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CN115161358A - Fermentation process for improving 5-HTP yield - Google Patents

Fermentation process for improving 5-HTP yield Download PDF

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CN115161358A
CN115161358A CN202210949191.0A CN202210949191A CN115161358A CN 115161358 A CN115161358 A CN 115161358A CN 202210949191 A CN202210949191 A CN 202210949191A CN 115161358 A CN115161358 A CN 115161358A
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glucose
fermentation
glycerol
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张山
李志�
肖运柱
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Shenzhen Upfo Biotech Co ltd
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
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    • C12P13/22—Tryptophan; Tyrosine; Phenylalanine; 3,4-Dihydroxyphenylalanine
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Abstract

The invention discloses a fermentation process for improving the yield of 5-HTP, and belongs to the field of production of amino acid derivatives. According to the fermentation process of the 5-HTP yield, the inoculum size is increased, the activity of the strain at the early stage of fermentation is enhanced, the fermentation lag phase is reduced, and the strain is accelerated to enter the logarithmic phase, so that the synthesis of the strain to the product is facilitated; on the other hand, the compound substrate is supplemented, the synthesis of cofactors (NADH and the like) in the product synthesis process is increased, the product yield can be effectively improved, and meanwhile, the reduction of byproducts also lightens the difficulty and the cost of the subsequent purification process to a certain extent, so that the yield of the 5-hydroxytryptophan reaches 4.78g/L. The benefits are improved, and the cost is reduced.

Description

Fermentation process for improving 5-HTP yield
Technical Field
The invention relates to a fermentation process for improving the yield of 5-HTP, and belongs to the field of production of amino acid derivatives.
Background
5-HTP, 5-hydroxytryptophan, chemical name 5-hydroxy-3-indolyl-a-aminopropionic acid, molecular formula C 11 H 12 N 2 O 3 Molecular weight is 220.23.5-HTP is a ginsengNatural amino acids synthesized with proteins. In mammals, 5-HTP is a precursor of the neurotransmitters serotonin and melatonin, has a regulating effect on physiological functions such as sleep, pain sensation, body temperature, appetite and behavior, and has been successfully used for the treatment of diseases such as depression, insomnia and migraine due to its good biological and pharmacological properties and advantages of safety and stability.
At present, the preparation method of 5-hydroxytryptophan mainly comprises plant extraction method, chemical synthesis method and fermentation method, wherein the 5-hydroxytryptophan produced and prepared on a large scale is basically extracted from Griffonia simplicifolia seeds. However, the raw material of the gardner seeds is mainly from western african countries such as gardner, cotdetwa and dorgo, so that the cost and process of the plant extraction method are greatly influenced by the raw material, and the large-scale industrial production of 5-hydroxytryptophan is severely limited. The chemical synthesis method has complex process, low yield and high cost, so the method is not suitable for large-scale production. Compared with a plant extraction method, the fermentation method is not limited by raw materials, and in addition, compared with a chemical synthesis method, the process route is simpler, the production period is shorter, and the cost is lower, so that the method for preparing the 5-hydroxytryptophan by the fermentation method has natural advantages compared with other two methods.
At present, researchers at home and abroad are always dedicated to research on the preparation of 5-hydroxytryptophan by a fermentation method, but the fermentation level of the 5-hydroxytryptophan is still low, so that great influence is caused on the industrialization promotion of the 5-hydroxytryptophan, and the analysis reason is mainly two points, namely, the fermentation yield and the content of the 5-hydroxytryptophan are low; on the other hand, the content of the remaining tryptophan in the fermentation system is higher, which can adversely affect the subsequent separation and purification. In order to solve the problems, CN113493761A and CN113373103A increase the yield of 5-HTP by changing the concentration of metal ions or reducing dissolved oxygen, the highest yield respectively reaches 2.6g/L and 1.93g/L, although the yield is improved, the improvement is relatively limited, and the yield is still at a lower level in large-scale industrial production; in addition, CN113563251A discloses a method for separating and extracting 5-hydroxytryptophan from escherichia coli fermentation liquor, the extraction steps of the method comprise fermentation liquor obtaining, thallus separation, pigment separation, byproduct separation, protein separation and drying to obtain a product, and the yield of 5-HTP is lower to 55% due to overhigh content of the byproduct tryptophan in the fermentation liquor, so that the industrial development of the 5-HTP is influenced.
Disclosure of Invention
[ problem ] to
The invention aims to provide a fermentation process for improving the yield of 5-HTP.
[ solution ]
In order to solve the above problems, the technical scheme provided by the invention is as follows:
the fermentation process for improving the yield of the 5-HTP provided by the invention has the advantages that on one hand, the activity of the strain is further increased, the fermentation lag phase is shortened, and the inoculation amount of seeds in a tank is changed; on the other hand, the product yield is effectively improved and the byproduct content is reduced by supplementing the mixed substrate after induction and controlling the supplementing rate.
The invention provides a method for fermenting 5-HTP, which comprises the following steps:
(1) Activating the 5-hydroxytryptophan producing strain;
(2) Inoculating the activated strain into a seed culture medium to be cultured into a secondary seed solution;
(3) Adding the secondary seed liquid into a fermentation tank according to the amount of 5-20% (v/v), culturing at 35-40 ℃, feeding glucose after the initial sugar in the fermentation system is completely consumed, and culturing to OD 600 Not less than 30 ℃, reducing the culture temperature to 30 ℃, supplementing 1% of arabinose at one time, and simultaneously feeding a mixed substrate of glucose and glycerol at a speed of 4-6 mL/L/h.
In one embodiment, the 5-Hydroxytryptophan-producing strain is a recombinant E.coli expressing Phenylalanine 4-Hydroxylase (P4H) from a prokaryotic microorganism, disclosed in Yuheng Lin et al, engineering Bacterial Phenylalanine 4-Hydroxylase for Microbial Synthesis of Human neuron Precursor 5-Hydroxytryptophan, ACS Synth.Biol., just Accepted Manual Publication Date (Web): 17Jun2014, downloaded from tp:// pubs. Acids.org on Jun 24,2014.
In one embodiment, the 5-HTP-producing strain containment tube is removed from the-80 ℃ freezer and the activated strain is streaked.
In one embodiment, a single clone is picked from the streaking plate, added to a primary seed shake flask, and after overnight incubation, the primary shake flask is inoculated with seed from a secondary shake flask and incubated to OD 600 ＝1±0.2。
In one embodiment, glucose is fed at a concentration of 50% (w/v) to the OD of the fermentation system after the initial sugar in the fermentation system is consumed 600 Not less than 30.
In one embodiment, the mixed substrate of glucose and glycerol is prepared by mixing glucose at a concentration of 20% to 50% (w/v) and glycerol at an equal volume ratio of 20% to 80% (w/v).
In one embodiment, the pH of the fermentation system is maintained at 7.0 + -0.5 for not less than 72h.
Preferably, the secondary seed liquid is added to the fermentor in an amount of 15% (v/v).
Preferably, the mixed substrate of glucose and glycerol is prepared by mixing glucose with a concentration of 50% (w/v) and glycerol with an equal volume ratio of 50%.
Preferably, the mixed substrate of glucose and glycerol is fed at a rate of 4 mL/L/h.
In one embodiment, the streaking plate medium (g/L) is: tryptone 5, yeast powder 15, naCl 20 and agar powder 15.
In one embodiment, the seed shake flask medium (g/L) is: tryptone 5, yeast powder 15 and NaCl 20;
in one embodiment, the fermentation medium (g/L) is: glucose 20,KH 2 PO 4 12，(NH 4 ) 2 SO 4 6，MgSO 4 .7H 2 O2, citric acid 5; EDTA 8.4, coCl 2 ·6H 2 O 2.5，MnCl 2 ·4H 2 O 15.0，CuCl 2 ·2H 2 O 1.5，H 3 BO 3 3.0，Na 2 MoO 4 ·2H 2 O 2.5，ZnSO 4 13.0, ferric citrate 100, hydrochloric acidAmmonium sulfate 4.5, feSO 4 ·7H 2 O8.0; the pH was maintained at about 7.0 using ammonia.
[ advantageous effects ]
According to the fermentation process of the 5-HTP yield, provided by the invention, the inoculum size is increased, the strain activity at the early stage of fermentation is enhanced, the fermentation lag phase is reduced, and the strain is accelerated to enter the logarithmic growth phase, so that the synthesis of a product by the strain is facilitated; on the other hand, the compound substrate is supplemented, the synthesis of cofactors (NADH and the like) in the product synthesis process is increased, the product yield can be effectively improved, and meanwhile, the reduction of byproducts also lightens the difficulty and the cost of the subsequent purification process to a certain extent, so that the yield of the 5-hydroxytryptophan reaches 4.78g/L. The benefits are improved, and the cost is reduced.
Drawings
FIG. 1 is a flow diagram of 5-HTP fermentation.
Detailed Description
The method used in the following examples to detect 5-hydroxytryptophan and tryptophan was: 5-hydroxytryptophan and tryptophan are simultaneously detected by HPLC, a chromatographic column used by HPLC is InfinityLab poroschel HPH-C18.6 x 150mm, a mobile phase is 75% of water and 25% of methanol, the flow rate is 1ml/min, the sample loading amount is 10uL, the detection wavelength is 254nm, the sample preparation concentration is 0.1g/L, the column temperature is 30 ℃, and the time is 15min, wherein the peak time of 5-hydroxytryptophan is 4min, and the peak time of tryptophan is 7.5min.
In the examples described below, the recombinant Escherichia coli capable of synthesizing 5-Hydroxytryptophan is a recombinant Escherichia coli expressing Phenylalanine 4-Hydroxylase (P4H) of prokaryotic Microbial origin (disclosed in Yuheng Lin et al, engineering Bacterial Phenylalanine 4-Hydroxylase for Microbial Synthesis of Human neuron preferer 5-Hydroxytryptophan, ACS Synth. Biol., just Accepted amplified Manual Publication Date (Web): 17Jun2014, downloaded from tp:// pubs. Acids. Org on June 24,2014) capable of converting tryptophan to 5-Hydroxytryptophan.
Examples1
A fermentation process for improving the yield of 5-HTP comprises the following specific steps:
(1) Activation culture: taking out a 5-HTP production strain protecting tube from a refrigerator at the temperature of-80 ℃, and streaking an activated strain on a plate, wherein a streaking plate culture medium (g/L) is as follows: tryptone 5, yeast powder 15, naCl 20 and agar powder 15;
(2) Seed culture: selecting a single clone in a streak plate, adding a first-stage seed shake flask, culturing overnight, and inoculating the seed solution of the first-stage shake flask to a second-stage shake flask according to the inoculation amount of 1%; second-stage shake culture to logarithmic growth phase of thallus, that is, concentration OD of thallus in seed 600 The culture medium can be inoculated into the fermentation tank when the concentration is about 1.0.
The seed shake flask culture medium (g/L) is: tryptone 5, yeast powder 15 and NaCl 20;
(3) Fermentation culture: inoculating the secondary seed to a fermenter at 15% (V/V) inoculum size, continuously culturing at 37 deg.C, continuously adding 50% (W/V) glucose at 6mL/L/h when the initial sugar is consumed, and culturing at OD 600 At a temperature of 30 ℃ or more, the culture temperature was lowered to 30 ℃ while supplementing arabinose at a final concentration of 1% (g/100 mL) and 50% (W/V) glucose to 50% (V/V) glycerol and 50% (W/V) glucose in a ratio of 1:1, gradually reducing the feeding rate of the mixed substrate obtained by mixing from 6mL/L/h to 4mL/L/h, sampling on time, and fermenting for 72h to obtain fermentation liquor;
the fermentation medium (g/L) is: glucose 20,KH 2 PO 4 12，(NH 4 ) 2 SO 4 6，MgSO 4 .7H 2 O2, citric acid 5; EDTA 8.4, coCl 2 ·6H 2 O 2.5，MnCl 2 ·4H 2 O 15.0，CuCl 2 ·2H 2 O 1.5，H 3 BO 3 3.0，Na 2 MoO 4 ·2H 2 O 2.5，ZnSO 4 13.0, ferric citrate 100, ammonium sulfate hydrochloride 4.5 4 ·7H 2 O8.0; the pH was maintained at about 7.0 using ammonia.
Examples2
The fermenter inoculum size was changed to 5% on the basis of example 1, and the remaining conditions were unchanged.
Examples3
The fermenter inoculum size was changed to 10% on the basis of example 1, and the remaining conditions were unchanged.
Examples4
The fermenter inoculum size was changed to 20% on the basis of example 1, and the remaining conditions were unchanged.
TABLE 1 Effect of different inoculum sizes on fermentation yields and Strain concentrations
Figure BDA0003788459290000041
As can be seen from Table 1, the difference of the inoculation amount has a great influence on the fermentation yield, and the yield of 5-hydroxytryptophan shows a trend of increasing first and then decreasing with the increase of the fermentation inoculation amount. And from the density OD of the cells 600 It can be seen that the delay period of the thallus fermentation can be obviously increased by low inoculation amount (5%), so that the later induction time is delayed, and the integral yield is limited; and the high inoculation amount (20%) effectively shortens the lag phase of the thalli, and the thalli can grow to a higher concentration in a shorter time after inoculation, so that the induction time is advanced, but the later fermentation data shows that the excessively high inoculation amount can cause the activity of strains in the middle and later stages of fermentation to be reduced, and the density of the thalli is obviously reduced. In addition, in view of the fermentation yield, too high inoculation amount of the bacterial cells failed to further increase the yield of 5-hydroxytryptophan. And in the embodiment 1 (15% inoculation amount), a better improvement effect is achieved, the fermentation lag phase is reduced to a certain extent, the strain activity is not reduced in advance in the later period, and the strain productivity is also greatly improved.
Examples5
The feed substrate was changed on the basis of example 1 to 20% (W/V) glucose and 50% (V/V) glycerol at a ratio of 1:1 the resulting mixed substrate was mixed, and the remaining conditions were not changed.
Examples6
The feed substrate was changed on the basis of example 1 to 30% (W/V) glucose and 50% (V/V) glycerol at a ratio of 1:1 the resulting mixed substrate was mixed, and the remaining conditions were unchanged.
Examples7
The feed substrate was changed on the basis of example 1 to 40% (W/V) glucose and 50% (V/V) glycerol at a ratio of 1:1 the resulting mixed substrate was mixed, and the remaining conditions were unchanged.
Examples8
The feed substrate was changed on the basis of example 1 to 50% (W/V) glucose and 20% (V/V) glycerol at a ratio of 1:1 the resulting mixed substrate was mixed, and the remaining conditions were unchanged.
Examples9
The feed substrate was changed on the basis of example 1 to 50% (W/V) glucose and 60% (V/V) glycerol at a ratio of 1:1 the resulting mixed substrate was mixed, and the remaining conditions were not changed.
Examples10
The feed substrate was changed on the basis of example 1 to 50% (W/V) glucose and 80% (V/V) glycerol in a ratio of 1:1 the resulting mixed substrate was mixed, and the remaining conditions were unchanged.
Examples11
The feed substrate was changed on the basis of example 1 to 50% (W/V) glucose and 100% glycerol at a 1:1 the resulting mixed substrate was mixed, and the remaining conditions were unchanged.
TABLE 2 Effect of different substrate concentrations on fermentation yield
Figure BDA0003788459290000051
As can be seen from Table 2, when the glucose concentration is lower, the 5-hydroxytryptophan produced by fermentation and the tryptophan production are lower, and the product concentration shows a tendency to increase with the increase of the glucose concentration. When the concentration of glycerol is higher, the yield of the product is not greatly improved and even tends to be reduced, and the content of the byproduct tryptophan is increased along with the increase of the concentration of the glycerol. Finally, it was found that 50% (W/V) glucose and 50% (V/V) glycerol in example 1 were mixed in a ratio of 1:1 the mixed substrate obtained by mixing is fed-batch relatively to have better effect. Therefore, the flow acceleration rate was investigated on the basis of this optimized feed substrate.
Examples12
The feeding rate of the composite substrate was changed to 1mL/L/h based on example 1, and the rest conditions were unchanged.
Examples13
The feeding rate of the composite substrate was changed to 2mL/L/h based on example 1, and the rest conditions were unchanged.
Examples14
The feeding rate of the composite substrate was changed to 3mL/L/h based on example 1, and the rest conditions were unchanged.
TABLE 3 Effect of different substrate feed rates on fermentation yield
Figure BDA0003788459290000061
As can be seen from Table 3, the 5-hydroxytryptophan production increased with the increase of the substrate feed rate. The increase of the substrate feeding rate can effectively improve the growth of microorganisms, the increase of the bacterial quantity can effectively improve the yield to a certain extent, in addition, the feeding rate is improved, the glycerol supplementation amount is also improved to a certain extent, the glycerol can provide a certain cofactor (reducing power) in the metabolic process, and the level of the reducing power in the catalytic process of hydroxylase can be further improved and enhanced through metabolic balance adjustment, so that the content of the byproduct tryptophan is effectively reduced.
In conclusion, the invention greatly improves the whole yield of the 5-hydroxytryptophan by improving the inoculation amount, supplementing the composite substrate and simultaneously controlling the components and the supplementing rate of the composite substrate in the later period, and simultaneously reduces the generation of byproducts to a certain extent, thereby being a better process which is worth reference and application.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by one skilled in the art without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (10)

1. A method of fermenting 5-HTP, said method comprising:
(1) Activating the strain producing 5-hydroxytryptophan;
(2) Inoculating the activated strain into a seed culture medium to be cultured into a secondary seed solution;
(3) Adding the secondary seed liquid into a fermentation tank according to the amount of 5-20% (v/v), culturing at 35-40 ℃, feeding glucose after the initial sugar in the fermentation system is completely consumed, and culturing to OD 600 Not less than 30 ℃, reducing the culture temperature to 28-30 ℃, supplementing 1% -5% of arabinose at one time, and simultaneously feeding a mixed substrate containing glucose and glycerol at a speed of 4-6 mL/L/h.
2. The method of claim 1, wherein the activated strain is added to a primary seed shake flask, and after overnight cultivation, the seeds from the primary shake flask are inoculated into a secondary shake flask and cultivated to OD 600 ＝1±0.2。
3. The method according to claim 1 or 2, wherein glucose is fed at a concentration of 50% (w/v) to OD in the fermentation system after the initial sugar in the fermentation system is consumed 600 Not less than 30.
4. The method according to any one of claims 1 to 3, wherein the mixed substrate of glucose and glycerol is prepared by mixing glucose at a concentration of 20% to 50% (w/v) and glycerol at an equal volume ratio of 20% to 80% (w/v).
5. The method according to any one of claims 1 to 4, wherein the pH of the fermentation system is maintained at 7.0. + -. 0.5, and the fermentation time is not less than 72 hours.
6. The method according to any one of claims 1 to 5, wherein the secondary seed liquid is added to the fermentor in an amount of 15% (v/v).
7. The method according to any one of claims 1 to 6, wherein the mixed substrate of glucose and glycerol is prepared by mixing glucose at a concentration of 50% (w/v) and glycerol at an equal volume ratio of 50%.
8. The method according to any one of claims 1 to 7, wherein the mixed substrate of glucose and glycerol is fed at a rate of 4 mL/L/h.
9. The method according to any one of claims 1 to 8, wherein the ratio of seed shake flask medium (g/L): tryptone 5.0-10.0, yeast powder 15.0-30.0 and NaCl 20.0-40.0.
10. The method according to any one of claims 1 to 9, wherein the fermentation medium (g/L): 15.0 to 20.0 portions of glucose and KH 2 PO 4 10.0～12.0，(NH 4 ) 2 SO 4 3～6，MgSO 4 .7H 2 1.0 to 2.0 percent of O and 2 to 5 percent of citric acid; EDTA 7 to 9, coCl 2 ·6H 2 O 1～5，MnCl 2 ·4H 2 O 10.0～20.0，CuCl 2 ·2H 2 O 1.0～3.0，H 3 BO 3 1.0～3.0，Na 2 MoO 4 ·2H 2 O 1.5～3.5，ZnSO 4 10.0 to 15.0 percent, 90.0 to 110.0 percent of ferric citrate, 3.5 to 5.5 percent of ammonium sulfate hydrochloride 4 ·7H 2 O 5.0～10.0。
CN202210949191.0A 2022-08-09 2022-08-09 Fermentation process for improving 5-HTP yield Pending CN115161358A (en)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170240939A1 (en) * 2014-05-16 2017-08-24 University Of Georgia Research Foundation, Inc. Microbial approach for the production of 5-hydroxytryptophan
CN113373103A (en) * 2021-06-18 2021-09-10 天津科技大学 Method for improving yield of 5-hydroxytryptophan

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20170240939A1 (en) * 2014-05-16 2017-08-24 University Of Georgia Research Foundation, Inc. Microbial approach for the production of 5-hydroxytryptophan
CN113373103A (en) * 2021-06-18 2021-09-10 天津科技大学 Method for improving yield of 5-hydroxytryptophan

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
YUHENG LIN等: "Engineering Bacterial Phenylalanine 4‑Hydroxylase for Microbial Synthesis of Human Neurotransmitter Precursor 5‑Hydroxytryptophan", ACS SYNTH. BIOL., vol. 3, 17 July 2014 (2014-07-17), pages 497 *
王海蛟: "代谢工程改造大肠杆菌合成5-羟基色氨酸的研究", 中国博士学位论文全文数据库基础科学辑, 15 March 2020 (2020-03-15), pages 84 - 87 *

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