CN115960974A

CN115960974A - Method for producing theanine by enzymatic method

Info

Publication number: CN115960974A
Application number: CN202310020262.3A
Authority: CN
Inventors: 刘珊珊; 刘辉; 周稳文; 秦永发
Original assignee: Beijing Tiankai Yida Biological Science & Technology Co ltd
Current assignee: Beijing Tiankai Yida Biological Science & Technology Co ltd
Priority date: 2017-06-15
Filing date: 2017-06-15
Publication date: 2023-04-14
Also published as: CN116042739A; CN116042745A; CN109136309B; CN109136309A

Abstract

The divisional application discloses a method for producing theanine by an enzymatic method, which comprises the following steps: (1) Reaction of theanine synthesis and ATP regeneration in a reaction tank; (2) separating theanine and ATP regenerating enzyme in the filter: separating theanine synthetase, ATP regenerating enzyme and AK enzyme from the reaction solution obtained in the step (1) by ultrafiltration through a filter, wherein the filtrate contains theanine, ATP, ADP, AMP and salt; the recovered theanine synthase, PPK enzyme, ADK enzyme and AK enzyme were detected, and used again for the reaction after adding a new enzyme. The invention has the following beneficial effects: 1) Adenosine is used for replacing ATP or AMP, so that a large amount of cost is saved for industrial production; 2) A stable enzyme recovery system is established, and the method is energy-saving and environment-friendly; 3) The byproducts ATP, ADP and AMP which are generated in small amount are directly used for the circulation reaction, or used for producing ATP, or are purified by methods such as filtration, ion exchange and the like, and the operation is simple.

Description

Method for producing theanine by enzymatic method

The present application is a divisional application of patent applications filed on 2017, 6/15/2017, with application number 201710453365.3, entitled "a production method for performing an enzymatic reaction using adenosine instead of ATP".

Technical Field

The invention relates to the technical field of biology, in particular to a production method for carrying out enzymatic reaction by using adenosine instead of ATP, and specifically relates to a method for producing theanine by an enzymatic method.

Background

Adenosine Triphosphate (ATP) consists of one adenosine and three phosphate groups, has a molecular weight of 507 and a molecular formula C ₁₀ H ₁₆ N ₅ O ₁₃ P ₃ . It is a converter and reservoir of biological energy and plays an irreplaceable important role in the enzymatic reactions that utilize and convert energy.

Because of the high cost of ATP, there is little benefit in the industrial production of ATP directly used for enzymatic reactions. Therefore, it is a research direction to use ATP for industrial production at present to recycle ATP during the reaction process by building a stable and effective ATP regeneration system by adding a small amount of ATP to the reaction production.

The method commonly used for regenerating ATP industrially is to utilize glycolysis pathway of yeast to regenerate ATP by substrate level phosphorylation. The method has the advantages of numerous enzymes participating in catalytic reaction, complex reaction process, difficult control of the reaction process and large quality difference among product batches. Meanwhile, the quality of yeast enzyme systems is greatly different due to different suppliers, different batches and even different seasons. In addition, a large amount of yeast cell enzyme liquid is required to be added in the reaction process, and a plurality of impurities such as protein, pigment and the like are introduced to bring certain difficulty to later-stage purification. In recent years, research on ATP regeneration has focused on the use of a single enzyme or a relatively simple enzyme system to achieve a highly efficient and stable regeneration effect. Among them, enzymes such as acetate kinase, ammonia kinase, pyruvate kinase, etc. can effectively regenerate ATP. However, the substrates used by these enzymes are expensive, such as phosphoenolpyruvate, which is used by pyruvate kinase; and the generated by-products have certain biological toxicity and pollution, for example, the products of the catalytic reaction of the acetate kinase and the ammonia kinase are respectively acetic acid and ammonia gas, so that the by-products are difficult to be used in large quantities in industrial production.

Patent CN201610268246.6 uses polyphosphoric acid or its salt as phosphate and energy donor, and uses polyphosphate kinase (PPK, EC 2.7.4.1), adenylate kinase (ADK, EC 2.7.4.3) and polyphosphate-adenylate transferase (PAP, EC 2.7.4), which is reported in literature to belong to one class of PPK enzymes, but IUBMB does not clearly classify the enzymes yet), three kinds of ATP regenerating enzymes are reasonably combined to regenerate ATP, and are applied to various ATP-requiring enzymatic reactions. The method has the advantages of relatively low reaction substrate price and less product pollution, and is suitable for industrial production. However, this method still requires the addition of a certain amount of ATP for the enzymatic reaction.

Disclosure of Invention

The invention provides a production method for enzymatic reaction by using adenosine to replace ATP, in particular to a method for producing theanine by an enzymatic method, which can further reduce the production cost, namely, adenosine kinase (AK, EC 2.7.1.20) is added into the original reaction system containing ATP regenerative enzyme, the enzyme can catalyze adenosine to generate AMP, and the enzymatic reaction can be carried out by combining other ATP regenerative enzymes, without using ATP, only adding a small amount of adenosine, thus being a great innovation in the field.

The technical problem to be solved by the invention is realized by the following technical scheme:

a production method for performing an enzymatic reaction using adenosine instead of ATP, the enzymatic reaction being an enzymatic reaction requiring ATP, comprising the steps of:

(1) In an enzymatic reaction system, adding ATP regenerating enzyme, AK enzyme and adenosine according to the proportion to carry out enzymatic reaction:

ATP regenerating enzyme and AK enzyme are obtained by genetic engineering modification, fermentation and purification, or are obtained by other modes such as natural extraction and the like. The ATP regenerating enzyme and the AK enzyme can be prepared into enzyme liquid or dry powder in the form of free enzyme; or further fixing on the immobilization carrier to obtain the immobilized ATP regenerating enzyme and AK enzyme.

In an enzymatic reaction system, ATP regenerating enzyme and AK enzyme are added in proportion, and adenosine is added to replace ATP to carry out enzymatic reaction. Wherein the reaction system is an aqueous solution containing adenosine, polyphosphoric acid or a salt thereof, and one or two of magnesium ions and manganese ions. In addition, the reaction system can also comprise one or more of potassium ions, sodium ions and ammonium ions, and one or more of Tris ions and phosphate ions. The added substrate, enzyme and various salts can be added into the reaction system at one time, and can also be added in batches and fed in a flowing manner according to the industrial production process flow.

(2) The immobilized ATP regenerating enzyme and AK enzyme are directly separated in a reaction tank, and the free ATP regenerating enzyme and AK enzyme are separated by an ultrafiltration membrane in a filter:

the immobilized ATP regenerating enzyme and AK enzyme are directly separated in a reaction tank. The separation can be carried out by a filter bag or directly in a reaction column. Or

The free ATP-regenerating enzyme and the AK enzyme were separated by ultrafiltration membrane in a filter. Wherein the filter is provided with a feed inlet, a discharge outlet and a reflux opening, and a trapped ultrafiltration membrane is arranged in the filter. The retentate passing through the filter is the recovered enzyme solution, and the filtrate is the reaction solution containing the product after the enzyme is separated.

(3) And (3) separating and purifying the filtrate obtained in the step (2) to obtain a product.

Preferably, in the above technical solution, the method further includes the steps of:

(4) Recovering the ATP regenerating enzyme and the AK enzyme, and reusing the ATP regenerating enzyme and the AK enzyme for the reaction in the step (1);

(5) A small amount of ATP, ADP or AMP generated in the reaction is separated by a filtration or ion exchange method to produce ATP; or recovering ATP, ADP or AMP, and reusing for the reaction in step (1).

Preferably, in the above technical solution, in the step (1), the ATP regenerating enzyme and the AK enzyme are immobilized enzymes or free enzymes, and the concentration of the AK enzyme is 0.01-8000U/L, wherein complete conversion of 1 μ M of the substrate within 1 minute is defined as 1 activity unit (U); the ATP regenerating enzyme is any two or three combinations of polyphosphate kinase (PPK), adenylate kinase (ADK) and polyphosphate-adenylate phosphotransferase (PAP), namely PPK and ADK, or ADK and PAP, or PPK, ADK and PAP; the enzyme addition amount is 0.01-5000U/L PPK enzyme concentration, 0.01-5000U/L ADK enzyme concentration and 0.01-5000U/L PAP enzyme concentration. The ATP regenerating enzyme and AK enzyme can be derived from any organism, or are artificially modified to have the same catalytic function.

Preferably, in the above technical scheme, the reaction conditions of step (1) are as follows:

the reaction temperature is 25-60 ℃, and the preferable temperature is 30-50 ℃;

the reaction pH is 5-10, preferably 6-9;

the reaction system comprises: adenosine; polyphosphoric acid or a salt thereof; one or two of magnesium ions and manganese ions;

adding ATP regenerating enzyme and AK enzyme in certain proportion in the enzyme reaction system to perform enzyme reaction.

Preferably, in the above technical solution, the reaction of step (1) further comprises:

one or more of ammonium ion, potassium ion or sodium ion; one or two combinations of Tris or phosphate ions; wherein, the concentration of potassium ion is 0.01-0.5M; the concentration of sodium ions is 0.01-0.5M; the concentration of ammonium ion is 0.01-0.3M; the concentration of Tris is 0.01-0.1M; the phosphate concentration is 0.01-0.1M.

Preferably, in the above technical scheme, the concentration of adenosine in the step (1) is 0.01-20g/L; the concentration of the polyphosphoric acid or salt thereof is 0.01-0.3M; the concentration of magnesium ions is 0.01-0.2M; the concentration of manganese ion is 0.005-0.15M.

Preferably, in the above technical solution, the magnesium ion is selected from one or more of magnesium chloride, magnesium sulfate, magnesium sulfite and magnesium nitrate; the manganese ions are selected from one or more of manganese chloride and manganese sulfate; the potassium ions are selected from one or more of potassium chloride, potassium sulfate, potassium nitrate, potassium hydroxide, potassium sulfite, potassium carbonate, potassium bicarbonate, potassium acetate, dipotassium hydrogen phosphate, potassium dihydrogen phosphate and potassium citrate; the sodium ions are selected from one or more of sodium chloride, sodium sulfate, sodium nitrate, sodium hydroxide, sodium sulfite, sodium carbonate, sodium bicarbonate, sodium acetate, disodium hydrogen phosphate, sodium dihydrogen phosphate and sodium citrate; the ammonium ion is selected from one or more of ammonium chloride, ammonium sulfate, ammonium nitrate, ammonia water, ammonium carbonate, ammonium bicarbonate, diammonium hydrogen phosphate, ammonium dihydrogen phosphate and ammonium acetate; the polyphosphoric acid or salt thereof is selected from one or more of sodium polyphosphate, potassium polyphosphate and ammonium polyphosphate.

Preferably, in the above technical solution, in the step (2), the ATP reproducing enzyme and the AK enzyme are immobilized on the immobilization carrier by: adsorption, entrapment, covalent bonding, conjugation, cross-linking, or combinations thereof; the immobilized carrier is selected from one or more of a macromolecular carrier, an inorganic carrier and a magnetic macromolecular microsphere carrier. Wherein the polymer carrier is selected from cellulose, glucose gel, agarose, polyacrylamide, polyamino acid, polystyrene, polyacrylic acid, sodium alginate, chitosan, starch, polyvinyl alcohol, gelatin, carrageenan, nylon or synthetic polymer, etc.; the inorganic carrier is selected from porous glass, silica gel, active carbon or diatomite.

Preferably, in the above technical solution, the ultrafiltration membrane is selected from a cellulose acetate membrane, a polysulfone membrane, a polyacrylonitrile membrane, a polyvinyl chloride membrane, a polyvinylidene fluoride membrane, a polyamide membrane, or a ceramic membrane.

Use of a production method for carrying out an enzymatic reaction using adenosine instead of ATP, for a variety of ATP-requiring enzymatic reactions, for synthesis of substances and cell-free protein expression.

Preferably, in the above technical scheme, the method is applied to a cell-free protein expression technology.

Preferably, in the above technical scheme, the enzymatic reaction requiring ATP is an enzymatic reaction involving a transferase (EC 2.7) transferring a phosphate group, and an enzymatic reaction involving a portion of ligase. The enzymatic reaction in which the transferase transferring phosphate groups (EC 2.7) takes part, for example: creatine phosphate, arginine phosphate, hexose 6-phosphate, 1,6-fructose phosphate, 3-glycerophosphate, oxidized coenzyme II, CT (D) P, GT (D) P, UT (D) P, and enzymatic reactions in which a part of ligase (EC 6) participates, for example: acetyl coenzyme A, carnosine, enteromycin, glutamine, L-theanine, phycocyanin, D-alanyl alanine and the like.

The catalytic enzymes and substrates required for the enzymatic synthesis reaction are shown in table 1 below. The catalytic enzymes listed in Table 1 may be derived from any organism or may be engineered to have the same catalytic function and are commercially available.

TABLE 1 enzymes and substrates for catalyzing enzymatic reactions according to the present invention

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Cell-free protein expression technology refers to protein synthesis in vitro from cell lysates containing components necessary for protein synthesis (ribosomes, transfer RNA, initiation/extension/termination factors, ATP, magnesium and potassium ions, etc.).

The technical scheme of the invention has the following beneficial effects:

1) The adenosine is used for replacing ATP or AMP, so that a large amount of cost is saved for industrial production, the selling price of the adenosine is only 10% of that of the ATP or about 30% of that of the AMP, the price is low, the source is wide, and the dosage of the adenosine in the reaction can be optimized to be less than 10% of that of the original ATP.

2) A stable enzyme recovery system is established, and both immobilized enzyme and free enzyme can be recycled in the whole reaction process, so that the method can be applied to large-scale continuous production, and is low in cost, energy-saving and environment-friendly;

3) The byproducts ATP, ADP and AMP generated in small amount are directly used for cyclic reaction, or used for producing ATP, or concentrated purified by methods such as filtration, ion exchange and the like, the operation is simple, and the purified finished product can be used as an additional product, thereby having economic benefit.

Drawings

FIG. 1 is an SDS-PAGE pattern of PPK enzyme, ADK enzyme, PAP enzyme and AK enzyme expressed in E.coli.

FIG. 2 is a flow chart of the reaction process using free enzyme according to the present invention.

FIG. 3 is a process flow diagram of the reaction of the present invention using immobilized enzymes.

Fig. 4 is a graph showing the residual amount of creatine and the amount of creatine phosphate produced by High Performance Liquid Chromatography (HPLC).

FIG. 5 is a SDS-PAGE pattern of the gshF enzyme expressed using cell-free extracts of the invention.

Detailed Description

Specific examples of the invention are described in detail below to facilitate a further understanding of the invention.

The various materials used in the following examples and comparative examples of the present invention are commercially available unless otherwise specified.

EXAMPLE 1 preparation of crude enzyme

The ATP regenerating enzyme and AK enzyme in the method of the invention can be obtained commercially, or enzymes with the same catalytic function after artificial modification.

The enzyme was prepared as follows:

primers are designed according to the gene sequences of PPK enzyme, ADK enzyme, PAP enzyme and AK enzyme, gene fragments are respectively amplified through PCR and are respectively connected to pET22b vectors (sold on the market), and after the sequencing is correct, the gene fragments are respectively transferred into E.coli BL21 (DE 3) strains (sold on the market).

And (3) inoculating the transformed E.coli BL21 (DE 3) monoclonal into an LB culture medium, after culturing to a logarithmic phase, adding 1mM isopropyl-beta-D-thiogalactopyranoside (IPTG) for induction, collecting thalli after induction for 5 hours, and screening high-expression strains by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Inoculating the screened high-expression strain into a seed culture medium under an aseptic condition, culturing until the strain reaches a logarithmic growth phase, carrying out expanded culture, finally inoculating into a fermentation tank containing 500L of fermentation culture medium, adding 1mM IPTG (isopropyl-beta-thiogalactoside) for induction for 5 hours when the strain is cultured until the OD600 value is 20-30, and centrifuging to collect thalli.

Wherein the LB culture medium comprises the following components: 1% peptone, 0.5% yeast powder and 1% NaCl; the seed culture medium comprises the following components: 1% peptone, 0.5% yeast powder and 1% sodium chloride; the fermentation medium comprises the following components: 1% peptone, 0.5% yeast powder, 1% sodium chloride, 5% disodium hydrogen phosphate, 1% sodium dihydrogen phosphate, 0.01% magnesium sulfate and 1% glycerol.

FIG. 1 is an SDS-PAGE pattern of PPK enzyme, ADK enzyme, PAP enzyme and AK enzyme expressed in E.coli. As shown in fig. 1: lane 1 is protein marker 14.4-116kDa (commercially available); lane 2 is PPK enzyme, 40kDa; lane 3 is ADK enzyme, 25kDa; lane 4 PAP enzyme, 55kDa; lane 5 is AK enzyme, 40kDa.

The harvested thalli is subjected to ultrasonic or high-pressure homogenate and bacterium breaking, and then the supernatant is collected by centrifugation. The crude enzyme is obtained by precipitation and filtration.

Example 2 enzymatic production of creatine phosphate

FIG. 2 is a flow chart of the reaction process using free enzyme according to the present invention. As shown in fig. 2, the procedure for preparing creatine phosphate by the enzyme method is as follows:

(1) Reaction for synthesizing creatine phosphate in reaction tank:

in the reaction tank, a 100L reaction system was a solution containing 2.0kg of creatine as a substrate, 0.2kg of adenosine, 1.8kg of sodium polyphosphate, 0.4kg of potassium chloride, 0.5kg of magnesium chloride hexahydrate and 0.3kg of disodium hydrogen phosphate, and was stirred uniformly during preparation to prevent precipitation. The pH value is adjusted to 7.0, and 1000U/L, PPK enzyme 500U/L, ADK enzyme 500U/L and AK enzyme 500U/L are added into the reaction system to start the reaction. During the reaction, the pH was controlled at 7.0 and the temperature at 35 ℃.

Fig. 4 is a graph showing the residual amount of creatine and the amount of creatine phosphate produced by High Performance Liquid Chromatography (HPLC). As shown in FIG. 4, the amount of creatine remaining and the amount of creatine phosphate produced were measured by High Performance Liquid Chromatography (HPLC) every 1 hour, and the amount of creatine phosphate produced was 30g/L after 6 hours of reaction. The HPLC detection conditions are as follows: a Kromasil C18 column (available from AKZO NOBEL Co.) (150X 4.6 mm) with a detection wavelength of 210nm, a detection temperature of 25 ℃ and a detection flow rate of 1ml/min was used as a mobile phase, and an aqueous solution (pH 6.6) containing 0.2% of potassium dihydrogen phosphate, 0.1% of tetrabutylammonium hydroxide and 5% of acetonitrile was used as a mobile phase.

(2) Isolation of creatine kinase, ATP regenerating enzyme and AK enzyme in filter:

the reaction solution of the reaction system of step (1) is passed through a filter to separate creatine kinase, ATP regenerating enzyme and AK enzyme by ultrafiltration method, the filter is packed with a membrane (available from Pall corporation, molecular weight cut-off 20 kDa), the filtrate is the reaction solution after enzyme separation, contains creatine phosphate, ATP, ADP, AMP and salts, etc., and can be further purified by ion exchange chromatography, etc.

The activities of the recovered creatine kinase, PPK enzyme, ADK enzyme and AK enzyme were detected to be decreased by 5% -20% compared to those before the reaction, and the recovered creatine kinase, PPK enzyme, ADK enzyme and AK enzyme were reused in the reaction of step (1) after adding the corresponding new enzyme.

EXAMPLE 3 enzymatic production of 1,6-fructose diphosphate (immobilized enzyme)

FIG. 3 is a process flow diagram of the reaction of the method of the present invention using immobilized enzymes. As shown in FIG. 3, the operation steps of the preparation of 1,6-fructose diphosphate by the immobilized enzyme method are as follows:

(1) Immobilization of catalytic enzymes, ATP regenerating enzymes and AK enzymes:

catalytic enzymes Fructokinase (FK) and Phosphofructokinase (PFK) were commercially available and immobilized on a commercial epoxy-based immobilization support LX1000EP along with the ADK enzyme, PAP enzyme and AK enzyme initially purified in example 1.

FK, PFK, ADK, PAP and AK enzymes were mixed in a ratio of 2:2:1:1:1 to prepare a mixed enzyme solution, wherein the activity of AK enzyme in the enzyme solution is 2000U/L. 2kg of LX1000EP wet carrier was added to the stirring vessel at a constant temperature, mixed with the above enzyme solution, and stirred at 20 ℃ and 150rpm for 12 hours. Filtering and collecting the carrier, and washing for 2 times by using 0.02M potassium phosphate buffer solution with the pH value of 8.0 to obtain the immobilized mixed enzyme.

(2) Producing 1,6-fructose diphosphate in the reaction column:

a reaction solution was prepared, each 100L of which contained 1.8kg of fructose as a substrate, 0.3kg of adenosine, 2.0kg of sodium hexametaphosphate, 0.5kg of ammonium chloride, 0.5kg of sodium chloride, 0.5kg of magnesium sulfate heptahydrate, 0.2kg of manganese sulfate, and 0.85kg of disodium hydrogenphosphate, and was stirred uniformly during preparation to prevent precipitation. The pH value is adjusted to 6.8 and the temperature is raised to 35-40 ℃.

And (2) loading 10kg of the mixed immobilized enzyme obtained in the step (1) into a reaction column, and discharging bubbles to obtain the enzyme reaction column. The reaction solution was slowly passed through the enzyme reaction column from bottom to top at a flow rate of 30L/h using a constant flow pump, the temperature being controlled at 37 ℃ during the reaction. After the cyclic reaction for 6 hours, the reaction solution was collected, and the amount of produced 1,6-fructose diphosphate was determined to be 30g/L.

The immobilized enzyme is circularly reacted for more than 20 times or stored for more than one month at the temperature of minus 4 ℃, the enzyme activity is reduced by 10 to 15 percent, and partial new enzyme is supplemented or replaced according to the proportion.

EXAMPLE 4 enzymatic production of carnosine

As shown in FIG. 2, the procedures for preparing carnosine by the enzyme method and coupling free ATP regenerating enzyme and AK enzyme are as follows:

(1) Reaction for the synthesis of carnosine in the reaction tank:

in the reaction tank, 100L of a reaction system was a solution containing 1.8kg of L-histidine as a substrate, 1.0kg of beta-alanine, and 0.25kg of adenosine, 1.0kg of sodium polyphosphate, 0.3kg of sodium chloride, 0.38kg of potassium chloride, 1.0kg of magnesium chloride hexahydrate, and 0.6kg of disodium hydrogen phosphate, and was stirred uniformly during preparation to prevent precipitation. The pH was adjusted to 8.0, and 1000U/L of carnosine synthase, 600U/L of ADK enzyme, 600U/L of PAP enzyme, and 800U/L of AK enzyme were added to the reaction system to start the reaction. During the reaction, the pH was controlled at 8.0 and the temperature at 37 ℃.

After 6 hours of reaction, the amount of carnosine produced was 21g/L. The HPLC detection conditions are as follows: kromasil C18 column (available from AKZO NOBEL Co.) (150X 4.6 mM), detection wavelength 210nm, detection temperature 25 ℃, detection flow rate 0.8ml/min, mobile phase containing 80mM phosphate buffer and 15% methanol aqueous solution.

(2) Isolation of carnosine synthase and ATP regenerator enzymes in the filter:

separating the reaction solution of the reaction system in the step (1) from the carnosine synthetase, ATP regenerating enzyme and AK enzyme by an ultrafiltration method through a filter, wherein the filter is internally provided with a membrane package (purchased from Pall company, with a molecular weight cut-off of 20 kDa), and the filtrate is the reaction solution after enzyme separation, contains carnosine, ATP, ADP, AMP, salt and other substances, and can be further purified by means of ion exchange chromatography and the like.

The activities of the recovered carnosine synthetase, ADK enzyme, PAP enzyme and AK enzyme were detected to be decreased by 10% -20% as compared with those before the reaction, and the recovered carnosine synthetase, ADK enzyme, PAP enzyme and AK enzyme were added to the reaction solution before the reaction, and the reaction solution was used again in the step (1).

EXAMPLE 5 enzymatic production of theanine

As shown in FIG. 2, the enzymatic preparation of theanine coupled with free ATP regenerating enzyme and AK enzyme comprises the following steps:

(1) Reaction for theanine synthesis and ATP regeneration in the reaction tank:

in a reaction tank, a 100L reaction system is a solution containing 1.5kg of sodium glutamate as a substrate, 0.7kg of ethylamine hydrochloride, 0.2kg of adenosine, 2.0kg of tetrapolyphosphoric acid, 1.0kg of magnesium chloride hexahydrate and 0.5kg of disodium hydrogen phosphate, and the solution is uniformly stirred during preparation to prevent precipitation. The pH was adjusted to 7.0, and 500U/L theanine synthetase, 500U/L PPK enzyme, 500U/L ADK enzyme, and 800U/L AK enzyme were added to the reaction system to start the reaction. During the reaction, the pH was controlled at 7.0 and the temperature at 30 ℃.

After 5 hours of the reaction, the amount of theanine produced was 14g/L. The HPLC detection conditions are as follows: kromasil C18 column (available from AKZO NOBEL Co.) (150X 4.6 mm), detection wavelength 203nm, detection temperature 30 ℃, detection flow rate 1ml/min, mobile phase containing 0.05% TFA and 5% acetonitrile in water solution.

(2) Separation of theanine and ATP regenerating enzymes in the filter:

separating theanine synthetase, ATP regenerating enzyme and AK enzyme from the reaction solution of the reaction system in the step (1) by ultrafiltration, wherein the filter is packed with a membrane (available from Pall corporation, molecular weight cut-off 20 kDa), and the filtrate is the reaction solution after enzyme separation, and contains theanine, ATP, ADP, AMP and salt, and can be further purified by ion exchange chromatography or the like.

The activities of the recovered theanine synthase, PPK enzyme, ADK enzyme and AK enzyme were detected to be decreased by 5-10% compared with those before the reaction, and the enzyme was added with a new enzyme and used again in the reaction of step (1).

Example 6 production of creatine phosphate by enzymatic Process

As shown in FIG. 2, the enzymatic preparation of creatine phosphate while coupling free ATP regenerating enzyme and AK enzyme is performed as follows:

(1) Reaction for synthesizing creatine phosphate in reaction tank:

in a reaction tank, a 100L reaction system is a solution containing 0.2kg of creatine substrate, 0.001kg of adenosine, 0.47kg of tetrapolyphosphoric acid, 0.053kg of ammonium chloride, 0.2kg of magnesium chloride hexahydrate, 0.072kg of manganese chloride monohydrate and 1.2kg of Tris, and the solution is uniformly stirred during preparation to prevent precipitation. The pH value is adjusted to 10.0, creatine kinase 1U/L, PPK enzyme 0.01U/L, ADK enzyme 0.01U/L, PAP enzyme 0.01U/L and AK enzyme 0.01U/L are added into the reaction system to start the reaction. During the reaction, the pH was controlled to 10.0 and the temperature was controlled to 55 ℃.

After 5 hours of reaction, the amount of creatine phosphate produced was 2g/L by HPLC. HPLC detection conditions were the same as in step (1) of example 2.

The activities of the recovered creatine kinase, PPK enzyme, ADK enzyme, PAP enzyme and AK enzyme were detected to be reduced by 30% -50% compared with those before the reaction, and the recovered creatine kinase, PPK enzyme, ADK enzyme, PAP enzyme and AK enzyme were reused in the reaction of step (1) after adding corresponding new enzyme.

Example 7 production of creatine phosphate by enzymatic Process

Referring to fig. 2, the procedure for enzymatic preparation of creatine phosphate while coupling free ATP-regenerating enzyme and AK enzyme is as follows:

(1) Reaction for synthesizing creatine phosphate in reaction tank:

in a reaction tank, 100L of a reaction system was a solution containing 2.0kg of creatine as a substrate, 2.0kg of adenosine, 14.0kg of tetrapolyphosphoric acid, 3.73kg of potassium chloride, 4.07kg of magnesium chloride hexahydrate and 1.56kg of sodium dihydrogen phosphate dihydrate, and was uniformly stirred during preparation to prevent precipitation. The pH value is adjusted to 5.0, creatine kinase 2000U/L, PPK enzyme 1000U/L, PAP enzyme 1000U/L and AK enzyme 1000U/L are added into the reaction system to start the reaction. During the reaction, the pH was controlled at 5.0 and the temperature at 25 ℃.

After 8 hours of reaction, the amount of creatine phosphate produced was 14g/L as measured by HPLC. HPLC detection conditions were the same as in step (1) of example 2.

The activities of the recovered creatine kinase, PPK enzyme, PAP enzyme and AK enzyme were detected to be reduced by 20% -40% compared with those before the reaction, and the recovered creatine kinase, PPK enzyme, PAP enzyme and AK enzyme were reused in the reaction of step (1) after adding corresponding new enzyme.

EXAMPLE 8 enzymatic production of 1,6-fructose diphosphate (immobilized enzyme)

Referring to FIG. 3, the procedure for preparing 1,6-fructose diphosphate by the immobilized enzyme method while coupling ATP regenerating enzyme and AK enzyme is as follows:

catalytic enzymes Fructokinase (FK) and Phosphofructokinase (PFK) were commercially available, and the ATP regenerating enzymes PPK and PAP, which were preliminarily purified in the same manner as in example 1, were immobilized on a commercially available epoxy group-immobilized carrier LX1000EP or an amino group-containing synthetic polymer carrier LX1000HA, respectively.

FK, PFK, PPK, PAP and AK enzymes were prepared into enzyme solutions, respectively, with the activity of each of the enzyme solutions, FK, PFK and AK, 8000U/L, and the activity of each of the enzyme solutions, PPK and PAP, 5000U/L.

1kg of LX1000EP wet carrier and 3L of FK enzyme solution were added to a constant temperature stirring tank, and stirred at 20 ℃ and 150rpm for 12 hours. Filtering and collecting the carrier, and washing with 0.02M pH 8.0 potassium phosphate buffer solution for 2 times to obtain the immobilized FK enzyme. The PFK enzyme was immobilized in the same manner.

1kg of LX1000HA wet carrier and 3L of PPK enzyme solution were added to a constant temperature agitation tank, and stirred at 150rpm for 12 hours at 20 ℃. Filtering and collecting the carrier, and washing for 2 times by using 0.02M pH 8.0 potassium phosphate buffer solution to obtain the immobilized PPK enzyme. The PAP and AK enzymes were immobilized in the same manner.

(2) Producing 1,6-fructose diphosphate in the reaction column:

a reaction solution was prepared containing 3.6kg of fructose as a substrate, 2.0kg of adenosine, 8.0kg of sodium hexametaphosphate, 1.6kg of ammonium chloride, 4.0kg of magnesium chloride hexahydrate and 1.56kg of sodium dihydrogen phosphate dihydrate per 100L, and was stirred uniformly during preparation to prevent precipitation. The pH was adjusted to 6.0 and the temperature increased to 50 ℃.

And (2) loading 10kg of the mixed immobilized enzyme obtained in the step (1) into a reaction column, and discharging bubbles to obtain the enzyme reaction column. The reaction solution slowly passes through the enzyme reaction column from bottom to top at a flow rate of 30L/h by using a constant flow pump, and the temperature is controlled to be 50 ℃ during the reaction. After the cyclic reaction is carried out for 6 hours, the reaction liquid is collected, and the generation amount of 1,6-fructose diphosphate is 25g/L by HPLC detection.

The immobilized enzyme is circularly reacted for more than 20 times or stored for more than one month at the temperature of minus 4 ℃, the enzyme activity is reduced by 20 to 40 percent, and partial new enzyme needs to be supplemented or replaced according to the proportion.

Example 9 cell-free protein expression of GshF enzyme

The operation steps of expressing GshF enzyme without cell protein and coupling ATP regenerative enzyme and AK enzyme at the same time are as follows:

(1) Construction of expression vector pIVEX2.4d-gshF:

designing a pair of amplification primers according to the sequence of the gshF gene, wherein the primer sequences are as follows:

gshF sense primer: 5'-TCCATGGCATTAAACCAACTTCTTCAAAAACTG-3'; and

gshF antisense primer: 5'-CGGATCCTTAAGTTTGACCAGCCACTATTTC-3';

the gshF gene fragment was amplified by PCR using the extracted Streptococcus thermophilus strain DNA as a template, digested simultaneously with Nco I and BamH I, ligated to pIVEX2.4d vector (purchased from Roche) to construct pIVEX2.4d-gshF vector, and sequenced correctly.

(2) Preparation of cell-free extract of Escherichia coli:

inoculating E.coli A19 (lacking nuclease I gene, not degrading exogenous gene) into LB culture medium, and culturingInoculating into fermentation tank containing 5L fermentation medium until logarithmic growth phase, and culturing to OD ₆₀₀ When the concentration reached 3, the cells were collected by centrifugation. See example 1 for LB medium and fermentation medium composition.

Resuspending the cells in S30 buffer, washing 3 times at 4 deg.C, collecting cells, and storing at-80 deg.C. The S30 buffer solution comprises the following components: 14mM magnesium acetate, 60mM potassium acetate, 1mM Dithiothreitol (DTT), and 10mM Tris (pH 8.1).

The cells were weighed, and 100ml of S30 buffer solution was added to 10 g of the cells to re-dissolve the cells, and 1mM DTT was added thereto. The cells were disrupted by high-pressure homogenization under a pressure of 75kPa, and then 1mM DTT was added thereto, followed by centrifugation at 4 ℃ to collect the supernatant. The cells were incubated for 30 minutes at 37 ℃ in a constant temperature shaker at 100rpm to obtain a cell-free extract of E.coli.

(3) Expression of proteins using cell-free extracts and coupling of ATP regenerating enzymes and AK enzymes

Adding 20 ul of the Escherichia coli cell-free extract obtained in the step (2) into a 100 ul protein expression system, adding 15 ug/ml of the expression vector pIVEX2.4d-gshF obtained in the step (1), adding 15mM magnesium acetate, 50mM ammonium acetate, 50mM HEPES-potassium hydroxide (pH 7.5), 2% PEG8000, 2mM DTT, 0.33mM NAD ⁺ 0.27mM coenzyme A, 4mM oxalic acid, 1U/. Mu. l T7 RNA polymerase, 2mM adenosine, 1mM GTP, 1mM CTP, 1mM UTP, and 2mM each of 20 amino acids were added while 1U/. Mu.l PPK enzyme, 1U/. Mu.l ADK enzyme, 1U/. Mu.l AK enzyme, and 10mM sodium polyphosphate were added for ATP production and regeneration. The reaction was carried out in a constant temperature shaker at 200rpm and 30 ℃ for 6 hours.

FIG. 5 is a SDS-PAGE graph of gshF enzyme expression using cell-free extracts, as shown in FIG. 5: lane 1 is protein marker 14.4-116kDa; lane 2 is cell-free expressed gshF enzyme, 85kDa.

Comparative example 1 production of creatine phosphate by enzymatic Process

The operation steps for preparing the creatine phosphate by the enzyme method are as follows:

in the reaction tank, a 100L reaction system was a solution containing 2.0kg of creatine as a substrate, 8.0kg of ATP, 0.4kg of potassium chloride, 0.5kg of magnesium chloride hexahydrate and 0.3kg of disodium hydrogenphosphate, and was uniformly stirred at the time of preparation to prevent precipitation. The pH value was adjusted to 7.0, and 1000U/L creatine kinase was added to the reaction system to start the reaction. During the reaction, the pH was controlled at 7.0 and the temperature at 35 ℃.

After 6 hours of reaction, the creatine phosphate production was 25g/L, and 90% or more of ATP was converted into ADP and AMP. HPLC detection conditions were the same as in step (1) of example 2.

As can be seen from comparative example 2: adenosine is used for replacing ATP, AK enzyme and ATP regeneration enzyme are added for enzymatic reaction, although the enzyme amount required by the reaction is increased, the adenosine dosage is less, the price is low, and a large amount of cost is saved for industrial production. The byproducts ATP, ADP and AMP which are generated in small amount are directly used for the circulation reaction or used for producing ATP, has certain economic benefit and is more suitable for industrial large-scale production.

Meanwhile, the ATP regeneration system described in the present invention can also be applied to various enzymatic reactions requiring ATP.

Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited thereto, and various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention.

Claims

1. An enzymatic method for producing theanine, comprising the steps of:

(1) Reaction for theanine synthesis and ATP regeneration in the reaction tank:

in a reaction tank, a 100L reaction system is a solution containing 1.5kg of sodium glutamate as a substrate, 0.7kg of ethylamine hydrochloride, 0.2kg of adenosine, 2.0kg of tetrapolyphosphoric acid, 1.0kg of magnesium chloride hexahydrate and 0.5kg of disodium hydrogen phosphate, and is uniformly stirred during preparation to prevent precipitation; adjusting the pH value to 7.0, adding 500U/L theanine synthetase, 500U/L PPK enzyme, 500U/L ADK enzyme and 800U/L AK enzyme into the reaction system to start reaction; controlling the pH value to be 7.0 and the temperature to be 30 ℃ during the reaction period;

(2) Separation of theanine and ATP regenerating enzymes in the filter:

separating theanine synthetase, ATP regenerative enzyme and AK enzyme from the reaction liquid of the reaction system in the step (1) by a filter through an ultrafiltration method, wherein the filter is internally provided with a membrane package, and the filtrate is the reaction liquid after separating the enzyme, contains theanine, ATP, ADP, AMP and salt, and is further purified by an ion exchange chromatography mode;

the activities of the recovered theanine synthase, PPK enzyme, ADK enzyme and AK enzyme are detected to be reduced by 5-10% compared with the activities before the reaction, and the recovered theanine synthase, PPK enzyme, ADK enzyme and AK enzyme are used for the reaction in the step (1) after corresponding new enzyme is added.