pick a single colony and add it to 50mL of LB medium (containing 20μg / mL kanamycin), incubate at 37℃ and 200r / min for 6h to obtain seed culture.

[0223] Shake-flask fermentation: Kanamycin at a final concentration of 20 μg / mL was added to a 250 mL Erlenmeyer flask containing 50 mL of fermentation medium prepared in step (1) with different carbon sources. Then, 2 mL of the activated seed culture was transferred to the flask and cultured in a shaker at 30℃ and 200 rpm for 72 h. After fermentation was stopped, the fermentation broth was centrifuged at 4℃ and 10000 rpm for 20 min, and the supernatant was collected to obtain recombinant MFA. PS -ΔCBM crude enzyme solution. The relationship between enzyme production and carbon source type of recombinant Bacillus subtilis under phosphate-limited conditions and SDS-PAGE electrophoresis analysis are shown in Figures 16 and 17, respectively.

[0224] The results show:

[0225] As shown in Figure 16, the enzyme activity, pH, and OD values ​​of the corn starch experimental group under phosphate-limited nutrient conditions were all higher than those of the control group. Protein electrophoresis also showed (Figure 17) that all bands under phosphate-limited nutrient conditions were thicker than those in the control group, further indicating that phosphate-limited nutrient conditions can promote the growth and protein secretion of recombinant Bacillus subtilis.

[0226] Example 7: Relationship between enzyme production and carbon source concentration of recombinant bacteria under phosphate-limited nutrient conditions

[0227] To facilitate scale-up cultivation and feed optimization in the fermenter system, the relationship between phosphate nutrient limitation and the concentrations of the main carbon sources, corn starch and glucose, was further investigated based on Example 6. The experimental results are shown in Figures 18 and 19.

[0228] The specific implementation method is the same as in Example 6, except that the carbon source is adjusted to corn starch and glucose, and the concentration of the carbon source is adjusted to 2g / L, 4g / L, 6g / L, 8g / L, and 10g / L.

[0229] Fermentation culture was carried out according to the method in Example 6.

[0230] The results show that:

[0231] (1) When the carbon source is corn starch:

[0232] The promoting effect of phosphate-limited environment on enzyme activity increases with increasing corn starch concentration. When the corn starch concentration is 10 g / L, the effect of phosphate restriction on enzyme activity is significant.

[0233] (2) When the carbon source is glucose:

[0234] The effect of phosphate restriction on enzyme activity gradually decreases with increasing glucose concentration. When glucose concentration exceeds 10 g / L, phosphate restriction has no promoting effect on enzyme activity; in fact, it has a negative impact when the concentration exceeds a critical level. This is presumably because when glucose concentration is too high, sufficient phosphorus is needed to promote pyruvate autophosphorylation and glycolysis, enabling nutrient absorption and utilization to maintain normal growth and metabolism.

[0235] Example 8: A Preliminary Exploration of a Horizontal Feeding Strategy for Shaking Flasks

[0236] Example 2 above demonstrated that an appropriate nitrogen source feeding strategy in a 15L fermentation system can improve the enzyme production capacity of recombinant Bacillus subtilis. Example 6 further demonstrated that under phosphate-limited nutrient conditions, glucose not only promotes the rapid growth of recombinant bacteria but also increases enzyme activity. Since corn starch needs to be kept in a gelatinized state during fermentation, glucose was chosen as the carbon source for industrial applications. Based on the experimental results of Example 7, the concentration of corn starch as the carbon source in the fermentation medium was 10 g / L.

[0237] Based on the above experimental results, two carbon source feeding strategies are proposed:

[0238] (1) Pre-feeding strategy, that is, from the start of the fermentation process until the end of the fermentation process, glucose is intermittently supplemented to control the glucose concentration in the culture medium at 0.2-0.4 g / L;

[0239] (2) Post-feeding strategy, when OD 600 When the condition is stable, glucose concentration in the culture medium is controlled at 0.2-0.4 g / L by intermittent glucose supplementation;

[0240] First, we conducted an initial exploration of these two feeding strategies at the shake-flask level.

[0241] The specific method is as follows:

[0242] (1) Prepare the fermentation medium:

[0243] The phosphate-containing medium formula is as follows: yeast extract 24 g / L, soybean peptone 6 g / L, K2HPO4·3H2O 16.43 g / L, KH2PO4 2.32 g / L, corn starch 10 g / L, and kanamycin at a final concentration of 20 μg / mL added before inoculation;

[0244] The phosphate-limited medium formula is: 24 g / L yeast extract, 6 g / L soybean peptone, 10 g / L corn starch, and kanamycin at a final concentration of 20 μg / mL added before inoculation.

[0245] Prepare glucose fed culture medium: glucose 50g / L.

[0246] Fermentation medium was autoclaved at 121°C for 30 minutes before use; glucose fed medium was autoclaved at 115°C for 30 minutes.

[0247] (2) Fermentation culture

[0248] Seed activation: Take a loopful of recombinant Bacillus subtilis from a glycerol tube stored at -80℃, streak it on a plate containing kanamycin resistance, incubate at 37℃ for 12h, pick a single colony and add it to 50mL of LB medium (containing 20μg / mL kanamycin), incubate at 37℃ and 200r / min for 6h to obtain seed culture.

[0249] Shake flask fermentation: Add kanamycin at a final concentration of 20 μg / mL to a 250 mL Erlenmeyer flask containing 50 mL of the fermentation medium (containing phosphate medium / phosphate restriction medium) prepared in step (1), and then transfer 2 mL of the activated seed liquid to the flask and culture it in a shaker at 30 °C and 200 rpm for 72 h.

[0250] Option 1: Pre-feeding strategy: Add glucose to the culture medium before fermentation to a final concentration of 0.3 g / L, and then take samples every four hours to measure the glucose content and feed the medium to keep the final glucose concentration at 0.2-0.4 g / L.

[0251] Option 2: Post-material replenishment strategy: In OD 600 When the fermentation time is stable (40 hours), glucose is added to the culture medium to a final concentration of 0.3 g / L. This process continues until the fermentation process is complete. Glucose concentration in the culture medium is controlled at 0.2-0.4 g / L by intermittently supplementing glucose. Then, glucose content is measured every four hours, and feed is added to maintain the final glucose concentration at 0.2-0.4 g / L.

[0252] The control group consisted of individuals who received no additional glucose during the fermentation process.

[0253] After fermentation was stopped, the fermentation broth was centrifuged at 4°C and 10,000 rpm for 20 minutes, and the supernatant was collected to obtain recombinant MFA. PS -ΔCBM crude enzyme solution.

[0254] The experimental results are shown in Figure 20.

[0255] As shown in Figure 20, adding glucose to the initial culture medium is beneficial to improving enzyme activity; under phosphate-limited nutrient conditions, the post-feeding method has a more significant effect on improving enzyme activity.

[0256] Example 9: Optimization of feeding conditions for recombinant bacteria in a 15L fermenter under different phosphorus nutrient environments

[0257] Based on Example 8, the effects of feeding methods and feeding time on the growth and enzyme production of recombinant bacteria in a 15L fermenter were further investigated. Three different feeding methods were designed: (1) a pre-feeding strategy, where carbon and nitrogen sources were added simultaneously after inoculation; (2) simultaneous addition of carbon and nitrogen sources when dissolved oxygen rebounded (around 24 hours); and (3) simultaneous addition of carbon and nitrogen sources when the OD value of the recombinant bacteria reached stability (around 36 hours). The flow rate of carbon source feeding was 20 mL / h, and the flow rate of nitrogen source feeding was dynamically adjusted according to the growth of recombinant Bacillus subtilis.

[0258] The specific method is as follows:

[0259] (1) Seed culture: Take a loopful of recombinant Bacillus subtilis from a glycerol tube stored at -80℃, streak it on a plate containing kanamycin resistance, and incubate at 37℃ for 12h. Pick a single colony and add it to 50mL of LB medium (containing 20μg / mL kanamycin), and incubate at 37℃ and 200r / min for 6h to obtain seed culture solution.

[0260] Secondary seed culture: wait until the seed culture medium grows to OD 600 The inoculum was 0.6-1.2, and 3-5% (v / v) was inoculated into the phosphate-limited secondary seed medium. The culture was then incubated in a water bath at 37°C and 200 r / min for 5 h to obtain the secondary seed culture solution.

[0261] (2) Fermentation culture in a 15L fermenter:

[0262] The phosphate-limited fermentation medium was filled to a volume of 10 L. After sterilization, the temperature was lowered to 30 °C, the initial pH was adjusted to 7.0, the initial stirring speed was 200 rpm, and the initial aeration rate was 1.5 VVM. After igniting the flame ring, 10 mL / L of trace element solution, 20 μg / mL of kanamycin, and 0.06 g / L of MgSO4 were added to the fermenter. Then, 10% (v / v) of secondary seed culture was added. After inoculation, dissolved oxygen was calibrated to 100%.

[0263] During fermentation, the fermentation temperature was maintained at 30℃, and the pH was maintained at 7.0 using 3% sulfuric acid and 10% ammonia solution. Dissolved oxygen was maintained at approximately 30% by adjusting stirring and aeration. The tank pressure was controlled at 0.03-0.05 MPa during fermentation.

[0264] The above-mentioned secondary seed culture and fermenter culture both used phosphate-containing samples as the control group (i.e., the difference between the control group and the experimental group is the addition of phosphate: K2HPO4·3H2O 16.43g / L, KH2PO4 2.32g / L), while the samples without phosphate were used as the experimental group.

[0265] Three different feeding methods were designed:

[0266] Method 1: Supplement carbon and nitrogen sources simultaneously after inoculation. Specifically, start the fermentation at 0 h with carbon source feed medium at a flow rate of 20 mL / h; during the fermentation stagnation period and the early to mid-logarithmic growth phase (0-40 h of fermentation), add nitrogen source feed medium at a flow rate of 24 mL / h; after 40 h of fermentation, add nitrogen source feed medium at a flow rate of 30 mL / h until the end of fermentation.

[0267] Method 2: When dissolved oxygen rebounds (24 hours of fermentation), carbon and nitrogen sources are simultaneously supplemented. Specifically, carbon source feed medium is added at a flow rate of 20 mL / h starting at 24 hours of fermentation; nitrogen source feed medium is added at a flow rate of 24 mL / h from 24 to 40 hours of fermentation; and nitrogen source feed medium is added at a flow rate of 30 mL / h after 40 hours of fermentation until fermentation ends.

[0268] Method 3: When the OD value of the recombinant bacteria reaches a stable level (around 36 hours of fermentation), begin simultaneously supplementing with carbon and nitrogen sources, making appropriate adjustments and simplifications. That is, starting at 36 hours of fermentation, add carbon source feed medium at a flow rate of 20 mL / h and nitrogen source feed medium at a flow rate of 30 mL / h until fermentation ends.

[0269] The results show:

[0270] Method 1:

[0271] The results of fed-batch fermentation are shown in Figure 21. When glucose was used as the initial carbon source in the fermentation medium, the OD values ​​of the recombinant bacteria in the experimental and control groups increased rapidly in the 15L fermenter and quickly reached stability. The enzyme activity of the experimental group also continued to increase under phosphate nutrient restriction conditions. However, when glucose was used as the main carbon source in the fermenter, the logarithmic growth phase of the recombinant bacteria was greatly shortened. Therefore, when the recombinant bacteria fermented in the 15L fermenter for 52 hours, the enzyme activity reached a maximum of only 724.33 U / mL. Thus, the fed-batch strategy cannot guarantee the complete utilization of nutrients in the original fermentation system, and the excessively long feeding time greatly increases the production cost, which is not conducive to the industrial production of linear maltotetrasaccharide generating enzyme.

[0272] Method 2:

[0273] Figure 22 shows the results of fed-batch fermentation during dissolved oxygen rebound. When corn starch was used as the initial carbon source in the fermentation medium, the OD values ​​of the recombinant bacteria in both the experimental and control groups stabilized after approximately 24 hours of fermentation in a 15L fermenter, at which point the dissolved oxygen level in the tank was also relatively high. According to literature, a rebound in dissolved oxygen in the fermenter indicates nutrient depletion and feeding should be initiated at this time. Therefore, carbon and nitrogen sources were simultaneously supplemented starting at 24 hours. However, the figure shows that under phosphate-limited conditions, the OD values ​​of the experimental group remained stable or showed slight fluctuations from 24 to 36 hours after feeding began. The residual sugar content in the tank also indicated that the recombinant bacteria consumed very little or no glucose within 12 hours after feeding, resulting in residual sugar accumulation. The phosphorus-containing control group only experienced a short period of stagnation after feeding began, and then resumed the logarithmic growth phase after 33 hours. Simultaneously, the residual sugar level in the tank began to decrease, indicating that glucose was the primary carbon source in the control group at this time.

[0274] When the experimental group fermented for 36 hours (12 hours after feeding), the OD value began to rise rapidly, accompanied by a rapid increase in enzyme activity. In contrast, the control group, after returning to normal logarithmic growth, only experienced a rapid increase in OD value, while enzyme activity increased slowly. This is similar to the results obtained in the shake-flask experiments: when the culture medium contains phosphorus, glucose only promotes the growth of recombinant bacteria but not enzyme production; when the recombinant bacteria are in a phosphorus-limited environment, glucose not only promotes the growth and reproduction of the recombinant bacteria but also increases enzyme activity. Therefore, the experimental group exhibited the experimental phenomenon of a rapid and simultaneous increase in OD value and enzyme activity.

[0275] The experimental group reached its highest enzyme activity of 824.51 U / mL at 63 hours of fermentation, with an OD value of 36.85. In contrast, the control group's enzyme activity was only 342.71 U / mL at 72 hours of fermentation, with an OD value of 37.23. However, considering the overall fermentation process, the transition from corn starch to glucose as the carbon source required a relatively long buffer period, indicating that the corn starch was not completely depleted at this point, resulting in a prolonged competition process. Comparing this to the control group (Example 5, phosphate-restricted, no feeding), the OD value only began to stabilize and gradually decrease at 36 hours of fermentation. This should be the point at which the corn starch was completely depleted. Adding glucose at this point might shorten the competition buffer period, achieving complete utilization of the corn starch and further increasing enzyme activity. Therefore, feeding when dissolved oxygen rebounds is not suitable for this study, while feeding when the OD value stabilizes is appropriate. This strategy ensures efficient and complete utilization of the carbon source in the fermentation system.

[0276] In comparison, the strategy of starting feeding when the OD value stabilizes (around 36 hours) resulted in 47.97% higher enzyme activity, a 15-hour shorter feeding time, and a 3-hour shorter fermentation time compared to the strategy of starting feeding when dissolved oxygen rebounds (around 24 hours). This indicates that appropriately delaying the feeding time not only promotes the complete utilization of corn starch, thereby facilitating a high-efficiency increase in enzyme activity, but also reduces the consumption of feeding medium, lowers production costs, and achieves the effect of improving quality and reducing costs.

[0277] Method 3:

[0278] When the OD value of the recombinant bacteria reached a stable level (36h), the results of fed-batch fermentation are shown in Figures 23 and 24. When the recombinant bacteria in the experimental group fermented in a 15L fermenter for 60h, the enzyme activity reached a maximum of 1220.04U / mL, which was 176% higher than that of the control group (normal phosphate addition, fermentation for 72h, enzyme activity reached a maximum of 442.18U / mL), and the fermentation time was shortened by 12h.

[0279] This indicates that starting to feed when the OD value reaches a stable level can not only promote the complete utilization of corn starch, thereby promoting the efficient improvement of enzyme activity, but also reduce the consumption of feeding medium, reduce production costs, and achieve the effect of improving quality and reducing costs.

[0280] Example 10: Effects of glucose starvation strategy on the growth and enzyme production of recombinant bacteria under different phosphorus nutrition environments

[0281] Based on Example 9, the effects of glucose starvation strategy on the growth and enzyme production of recombinant bacteria in a 15L fermenter were further investigated.

[0282] Based on the experimental results of previous studies:

[0283] The initial culture medium used 10 g / L corn starch as the carbon source and 24 g / L yeast extract and 6 g / L soybean peptone as the nitrogen source.

[0284] The control group culture medium was supplemented with phosphate, while the experimental group was not supplemented with phosphate; the liquid volume was 10L.

[0285] The seed culture inoculum was 10%, dissolved oxygen was adjusted to 100% under fermentation conditions, pH was kept constant at 7.0, initial fermentation temperature was set at 30℃, initial stirring speed was 200 r / min, and initial aeration rate was 1.5 VVM. Subsequently, dissolved oxygen was maintained at approximately 30% by controlling the stirring speed and aeration rate. The tank pressure was controlled between 0.03 and 0.05 MPa during fermentation. Carbon and nitrogen sources were simultaneously supplemented when the OD value of the recombinant bacteria reached stability (around 36 hours). A 4-hour glucose starvation phase was introduced in the later stages of fermentation (around 60 hours). The experimental results are shown in Figure 25. The nitrogen source feeding rate was 30 mL / h.

[0286] The specific method is as follows:

[0287] (1) Seed culture: Take a loopful of recombinant Bacillus subtilis from a glycerol tube stored at -80℃, streak it on a plate containing kanamycin resistance, and incubate at 37℃ for 12h. Pick a single colony and add it to 50mL of LB medium (containing 20μg / mL kanamycin), and incubate at 37℃ and 200r / min for 6h to obtain seed culture solution.

[0288] Secondary seed culture: wait until the seed culture medium grows to OD 600 The inoculum was 0.6-1.2, and 3-5% (v / v) was inoculated into the phosphate-limited secondary seed medium. The culture was then incubated in a water bath at 37°C and 200 r / min for 5 h to obtain the secondary seed culture solution.

[0289] (2) Fermentation culture in a 15L fermenter:

[0290] The phosphate-limited fermentation medium was filled to a volume of 10 L. After sterilization, the temperature was lowered to 30 °C, the initial pH was adjusted to 7.0, the initial stirring speed was 200 rpm, and the initial aeration rate was 1.5 VVM. After igniting the flame ring, 10 mL / L of trace element solution, 20 μg / mL of kanamycin, and 0.06 g / L of MgSO4 were added to the fermenter. Then, 10% (v / v) of secondary seed culture was added. After inoculation, dissolved oxygen was calibrated to 100%.

[0291] During fermentation, the fermentation temperature was maintained at 30℃, and the pH was maintained at 7.0 using 3% sulfuric acid and 10% ammonia solution. Dissolved oxygen was maintained at approximately 30% by adjusting stirring and aeration. The tank pressure was controlled at 0.03-0.05 MPa during fermentation.

[0292] The above-mentioned secondary seed culture and fermenter culture both used phosphate-containing samples as the control group (i.e., the difference between the control group and the experimental group is the addition of phosphate: K2HPO4·3H2O 16.43g / L, KH2PO4 2.32g / L), while the samples without phosphate were used as the experimental group.

[0293] A material replenishment method was designed:

[0294] When the OD value of the recombinant bacteria reached a stable level (around 36 hours of fermentation), carbon and nitrogen sources were simultaneously added. Specifically, starting at 36 hours of fermentation, carbon source feed medium was added at a flow rate of 20 mL / h, and nitrogen source feed medium was added at a flow rate of 30 mL / h until the end of fermentation. In the later stage of fermentation (around 60 hours of fermentation), a 4-hour glucose starvation phase was introduced. The experimental results are shown in Figure 25.

[0295] The results show:

[0296] When the recombinant bacteria in the experimental group fermented in a 15L fermenter for 64 hours, the enzyme activity reached a maximum of 1393.51 U / mL, which was 186.06% higher than that of the control group (with normal phosphate addition, fermentation for 69 hours, enzyme activity reached a maximum of 487.13 U / mL), and the fermentation time was shortened by 5 hours.

[0297] This demonstrates that the feeding strategy determined in Example 10, combined with the glucose starvation strategy determined in previous studies, can not only promote the complete utilization of residual nutrients in the tank and reduce costs, but also promote the efficient improvement of enzyme activity levels, shorten feeding time, and reduce the consumption of feeding culture medium, thereby achieving the effect of improving quality and reducing costs.

[0298] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A method for producing linear maltodextrose synthase by fermentation of Bacillus subtilis, characterized in that, Includes the following steps: Step S1: Activate recombinant Bacillus subtilis to obtain seed culture medium. The recombinant Bacillus subtilis uses pP43nmk as a vector and employs the signal peptide SP. Bgls Heterologous expression of recombinant linear maltotetrasaccharide-generating enzyme with starch-binding module removed; Step S2: Inoculate the seed culture medium into the secondary seed culture medium to obtain the secondary seed culture medium; Step S3: Inoculate the secondary seed culture medium into the fermentation medium for fermentation culture. The fermentation medium consists of yeast powder, peptone, carbon source, magnesium sulfate and antifoaming agent. Step S4: Nitrogen source feeding begins from the 20th to 36th hour of fermentation, and carbon source feeding begins from the 24th to 36th hour.

2. The method according to claim 1, characterized in that: In step S1, the expression host of the recombinant Bacillus subtilis is Bacillus subtilis WB600.

3. The method according to claim 1, characterized in that: In step S1, the nucleotide sequence of the recombinant linear maltotetrasaccharide generating enzyme is shown in SEQ ID NO.

1.

4. The method according to claim 1, characterized in that: In step S2, the inoculation amount of the seed culture solution is 3-5% (v / v).

5. The method according to claim 1, characterized in that: In step S2, the secondary seed culture medium consists of a nitrogen source and a carbon source.

6. The method according to claim 5, characterized in that: The secondary seed culture medium does not contain added phosphate.

7. The method according to claim 5, characterized in that: The carbon source is corn starch.

8. The method according to claim 1, characterized in that: In step S3, the inoculation amount of the secondary seed culture medium is 10-15% (v / v).

9. The method according to claim 1, characterized in that: In step S3, the carbon source is corn starch.

10. The method according to claim 1, characterized in that: In step S3, trace elements are also added to the fermentation medium, including iron, calcium, manganese, cobalt, sodium, molybdenum, zinc, aluminum, copper and boron.

11. The method according to claim 1, characterized in that: In step S3, no phosphate is added to the fermentation medium.

12. The method according to claim 1, characterized in that: In step S4, the nitrogen source includes yeast powder and peptone, and the carbon source is glucose.

13. The method according to claim 1, characterized in that: In step S4, the flow rate of the nitrogen source feed is 24-36 mL / h.

14. The method according to claim 1, characterized in that: In step S4, the flow rate of the carbon source feed is 18-22 mL / h.

15. The method according to claim 1, characterized in that: In step S4, a carbon source starvation phase is introduced during the 55th to 65th hour of fermentation, during which carbon source feeding is stopped.

16. The method according to claim 15, characterized in that: The carbon source starvation phase lasts for 4-6 hours.

17. The method according to claim 1, characterized in that: Step S4 involves feeding nitrogen and carbon sources starting at the 36th hour of fermentation. A carbon source starvation phase is introduced at the 60th hour of fermentation, during which carbon source feeding is stopped and lasts for 4 hours.

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