CN110564789A - Method for producing L-theanine by using escherichia coli fermentation - Google Patents
Method for producing L-theanine by using escherichia coli fermentation Download PDFInfo
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Abstract
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.
Description
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.
Example 1
inoculating an original strain of escherichia coli to a slant, culturing for 20h at 37 ℃ in a biochemical incubator, then performing gradient dilution, coating on a complete culture medium (LB), selecting single colonies, respectively inoculating to the slant, culturing for 20h at 35 ~ 40 ℃ in the biochemical incubator, selecting the single colonies, inoculating to a 500mL seed bottle, and culturing under the conditions that the temperature is 35 ~ 40 ℃ and the OD value is 10.0 after the culture is performed at the rotating speed of a shaking table of 200 rmp.
inoculating the mixture into a fermentation medium, wherein the inoculation amount is 7%, the culture conditions comprise that the initial fermentation temperature is 35 ~ 40 ℃, the rotating speed of a shaking table is 200rmp, when the culture OD value reaches 20, D ~ xylose is added to enable the concentration of the D ~ xylose to be 5g/L, the temperature is reduced to 28 ~ 35 ℃, after induction is carried out for 4 ~ 5 hours, an ethylamine solution with the volume concentration of 40% is supplemented at a constant flow rate with the volume ratio of a fermentation liquid to the ethylamine solution being 100:1, fermentation culture is continuously carried out for 20 ~ 30 hours under the conditions that the pH is 7.0 ~ 7.2, the temperature is 35 ~ 40 ℃, and the dissolved oxygen is 10 ~ 40%, and after the fermentation is finished, the L ~ theanine content is detected to be 38.37 g/L.
as a comparison, the same fermentation method is adopted, the ethylamine hydrochloride solution with the same concentration is supplemented in the fermentation stage, and the content of L-theanine is detected to be 25.65 g/L.
Example 2
A ring of escherichia coli is picked from a fresh inclined plane and inoculated into a 500mL seed bottle under the culture conditions that the temperature is 35 ~ 40 ℃, the rotating speed of a shaking table is 200rmp, when the OD value is 15.0, the shake flask seeds are inoculated into a 5L full ~ automatic fermentation tank filled with 3L fermentation medium in an inoculation amount of 10% for secondary seed culture, the culture temperature is 35 ~ 40 ℃, the tank pressure is 0.01 ~ 0.05MPa, the air flow is 1 ~ 8L/min, the stirring speed is 200 ~ 800r/min, so that the dissolved oxygen is maintained at 10 ~ 50%, the pH value is controlled at 6.5 ~ 7.5, and the culture time is 5 ~ 8 h.
inoculating 10% of secondary seeds into a 30L full-automatic fermentation tank filled with 18L of fermentation medium for acid-producing fermentation, controlling the initial fermentation temperature to be 35 ~ ℃, the tank pressure to be 0.01-0.15 MPa, the air flow to be 5-50L/min and the stirring speed to be 200-800 r/min to keep the dissolved oxygen at 10 ~%, controlling the fermentation pH value to be 6.5-7.5, controlling the shaking table rotating speed to be 200rmp, adding D-xylose to ensure that the concentration of the D-xylose is 15g/L when the culture OD value reaches 25, cooling to 28-35 ℃ for induction for 4-5 h, beginning to supplement 70% of ethylamine solution at a constant flow rate of which the volume ratio of fermentation liquor to the ethylamine solution is 300:1, continuing to supplement the ethylamine solution at a pH value of 6.5-7.2, controlling the temperature to be 35 ~ ℃, testing the L-theanine content to be 120.33g/L after the fermentation is finished, testing the ethylamine solution content to be 67.24% of the theanine content, and comparing the invention with 78.96% of the invention.
Example 3
selecting a ring of Escherichia coli from a fresh inclined plane, inoculating into a 500mL seed bottle under the culture conditions of 35 ~ 40 deg.C and shaking table rotation speed of 200rmp, inoculating the seed into a flask containing 0.6m of Escherichia coli when OD value reaches about 20.032m of fermentation Medium3performing secondary seed culture in a fermentation tank, wherein the culture temperature is 35 ~ 40 ℃, the tank pressure is 0.01 ~ 0.05MPa, and the air flow is 0.1 ~ 0.6m3the dissolved oxygen is maintained at 10 ~ 50% at a stirring speed of 100 ~ 300r/min, the pH value is controlled at 6.5 ~ 7.5, and the culture time is 5 ~ 8 h.
Respectively inoculating the second-level seeds with an inoculum size of 10% to a container containing 6m310 m of fermentation Medium3performing acid ~ producing fermentation in a fermentation tank, wherein the initial fermentation temperature is 35 ~ 40 ℃, the tank pressure is 0.01 ~ 0.15MPa, the dissolved oxygen is maintained 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 30, D ~ xylose is added to ensure that the concentration of the D ~ xylose is 20g/L, the temperature is reduced to 28 ~ 35 ℃ for induction for 4 ~ 5h, then, ethylamine solution with the volume concentration of 80% is supplemented at a constant flow rate of 200:1 volume ratio of fermentation liquor to ethylamine solution, the fermentation culture is continued for 20 ~ 30h under the conditions of pH6.5 ~ 7.2, temperature of 35 ~ 40 ℃ and dissolved oxygen of 10 ~ 40%, and after the fermentation is finished, the content of L ~ theanine is detected to be 117.86 g/L.
It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.
Claims (4)
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.
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| CN114480522A (en) * | 2021-12-16 | 2022-05-13 | 精晶药业股份有限公司 | Method for preparing L-theanine by microbial fermentation |
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