A method for producing linear maltotetraose generating enzyme by fermentation of Bacillus subtilis

Abstract

The present invention relates to a method for producing linear maltotetraose-forming enzyme by Bacillus subtilis fermentation, belonging to the technical field of fermentation engineering. The steps include activating recombinant Bacillus subtilis to obtain a seed culture solution; inoculating the seed culture solution into a secondary seed culture medium to obtain a secondary seed culture solution; inoculating the secondary seed culture solution into a fermentation medium for fermentation culture. Nitrogen source feeding starts at the 20th to 36th hour of fermentation culture, carbon source feeding starts at the 24th to 36th hour, and a carbon source starvation stage is introduced at the 55th to 65th hour, during which the carbon source feeding is stopped. The recombinant Bacillus subtilis used in the present invention uses pP43nmk as a vector and selects signal peptide SP Bgls , and heterologously expresses a recombinant linear maltotetraose-forming enzyme with the starch-binding module truncated. In this fermentation system, the enzyme activity of the recombinant Bacillus subtilis in a 15 L fermenter reaches a maximum of 1393.51 U / mL, which is 450% of the enzyme activity in shake flask fermentation.

Description

Technical Field

[0001] The invention relates to the technical field of fermentation engineering, in particular to a method for producing linear maltotetraose generating enzyme by fermenting Bacillus subtilis. Background Art

[0002] Linear maltotetraose is one of the most promising maltooligosaccharides and a new functional sugar source. It not only has good food processing characteristics and unique physiological effects, but also plays a special role in a variety of biological processes. Therefore, it has broad application prospects in the fields of food, medicine, and chemical industry. Studies have shown that it may help maintain intestinal health, improve microbial balance, and even have antioxidant properties to protect the human body from oxidative stress. Further studies have also found that linear maltotetraose may be beneficial to the regulation of the immune system and is expected to play a role in the prevention and treatment of immune-related diseases. However, due to the low production efficiency and high price of linear maltotetraose, its functional value and industrial potential have not been fully explored, which seriously restricts its industrial production and application.

[0003] The production of linear maltotetraose mainly adopts enzymatic process, and linear maltotetraose generating enzyme (EC 3.2.1.60) is the key enzyme preparation in its production, which has been monopolized by foreign countries for a long time. As a member of GH13 family, linear maltotetraose generating enzyme has significant exonuclease characteristics, which can specifically act on the fourth α-1,4 glycosidic bond from the non-reducing end of starch, thereby efficiently generating maltooligosaccharides mainly composed of linear maltotetraose. The performance of this enzyme is one of the core factors that determine the industrial value of maltotetraose, which directly affects the yield and production cost of the target product. The protein expression of most linear maltotetraose generating enzymes reported at home and abroad is not ideal, which limits its industrial application. In addition to directly screening new enzymes, fermentation optimization with wild bacteria to improve enzyme activity, or expression in heterologous hosts and optimization of fermentation process are two common strategies to increase the expression of target proteins. Among them, the latter has the advantages of higher efficiency and greater universality.

[0004] The applicant has successfully achieved the linear maltotetraose synthase (MFA) from Pseudomonas saccharophila STB07. PS The enzyme was recombinantly expressed in Bacillus subtilis, and the enzyme had excellent stability and product specificity. On this basis, a mutant (MFA) with significantly improved substrate specificity and substrate conversion rate was constructed. PS-ΔCBM enzyme). However, the protein expression of this enzyme is still at a low level, which is far behind that of industrial enzymes, limiting its industrial application. The stability of the enzyme has a direct impact on production efficiency and product quality, which means that it is necessary to deeply understand the working mechanism of the enzyme in order to effectively improve and optimize it. Secondly, how to achieve large-scale and low-cost production of enzymes while ensuring enzyme activity and stability is a key issue to be solved. The production process of enzymes requires human costs and a large amount of material costs such as water, steam and energy. It is also a problem that needs to be solved at present to shorten the fermentation time as much as possible while ensuring enzyme activity and achieve green and environmentally friendly production of enzymes. Summary of the invention

[0005] In order to solve the above technical problems, the present invention provides a method for producing linear maltotetraose generating enzyme by fermentation of Bacillus subtilis, optimizes the fermentation conditions of recombinant Bacillus subtilis, limits the phosphate nutrition of the culture medium during the fermentation process, and introduces a carbon source starvation stage, which greatly improves the enzyme activity level of recombinant linear maltotetraose generating enzyme produced by recombinant Bacillus subtilis. The steps include activating the recombinant Bacillus subtilis to obtain a seed culture solution; inoculating the seed culture solution into a secondary seed culture medium to obtain a secondary seed culture solution; inoculating the secondary seed culture solution into a fermentation medium for fermentation culture, starting nitrogen source feeding at the 20th to 36th hour of the fermentation culture, starting carbon source feeding at the 24th to 36th hour, and introducing a carbon source starvation stage at the 55th to 65th hour, during which carbon source feeding is stopped. The recombinant Bacillus subtilis used in the present invention uses pP43nmk as a vector, and uses a signal peptide SP Bgls , heterologously expressing recombinant maltotetraose-producing enzyme with starch binding module truncated.

[0006] The first object of the present invention is to provide a method for producing linear maltotetraose generating enzyme by fermentation of Bacillus subtilis, comprising the following steps:

[0007] Step S1, activating recombinant Bacillus subtilis to obtain seed culture solution, wherein the recombinant Bacillus subtilis uses pP43nmk as a vector and uses a signal peptide SP Bgls , heterologously expressing a recombinant linear maltotetraose generating enzyme with the starch binding module truncated;

[0008] Step S2, inoculating the seed culture solution into the secondary seed culture medium to obtain the secondary seed culture solution;

[0009] Step S3, inoculating the secondary seed culture solution into the fermentation medium for cultivation;

[0010] Step S4: nitrogen source supplementation is started at 20-36 hours into the fermentation culture, and carbon source supplementation is started at 24-36 hours into the fermentation culture.

[0011] Furthermore, in step S1, the expression host of the recombinant Bacillus subtilis is Bacillus subtilis WB600.

[0012] Furthermore, in step S1, the nucleotide sequence of the recombinant linear maltotetraose producing enzyme is as shown in SEQ ID NO.1.

[0013] Furthermore, in step S2, the inoculation amount of the seed culture solution is 3-5% (v / v).

[0014] Furthermore, in step S2, the secondary seed culture medium consists of a nitrogen source and a carbon source.

[0015] Furthermore, no phosphate is added to the secondary seed culture medium.

[0016] Furthermore, the carbon source is corn starch.

[0017] Furthermore, in step S3, the inoculation amount of the secondary seed culture solution is 10-15% (v / v).

[0018] Furthermore, in step S3, the fermentation medium consists of yeast powder, peptone, a carbon source, magnesium sulfate and a defoaming agent.

[0019] Furthermore, no phosphate is added to the fermentation medium.

[0020] Furthermore, the carbon source is corn starch.

[0021] Furthermore, in step S3, trace elements are added to the fermentation medium, and the trace elements include iron, calcium, manganese, cobalt, sodium, molybdenum, zinc, aluminum, copper and boron.

[0022] Specifically, the fermentation medium is added with a trace element liquid, wherein the trace element liquid contains FeSO 4 , CaCl 2 、MnSO 4 、CoCl 2 、NaMoO 4 、ZnSO 4 、AlCl 3 , CuCl 2 and H 3 BO 4 .

[0023] Furthermore, in step S4, the nitrogen source includes yeast powder and peptone, and the carbon source is glucose.

[0024] Furthermore, in step S4, the flow rate of the nitrogen source feed is 24-36 mL / h.

[0025] Preferably, the flow rate of the nitrogen source is 30 mL / h.

[0026] Furthermore, in step S4, the flow rate of the carbon source feed is 18-22 mL / h.

[0027] Furthermore, in step S4, a carbon source starvation phase is introduced at 55-65 hours of fermentation culture, during which carbon source feeding is stopped.

[0028] Preferably, the carbon source starvation phase begins at the 60th hour of fermentation.

[0029] Furthermore, the carbon source starvation phase lasts for 4-6 hours.

[0030] Preferably, the glucose starvation phase lasts for 4 hours.

[0031] Beneficial effects of the present invention:

[0032] The present invention improves the expression of the target protein through a fermentation regulation strategy, realizes large-scale, low-cost production of the enzyme, and provides a strong guarantee for the industrial production of tetrasaccharides. In the fermentation system provided by the present invention, the enzyme activity of the recombinant Bacillus subtilis in a 15L fermenter reaches up to 1393.51U / mL, which is 450% of the shake flask fermentation level, and the overall fermentation time is shortened, the production efficiency is improved, and the raw material cost is reduced. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein

[0034] Figure 1 The growth and enzyme production rules of the recombinant Bacillus subtilis in a 15L fermenter in Example 1 of the present invention;

[0035] Figure 2 This is an SDS-PAGE image of the fermentation supernatant of the recombinant Bacillus subtilis in Example 1 of the present invention in a 15L fermenter, wherein lane M is the standard protein and lane 1 is the fermenter supernatant;

[0036] Figure 3 This is the growth curve of the recombinant Bacillus subtilis in Example 2 of the present invention at different nitrogen source feeding times;

[0037] Figure 4 The enzyme activity change curve of the recombinant Bacillus subtilis at different nitrogen source feeding times in Example 2 of the present invention;

[0038] Figure 5 is the growth curve of the recombinant Bacillus subtilis at different nitrogen source feed flow rates in Example 2 of the present invention;

[0039] Figure 6 is the change curve of enzyme activity of the recombinant Bacillus subtilis at different nitrogen source feed flow rates in Example 2 of the present invention;

[0040] Figure 7 It is the nitrogen source feeding strategy in Example 2 of the present invention;

[0041] Figure 8 is the growth and enzyme activity curve of the recombinant Bacillus subtilis under the optimized nitrogen source feeding strategy in Example 2 of the present invention;

[0042] Fig. 9 is the growth curve of the recombinant Bacillus subtilis in Example 3 of the present invention at different starvation times;

[0043] Fig.10 is the enzyme activity curve of the recombinant Bacillus subtilis at different starvation times in Example 3 of the present invention;

[0044] Fig.11 is the enzyme production curve of the recombinant Bacillus subtilis in Example 3 of the present invention at different starvation times (20h starvation);

[0045] Fig.12 is the enzyme production curve of the recombinant Bacillus subtilis in Example 3 of the present invention at different starvation times (60h starvation);

[0046] Fig.13 is the growth and enzyme production curve of the recombinant Bacillus subtilis under the glucose starvation strategy in Example 3 of the present invention;

[0047] Fig.14 The amount of phosphate added to MFA in Example 4 of the present invention PS -ΔCBM enzyme activity, where A is K 2 HPO 4 , B is KH 2 PO 4 ;

[0048] Fig.15 This is the growth and enzyme production curve of the recombinant Bacillus subtilis in Example 5 of the present invention in a 15L fermenter under different phosphate nutrition environments;

[0049] Fig.16 is the relationship between the enzyme production of the recombinant Bacillus subtilis in different phosphate nutrition environments and the carbon source in Example 6 of the present invention;

[0050] Fig.17 is an SDS-PAGE image of the fermentation supernatant of the recombinant Bacillus subtilis in Example 6 of the present invention in different phosphate nutrition environments and different carbon source types of culture media, and the lane M is the standard protein;

[0051] Fig.18 is the relationship between the enzyme production of the recombinant Bacillus subtilis in different phosphate nutrition environments and the corn starch concentration in Example 7 of the present invention;

[0052] Fig.19 is the relationship between the enzyme production of the recombinant Bacillus subtilis in Example 7 of the present invention under different phosphate nutrition environments and the glucose concentration;

[0053] Fig. 20 The effect of the timing of glucose addition on the growth and enzyme production of recombinant Bacillus subtilis under different phosphate nutrition environments in Example 8 of the present invention;

[0054] Fig.21 This is the growth and enzyme production curve of the recombinant Bacillus subtilis in Example 9 of the present invention under the pre-feed strategy in different phosphate nutritional environments;

[0055] Fig. 22 This is the growth and enzyme production curve of the recombinant Bacillus subtilis in Example 9 of the present invention under the feeding strategy when the dissolved oxygen rebounds (24h) in different phosphate nutrition environments;

[0056] Fig.23 This is the growth and enzyme production curve of the recombinant Bacillus subtilis in Example 9 of the present invention under the 36h feeding strategy in different phosphate nutrition environments;

[0057] Fig.24 is an SDS-PAGE image of the fermentation supernatant of the recombinant Bacillus subtilis in Example 9 of the present invention under the 36h feeding strategy in a phosphate nutrient-limited environment, wherein lane M is the standard protein and lane Y is the supernatant of the shake flask control group;

[0058] Fig.25 The growth and enzyme production of the recombinant Bacillus subtilis in Example 10 of the present invention were carried out in a 15 L fermentor under phosphate nutrient limitation conditions and glucose starvation. DETAILED DESCRIPTION

[0059] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0060] The recombinant Bacillus subtilis described in the example is disclosed in "Expression, modification and application of linear maltotetraose-producing enzyme from Pseudomonas saccharophila STB07, Duan Kaiwen, Jiangnan University", using Bacillus subtilis WB600 as the host, pP43nmk as the vector, and the signal peptide SP Bgls, heterologous expression of linear maltotetraose synthase MFA from Pseudomonas saccharophila STB07 PS , and truncated the starch binding module CBM to obtain the recombinant linear maltotetraose generating enzyme pP43nmk-SP Bgls- MFA PS -ΔCBM (hereinafter referred to as MFA PS -ΔCBM).

[0061] (I) Culture medium and reagent formula

[0062] LB liquid medium (g / L): 5 g of analytical grade yeast powder, 10 g of analytical grade tryptone, and 10 g of NaCl.

[0063] LB solid medium: Add 1.5-2% (w / v) agar powder to LB liquid medium.

[0064] Shake flask fermentation medium (g / L): 24g of analytical grade yeast powder, 12g of analytical grade tryptone, 10g of corn starch, 10g of KH 2 PO 4 0-34mmol / L, K 2 HPO 4 ·3H 2 O 0-110mmol / L (total phosphate concentration range 0-144mmol / L), kanamycin was added to a final concentration of 20μg / mL before inoculation.

[0065] Secondary culture medium (g / L): industrial yeast powder 24, industrial soy peptone 6, corn starch 5, glucose 4, K 2 HPO 4 ·3H 2 O 16.43, KH 2 PO 4 2.32, add kanamycin to a final concentration of 20 μg / mL before inoculation.

[0066] Phosphate-limited secondary seed culture medium (g / L): 24 g of industrial yeast powder, 6 g of industrial soy peptone, 10 g of corn starch, and kanamycin at a final concentration of 20 μg / mL before inoculation.

[0067] Fermentation medium (g / L): industrial yeast powder 24, industrial soy peptone 6, corn starch 5, glucose 4, K 2 HPO 4 ·3H 2 O 16.43, KH 2 PO 4 2.32,MgSO 40.06, trace element solution 10mL / L, add appropriate amount of defoaming agent, and add kanamycin with a final concentration of 20μg / mL before inoculation.

[0068] Phosphate-limited fermentation medium (g / L): industrial yeast powder 24, industrial soy peptone 6, corn starch 10, MgSO 4 0.06, trace element solution 10mL / L, add appropriate amount of defoaming agent, and add kanamycin with a final concentration of 20μg / mL before inoculation.

[0069] Trace element solution (g / L): FeSO 4 7H 2 O 4, CaCl 2 4. MnSO 4 ·5H 2 O 1, CoCl 2 6H2O0.4,NaMoO 4 ·2H 2 O 0.2, ZnSO 4 7H 2 O 0.2, AlCl 3 6H 2 O 0.1, CuCl 2 ·H 2 O0.1,H 3 BO 4 0.05, dispense into 50mL centrifuge tubes and sterilize, then add before inoculation.

[0070] Carbon source feed (g / L): 550 g / L glucose monohydrate.

[0071] Nitrogen source feed (g / L): industrial grade yeast powder 240, industrial grade soy peptone 60.

[0072] (II) Preparation of DNS solution and drawing of standard curve

[0073] Preparation of DNS solution (1L): Weigh 182g of sodium potassium tartrate, add 500mL of distilled water, heat in a water bath, add 6.3g of DNS (3,5-dinitrosalicylic acid) while hot, then quickly add 21g of NaOH (weighed in a beaker and dissolved in a small amount of water in advance), add 5g of phenol at the same time, then add 5g of Na 2 SO 3 After stirring to dissolve, cool with ice water and make up to 1L. Store in a brown bottle away from light. Open after 7 days. The shelf life is 45 days.

[0074] 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 -80°C refrigerator.

[0075] Steps for drawing the glucose standard curve: measure 0mL, 0.05mL, 0.1mL, 0.15mL, 0.2mL, 0.25mL, 0.3mL, 0.4mL, and 0.5mL of glucose standard solution respectively, add distilled water to make up to 1mL, then add 1.0mL of DNS to mix, boil in boiling water for 5min, immediately cool in ice water, then add 2mL of distilled water, vortex and mix, and measure the absorbance value at a wavelength of 540nm. Use glucose mass (mg) as the horizontal axis and absorbance value as the vertical axis to draw the curve using a scatter plot.

[0076] (III) Definition and determination method of enzyme activity

[0077] Definition of enzyme activity: The amount of enzyme required to generate 1 μmol of reducing sugar (calculated as glucose) per minute is 1 unit of enzyme activity (U).

[0078] The hydrolytic activity of the enzyme was characterized by measuring the change in reducing sugar content using the DNS method. A 1% (w / v) completely gelatinized soluble starch solution was prepared in deionized water or PBS buffer (pH=7.0) as the substrate, 100 μL of the enzyme solution was diluted a certain multiple and added to 900 μL of the substrate, reacted at 50°C for 15 minutes, and then 1 mL of DNS solution was added to terminate the reaction. The solution was heated in a boiling water bath for 5 minutes to develop color, immediately placed in an ice water bath to cool to room temperature, and then 2 mL of deionized water was added. The solution was vortexed and mixed, and the absorbance value was measured at 540 nm.

[0079] (IV) Determination of bacterial concentration

[0080] The bacterial concentration was measured by spectrophotometer at 600 nm. 600 express.

[0081] Example 1: Growth and enzyme production of recombinant Bacillus subtilis in a 15L fermenter

[0082] Seed culture: Dip a ring of recombinant Bacillus subtilis from a glycerol tube stored at -80°C, streak it on an LB solid medium plate containing kanamycin resistance, culture at 37°C for 12 h, pick a single colony to 50 mL of LB liquid medium (containing 20 μg / mL kanamycin), culture at 37°C, 200 r / min, for 6 h to obtain the seed culture solution.

[0083] Secondary seed culture: wait until the seed culture solution grows to OD 600 The concentration of 0.6-1.2 is inoculated into the secondary seed culture medium at an inoculum amount of 3-5% (v / v), and cultured in a water bath shaker at 37°C and 200r / min for 5 hours to obtain the secondary seed culture solution.

[0084] Fermentation culture in 15L fermenter: The fermentation medium volume is 10L. After sterilization, wait for the temperature to drop to 30°C and the initial pH to 7.0. After lighting the fire ring, add 10mL / L of trace element solution, kanamycin with a final concentration of 20μg / mL, and MgSO with a final concentration of 0.06g / L to the fermenter. 4 and 4g / L glucose, and then add 10% (v / v) secondary seed culture solution, and calibrate the dissolved oxygen to 100% after inoculation. During the fermentation process, the fermentation temperature was maintained at 30°C, and the pH was maintained at 7.0 with 40% phosphoric acid and 29% ammonia water. The dissolved oxygen was maintained at about 30% by adjusting the stirring and air intake. The culture was terminated when the activity of the linear maltotetraose production enzyme decreased. The growth and enzyme production rules of recombinant Bacillus subtilis without feeding are as follows Figure 1 The protein electrophoresis diagram is shown in Figure 2 As shown, lane M is the standard protein and lane 1 is the supernatant after the fermentation tank culture is completed.

[0085] Depend on Figure 1 It can be seen that 0-4h is the stagnant period, 4-60h the bacteria are in a vigorous logarithmic growth period, and enter the stable period around 60h. During the logarithmic growth period of the bacteria, the enzyme production of the bacteria showed a consistent trend with the growth of the bacteria. When the growth of the bacteria entered the stable period, that is, around 60h of fermentation, the enzyme production tended to peak. Under the condition of no feed, when the recombinant bacteria was fermented in a 15L fermenter for 60h, MFA PS -ΔCBM had a maximum enzyme activity of 493.04 U / mL, which was 1.59 times the highest level of shake flask optimization. 600 Reached 19.33.

[0086] Example 2: Determination of nitrogen source feeding strategy

[0087] (1) Effect of nitrogen source supplementation timing on the growth and enzyme production of recombinant Bacillus subtilis

[0088] Fermentation was carried out in a 15L fermenter according to the method of Example 1. Nitrogen source was supplemented at a flow rate of 30 mL / h at the early logarithmic growth stage (4th hour), the middle logarithmic growth stage (20th hour) and the late logarithmic growth stage (60th hour) of bacterial growth, and fermentation was stopped at 72 hours. The growth and enzyme production curves of the recombinant bacteria at different feeding times are shown in Figure 2. Figure 3 and Figure 4 shown.

[0089] The best effect was achieved when nitrogen source was added in the middle of the logarithmic growth period (20h), and the enzyme activity reached 602.48U / mL after 60h of fermentation. It was not conducive to improve the enzyme activity when nitrogen source was added in the early (4h) and late (60h) logarithmic growth period.

[0090] (2) Effect of nitrogen source feed flow rate on the growth and enzyme production of recombinant Bacillus subtilis

[0091] The 15L fermentation tank was cultured according to the method of Example 1. Nitrogen source was supplemented at 24 mL / h, 30 mL / h, and 36 mL / h in the middle of the logarithmic growth period of the bacteria (20 h), and the fermentation was stopped at 72 h. The growth and enzyme production curves of the recombinant bacteria at different nitrogen source feed flow rates are shown in Figure 1. Figure 5 and Figure 6 shown.

[0092] From the bacterial growth curve ( Figure 5 ) shows that in the middle of the logarithmic growth period of the bacteria (20-40h), there is no obvious difference in the growth of the bacteria under the three nitrogen source feed flow rates. After 40h, the higher the feed flow rate, the faster the bacteria grow. Figure 6 ) It can be observed that the enzyme activity levels of the experimental groups fed at 24 mL / h and 30 mL / h in the first 40 h were similar, and the differences between the different feeding flow rates were quite obvious after 40 h. Finally, the enzyme activity levels of the experimental groups fed at nitrogen source flow rates of 30 mL / h and 36 mL / h were slightly different.

[0093] (3) Development of nitrogen source supplementation strategy and its effect on the growth and enzyme production of recombinant Bacillus subtilis

[0094] The 15 L fermentation tank was cultured according to the method of Example 1. In order to save costs, the following was formulated based on the above-mentioned optimization of nitrogen source feed flow rate: Figure 7 The nitrogen source feeding strategy shown in the figure is to supplement nitrogen source at 24 mL / h in the middle of logarithmic growth (20-40 h), and supplement nitrogen source at 30 mL / h in the middle and late of logarithmic growth (after 40 h). The culture is terminated when the activity of linear maltotetraose production enzyme decreases. The growth and enzyme production curves of the recombinant bacteria under this nitrogen source feeding strategy are shown in Figure 2. Figure 8 shown.

[0095] Under this feeding strategy, the enzyme activity of recombinant Bacillus subtilis reached a maximum level of 679.05U / mL at 62h of fermentation, which was 119% higher than that of the starting strain. This feeding strategy achieved a higher enzyme activity level with a lower feed amount, and the flow rate switching was easy to control, which was conducive to industrial production.

[0096] Example 3: Determination of glucose starvation strategy

[0097] (1) Effects of glucose starvation timing on the growth and enzyme production of recombinant Bacillus subtilis

[0098] The 15L fermentation tank was cultured according to the method of Example 1. Nitrogen source was supplemented at 24 mL / h in the middle of logarithmic growth (20-40 h), and at 30 mL / h in the middle and late of logarithmic growth (after 40 h). A 4-h glucose starvation phase was introduced at the 20th, 40th and 60th h of logarithmic growth of the bacteria, and the culture was terminated when the activity of the linear maltotetraose production enzyme decreased. The growth and enzyme production curves of the recombinant bacteria at different starvation times are shown in Figure 1. Fig. 9 , 10 shown.

[0099] Depend on Fig.10 It can be seen that the introduction of starvation stages at the 20th and 60th hours was beneficial to the improvement of enzyme activity levels, reaching 735.05U / mL and 759.29U / mL at the 60th and 64th hours of fermentation, respectively, which were 137% and 145% higher than the enzyme activity of the starting strain.

[0100] (2) Effects of glucose starvation duration on the growth and enzyme production of recombinant Bacillus subtilis

[0101] The 15L fermentation tank was cultured according to the method of Example 1. Nitrogen source was supplemented at 24 mL / h in the middle of logarithmic growth (20-40 h), and at 30 mL / h in the middle and late of logarithmic growth (after 40 h). Glucose starvation phases of 2, 4, and 6 h were introduced at the 20th hour of fermentation, and the culture was terminated when the activity of linear maltotetraose production enzyme decreased. The enzyme production of recombinant Bacillus subtilis under different starvation periods is shown in Figure 1. Fig.11 At 60h, glucose starvation stages of 2, 4, and 6h were introduced, and the culture was terminated when the activity of the linear maltotetraose-producing enzyme decreased. The growth and enzyme production curves of the recombinant Bacillus subtilis at different starvation times are shown in Figure 2. Fig.12 shown.

[0102] The introduction of a 2-hour starvation phase at the 20th hour of fermentation was most conducive to the improvement of enzyme activity, and the enzyme activity reached a maximum of 725.05 U / mL at the 62nd hour of fermentation, which was 134% higher than that of the starting strain. The introduction of a 4-hour starvation phase at the 60th hour of fermentation was most conducive to the improvement of enzyme activity, and the enzyme activity reached 762.05 U / mL at the 64th hour of fermentation, which was 146% higher than that of the starting strain.

[0103] (3) Development of glucose starvation strategy and its effect on the growth and enzyme production of recombinant Bacillus subtilis

[0104] The 15L fermentation tank was cultured according to the method of Example 1. Nitrogen source was supplemented at 24 mL / h in the middle of logarithmic growth (20-40 h), and at 30 mL / h in the middle and late of logarithmic growth (after 40 h). At the same time, a glucose starvation phase of 2 h and 4 h was introduced at 20 h and 60 h, respectively. The culture was terminated when the activity of linear maltotetraose production enzyme decreased. The growth and enzyme production curves of the recombinant bacteria under the fermentation conditions are shown in Figure 2. Fig.13 shown.

[0105] Under the fermentation conditions, the enzyme activity of the recombinant bacteria reached a maximum level of 850.05U / mL when the fermentation lasted for 64 hours, which was 174% higher than that of the starting strain. This fermentation process can not only significantly improve the enzyme activity, but also shorten the fermentation cycle, realize the comprehensive and efficient utilization of nutrients in the fermentation system, solve the problems of low enzyme activity and high production cost in the existing fermentation process, and is suitable for large-scale industrial production.

[0106] Example 4: Effect of Phosphate Addition on MFA PS -ΔCBM enzyme activity

[0107] To further improve MFA based on the above embodiments PS -ΔCBM enzyme activity, to explore the relationship between the amount of phosphate added in the culture medium and enzyme activity.

[0108] Seed activation: Dip a loop of recombinant Bacillus subtilis from a glycerol tube stored at -80°C, streak it on a plate containing kanamycin resistance, culture at 37°C for 12 h, pick a single colony and add it to 50 mL of LB medium (containing 20 μg / mL kanamycin), culture at 37°C, 200 r / min, for 6 h to obtain seed solution.

[0109] Shake flask fermentation: Add kanamycin at a final concentration of 20 μg / mL to a 250 mL conical flask containing 50 mL of shake flask fermentation medium, then transfer 2 mL of activated seed solution to the flask and culture in a shaker at 30°C and 200 rpm / min for 72 hours. After stopping the fermentation, centrifuge the fermentation liquid at 4°C and 10,000 rpm / min for 20 minutes, and collect the supernatant to obtain the recombinant MFA. PS -ΔCBM crude enzyme solution.

[0110] Phosphate addition in culture medium and MFA PS -ΔCBM enzyme activity Fig.14 shown.

[0111] The experimental results showed that the recombinant Bacillus subtilis produced MFA PSThe enzyme activity of -ΔCBM enzyme decreased with the increase of phosphate addition, and the enzyme activity was the highest when no phosphate was added, indicating that phosphate nutrient limitation would promote the metabolism of recombinant bacteria and the secretory expression of proteins. The possible reason for this is that Bacillus subtilis belongs to soil bacteria and often faces nutrient depletion. When Bacillus subtilis enters the steady state from exponential growth, the previously silent genes are activated and provide the products necessary for survival under stress conditions.

[0112] Example 5: Scale-up culture of recombinant bacteria in a 15L fermenter under different phosphorus nutrition environments and phenomenon verification

[0113] Based on Example 4, amplification culture and phenomenon verification were performed in a 15L fermenter. The steps of fermenting and culturing recombinant Bacillus subtilis in a 15L fermenter are as follows:

[0114] Seed culture: Dip a ring of recombinant Bacillus subtilis from a glycerol tube stored at -80°C, streak it on a plate containing kanamycin resistance, culture at 37°C for 12 h, pick a single colony and add it to 50 mL of LB medium (containing 20 μg / mL kanamycin), culture at 37°C, 200 r / min, for 6 h to obtain seed culture solution.

[0115] Secondary seed culture: wait until the seed culture solution grows to OD 600 The inoculum concentration was 0.6-1.2, and the inoculum concentration was 3-5% (v / v) into the phosphate-limited secondary seed culture medium, and the culture was carried out in a water bath shaker at 37° C. and 200 r / min for 5 hours to obtain the secondary seed culture solution.

[0116] Fermentation culture in 15L fermenter: The volume of phosphate-limited fermentation medium is 10L. After sterilization, wait until the temperature drops to 30°C and the initial pH reaches 7.0. After lighting the fire ring, add 10mL / L of trace element solution, kanamycin with a final concentration of 20μg / mL, and MgSO with a final concentration of 0.06g / L to the fermenter. 4 , then add 10% (v / v) of the secondary seed culture solution, and calibrate the dissolved oxygen to 100% after inoculation. During the fermentation process, the fermentation temperature was maintained at 30°C, and the pH was maintained at 7.0 with 3% sulfuric acid and 10% ammonia water. The dissolved oxygen was maintained at around 30% by adjusting the stirring and air intake. The above-mentioned secondary seed culture and fermentation tank culture were both controlled by the phosphate-containing control group and the phosphate-free group as the experimental group. The growth and enzyme production of recombinant Bacillus subtilis in a 15L fermentation tank under different phosphorus nutrition environments are shown in Figure 2. Fig.15 shown.

[0117] Experimental results show that, under phosphate nutrient restriction, when recombinant Bacillus subtilis was fermented to 52h in a 15L fermentor, the enzyme activity reached a maximum of 729.15U / mL, which was 47.88% higher than that of the control group (normal addition of phosphate, fermentation for 64h, enzyme activity up to 493.04U / mL, see Example 1), and the fermentation time was shortened by 12h. This shows that the phenomenon that phosphate nutrient restriction promotes enzyme activity improvement still exists in a larger fermentation system. And the recombinant bacteria enters the stationary phase faster under phosphate nutrient restriction, so that the fermentation time is shortened, which shows that phosphate nutrient restriction can significantly affect the growth metabolism and protein secretion of recombinant Bacillus subtilis, and thus phosphate nutrient restriction can promote the improvement of heterologous expression enzyme activity.

[0118] Example 6: Relationship between enzyme production and carbon source type of recombinant bacteria under phosphate nutrient limitation conditions

[0119] Since phosphate is often closely related to the transportation and utilization of carbon sources, the relationship between phosphate nutritional limitation and carbon source types was investigated. That is, under different phosphate nutritional environments, recombinant Bacillus subtilis was cultured with 5 g / L glycerol, corn starch, glucose, and a mixture of glucose and corn starch as the carbon source in the fermentation system, and the culture steps were as described in Example 4. The relationship between the enzyme production of recombinant Bacillus subtilis under phosphate nutritional limitation and the type of carbon source and SDS-PAGE electrophoresis analysis are shown in Figure 4. Fig.16 , 17 shown.

[0120] Fig.16 It can be seen that the enzyme activity, pH and OD values ​​of the corn starch experimental group under phosphate nutrient restriction conditions were higher than those of the control group. Protein electrophoresis also showed that ( Fig.17 ), all bands under phosphate limitation conditions were thicker than those in the control group, further indicating that phosphate limitation can promote the growth and protein secretion of recombinant Bacillus subtilis.

[0121] Example 7: Relationship between enzyme production and carbon source concentration of recombinant bacteria under phosphate nutrient limitation conditions

[0122] In order to facilitate the scale-up culture and feed optimization in the fermentation tank system, the relationship between phosphate nutrient limitation and the concentration of the main carbon sources corn starch and glucose was further investigated on the basis of Example 6. The experimental results are as follows: Fig.18 , Fig.19 shown.

[0123] The results showed that the promoting effect of phosphate nutrient limitation on enzyme activity increased with the increase of corn starch concentration. When the corn starch concentration was 10g / L, the improvement of enzyme activity by phosphate nutrient limitation was obvious. In contrast, the improvement of enzyme activity by phosphate nutrient limitation gradually decreased with the increase of glucose concentration. When the glucose concentration was higher than 10g / L, phosphate nutrient limitation had no promoting effect on enzyme activity, and even when it exceeded the critical concentration, phosphate nutrient limitation had a negative impact on enzyme activity. It is speculated that when the glucose concentration is too high, sufficient phosphorus is needed to promote the autophosphorylation and glycolysis of pyruvate, realize the absorption and utilization of nutrients, and maintain normal growth metabolism.

[0124] Example 8: Preliminary exploration of horizontal feeding strategy in shake flasks

[0125] The above-mentioned Example 2 has proved that the appropriate nitrogen source supplementation strategy in the 15L fermentation system can improve the enzyme production capacity of recombinant Bacillus subtilis. Example 6 further proves that under the phosphate nutrient restriction environment, glucose can not only promote the rapid growth of recombinant bacteria, but also promote the increase of enzyme activity level. Since the use of corn starch for supplementation during the fermentation production process requires that the corn starch be kept in a gelatinized state, glucose is selected as the supplementary carbon source for industrial application considerations. According to the experimental results of Example 7, the concentration of carbon source corn starch in the fermentation medium is 10g / L.

[0126] Based on the above experimental results, two carbon source feeding strategies were proposed: (1) pre-feeding strategy, that is, glucose is added to the fermentation system at a low rate (20 mL / h) from the beginning of the fermentation process until the end of the fermentation process; (2) post-feeding strategy, that is, glucose is added to the fermentation system at a low rate (20 mL / h) from the middle and late stages of fermentation when the bacteria reach the stable period until the end of the fermentation process. First, these two feeding strategies were explored at the shake flask level. The experimental results are as follows. Fig. 20 shown.

[0127] Depend on Fig. 20 It can be seen that adding glucose to the initial culture medium is beneficial to the improvement of enzyme activity levels; under the condition of phosphate nutrient limitation, the post-feeding method has a more significant effect on improving enzyme activity.

[0128] Example 9: Optimization of feeding conditions of recombinant bacteria in a 15L fermenter under different phosphorus nutrition environments

[0129] On the basis of Example 8, the effects of feeding method and feeding time on the growth and enzyme production of the recombinant bacteria in a 15 L fermenter were further investigated.

[0130] According to the experimental results of the previous study, the initial culture medium used 10g / L corn starch as the carbon source; 24g / L yeast powder and 6g / L soy peptone as the nitrogen source; phosphate was added to the culture medium of the control group, and no phosphate was added to the experimental group; the liquid volume was 10L. The seed liquid inoculation amount was 10%, the dissolved oxygen was corrected to 100% under the fermentation environment, the pH was set constant to 7.0, the initial fermentation temperature was set to 30℃, the initial stirring speed was 200r / min, and the initial ventilation volume was 1.5VVM. After that, the dissolved oxygen was maintained at 30% as much as possible by controlling the stirring speed and ventilation volume, and the tank pressure was controlled at 0.03-0.05MPa during the fermentation process.

[0131] Three different feeding methods were designed, namely (1) pre-feeding strategy, supplementing carbon source and nitrogen source at the same time after inoculation; (2) starting to supplement carbon source and nitrogen source at the same time when dissolved oxygen rebounds (around 24 hours); (3) starting to supplement carbon source and nitrogen source at the same time when the OD value of the recombinant bacteria reaches stability (around 36 hours). The flow rate of carbon source feeding is 20mL / h, and the flow rate of nitrogen source feeding is 30mL / h.

[0132] (1) The results of pre-feed fermentation are as follows Fig.21 As shown in the figure, when glucose was used as the initial carbon source of the fermentation medium, the OD values ​​of the recombinant bacteria in the experimental group and the control group increased rapidly in the 15L fermentor and soon reached stability. The enzyme activity of the experimental group also continued to increase under phosphate nutrient limitation conditions. However, when glucose was used as the main carbon source in the fermentor, the logarithmic growth period of the recombinant bacteria was greatly shortened. Therefore, when the recombinant bacteria was fermented in the 15L fermentor for 52 hours, the enzyme activity reached a maximum of only 724.33U / mL. Therefore, the pre-feeding strategy cannot guarantee the complete utilization of nutrients in the original fermentation system, and the long feeding time greatly increases the production cost, which is not conducive to the industrial production of linear maltotetraose producing enzyme.

[0133] (2) The results of fed-batch fermentation when dissolved oxygen rebounds are as follows Fig. 22As shown in the figure, when corn starch is used as the initial carbon source of the fermentation medium, the OD values ​​of the recombinant bacteria in the experimental group and the control group tend to be stable when the fermentation is carried out in a 15L fermenter for about 24 hours, and the dissolved oxygen in the tank is also at a high level. According to literature reports, when the dissolved oxygen in the fermenter rebounds, it means that the nutrients in the system are exhausted and feeding should be carried out at this time. Therefore, it is chosen to supplement the carbon source and nitrogen source at the same time starting from the 24th hour. However, it can be observed from the figure that the OD value of the experimental group under phosphate nutrient limitation conditions is always stable or slightly fluctuates from the 24th hour to the 36th hour after the start of feeding, and the residual sugar content in the tank also shows that the recombinant bacteria consume little or no glucose within 12 hours after feeding, resulting in the accumulation of residual sugar. The control group containing phosphorus only had a short stagnation period after the start of feeding, and it began to enter the logarithmic growth period again after the 33rd hour. At the same time, it can be observed that the residual sugar level in the tank began to decline at this time, indicating that the main carbon source in the tank of the control group was glucose.

[0134] When the experimental group fermented to 36 hours, that is, the 12th hour of feeding, the OD value began to rise rapidly and was accompanied by a rapid increase in enzyme activity. After the control group resumed normal logarithmic growth, only the OD value rose rapidly, but the enzyme activity level increased slowly. This is similar to the experimental results obtained at the shake flask level in the early stage: when the culture medium contains phosphorus, glucose can only promote the growth of recombinant bacteria but not its enzyme production; when the recombinant bacteria are in a phosphorus-limited environment, glucose can not only promote the growth and reproduction of the recombinant bacteria but also promote the improvement of enzyme activity. Therefore, the experimental group showed an experimental phenomenon in which the OD value and enzyme activity increased rapidly at the same time.

[0135] The experimental group reached a maximum of 824.51U / mL in the 63rd hour of fermentation, and the OD value reached 36.85 at this time, and the enzyme activity of the control group was only 342.71U / mL when the fermentation was 72h, and the OD value was 37.23 at this time. However, from the perspective of the overall fermentation process, a longer buffer period is required when the carbon source in the fermentation system is switched from corn starch to glucose, indicating that corn starch is not completely consumed in the system at this time, so the competition process time is longer. Contrast the control group (embodiment 5, phosphate nutrition limitation, no feed supplement) when not supplementing feed, it can be found that the OD value begins to reach stability and gradually declines when the fermentation is the 36th hour, and it should be the opportunity that corn starch is completely consumed at this time, if glucose is supplemented at this time, the competition buffer time may be shortened, and the further improvement of the full utilization of corn starch and the enzyme activity level is realized. Therefore, starting to supplement feed when dissolved oxygen rebounds is not applicable to this research object, and supplementing feed when OD value reaches stability, applicable to this research object, this strategy can ensure the efficient and complete utilization of carbon source in the fermentation system.

[0136] Compared with the strategy of starting to feed when the OD value reaches stability (around 36h), the enzyme activity increased by 47.97%, the feeding time was shortened by 15h, and the fermentation time was shortened by 3h. This shows that appropriately delaying the feeding time can not only promote the complete utilization of corn starch, and then promote the efficient improvement of enzyme activity level, but also reduce the consumption of feed culture medium, reduce production costs, and achieve the effect of improving quality and reducing costs.

[0137] (3) When the OD value of the recombinant bacteria reached a stable state (36 h), the results of the fed-batch fermentation were as follows: Fig.23 , Fig.24 As shown, when the experimental group of recombinant bacteria was fermented in a 15L fermenter for 60 hours, the enzyme activity reached a maximum of 1220.04U / mL, which was 176% higher than that of the control group (normal addition of phosphate, fermentation for 72 hours, enzyme activity up to 442.18U / mL), and the fermentation time was shortened by 12 hours. This shows that starting to feed when the OD value reaches a stable state can not only promote the complete utilization of corn starch, and then promote the efficient improvement of enzyme activity level, but also reduce the consumption of feed culture medium, reduce production costs, and achieve the effect of improving quality and reducing costs.

[0138] Example 10: Effects of glucose starvation strategy on growth and enzyme production of recombinant bacteria under different phosphorus nutrition environments

[0139] On the basis of Example 9, the effect of glucose starvation strategy on the growth and enzyme production of recombinant bacteria in a 15L fermenter was further investigated. According to the experimental results of previous studies, the initial culture medium used 10g / L corn starch as the carbon source; 24g / L yeast powder and 6g / L soy peptone as the nitrogen source; phosphate was added to the culture medium of the control group, and no phosphate was added to the experimental group; the liquid volume was 10L. The seed liquid inoculation amount was 10%, the dissolved oxygen was corrected to 100% under the fermentation environment, the pH was set constant to 7.0, the initial fermentation temperature was set to 30°C, the initial stirring speed was 200r / min, and the initial ventilation volume was 1.5VVM. Thereafter, the stirring speed and ventilation volume were controlled to maintain the dissolved oxygen at 30% as much as possible, and the tank pressure was controlled at 0.03-0.05MPa during the fermentation process. When the OD value of the recombinant bacteria reached stability (around the 36th hour), the carbon source and nitrogen source were supplemented simultaneously, and a 4h glucose starvation stage was introduced in the late fermentation period (around the 60th hour). The experimental results are as follows Fig.25 As shown, the flow rate of nitrogen source feed was 30 mL / h.

[0140] When the recombinant bacteria in the experimental group were fermented in a 15L fermenter for 64 hours, the enzyme activity reached a maximum of 1393.51U / mL, which was 186.06% higher than that of the control group (normal addition of phosphate, fermentation for 69 hours, enzyme activity up to 487.13U / mL), and the fermentation time was shortened by 5 hours. It is shown 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 the feeding time, and reduce the consumption of feeding medium, so as to achieve the effect of improving quality and reducing costs.

[0141] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A method for producing linear maltotetraose generating enzyme by fermentation of Bacillus subtilis, characterized in that: The following steps are involved: Step S1, activating recombinant Bacillus subtilis to obtain seed culture solution, wherein the recombinant Bacillus subtilis uses pP43nmk as a vector and uses a signal peptide SP Bgls , heterologously expressing a recombinant linear maltotetraose generating enzyme with the starch binding module truncated; Step S2, inoculating the seed culture solution into the secondary seed culture medium to obtain the secondary seed culture solution; Step S3, inoculating the secondary seed culture solution into a fermentation medium for fermentation culture, wherein the fermentation medium consists of yeast powder, peptone, a carbon source, magnesium sulfate and a defoaming agent; Step S4, starting nitrogen source and carbon source feeding at the 36th hour of fermentation culture, introducing a carbon source starvation stage at the 60th hour of fermentation culture, during which carbon source feeding is stopped and the carbon source starvation stage lasts for 4 hours.

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 maltotetraose generating enzyme is shown as 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 carbon source is corn starch.

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

8. The method according to claim 1, characterized in that: The carbon source is corn starch.

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

10. 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.

11. 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.

12. 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.

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