阅读说明：本技术 一种利用大肠杆菌发酵生产l-茶氨酸的方法 (Method for producing L-theanine by using escherichia coli fermentation ) 是由 曹华杰 谢沛 李静 张孟涛 封浪 刘晓东 于 2019-09-12 设计创作，主要内容包括：本发明涉及一种利用大肠杆菌发酵生产L-茶氨酸的方法,属于生物工程技术领域,所述方法是在发酵培养过程中,将发酵罐中的大肠杆菌培养至OD为20-30时,向发酵罐中添加D~木糖使D~木糖的浓度为5~20g/L并调温至28~35℃诱导4~5h；然后以发酵液与乙胺溶液体积比为100~300:1的恒定流速补加体积浓度为40-80%的乙胺溶液,继续在pH6.5~7.5、温度35~40℃、溶氧10~40%的条件下发酵培养20-30h,合成L-茶氨酸。发酵周期短、产酸效率高、菌体密度高；底物转化率高；而且工艺简单,环境污染低,降低了L~茶氨酸的生产成本,有利于工业化生产的推广和应用。(the invention relates to a method for producing L ~ theanine by fermenting escherichia coli, which belongs to the technical field of bioengineering, and the method comprises the steps of culturing escherichia coli in a fermentation tank until OD is 20 ~ 30 in the fermentation culture process, adding D ~ xylose into the fermentation tank to enable the concentration of the D ~ xylose to be 5 ~ 20g/L, adjusting the temperature to 28 ~ 35 ℃, inducing for 4 ~ 5 hours, then supplementing 40 ~ 80% of ethylamine solution at a constant flow rate with the volume ratio of fermentation liquor to ethylamine solution being 100 ~ 300:1, continuously fermenting and culturing for 20 ~ 30 hours under the conditions of pH6.5 ~ 7.5, temperature being 35 ~ 40 ℃ and dissolved oxygen being 10 ~ 40%, and synthesizing the L ~ theanine.)

1. A method for producing L ~ theanine by using escherichia coli through fermentation is characterized in that in the fermentation culture process, when escherichia coli in a fermentation tank is cultured until OD is 20 ~ 30, D ~ xylose is added into the fermentation tank to enable the concentration of the D ~ xylose to be 5 ~ 20g/L, the temperature is adjusted to 28 ~ 35 ℃ for induction for 4 ~ 5 hours, then ethylamine solution with the volume concentration of 40 ~ 80% is supplemented at a constant flow rate with the volume ratio of fermentation liquor to ethylamine solution being 100 ~ 300:1, and fermentation culture is continued for 20 ~ 30 hours under the conditions that pH is 6.5 ~ 7.5, the temperature is 35 ~ 40 ℃ and dissolved oxygen is 10 ~ 40%, so that L ~ theanine is synthesized.

2. The method for producing L-theanine by fermentation using Escherichia coli as claimed in claim 1, wherein: the method comprises strain activation, seed culture and fermentation culture;

the seed culture is to inoculate an activated strain into a seed culture medium for culture under the conditions that the temperature is 35 ~ 40 ℃, the rotating speed of a shaking table is 200rmp, and the OD is 10 ~ 20;

the fermentation culture is to inoculate a strain to a fermentation medium by 5 ~ 15% of inoculation amount and then carry out acid ~ producing fermentation under the conditions that the initial fermentation temperature is 35 ~ 40 ℃, the air flow is 5 ~ 50L/min, the stirring speed is 200 ~ 800r/min so that the dissolved oxygen is maintained at 10 ~ 40%, the fermentation pH value is controlled to be 6.5 ~ 7.5, and the rotating speed of a shaking table is 200 rmp.

3. the method for producing L ~ theanine by using Escherichia coli fermentation as claimed in claim 2, wherein the seed culture medium comprises glucose 20 ~ 30g/L and K₂HPO₄ 10-20g/L, MgSO₄·7H₂O 0.5~3.0g/L，KH₂PO₄ 10-20g/L, yeast powder 0.5-5.0g/L, (NH)₄)₂SO₄5.0 ~ 10.0g/L, 5 ~ 10.0mL/L of trace elements, and 67 pH.

4. the method for producing L ~ theanine by fermentation of Escherichia coli as claimed in claim 2, wherein the fermentation medium comprises glucose 20 ~ 30g/L and K₂HPO₄ 10-20g/L，MgSO₄·7H₂0.5-3.0g/L of O, 0.5-3.0g/L of yeast powder, (NH)₄)₂SO₄5.0 ~ 10.0g/L, 5 ~ 10.0mL/L of trace elements, and initial pH of 7.0.

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a method for producing L-theanine by utilizing escherichia coli fermentation. .

background

L ~ theanine (L ~ theanine) is an amino acid existing in tea, can promote the formation of cerebral epidermal alpha waves to relax people, can reduce the concentration of 5 ~ hydroxytryptamine in human bodies to play a role in reducing blood pressure, and has wide application in the aspects of tumor treatment, neuroprotection and the like.

Gamma ~ glutamyltranspeptidase (gamma ~ glutamyltripeptidase, GGT, EC 2.3.2.2) is a key enzyme in a glutathione metabolic pathway, is a heterodimer consisting of large subunits and small subunits, and has 2 types of catalytic reactions, namely a hydrolysis reaction for catalyzing gamma ~ glutamyl compounds, and a transpeptidation reaction for catalyzing gamma ~ glutamyl in gamma ~ glutamyl compounds to transfer amino acids or polypeptides, Suzuki and the like firstly report that GGT from E.coli K ~ 12 SH642 can catalyze L ~ glutamine and ethylamine to generate L ~ theanine, the reaction does not need protection and deprotection of reactants, the reaction process does not need participation of ATP, so that the catalytic synthesis of L ~ theanine by GGT becomes a hotspot of current research, E.coli BL21 (DE 3)/pET 32a ~ GGT catalyzes L ~ glutamine and ethylamine to synthesize L ~ theanine by Escherichia coli BL21 (DE 3)/BLT 32a ~ GGT, the L ~ glutamine and ethylamine by utilizing the substrate of Bsanguis 7.80 g, the engineering bacteria can synthesize L ~ glutamine by catalyzing L ~ glutamine at 37 ℃ and the substrate of Shuihe ~ 7 ~ 80 ℃ and the substrate of Bsanguis ~ 7.10 g, the L ~ 80 ~ 8 ~ g of Bluis ~ L ~ glutamine.

Disclosure of Invention

In order to solve the defects in the prior art, the invention aims to provide a method for producing L-tryptophan by using escherichia coli through fermentation.

In order to achieve the purpose, the invention adopts the specific scheme that:

A method for producing L ~ theanine by using escherichia coli through fermentation comprises the steps of culturing escherichia coli in a fermentation tank until OD is 20 ~ 30 in the fermentation culture process, adding D ~ xylose into the fermentation tank to enable the concentration of the D ~ xylose to be 5 ~ 20g/L, adjusting the temperature to 28 ~ 35 ℃, inducing for 4 ~ 5 hours, supplementing 40 ~ 80% of ethylamine solution at a constant flow rate with the volume ratio of fermentation liquor to ethylamine solution being 100 ~ 300:1, continuing fermentation culture for 20 ~ 30 hours under the conditions of pH6.5 ~ 7.5, temperature being 35 ~ 40 ℃ and dissolved oxygen being 10 ~ 40%, and synthesizing the L ~ theanine.

As a further optimization of the scheme, the method comprises strain activation, seed culture and fermentation culture;

the seed culture is to inoculate an activated strain into a seed culture medium for culture under the conditions that the temperature is 35 ~ 40 ℃, the rotating speed of a shaking table is 200rmp, and the OD is 10 ~ 20;

the fermentation culture is to inoculate a strain to a fermentation medium by 5 ~ 15% of inoculation amount and then carry out acid ~ producing fermentation under the conditions that the initial fermentation temperature is 35 ~ 40 ℃, the air flow is 5 ~ 50L/min, the stirring speed is 200 ~ 800r/min so that the dissolved oxygen is maintained at 10 ~ 40%, the fermentation pH value is controlled to be 6.5 ~ 7.5, and the rotating speed of a shaking table is 200 rmp.

As a further optimization of the scheme, the formula of the seed culture medium is that the glucose is 20 ~ 30g/L, and the K is₂HPO₄ 10-20g/L, MgSO₄·7H₂O 0.5~3.0g/L，KH₂PO₄ 10-20g/L, yeast powder 0.5-5.0g/L, (NH)₄)₂SO₄5.0 ~ 10.0g/L, 5 ~ 10.0mL/L of trace elements, and pH 6.7.

As a further optimization of the scheme, the formula of the fermentation medium is that the glucose is 20 ~ 30g/L, and the K is₂HPO₄ 10-20g/L，MgSO₄·7H₂0.5-3.0g/L of O, 0.5-3.0g/L of yeast powder, (NH)₄)₂SO₄5.0 ~ 10.0g/L, 5 ~ 10.0mL/L of trace elements, and initial pH of 7.0.

has the advantages that:

1. The method adopts escherichia coli fermentation to produce the L-theanine, the strain fermentation is aerobic fermentation, and the thallus grows fast; in the acid-producing fermentation stage, D-xylose is firstly adopted for low-temperature induction, then ethylamine is added to provide a raw material for synthesizing L-theanine, and theanine synthetase has high specificity and can only catalyze glutamic acid and ethylamine to synthesize theanine; then the yield of the L-theanine is obviously increased under the regulation and control of the whole fermentation system.

2. the fermentation method has the advantages of short fermentation period, high thallus density, high L-theanine yield, high saccharic acid conversion rate, less metabolic byproducts, low production cost and convenience in extraction.

Detailed Description

a method for producing L ~ theanine by using an escherichia coli strain fermentation method comprises the following specific steps:

1. method for producing L-theanine by using escherichia coli strain fermentation method

and (3) slant culture, namely activating an original escherichia coli strain by using a slant, culturing for 20 hours in a biochemical incubator at 37 ℃, performing gradient dilution, coating on a complete culture medium (LB), selecting single colonies, respectively inoculating the single colonies onto the slant, and culturing for 12-20 hours in the biochemical incubator at 35-40 ℃.

and (3) seed culture, wherein a single colony is selected and inoculated into a seed culture medium under the culture conditions that the temperature is 35 ~ 40 ℃, the rotating speed of a shaking table is 200rmp, and the culture OD value is 10.0 ~ 20.0.

and (2) fermentation culture, namely inoculating the mixture into a fermentation medium, wherein the inoculation amount is 5 ~ -15%, the culture conditions comprise that the initial fermentation temperature is 35 ~ -40 ℃, the air flow is 5 ~ -50L/min, the stirring speed is 200 ~ -800 r/min, the dissolved oxygen is kept at 10 ~ -40%, the fermentation pH value is controlled at 6.5 ~ -7.5, the rotating speed of a shaking table is 200rmp, when the culture OD value reaches 20 ~ -30, D ~ -xylose is added to enable the concentration of the D ~ -xylose to be 5 ~ -20g/L, the temperature is reduced to 28 ~ -35 ℃, after induction is carried out for 4 ~ -5 hours, ethylamine solution with the volume concentration of 40 ~ -80% is added at a constant flow rate of 100 ~ -300: 1, the fermentation solution and the ethylamine solution continue to be fermented and cultured for 20 ~ -30 hours under the conditions of pH6.5 ~ -7.5, the temperature is 35 ~ -40 ℃ and the dissolved oxygen is 10 ~ -40%, and fermentation broth containing L ~ -theanine is obtained after the fermentation is finished.

The seed culture medium comprises the following components in percentage by weight:glucose 20 ~ 30g/L, K₂HPO₄10~20g/L, MgSO₄·7H₂O 0.5~3.0g/L，KH₂PO₄ 10 ~ 20g/L, yeast powder 0.5 ~ 5.0g/L, (NH)₄)₂SO₄5 ~ 10.0g/L, 5 ~ 10.0mL/L of trace elements, and pH 6.7.

the fermentation medium comprises 20 ~ 30g/L of glucose and K₂HPO₄ 10~20g/L, MgSO₄·7H₂0.5 ~ 3.0g/L of O, 0.5 ~ 3.0g/L of yeast powder, (NH)₄)₂SO₄5 ~ 10.0g/L, 5 ~ 10.0mL/L of trace elements, and 7.0 of initial pH.

2. method for extracting L ~ theanine from escherichia coli fermentation liquor

heating the obtained fermentation liquor to 40 ~ 60 ℃, adjusting the pH value to 2 ~ 5 by using 2mo1/L hydrochloric acid, adding anhydrous sodium bisulfite, performing microfiltration by using a 30nm ceramic membrane, cleaning by using purified water for 2 ~ 3 times to obtain a supernatant, taking the supernatant, passing the supernatant through a hollow fiber bundle membrane with the molecular weight of 1000 ~ 5000, wherein the recovery rate of L ~ theanine is 92.3 ~ 95.4%, passing the supernatant through a strong acid cation exchange column under the condition of static adsorption, adding cation resin according to the volume ratio of 20%, stirring and adsorbing for 1h, performing dynamic adsorption, namely loading the column resin according to the volume ratio of 15%, feeding the column resin at the flow rate of 2BV/h, washing a BV column with 0.5 ~ 1BV water in the final stage, collecting the supernatant, adding activated carbon according to the volume ratio of 0.5 ~ 1% into the supernatant for 60 ℃ decolorization for 0.5 ~ 2h, filtering and removing the carbon, collecting the L ~ theanine supernatant, performing vacuum concentration, wherein the vacuum degree of vacuum is 0.1MPa, the rotation speed is 100 ~ 200rpm, the temperature is 50 ~ 80 ℃, and the L ~ 80 ℃ is used for cooling, and the L ~ 70% of the concentrated thea.

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.