Method for increasing fermentation yield of tryptophan synthetase by regulating ORP (Oxidation Reduction Potential)

Abstract

The invention discloses a method for increasing the fermentation yield of tryptophan synthetase by regulating ORP, and belongs to the technical field of fermentation engineering. According to the method, through feedback of different gradient ORP fermentation OD and enzymatic yield, on the basis of ensuring that the fermentation condition reaches the standard, the optimal ORP in the fermentation process is determined, the tryptophan synthetase catalytic capacity of thalli of unit mass is improved, the problems that the total amount of thalli is high, the enzyme production capacity of the thalli of unit mass is low, the enzymatic yield is unstable and the like in a traditional high-density fermentation mode are solved, and the method is suitable for industrial production. The yield of tryptophan synthetase in the fermentation liquor (in terms of wet thalli) is within the range of 160-185 g / L, and the yield is increased to 88% or above.

Description

TECHNICAL FIELD

[0001] The present application relates to a method for increasing the fermentation yield of tryptophan synthetase by regulating ORP, and belongs to the technical field of fermentation engineering. BACKGROUND

[0002] Cystine is used as a raw material for producing detoxicants and expectorants in medicine; as a milk additive and a bread quickening agent in food industry, which can produce various special flavors after heating with sugar in food and prevent oil oxidation, and has a wide range of uses in food processing; and as a raw material for producing cold perm in cosmetic industry. Tryptophan synthetase is a key enzyme for synthesizing cystine, and increasing the fermentation yield of tryptophan synthetase is an effective way to increase the yield of cystine.

[0003] Oxidation-reduction potential (ORP) not only directly reflects the oxidation-reduction ability of the environment where microorganisms are located, but also indirectly represents the metabolic activity of microorganisms. And ORP has universality and sensitivity in various states similar to pH, temperature and other fermentation parameters, which can overcome the limitations of the application of dissolved oxygen electrodes in micro-aerobic and anaerobic fermentation, and can monitor the growth state of microorganisms in real time. Therefore, online monitoring and control of ORP can effectively improve the fermentation results. However, there are few methods for increasing product yield by controlling ORP in aerobic fermentation. With the growth of bacteria in the production fermentation process, ORP also changes. At present, the optimal oxidation-reduction potential in the fermentation process of producing tryptophan synthetase is not known, which limits the further improvement of tryptophan synthetase. SUMMARY

[0004] The present application provides a method for increasing the fermentation yield of tryptophan synthetase by dynamically regulating ORP, which is a method for controlling carbon source feeding parameters in stages; the stage is to add Yg glucose (Y=aX+b) per liter of fermentation broth per hour for feeding from the 4th hour of fermentation, until the temperature is reduced to 25-28℃ after the ORP reaches the minimum ORP range, the inducer is added and the feeding is constant, until the fermentation is completed. Wherein, Y represents the feeding amount of unit time unit volume of fermentation broth (g / (L·h)), and X represents the feeding time (h); wherein, a is 0.5-0.6, and b is 8-10.

[0005] In an embodiment of the present application, the feeding time is calculated from the start of feeding.

[0006] In an embodiment, the minimum ORP range includes -120 mV to -140 mV, -140 mV to -160 mV, -160 mV to -180 mV; preferably -140 mV to -160 mV.

[0007] In an embodiment, the feed is a carbon source; the carbon source includes but is not limited to glucose.

[0008] In an embodiment, the method is fermentation using E. coli, including but not limited to E. coli ALE8-8 (DE3).

[0009] In an embodiment, the inducer includes but is not limited to IPTG; the final concentration of the inducer is 0.1-1.0 mM, calculated based on the initial fermentation volume.

[0010] The method of the present application does not have a special limitation on the fermentation volume, and can be various fermentation volumes used in current conventional production or research The present application also provides a method for preparing tryptophan synthetase, which comprises culturing E. coli in a culture medium at 35-40℃, and regulating ORP to a specified range by feeding, specifically: (1) At the 4th hour of fermentation, feeding according to the relationship Y=0.55x+9; wherein Y represents the feeding amount of the fermentation broth per unit volume per unit time (g / (L·h)), and X represents the feeding time (h); (b) When the minimum ORP reaches the control range, the fermentation temperature is reduced to 25-28℃, an inducer is added, and feeding is performed at a constant rate of 10 g / (L·h).

[0011] In an embodiment, the feed is a carbon source; the carbon source includes but is not limited to glucose.

[0012] In an embodiment, the method comprises culturing E. coli in a culture medium at 37℃, and during the culturing, first feeding according to the relationship Y=0.55x+9; wherein Y represents the feeding amount of the fermentation broth per unit volume per unit time (g / (L·h)), and X represents the feeding time (h); when the minimum ORP reaches the required range, an inducer is added, and feeding is performed at a constant rate of 10 g / (L·h). In an embodiment, the inducer includes but is not limited to IPTG; the concentration of the inducer is 0.1 mM-1 mM.

[0013] In an embodiment, the induction is performed at 25-28℃.

[0014] The present application also provides the use of the method in the preparation of tryptophan synthetase.

[0015] Advantages: The present application determines the optimal ORP of the fermentation process by the feedback of different gradient ORP fermentation OD and enzymatic yield, improves the tryptophan synthetase synthesis efficiency and catalytic activity of unit microorganism on the basis of ensuring the fermentation condition, makes the enzymatic yield reach more than 88%, effectively improves the tryptophan synthetase yield in the fermentation liquor, and ensures the stable and preset requirement of the enzymatic reaction yield. DETAILED DESCRIPTION

[0016] Seed medium: yeast extract powder 5 g / L, peptone 10 g / L, sodium chloride 5 g / L. Sterilize at 121 DEG C for 20 min, and cool down for standby.

[0017] Fermentation medium: glucose 10 g / L, potassium dihydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.2 g / L, ammonium sulfate 1 g / L, yeast extract powder 2 g / L, ferrous sulfate heptahydrate 0.1 g / L, citric acid 1 g / L. Sterilize at 121 DEG C for 30 min, and cool down for standby. Supplemental medium: glucose aqueous solution with a concentration of 480 g / kg.

[0018] Method for determining the catalytic reaction capacity of tryptophan synthetase in unit volume of fermentation liquor: the catalytic reaction capacity of tryptophan synthetase in unit volume of fermentation liquor is calculated by the yield of catalyzing L-serine to cystine. The specific determination method is as follows: (1) Take 50 mL of fermentation liquor, centrifuge at 12000 rpm for 5 min to prepare wet microorganism for standby; (2) In a 250 mL baffled Erlenmeyer flask, weigh 100 g of 8% serine solution (8 g of serine pure product); (3) Weigh 1.5 times of the molar number of serine pure product in the solution; (4) Add all the wet microorganism obtained in step (1) to the solution; (5) Add 0.01 g of pyridoxal phosphate (add 0.01 g if the addition amount is less than 0.01 g) accounting for 5 parts per million of the mass of the wet microorganism; (6) React the reaction system at 37 DEG C, 220 rpm for 6 h; (7) During the reaction process, take sample every 2 h, determine the content of the substrate serine and the product cystine by HPLC, and calculate the yield. HPLC conditions: Agilent C18, column temperature 35 DEG C, detection wavelength 192 nm, mobile phase uses 5 mmol / L sodium dodecyl sulfate: acetonitrile = 80:20.

[0019] Yield calculation: yield (%) = (2 x molar amount of cystine after enzymatic reaction) ÷ molar amount of serine before enzymatic reaction x 100%.

[0020] Example 1 (1) Seed liquid preparation: The engineering bacteria ALE8-8 (DE3) (disclosed in the patent with publication number CN107406864B) were cultured in a seed culture medium at 37°C in a shaker at 220 rpm for 6.5 h to obtain a seed liquid.

[0021] (2) Fermentation culture: The seed liquid prepared in step (1) was inoculated into a fermentation culture medium at a volume ratio of 2%-5% for stirring and aeration fermentation, the culture temperature was 37°C, the pH was controlled at 6.8-7.2, the dissolved oxygen was controlled at 20%-30%, and the rotation speed was adjusted according to the dissolved oxygen range. The feeding started at the 4th hour of fermentation. Taking a fermentation volume of 2 L as an example, the feeding mode was as follows: ① Linear feeding stage: taking the time of starting feeding as the feeding 0 time, from the 0 time, the feeding equation was set in the mode of Y=aX+b, wherein a=0.55, indicating the slope of the feeding amount increase, b=9, indicating the rate at the start of feeding, Y represents the feeding speed per unit time per unit volume (g / (L·h)), and X represents the feeding time (h); ② Constant speed feeding stage: after the ORP of fermentation reached-120 mV to-140 mV at 8 h, the temperature was reduced to the induction temperature of 28°C, the inducer IPTG with a final concentration of 0.5 mM was added, the feeding amount was set to a constant speed of 10 g / (L·h), and the fermentation ended after the cumulative fermentation time reached 28 h.

[0022] Example 2 The seed liquid prepared in Example 1 was used for fermentation culture. The fermentation culture medium and culture conditions were the same as in Example 1, except that when the ORP reached-140 mV to-160 mV at about 10 h, the temperature was reduced to the induction temperature of 28°C, the inducer IPTG with a final concentration of 0.5 mM was added, the feeding amount was set to a constant speed of 10 g / (L·h), and the fermentation ended after the cumulative fermentation time reached 28 h.

[0023] Example 3 The seed liquid prepared in Example 1 was used for fermentation culture. The fermentation culture medium and culture conditions were the same as in Example 1, except that when the ORP reached-160 mV to-180 mV, the temperature was reduced to the induction temperature of 28°C, the inducer IPTG with a final concentration of 0.5 mM was added, the feeding amount was set to a constant speed of 10 g / (L·h), and the fermentation ended after the cumulative fermentation time reached 28 h. Comparative Example 1: The seed liquid prepared in Example 1 was used for fermentation culture, and the fermentation medium was the same as that in Example 1, except that in the feeding formula Y = aX + b, a = 1, b = 12, after the minimum ORP reached-140 mV~ -160 mV and the temperature was induced, the feeding amount was set to a constant speed of 15 g / (L·h). The fermentation time was the same, and the results are shown in Table 1. Although the biomass in the fermentation broth was obviously improved, the synthesis of tryptophan synthetase was affected due to the too fast growth of the bacteria, the tryptophan synthetase could not be correctly folded, and the catalytic ability of the fermentation broth could not reach the ideal level.

[0024] Table 1: OD and yield of examples and comparative examples

[0025] Note: Enzyme yield (g / L) represents the concentration of wet bacteria with tryptophan synthetase catalytic ability in the fermentation broth.

[0026] Although the present application has been disclosed with reference to the preferred embodiments as above, it is not intended to limit the present application, and any person skilled in the art can make various modifications and modifications without departing from the spirit and scope of the present application, therefore the protection scope of the present application should be defined by the claims.

Claims

1. A method for increasing the fermentation yield of tryptophan synthase, characterized in that, Using *Escherichia coli* producing tryptophan synthase as the fermentation strain, carbon source feeding parameters were controlled in stages. The stages involved feeding starting at the 4th hour of fermentation, with the feeding rate controlled according to Y=0.55x+9 until the current ORP reached the minimum ORP range. The temperature was then lowered to 25-28℃, an inducer was added, and feeding was continued at a constant rate until fermentation ended. Here, Y represents the amount of feed per unit volume per unit time, and X represents the feeding duration. The minimum ORP range included -120 mV to -140 mV, -140 mV to -160 mV, and -160 mV to -180 mV.

2. The method according to claim 1, characterized in that, The method involves first fermenting at 35-40℃ for 3 hours, then feeding at Y=0.55x+9 from the 4th to the 8th hour until the current ORP reaches the range of -120 mV to -140 mV. The temperature is then lowered to 25-28℃, an inducer is added, and feeding is continued at a constant rate of 10 g / (L·h) until fermentation is complete.

3. The method according to claim 1, characterized in that, The method involves first fermenting at 35-40℃ for 3 hours, then feeding at Y=0.55x+9 from the 4th to the 10th hour until the current ORP reaches the range of -140 mV to -160 mV. The temperature is then lowered to 25-28℃, an inducer is added, and feeding is continued at a constant rate of 10 g / (L·h) until fermentation is complete.

4. The method according to claim 1, characterized in that, The method involves first fermenting at 35-40℃ for 3 hours, then feeding at Y=0.55x+9 from the 4th to the 12th hour until the current ORP reaches the range of -160 mV to -180 mV. The temperature is then lowered to 25-28℃, an inducer is added, and feeding is continued at a constant rate of 10 g / (L·h) until fermentation is complete.

5. The method according to any one of claims 1 to 4, characterized in that, The supplementary material is the addition of a carbon source; the carbon source includes, but is not limited to, glucose.

6. The method according to any one of claims 1 to 5, characterized in that, The Escherichia coli that produces tryptophan synthase includes, but is not limited to, Escherichia coli ALE8-8(DE3).

7. A method for producing tryptophan synthase by fermentation, characterized in that, Fermentation was carried out using Escherichia coli as the fermenting microorganism under the following conditions: (1) Ferment at 35~40℃ for the first 3 hours of fermentation; (2) Feeding is carried out from the 4th to the 12th hour of fermentation. The feeding rate is based on Y=0.55X+9; where Y represents the amount of feed per unit volume of fermentation liquid per unit time, and X represents the feeding time. When the current ORP reaches the control range, the temperature is reduced to 25~28℃, an inducer is added, and feeding is carried out at a constant rate until the fermentation ends.

8. The method according to claim 7, characterized in that, Induction was performed at 25-28℃.

9. The method according to claim 7 or 8, characterized in that, The inducer includes, but is not limited to, IPTG; the concentration of the inducer is 0.1 mM to 1 mM.

10. The use of the method according to any one of claims 1 to 9 in the preparation of products containing tryptophan synthase.

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