CN110564789B - 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 utilizing escherichia coli fermentation, 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 5-20 g/L, and adjusting the temperature to 28-35 ℃ for induction for 4-5 hours; then, supplementing 40-80% by volume of ethylamine solution at a constant flow rate with the volume ratio of fermentation liquor to ethylamine solution being 100-300: 1, and continuously performing fermentation culture for 20-30h under the conditions of pH 6.5-7.5, temperature being 35-40 ℃ and dissolved oxygen being 10-40% to synthesize the L-theanine. The fermentation period is short, the acid production efficiency is high, and the thallus density is high; the conversion rate of the substrate is high; and the process is simple, the environmental pollution is low, the production cost of the L-theanine is reduced, and the popularization and the application of industrial production are facilitated.
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 present in tea leaves. The composition 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. The U.S. Food and Drug Administration (FDA) has identified L-theanine as a Generally Recognized As Safe (GRAS) product.
Gamma-glutamyltranspeptidase (GGT, EC 2.3.2.2) is a key enzyme in the glutathione metabolic pathway, and is a heterodimer composed of large and small subunits, and the reactions catalyzed by the heterodimer are of 2 types: one is a reaction for catalyzing hydrolysis of a gamma-glutamyl compound, and the other is a reaction for catalyzing transpeptidation in which a gamma-glutamyl group in a gamma-glutamyl compound is transferred to an amino acid or a polypeptide. Suzuki et al first reported that E.coli K-12 SH 642-derived GGT can catalyze L-glutamine and ethylamine to produce L-theanine. According to the reaction, the protection and deprotection of reactants are not needed, and the participation of ATP is not needed in the reaction process, so that the catalytic synthesis of L-theanine by using GGT becomes a hotspot of the current research. Coliform BL21 (DE 3)/pET 32 a-GGT are used by Wangli Mandarin and the like to catalyze L-glutamine and ethylamine to synthesize L-theanine, and the yield of the engineering bacteria which can catalyze and synthesize the L-theanine at the temperature of 32-37 ℃ is 20.9 g/L at the pH of 9.5. GGT from Bacillus subtilis SK 11.004 such as Digitalis concisely takes 20 mmol/L L-glutamine as a substrate at the temperature of 37 ℃ and the pH value of the GGT is 10.0, and the molar conversion rate can reach 95%. ShrutiBindal and the like utilize rBLGGT from Bacillus licheniformis ER-15 and 80 mmol/L L-glutamine as initial substrates, and can catalyze and synthesize 35.2 g/L-theanine after 3 times of supplement.
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 utilizing escherichia coli 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 5-20 g/L, and the temperature is adjusted to 28-35 ℃ for induction for 4-5 hours; then, supplementing 40-80% by volume of ethylamine solution at a constant flow rate with the volume ratio of fermentation liquor to ethylamine solution being 100-300: 1, and continuously performing fermentation culture for 20-30h under the conditions of pH 6.5-7.5, temperature being 35-40 ℃ and dissolved oxygen being 10-40% to synthesize 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, and the culture conditions are as follows: culturing at 35-40 deg.C and shaking table rotation speed of 200rmp until OD is 10-20;
the fermentation culture is to inoculate a strain to a fermentation medium by 5-15% of inoculation amount and then perform acidogenic fermentation, wherein the culture conditions are as follows: 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 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 above scheme, the formula of the seed culture medium is as follows: glucose 20-30 g/L, K2HPO4 10-20g/L, MgSO4·7H2O 0.5~3.0g/L，KH2PO4 10-20g/L, yeast powder 0.5-5.0g/L, (NH)4)2SO4 5.0~10.0g/L，5-10.0 mL/L of trace elements and pH 6.7.
As a further optimization of the scheme, the formula of the fermentation medium is as follows: glucose 20-30 g/L, K2HPO4 10-20g/L，MgSO4·7H20.5-3.0g/L of O, 0.5-3.0g/L of yeast powder, (NH)4)2SO45.0-10.0 g/L, 5-10.0 mL/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 of the invention has short fermentation period and high thallus density. The L-theanine has the advantages of high yield and yield, high saccharic acid conversion rate, less metabolic byproducts, low production cost and convenient extraction.
Detailed Description
A method for producing L-theanine by using an escherichia coli strain fermentation method. The method comprises the following specific steps:
1. method for producing L-theanine by using escherichia coli strain fermentation method
Slant culture: the original strain of escherichia coli is activated by a slant, cultured for 20 hours at 37 ℃ in a biochemical incubator, then diluted in a gradient manner, coated on a complete culture medium (LB), single colonies are selected and respectively inoculated on the slant, and cultured for 12-20 hours at 35-40 ℃ in the biochemical incubator.
Seed culture: selecting a single colony to be inoculated into a seed culture medium, wherein the culture conditions are as follows: the temperature is 35-40 ℃, the rotating speed of a shaking table is 200rmp, and the culture OD value is 10.0-20.0.
Fermentation culture: inoculating into a fermentation medium, wherein the inoculation amount is as follows: 5 to 15 percent, and culture conditions are as follows: the initial fermentation temperature is 35-40 ℃, the air flow is 5-50L/min, the stirring speed is 200-800 r/min to keep the dissolved oxygen at 10-40%, the fermentation pH value is controlled between 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 ensure that the concentration of the D-xylose is 5-20 g/L, the temperature is reduced to 28-35 ℃ for induction for 4-5 h, then, 40-80% of ethylamine solution is supplemented at a constant flow rate with the volume ratio of the fermentation liquid to the ethylamine solution being 100-300: 1, the fermentation culture is continued for 20-30h under the conditions of the pH value being 6.5-7.5, the temperature being 35-40 ℃ and the dissolved oxygen being 10-40%, and the fermentation liquid containing the L-theanine is obtained after the fermentation is finished.
The seed culture medium comprises the following components in percentage by weight: glucose 20-30 g/L, K2HPO410~20g/L, MgSO4·7H2O 0.5~3.0g/L，KH2PO4 10-20g/L, yeast powder 0.5-5.0g/L, (NH)4)2SO45-10.0 g/L, 5-10.0 mL/L of trace elements, and pH 6.7.
The fermentation medium comprises the following components in percentage by weight: glucose 20-30 g/L, K2HPO4 10~20g/L, MgSO4·7H20.5-3.0g/L of O, 0.5-3.0g/L of yeast powder, (NH)4)2SO45-10.0 g/L, 5-10.0 mL/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, and cleaning purified water for 2-3 times to obtain a supernatant; taking the supernatant to pass through a hollow fiber bundle membrane with the molecular weight of 1000-5000, wherein the recovery rate of the L-theanine is 92.3-95.4%. Passing the supernatant through a strong acid cation exchange column under the conditions of static adsorption: adding cationic resin according to the volume ratio of 20%, stirring and adsorbing for 1 h; dynamic adsorption: filling column resin according to 15% volume, feeding at the flow rate of 2BV/h, and flushing the column with 0.5-1 BV water at the final stage; the supernatant was collected. Adding activated carbon into the supernatant according to the volume ratio of 0.5-1% for decoloring for 0.5-2 h at 60 ℃, and filtering to remove carbon. Collecting L-theanine supernatant, and performing vacuum concentration according to the following conditions: vacuum degree of 0.1MPa, rotation speed of 100-200 rpm, and concentration at 50-80 ℃. And cooling and crystallizing the L-theanine concentrated solution. The extraction yield is 70.0-88.5%.
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, performing gradient dilution, coating on a complete culture medium (LB), selecting single colonies, respectively inoculating to the slant, and culturing for 20h at 35-40 ℃ in the biochemical incubator. And (3) selecting a single colony to be inoculated into a 500mL seed bottle, and culturing conditions are as follows: the culture OD value is 10.0 at 35-40 deg.C and 200rmp of shaking table rotation speed.
Inoculating into a fermentation medium, wherein the inoculation amount is as follows: 7%, culture conditions: the initial fermentation temperature is 35-40 ℃, the rotation 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 ℃ for induction for 4-5 h, then 40% ethylamine solution is supplemented at a constant flow rate with the volume ratio of fermentation liquor to ethylamine solution being 100:1, the fermentation culture is continued for 20-30h under the conditions that the pH value is 7.0-7.2, the temperature is 35-40 ℃ and the dissolved oxygen is 10-40%, and the L-theanine content is detected to be 38.37g/L after the fermentation is finished.
And as a comparison, adopting the same fermentation method, supplementing the ethylamine hydrochloride solution with the same concentration in the fermentation stage, and detecting that the content of L-theanine is 25.65 g/L. Compared with the contrast, the method of the invention improves the yield by 49.95%.
Example 2
Picking a ring of escherichia coli from a fresh slant and inoculating the escherichia coli into a 500mL seeding bottle, and culturing conditions are as follows: the temperature is 35-40 ℃, the rotating speed of a shaking table is 200rmp, and when the OD value is 15.0, the seeds in the shaking bottle 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 0.01-0.05 MPa, the air flow is 1-8L/min, the stirring speed is 200-800 r/min, 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.
And inoculating the secondary seeds into a 30L full-automatic fermentation tank filled with 18L fermentation medium by 10 percent of inoculation amount to perform acidogenic fermentation. The initial fermentation temperature is 35-40 ℃, the tank pressure is 0.01-0.15 MPa, the air flow is 5-50L/min, the stirring speed is 200-800 r/min, the dissolved oxygen is maintained at 10-40%, the fermentation pH value is controlled to be 6.5-7.5, the rotating speed of a shaking table is 200rmp, when the culture OD value reaches 25, D-xylose is added to enable the concentration of the D-xylose to be 15g/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 70% is added at a constant flow rate of 300:1 of the volume ratio of fermentation liquor to the ethylamine solution, fermentation culture is continued for 20-30 hours under the conditions of pH 6.5-7.2, the temperature is 35-40 ℃ and the dissolved oxygen content is 10-50%, and the L-theanine content is detected to be 120.33g/L after the fermentation is finished. As a control, the same method is adopted, the ethylamine hydrochloride solution with the same concentration is added in the fermentation stage, and the content of L-theanine is detected to be 67.24 g/L. Compared with the control, the method of the invention improves the yield by 78.96%.
Example 3
Picking a ring of escherichia coli from a fresh slant and inoculating the escherichia coli into a 500mL seeding bottle, and culturing conditions are as follows: the temperature is 35-40 ℃, the rotating speed of a shaking table is 200rmp, when the OD value is about 20.0, the seeds in the shaking bottle are inoculated with the solution with the volume of 0.6m32m of fermentation Medium3And performing secondary seed culture in a fermentation tank. The culture temperature is 35-40 ℃, the tank pressure is 0.01-0.05 MPa, and the air flow is 0.1-0.6 m3The dissolved oxygen is maintained at 10-50% at a stirring speed of 100-300 r/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 Medium3Acid-producing fermentation is carried out in a fermentation tank. The initial fermentation temperature is 35-40 ℃, the tank pressure is 0.01-0.15 MPa, the dissolved oxygen is maintained at 10-40%, the fermentation pH value is controlled at 6.5-7.5, the rotation speed of a shaking table is 200rmp, when the culture OD value reaches 30, D-xylose is added to enable the concentration of the D-xylose to be 20g/L, the temperature is reduced to 28-35 ℃ for induction for 4-5 h, an ethylamine solution with the volume concentration of 80% is supplemented at a constant flow rate with the volume ratio of a fermentation liquid to the ethylamine solution being 200:1, the fermentation culture is continued for 20-30h under the conditions that the pH is 6.5-7.2, the temperature is 35-40 ℃, and the dissolved oxygen content is detected to be 117.86g/L after the fermentation is finished. As a comparison, the same 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 61.25 g/L. Compared with the contrast, the method of the invention improves the yield by 91.42%.
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 (3)
1. A method for producing L-theanine by utilizing escherichia coli fermentation is characterized by comprising the following steps: in the method, in the fermentation culture process, when escherichia coli in a fermentation tank is cultured until OD is 25-30, D-xylose is added into the fermentation tank to enable the concentration of the D-xylose to be 5-20 g/L, and the temperature is adjusted to 28-35 ℃ for induction for 4-5 hours; then, supplementing 40-80% of ethylamine solution by volume concentration at a constant flow rate with the volume ratio of fermentation liquor to ethylamine solution being 100-300: 1, and continuously fermenting for 20-30h under the conditions of pH 6.5-7.5, temperature being 35-40 ℃ and dissolved oxygen being 10-40% to synthesize L-theanine;
the formula of the fermentation medium is as follows: glucose 20-30 g/L, K2HPO4 10-20g/L，MgSO4·7H20.5-3.0g/L of O, 0.5-3.0g/L of yeast powder, (NH)4)2SO45.0-10.0 g/L, 5-10.0 mL/L of trace elements, and initial pH of 7.0.
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, and the culture conditions are as follows: culturing at 35-40 deg.C and shaking table rotation speed of 200rpm until OD is 10-20;
the fermentation culture is to inoculate a strain to a fermentation medium by 5-15% of inoculation amount and then perform acidogenic fermentation, wherein the culture conditions are as follows: 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 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 rpm.
3. The method for producing L-theanine by fermentation using Escherichia coli as claimed in claim 2, wherein: the formula of the seed culture medium is as follows: glucose 20-30 g/L, K2HPO4 10-20g/L, MgSO4·7H2O 0.5~3.0g/L，KH2PO4 10-20g/L, yeast powder 0.5-5.0g/L, (NH)4)2SO45.0-10.0 g/L, 5-10.0 mL/L of trace elements, and pH 6.7.
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