CN109593801A

CN109593801A - A kind of technique of fermenting and producing L-Trp

Info

Publication number: CN109593801A
Application number: CN201811542814.2A
Authority: CN
Inventors: 徐庆阳; 李德衡; 王健; 刘元涛; 包鑫; 张宗华; 杨瑞丽; 边恩来; 王飞; 米永花
Original assignee: XINJIANG FUFENG BIOTECHNOLOGY CO Ltd; Jilin University; Tianjin University of Science and Technology
Current assignee: XINJIANG FUFENG BIOTECHNOLOGY CO Ltd; Jilin University; Tianjin University of Science and Technology
Priority date: 2018-12-17
Filing date: 2018-12-17
Publication date: 2019-04-09

Abstract

The invention belongs to technical field of amino acid production, disclose a kind of technique of fermenting and producing L-Trp, it includes the following steps: to produce L-Trp Escherichia coli seed liquor in 10% inoculum concentration access fermentation medium, 36.8~37.0 DEG C of cultivation temperature, dissolved oxygen amount is 20%, through pH in Feeding ammonia water maintenance tank between 7.0~7.2；When fermentation to for 24 hours when, nutrition liquid stream is added in tank, maintaining sugared concentration in tank is 0.5-1g/L, until fermentation ends, total fermentation time 42-48h obtain L-Trp fermentation liquid.Present invention process is simple, and fermentation efficiency and saccharic acid conversion ratio are high.

Description

A kind of technique of fermenting and producing L-Trp

Technical field

The invention belongs to amino acid fermentation technical fields, and in particular to a kind of technique of fermenting and producing L-Trp.

Background technique

The molecular formula of L-Trp is C₁₁H₁₂O₂N₂, molecular weight 204.21, nitrogenous 13.72%.L-Trp be containing The neutral aromatic amino acid of indyl, it is odorless in silky lustre, hexagonal plate from color crystal, it is pleasantly sweet.Solubility in water 1.14g/L (25 DEG C), is dissolved in diluted acid or diluted alkaline, more stable in lye, decomposes in strong acid, is slightly soluble in ethyl alcohol, insoluble in chloroform, Ether.

L-Trp is one of eight kinds of essential amino acids in human body and animal life activity, is sent out the growth of humans and animals Educate, metabolism plays an important role, referred to as the second essential amino acid, it is the third after methionine and lysine Big feed addition amino acid, is widely used in feedstuff industry.

The production of L-Trp relies primarily on protein Hydrolyze method and chemical synthesis earliest, but with to microbial method Production L-Trp research deepens continuously, and microbial method has moved towards practical and in leading position.Microbial method again may be used It is divided into direct fermentation, microbe transformation method and enzyme process, L-Trp production at present is mainly based on Production by Microorganism Fermentation. Microbe fermentation method has many advantages, such as that low in raw material price, technology controlling and process be simple, reliable product quality.But with fermentation industry Fast development, fermentation production of L-tryptophan propose higher want to the reasonability of medium nutrient content and fermentation control It asks.Excellent L-Trp production bacterial strain, reasonable culture medium composition and fermentation control strategy appropriate is conducive to improve L- color The acid yield of propylhomoserin.

In recent years, for L-Trp as essential amino acid, price is higher always, and L-Trp prefers to Cheap highly effective Production and extracting method, to promote the application of L-Trp.As other biological engineering product, L-Trp industrial production Also it usually will receive the restriction of production cost, and in the composition of production cost, the cost of the downstream engineerings such as separation and Extraction is occupied Considerable proportion.Therefore the method that research improves L-Trp fermentation and extraction yield has important theory significance and practical valence Value.Patented technology " CN104694614A " before applicant discloses a kind of novel technology for extracting of L-Trp, comprising: bacterium Liquid fermentation, three columns in series ion-exchange column ion-exchange, ultrafiltration decoloration, reverse osmosis, double effect concentration, crystallization, is done the filtering of L-Trp mocromembrane The techniques such as dry, packaging obtain fine work L-Trp product.The present invention is using three columns in series ion-exchange column ion-exchange, ultrafiltration membrane decoloration and one Secondary crystallization obtains L-Trp product, simple process and can be continuously produced, and process residue can produce egg by integrated treatment White feed, it is environmentally protective；This method improves the sour efficiency of production by the way of mixed fermentation, opposite but there are zymotechniques The problems such as complicated, fermentation parameter is difficult to control.In view of the foregoing drawbacks, the patented technology " CN201711289822 " of applicant is open By the way of single bacterial strain fermentation, fermentation and acid amount is improved by addition tourmaline and using Dialysis culture base；It should Method improves fermentation and acid amount, but needs replacing culture medium, and operation difficulty is larger, be easy to cause part bacterial strain dead.? On the basis of above-mentioned patented technology, applicant continues that single bacterial strain fermentation condition is improved and optimized.

Summary of the invention

To solve the above problems, the present invention provides a kind of productions and extraction L- color in place of overcome the deficiencies in the prior art The method of propylhomoserin, this method improve fermentation efficiency, and extracting method is optimized by reasonable compatibility culture medium.

The purpose of the present invention is what is be achieved through the following technical solutions:

A kind of technique of fermenting and producing L-Trp comprising following steps: L-Trp Escherichia coli seed liquor will be produced with 10% Inoculum concentration access fermentation medium in, 36.8~37.0 DEG C of cultivation temperature, dissolved oxygen amount 20% maintains tank by Feeding ammonia water Interior pH is between 7.0~7.2；When fermentation to for 24 hours when, nutrition liquid stream is added in tank, maintaining sugared concentration in tank is 0.5-1g/L, Until fermentation ends, total fermentation time 42-48h obtain L-Trp fermentation liquid.

Preferably,

Choline chloride is added in the fermentation medium；Indoles and inositol are added in the nutrient solution.

Preferably,

The fermentation medium are as follows: glucose 20g/L, dipotassium hydrogen phosphate 9g/L, yeast extract 5g/L, citric acid 4.6g/L, sulfuric acid Ammonium 1.8g/L, magnesium sulfate 1.6g/L, choline chloride 0.4-0.6g/L, ferrous sulfate 65mg/L, biotin 0.2mg/L；PH control In 6.5-7.0, sterilize 15min at 115 DEG C.

Preferably,

The nutrient solution are as follows: the concentration of glucose is 100g/L, and the concentration of indoles is 1-1.5g/L, and the concentration of inositol is 0.5- 1g/L, sterilize at 115 DEG C 15min.

Preferably,

The choline chloride is 0.4-0.5g/L.

Preferably,

The concentration of the indoles is 1g/L.

Preferably,

The concentration of the inositol is 0.5g/L.

The present invention be also claimed it is above-mentioned be allowed to one described in technique preparation L-Trp.

Compared with prior art, technical solution of the present invention has the advantages that following prominent and uniqueness:

Tryptophan synthetic pathway is complicated, is influenced by more multifactor, and Escherichia coli are transformed by metabolic engineering means and accumulate color ammonia When sour, it often will appear growth question, this research improves cellular metabolic pathways by optimizing nutriment, and tryptophan accumulates To promotion；For the cell growth arrest phase for avoiding bacterial strain from occurring by nitrogen source switching, choline chloride is added, in the medium with thorn Swash thalli growth；To solve thallus early ageing and producing sour low efficiency, middle and later periods thallus vigor, selection stream plus glucose, indoles are improved And inositol, and the addition time is groped, later period cell acid producing ability is improved, tryptophan yield is further increased.With The increase of fermentation time, Escherichia coli can generating unit sub-wire propylhomoserin, by stream plus indoles, so as to utilize Escherichia coli color Propylhomoserin synzyme catalytic serine and indole synthesis tryptophan, improve the yield of tryptophan；Suitable inositol can strengthen CO2 Fixed reaction, promotes the accumulation of amino acid, improves fermentation conversion rate；Many factors mutually cooperate with, and improve the fermentation of tryptophan Yield.

Figure of description

Fig. 1: influence of the Additive Amount of Choline Chloride to Fungal biodiversity in fermentation liquid and tryptophan yield；

Fig. 2: influence of the indoles additive amount to tryptophan yield in fermentation liquid.

Specific embodiment

Those skilled in the art can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that All similar substitutions and modifications are apparent to those skilled in the art, they are considered as being included in this hair It is bright.Product and method of the invention is described by preferred embodiment, and related personnel can obviously not depart from this hair Product as described herein and method are modified in bright content, spirit and scope or appropriate changes and combinations, to realize and answer Use the technology of the present invention.For a further understanding of the present invention, the following describes the present invention in detail with reference to examples.

Embodiment 1

A kind of technique of fermenting and producing L-Trp comprising following steps:

Prepare fermentation medium: glucose 20g/L, dipotassium hydrogen phosphate 9g/L, yeast extract 5g/L, citric acid 4.6g/L, ammonium sulfate 1.8g/L, magnesium sulfate 1.6g/L, choline chloride 0.4g/L, ferrous sulfate 65mg/L, biotin 0.2mg/L；PH control 6.8, Sterilize 15min at 115 DEG C；

Prepare stream plus nutrient solution: the concentration of glucose is 100g/L, and the concentration of indoles is 1g/L, and the concentration of inositol is 0.5g/L, Sterilize 15min at 115 DEG C；

With coli strainE.coli For TRTH, with 10% inoculum concentration by seed liquor (OD₆₀₀Value is 10.8) access fermentation In culture medium, 36.8~37.0 DEG C of cultivation temperature, dissolved oxygen amount 20%, by being in 25% ammonium hydroxide of numerical control auto-feeding maintenance tank PH unite between 7.0~7.2, stream plus defoaming agent defoaming；When fermentation to for 24 hours when, nutrition liquid stream is added in tank, maintain tank in sugar Concentration is 0.5-1g/L；Until fermentation ends, total fermentation time is 42h to get rich in L-Trp fermentation liquid.

Embodiment 2

Prepare fermentation medium: glucose 20g/L, dipotassium hydrogen phosphate 9g/L, yeast extract 5g/L, citric acid 4.6g/L, ammonium sulfate 1.8g/L, magnesium sulfate 1.6g/L, choline chloride 0.5g/L, ferrous sulfate 65mg/L, biotin 0.2mg/L；PH control 6.6, Sterilize 15min at 115 DEG C；

Prepare stream plus nutrient solution: the concentration of glucose is 100g/L, and the concentration of indoles is 1.5g/L, and the concentration of inositol is 0.8g/ L, sterilize at 115 DEG C 15min；

With coli strainE.coli For TRTH, with 10% inoculum concentration by seed liquor (OD₆₀₀Value is 10.7) access fermentation In culture medium, 36.8~37.0 DEG C of cultivation temperature, dissolved oxygen amount 20%, by being in 25% ammonium hydroxide of numerical control auto-feeding maintenance tank PH unite between 7.0~7.2, stream plus defoaming agent defoaming；When fermentation to for 24 hours when, nutrition liquid stream is added in tank, maintain tank in sugar Concentration is 0.5-1g/L；Until fermentation ends, total fermentation time is 48h to get rich in L-Trp fermentation liquid.

Embodiment 3

One, influence of the Additive Amount of Choline Chloride to Fungal biodiversity in fermentation liquid and tryptophan yield.

Mode of operation is 0,0.2,0.4,0.6,0.8,1(g/L referring to embodiment 1, the additive amount of selective chlorination choline)；Such as Shown in Fig. 1-2, with the increase of Additive Amount of Choline Chloride, Fungal biodiversity is stepped up, and is mentioned when choline chloride addition concentration When rising to 0.4 g/L, cell growth status be improved significantly, cell fast-growth, tryptophan accumulation rate also obtains express delivery It is promoted, continues growing the additive amount of choline chloride, Fungal biodiversity and tryptophan yield are not obviously improved, and select 0.4- The additive amount of 0.6g/L is more appropriate.

Two, indoles and inositol are to Fungal biodiversity, tryptophan yield, saccharic acid conversion ratio and fermentation and acid speed in fermentation liquid The influence of rate.

Control group is set, wherein control group 1: not adding indoles and inositol, remaining is the same as embodiment 1；Control group 2: it does not add Indoles, remaining is the same as embodiment 1；Control group 3: not adding inositol, remaining is the same as embodiment 1；Experimental group is embodiment 1.Specifically it is shown in Table 1:

Table 1

Group	Experimental group	Control group 1	Control group 2	Control group 3
					Fungal biodiversity g/L	44.9	41.1	44.7	41.5
Tryptophan yield g/L	50.3	38.6	44.8	42.9
					Saccharic acid conversion ratio %	22.6	18.7	20.3	19.8
Fermentation and acid rate g/Lh	1.19	0.92	1.07	1.02

Conclusion: tryptophan synthetic pathway is complicated, when Escherichia coli accumulation tryptophan is transformed by metabolic engineering means, often goes out Existing growth question, this research gradually improve cellular metabolic pathways by optimization trophic factors, and tryptophan accumulation gets a promotion. Indoles is added in 24 h that ferment and inositol, two kinds of components mutually cooperate with, and compared with control group 1, Fungal biodiversity increases, color Propylhomoserin throughput rate maintains 1.19g/Lh, and final tryptophan yield improves 30.3% in 50.3 g/L, compared with control group 1；It is comprehensive The experimental result of control group 2 and control group 3 it is found that indoles on Fungal biodiversity influence and it is little, the yield of tryptophan is It improves, and inositol plays Pasitive Regulation Effect of Genseng to Fungal biodiversity and tryptophan yield.

The present invention also has detected the influence of indoles additive amount tryptophan yield, and the additive amount that indoles is arranged is respectively 0, 0.5,1,1.5,2,2.5(g/L), as shown in Fig. 2, with indoles additive amount increase, the yield of tryptophan increases sharply, After increasing 1g/L, amplification slows down, and then tends to be steady, and therefore, selects the concentration of indoles for 1-1.5g/L.

Although above-mentioned be described a specific embodiment of the invention in conjunction with the embodiments, not the present invention is protected The limitation of range, those skilled in the art should understand that, this to those skilled in the art should be very clear, without departing from These modifications or improvements on the basis of spirit of that invention, belong to the scope of protection of the invention.

Claims

1. a technology for producing L-tryptophan by fermentation, which comprises the steps: the L-tryptophan Escherichia coli seed liquid is inserted into the fermentation medium with an inoculum size of 10%, and the culture temperature is 36.8～37.0 ℃, The dissolved oxygen content is 20%, and the pH in the tank is maintained between 7.0 and 7.2 by adding ammonia water; when the fermentation reaches 24h, the nutrient liquid flow is added into the tank, and the sugar concentration in the tank is maintained at 0.5-1g/L until After the fermentation, the total fermentation time is 42-48h, and L-tryptophan fermentation broth is obtained.

2 . The process according to claim 1 , wherein choline chloride is added in the fermentation medium; indole and inositol are added in the nutrient solution. 3 .

3. technology according to claim 1 and 2 is characterized in that, described fermentation medium is: glucose 20g/L, dipotassium hydrogen phosphate 9g/L, yeast extract 5g/L, citric acid 4.6g/L, Ammonium sulfate 1.8g/L, magnesium sulfate 1.6g/L, choline chloride 0.4-0.6g/L, ferrous sulfate 65mg/L, biotin 0.2mg/L; pH controlled at 6.5-7.0, at 115°C Sterilize for 15 minutes.

4. technology according to claim 1 and 2 is characterized in that, described nutrient solution is: the concentration of glucose is 100g/L, the concentration of indole is 1-1.5g/L, the concentration of inositol is 0.5- 1g/L, sterilize at 115°C for 15min.

5. technique according to claim 3 is characterized in that, described choline chloride is 0.4-0.5g/L.

6. technology according to claim 4 is characterized in that, the concentration of described indole is 1g/L.

7. technology according to claim 4 is characterized in that, the concentration of described inositol is 0.5g/L.

8. The L-tryptophan prepared by the process according to any one of claims 1-7.