CN114875011B

CN114875011B - AMP phosphotransferase mutant, coding gene thereof and application thereof in ATP synthesis

Info

Publication number: CN114875011B
Application number: CN202210500299.1A
Authority: CN
Inventors: 秦浩杰; 任丽梅; 杨梦茜; 张蕴之; 米雅萱; 王素霞; 刘�东; 高文杲
Original assignee: Meibangmeihe Biotechnology Co ltd
Current assignee: Meibangmeihe Biotechnology Co ltd
Priority date: 2022-05-10
Filing date: 2022-05-10
Publication date: 2024-02-27
Anticipated expiration: 2042-05-10
Also published as: CN114875011A

Abstract

The invention discloses an AMP phosphotransferase mutant, a coding gene thereof and application thereof in ATP synthesis. The amino acid sequence of the mutant consists of SEQ: ID: the sequence shown in the NO. 1 is obtained by one or more mutations. Comprises one or more mutation sites of the 124 th (A124L), 312 th (G312M) and 393 (F393K) mutation sites. The invention also provides a composite biocatalyst comprising an adenosine kinase and an AMP phosphotransferase mutant and its use in ATP synthesis. The mutant has higher enzyme activity, higher stability and higher ATP generation ratio, and the repeated batch after immobilization is more than 100 batches, so that the cost for producing ATP by a full-enzyme method can be further reduced, and the invention can reduce the enzyme types to 2, can complete the ATP synthesis reaction by the integral adenosine phosphate, and is beneficial to large-scale industrial application.

Description

AMP phosphotransferase mutant, coding gene thereof and application thereof in ATP synthesis

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an AMP phosphotransferase mutant, a coding gene thereof and application thereof in ATP synthesis.

Background

Adenosine triphosphate, ATP for short, is an unstable high-energy compound which is mutually converted with ADP in cells to realize energy storage and energy release, thereby ensuring the energy supply of various vital activities of the cells. ATP plays an important role in human energy metabolism, and is involved in metabolism of carbohydrates, proteins, nucleic acids, fats, and the like in the living body as an intermediate of metabolism and a coenzyme. In clinical application, the traditional Chinese medicine composition has good treatment and auxiliary treatment effects on muscular atrophy, apoplexy sequela, myocardial infarction, coronary arteriosclerosis, hepatitis and other diseases.

The synthesis of ATP mainly includes chemical and biological methods, and biological methods also include microbial fermentation and biological enzyme catalysis. The microbial fermentation method is to synthesize ATP by glycolysis and substrate level phosphorylation by using a yeast enzyme system and using an in-vivo enzyme of yeast as a catalyst. Although the method has good production effect and low cost, the method has the problems of complex reaction process, numerous enzyme systems participating in catalytic reaction, difficult control of the reaction process, large quality difference among product batches, complex separation and purification and the like.

The biological enzyme catalysis method takes biological enzyme as a catalyst, and is in direct contact with a substrate, so that the substrate conversion rate is high, the production cost is low, and the production process is more efficient and stable, easy to control, energy-saving and environment-friendly. In terms of raw material cost, the fermentation method needs to consume a large amount of yeast mud, glucose and phosphate besides a substrate and a certain amount of inorganic salt such as magnesium salt, and the biological enzyme method needs to be completed by adding a certain amount of phosphate donor and corresponding catalytic enzyme.

Adenosine Kinase (AK), which belongs to the family of ribokinase enzymes and catalyzes the phosphorylation of Adenosine to AMP, and also can catalyze reactions with ATP as a phosphate donor, is the first enzyme of the Adenosine pathway, and thus plays a key role in regulating intracellular and extracellular Adenosine levels. AMP phosphotransferase (AMP phosphotransferase, AMP-PPT) is an enzyme that phosphorylates AMP to ADP using polyphosphoric acid as a phosphate donor. The AMP phosphotransferase mutant (AMP-PPTM) is a mutant with high ATP proportion, which is obtained by mutating the gene of the AMP phosphotransferase mutant by utilizing a PCR technology based on the original strain and screening out the enzyme activity, stability and the generated ATP proportion, so that the original functions are changed, and the double functions of the AMP phosphosynthesis ADP and the ADP phosphosynthesis ATP are achieved.

Compared with the fermentation method, the enzyme catalysis method has the advantages of high efficiency, stability, simple reaction system, easy operation of the reaction process and the like. In 2001, akihiko MARUYAMA et al reported that ATP was synthesized under the catalysis of amino acid-producing bacteria using adenine as a substrate, and that the ATP production amount was 70.6mg/mL after 28 hours of reaction, and the substrate conversion rate was 82%. A method for producing Adenosine Triphosphate (ATP) is proposed in patent CN 104762347A. The process is complex, the reaction process is not easy to control, the cost is high, the activity of the enzyme system is fast to drop, the concentration of substrate adenosine is 30g/L, the reaction is carried out for 4.5 hours, the adenosine conversion rate is 95.3%, the ATP generation amount is about 42.5g/L, the ATP generation rate is 89.57%, and the total yield is 81.2%. The patent CN110777180A discloses a method for preparing adenosine triphosphate by an immobilized enzyme method, wherein the method adopts three enzyme combinations for immobilization, the concentration of adenosine is 30g/L, the reaction is carried out for 6 hours, and 445g of adenosine triphosphate dry product is obtained through separation and purification. The reaction time is relatively longer, the enzyme combination variety is more, and the addition amount of the immobilized enzyme is large, so that the production cost is improved to a certain extent. Patent CN106191170a discloses a multi-enzyme participated ATP synthesis process. The process takes adenosine as a substrate, adopts four enzymes of AK enzyme, ppk2 enzyme, AK enzyme and Pap enzyme to react in combination to synthesize ATP, the concentration of the substrate is 30g/L, the adenosine conversion rate is 85% after 8 hours of reaction, the ATP generation rate is 87.71%, the generation amount is about 50g/L, and the total yield is 80%. The process enzyme combination consists of four enzymes, so that the cost is increased, side reactions are increased along with the increase of enzyme types, the effects on the later separation and purification are greatly increased, and meanwhile, the reaction is inhibited to a certain extent due to the increase of the enzyme types, so that a series of problems such as incomplete substrate conversion occur.

In summary, in order to further achieve the purposes of reducing the production cost, simplifying the process flow, improving the substrate conversion rate, improving the product quality and being environment-friendly in the production process, a new catalytic enzyme with better activity and stronger stability and a simpler and more efficient ATP production process need to be researched and developed.

Disclosure of Invention

The invention aims to provide an AMP phosphotransferase mutant, a coding gene thereof and application thereof in ATP synthesis, so as to solve the problems of low enzyme activity, multiple enzyme types, high cost, difficult process control and the like in the existing ATP synthesis technology.

The invention aims at realizing the following technical scheme:

selection and evolutionary screening of (one) AMP-PPTM enzyme

The invention provides an AMP phosphotransferase mutant, which is any one of the following mutants:

mutant 1, said mutant 1 being as set forth in SEQ: ID: on the basis of the amino acid sequence shown in the No. 1, the 124 th Ala is mutated into Leu;

mutant 5, said mutant 5 being in SEQ id no: ID: on the basis of the amino acid sequence shown in the NO. 1, the 124 th Ala of the amino acid sequence is mutated into Leu, and the 312 th Gly of the amino acid sequence is mutated into Met;

mutant 6, said mutant 6 being as set forth in SEQ id no: ID: on the basis of the amino acid sequence shown in the NO. 1, the 124 th Ala of the amino acid sequence is mutated into Leu, and the 177 th Gly of the amino acid sequence is mutated into Ala;

mutant 7, said mutant 7 being as set forth in SEQ: ID: on the basis of the amino acid sequence shown in the No. 1, the 124 th Ala of the amino acid sequence is mutated into Leu, and the 393 rd Phe of the amino acid sequence is mutated into Lys;

mutant 8, said mutant 8 being as set forth in SEQ id no: ID: on the basis of the amino acid sequence shown in the No. 1, the 124 th Ala is mutated into Leu, the 177 th Gly is mutated into Ala, and the 393 th Phe is mutated into Lys;

mutant 9, said mutant 9 being as set forth in SEQ id no: ID: on the basis of the amino acid sequence shown in the No. 1, the 124 th Ala is mutated into Leu, the 312 th Gly is mutated into Met, and the 393 th Phe is mutated into Lys;

mutant 10, said mutant 10 being as set forth in SEQ id no: ID: based on the amino acid sequence shown in NO. 1, gly at position 177 is mutated to Ala, gly at position 312 is mutated to Met, and Phe at position 393 is mutated to Lys.

The invention also provides genes encoding the above AMP phosphotransferase mutants.

The invention selects Pseudomonas aeruginosa (Paeruginosa pseudomonas) species, NCBI sequence No.: 553898144 is the original sequence, and forward mutation points with better vitality are obtained by using a vitality screening method after random mutation. And (3) carrying out accumulated mutation on the mutant with high initial screening activity, namely overlapping forward mutation points through a molecular biological means to improve the original ATP synthesis activity. Of the many mutations, 10 representative forward mutants were screened. Found in the late-phase accumulation experiments of these 10 representative mutants. Wherein, the mutant 9 containing the 124 th (A124L), 312 th (G312M) and 393 (F393K) mutation sites can improve the synthesis activity of ATP by about 80 times, the temperature stability can be improved by 20 times at 50 ℃ for 15min, the effect of synthesizing ATP with high single enzyme capacity can be achieved, and the mutant is named AMP-PPTM again.

(II) preparation of a composite biocatalyst: the composite biocatalyst includes adenosine kinase and the above AMP phosphotransferase mutant, preferably an AMP-PPTM mutant.

AK enzyme source: the adenosine kinase genome (GenBank: GHM 90234.1) was extracted from the Saccharomyces cerevisiae genome and subjected to PCR amplification, and the recovered enzyme gene fragment was ligated to a pET28a plasmid vector to obtain a recombinant plasmid pET28a-AK. The recombinant plasmid was transferred into Escherichia coli DH5 alpha competent cells for preservation. Transferring the recombinant plasmid into Escherichia coli BL (DE 3) competent cells, screening positive clones, culturing, inducing centrifugal disruption and obtaining AK enzyme.

AMP-PPTM enzyme source: and (3) connecting the gene fragment amplified by the mutant to a pET28a plasmid vector to obtain a recombinant plasmid pET28a-AMP-PPTM. The recombinant plasmid was transferred into Escherichia coli DH5 alpha competent cells for preservation. The recombinant plasmid is transferred into Escherichia coli BL (DE 3) competent cells, positive clones are screened, and the AMP-PPTM enzyme is obtained by culturing, inducing, centrifuging and crushing.

The complex biocatalyst used was a combination of adenosine kinase (AK enzyme) and AMP phosphotransferase mutant enzyme. The novel composite biocatalyst is obtained through genetic engineering modification, shake flask small-scale test, microbial liquid fermentation, protein disruption and combination according to the enzyme activity ratio of 0.6:1; the composite biocatalyst can participate in catalytic reaction in the form of free enzyme or freeze-dried powder, or can be immobilized to prepare immobilized enzyme to participate in the reaction.

(III) catalytic reaction: the composite biocatalyst is applied to ATP synthesis.

In the enzyme catalytic reaction, the concentration of substrate adenosine is 20-30g/L, the concentration of ATP salt is 0.5-1.5g/L, the concentration of magnesium ion is 5-15g/L, the concentration of sodium hexametaphosphate is 20-40g/L, and the concentration of free composite biocatalyst is 2000-3000U/L; the reaction pH is 6.0-8.0, the temperature is 30-40 ℃ and the time is 3-5h. Or alternatively

The concentration of the substrate adenosine is 20-30g/L, the ATP salt is 0.5-1.5g/L, the magnesium ion is 5-15g/L, the sodium hexametaphosphate is 20-40g/L, and the immobilized composite biocatalyst is 0.3-0.5kg/L; the reaction pH is 6.0-8.0, the temperature is 30-40 ℃ and the time is 3-5h. The substrate, enzyme and salt added in the invention can be added at one time, or can be added in batches according to industrial operation.

(IV) separating the product ATP and the composite biocatalyst:

the immobilized composite biocatalyst can be directly separated in a constant temperature reaction kettle by centrifugation and filtration;

the free composite biocatalyst is obtained by tangential flow system treatment. The tangential flow system comprises a feed inlet, a discharge outlet and a reflux outlet, a 10KD/30KD interception membrane is arranged in the tangential flow system, the interception liquid after the reaction conversion liquid enters the tangential flow system for treatment is recovered enzyme liquid, and the filtrate is ATP-containing reaction liquid after enzyme separation (after enzyme separation).

And (V) preparing a finished product:

and (3) carrying out enzyme separation (enzyme separation) on the reaction solution containing ATP obtained in the step (IV), and carrying out decolorization, ion exchange chromatography, concentration crystallization, drying and other treatments to obtain an ATP product.

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

(1) Develops a novel ATP composite biocatalyst, and the enzyme types are only two (AK enzyme+AMP-PPTM enzyme);

(2) The novel ATP composite biocatalyst is efficient, stable and cheap, and the ATP production process is simple and easy to operate;

(3) Adenosine is used as a substrate, the cost for producing ATP is reduced to a certain extent, and the product yield and the total yield are higher;

(4) An enzyme recovery system is established, the pollution is small, the impurity pigment is less in the production process, and the subsequent purification of ATP is easy.

Drawings

Fig. 1: the reaction was carried out for 30min in example 3.

Fig. 2: the reaction was carried out for 2 hours in example 3.

Fig. 3: the reaction was carried out for 3h in example 3.

Detailed Description

The following examples serve to further illustrate the invention in detail, but do not limit it in any way. The procedures and methods not described in detail in the examples below are conventional methods well known in the art, and the reagents used in the examples are either commercially available or prepared by methods well known to those of ordinary skill in the art. The following examples all achieve the object of the invention.

EXAMPLE 1 method for obtaining AMP phosphotransferase mutant

Random mutation is introduced by a random mutation PCR kit, and the difference of ATP synthesis capacity between the mutant and the wild type is screened by combining an HPLC method. Selecting forward mutant monoclonal strains, extracting plasmids, sequencing to obtain mutation points, and obtaining 4 single-point mutations, namely mutants 1-4. And constructing mutants 5-10 by introducing site-directed mutation through primer design in the later stage, and testing the activity.

Ten strains of high-activity and high-stability strains are obtained by the activity screening method under the same condition, and are respectively named as mutants 1-10, wherein mutant 9 (named as AMP-PPTM enzyme) containing 124 th (A124L), 312 th (G312M) and 393 (F393K) can be improved by about 80 times on ATP synthesis activity, can be improved by about 20 times on temperature stability, and is most remarkable in activity stability, and the result is shown in Table 1.

Table 1: influence of amino acid sequence mutation position on enzyme activity and the like

Example 2: determination of AMP-PPTM enzyme Properties

Respectively transforming plasmids containing AMP phosphotransferase and mutant thereof into Escherichia coli BL (DE) to obtain mutants, inoculating the strains into LB culture medium, and waiting for bacterial OD ₆₀₀ When the value reaches 0.6, the final concentration of 0.1mM IPTG is added to induce the expression of AMP phosphotransferase and mutant thereof, and the induction is carried out overnight at 20 ℃. The amount of horizontally collected thalli in the shake flask is 5g/L. The thalli are crushed to prepare crude enzyme liquid.

The high quality strain is determined based on the activity value of AMP-PPTM enzyme. Enzyme activity test 1mL reaction system: the substrate adenosine (13.5 mM/L), ATP disodium salt (5 mM/L), magnesium ion (50 mM/L) and sodium hexametaphosphate (50 mM/L), pH7.0, and 37℃were reacted for 30 minutes, and the ATP production amount and the substrate adenosine reduction were measured by high performance liquid chromatography, and the AMP-PPTM enzyme activity was evaluated by calculation. Definition of enzyme activity: under specific conditions, the amount of enzyme required to convert 1 micromolar substrate to one activity unit (U) within 1 minute.

Example 3: determination of detection method of substances in catalytic reaction

And detecting the residual and product formation conditions of the catalytic reaction substrate by using a high performance liquid chromatography. Chromatographic conditions: c18 reverse phase column, mobile phase phosphate buffer salt-methanol, flow rate 1.2mL/min, column temperature 30 ℃, wavelength 254nm. 100mL of the reaction system is tested and reacted for 30min, and the conditions of various substances in the system are detected, as shown in figure 1: the ATP production rate was 33%, and some AMP and ADP were produced; reaction for 2h, detection of ATP production, as shown in fig. 2: ATP production rate was 81%, and AMP was secondary; after 3 hours of reaction, the amount of ATP produced was measured, and as shown in FIG. 3, the substrate was substantially completely consumed, and the ATP production rate was 97%.

Example 4: high-density fermentation culture of AK enzyme and AMP-PPTM enzyme engineering bacteria

Culturing of seed solution of AMP-PPTM enzyme engineering bacteria

1. The AMP-PPTM enzyme gene is cloned into pET28a expression vector to obtain a plasmid for expressing the AMP-PPTM enzyme, the plasmid is named as pET28a-AMP-PPTM plasmid, and the plasmid is transformed into BL21 (DE 3) cells to obtain the genetically engineered bacterium BL21 (DE 3) positive to the AMP-PPTM enzyme gene [ pET28a-AMP-PPTM ].

2. Taking the prepared genetically engineered bacterium BL21 (DE 3) [ pET28a-AMP-PPTM ], inoculating the genetically engineered bacterium BL21 (DE 3) [ pET28a-AMP-PPTM ] on a culture dish plate to activate strains, and culturing for 12 hours; single colonies were picked from the plates and inoculated into LB medium (peptone 10g/L, yeast powder 5g/L, naCl 10 g/L), and cultured in a constant temperature shaker at 37℃and 220r for 12 hours to obtain a fermented seed culture solution.

High-density fermentation culture of (II) AMP-PPTM enzyme engineering bacteria

A5L fermenter was used for high-density fermentation of AMP-PPTM enzyme, with a liquid loading of 3L. Culturing the aboveThe obtained seed solution is inoculated into fermentation culture medium (peptone 30g/L, yeast powder 30g/L, glycerol 50g/L, na) according to 5% inoculation amount ₂ HPO ₄ 16.4g/L，KH ₂ PO ₄ 8g/L, 0.6g/L anhydrous magnesium sulfate and 10g/L NaCl), the temperature is controlled at 37 ℃, the rotating speed and ventilation linkage are controlled at 25%, and the pH value is controlled at 6.5-7.5 by automatic feeding of ammonia water. After about 8 hours of fermentation, when dissolved oxygen is mutated, feeding of feed medium (glycerol 50% (v/v), peptone 40g/L, yeast powder 40 g/L) is started, and the feeding rate is 30mL/h; when cultured for about 6 hours, OD ₆₀₀ At 24 hours, the culture temperature is regulated to 20 ℃, 0.1mM IPTG is added for induction, and the induction is carried out for 10 hours (sampling is carried out every 1 hour for subsequent protein expression analysis); after fermentation, the cell OD ₆₀₀ Finally, 110 g/L of the obtained thallus is obtained.

The high-density fermentation culture method of AK enzyme engineering bacteria is basically consistent with the method.

Example 5: method for preparing adenosine triphosphate by free enzyme method

The method specifically comprises the following steps:

preparing a composite biocatalyst: preparing high-yield AK enzyme and AMP-PPTM enzyme strains, and fermenting and centrifuging to obtain wet thalli; after being suspended in PBS buffer solution of pH7.0 mM at a ratio of 1:10, the mixture was broken by a high-pressure homogenizer, and a crude enzyme solution was obtained by high-speed centrifugation, and AK enzyme and AMP-PPTM enzyme were added at a ratio of 0.6:1 to prepare the composite biocatalyst.

Secondly, 250g of adenosine, 10g of ATP sodium salt, 100g of magnesium chloride hexahydrate and 300g of sodium hexametaphosphate are added into a 20L constant temperature reaction kettle, 10L of reaction system, dissolved by proper purified water, evenly stirred to prevent precipitation in the preparation process, the pH value is regulated to 7.0, and 2000U/L of composite biocatalyst is added into the reaction system to start the reaction. The pH was controlled to 7.0 during the reaction and the temperature was 37 ℃. After 3 hours of reaction, the conversion of adenosine was 98%, the ATP production rate was 95.47%, and the yield was 45.3g/L.

And thirdly, separating AK enzyme and AMP-PPTM enzyme from the reaction liquid in the step two by a tangential flow system through centrifugation, ultrafiltration and other methods, wherein the permeate liquid is clear liquid containing ATP, ADP, AMP and salt ions, and the concentrate liquid is the enzyme and other components separated by us.

And (3) measuring the enzyme activity of the enzyme residue in the concentrated solution, detecting the relative reduction of the enzyme residue by 12% before the reaction, and repeating the step (two) by adding a proper amount of new enzyme.

(IV) separating ATP: decolorizing, ion exchange chromatography, concentrating, crystallizing and drying the permeate liquid to obtain ATP product with purity up to 99% and total yield up to 85.92%.

Example 6: effect of pH8.0 on free enzymatic preparation of adenosine triphosphate

The method for preparing the adenosine triphosphate by the free enzyme method at the pH of 8.0 specifically comprises the following steps:

Secondly, 200g of adenosine, 10g of ATP sodium salt, 150g of magnesium chloride hexahydrate and 400g of sodium hexametaphosphate are added into a 20L constant temperature reaction kettle, 10L of reaction system, dissolved by proper purified water, evenly stirred to prevent precipitation in the preparation process, the pH value is regulated to 8.0, and 2500U/L of composite biocatalyst is added into the reaction system to start the reaction. The pH was controlled to 8.0 and the temperature to 37℃during the reaction. After 5 hours of reaction, the adenosine conversion was 95%, the ATP production rate was 90.62%, and the yield was 34.4g/L.

And (3) measuring the enzyme activity of the enzyme residue in the concentrated solution, detecting the relative reduction of 13% before the reaction, and repeating the operation of the step (two) by adding a proper amount of new enzyme.

(IV) separating ATP: decolorizing, ion exchange chromatography, concentrating, crystallizing and drying the permeate liquid to obtain ATP product with purity up to 97% and total yield up to 84.27%.

Example 7: effect of pH6.0 on free enzymatic preparation of adenosine triphosphate

The method for preparing the adenosine triphosphate by the free enzyme method at the pH of 6.0 specifically comprises the following steps:

Secondly, 250g of adenosine, 10g of ATP sodium salt, 50g of magnesium chloride hexahydrate and 400g of sodium hexametaphosphate are added into a 20L constant temperature reaction kettle, 10L of reaction system, dissolved by proper purified water, evenly stirred to prevent precipitation in the preparation process, the pH value is regulated to 6.0, and 2000U/L of composite biocatalyst is added into the reaction system to start the reaction. The pH was controlled to 6.0 and the temperature to 37℃during the reaction. After 4 hours of reaction, the conversion of adenosine was 96%, the ATP production rate was 92.94%, and the yield was 44.1g/L.

And (III) separating AK enzyme and AMP-PPTM enzyme from the reaction liquid in the step (I) by a tangential flow system through centrifugation, ultrafiltration and other methods, wherein the permeate liquid is clear liquid containing ATP, ADP, AMP and salt ions, and the concentrate liquid is the enzyme and other components separated by us.

Separating ATP, decolorizing, ion exchange chromatography, concentrating, crystallizing and drying the permeate liquid to obtain ATP product with purity up to 98% or higher and total yield up to 83.65%.

Example 8: novel enzymatic preparation of adenosine triphosphate at 30 DEG C

The novel method for preparing the adenosine triphosphate by the enzymatic method at 30 ℃ comprises the following steps:

Secondly, 250g of adenosine, 10g of ATP sodium salt, 150g of magnesium chloride hexahydrate and 300g of sodium hexametaphosphate are added into a 20L constant temperature reaction kettle, 10L of reaction system, dissolved by proper purified water, evenly stirred to prevent precipitation in the preparation process, the pH value is regulated to 7.0, and 3000U/L of composite biocatalyst is added into the reaction system to start the reaction. The pH was controlled to 7.0 during the reaction and the temperature was 30 ℃. After 6 hours of reaction, the ATP production rate was 94.20%, the yield was 44.7g/L, and the adenosine conversion rate was 97%.

And (3) measuring the enzyme activity of the enzyme residue in the concentrated solution, detecting the relative reduction of the enzyme residue by 11% before the reaction, and repeating the step (two) by adding a proper amount of new enzyme.

(IV) separating ATP: decolorizing, ion exchange chromatography, concentrating, crystallizing and drying the permeate liquid to obtain ATP product with purity up to 99% and total yield of 84.78%.

Example 9: novel enzymatic preparation of adenosine triphosphate at 40 DEG C

The novel method for preparing the adenosine triphosphate by the enzymatic method at 40 ℃ comprises the following steps:

Secondly, adding 300g of adenosine, 10g of ATP sodium salt, 150g of magnesium chloride hexahydrate and 300g of sodium hexametaphosphate into a 20L constant temperature reaction kettle, dissolving with proper purified water, uniformly stirring to prevent precipitation in the preparation process, adjusting the pH value to 7.0, and adding 2500U/L of composite biocatalyst into the reaction system to start reaction. The pH was controlled to 7.0 during the reaction and the temperature was 40 ℃. After 3 hours of reaction, the ATP production rate was 94.93%, the yield was 54.05g/L, and the adenosine conversion rate was 98%.

(IV) separating ATP: decolorizing, ion exchange chromatography, concentrating, crystallizing and drying the permeate liquid to obtain ATP product with purity up to 99% and total yield up to 85.44%.

Example 10: preparation method of immobilized enzyme

AK enzyme and AMP-PPTM enzyme were prepared into a 20L mixture at an enzyme activity ratio of 0.6:1. 5kg of epoxy immobilization carrier LX-1000EP wet carrier is added into a constant temperature reaction kettle to be mixed with the enzyme liquid, a shaking table is controlled to shake slowly for 24 hours at 28 ℃, the carrier is collected by filtration, and the immobilized composite biocatalyst is obtained by washing 2-3 times with pH7.0, 20mM phosphate buffer and water respectively.

Example 11: method for preparing adenosine triphosphate by immobilized enzyme method

The method for preparing the adenosine triphosphate by the immobilized enzyme method specifically comprises the following steps:

in a reaction kettle, 250g of substrate adenosine, 10g of ATP salt, 100g of magnesium chloride hexahydrate, 300g of sodium hexametaphosphate and the balance of purified water are added into a 10L system. Adjusting the pH to 7.0 by using 10M sodium hydroxide, adding 0.4kg of the immobilized ATP novel composite biocatalyst, reacting for 4 hours at 37 ℃ and 300r, and detecting the residual and product generation conditions of the substrate by sampling every 1 hour by using high performance liquid chromatography. After the reaction is finished, the reaction solution is filtered to recycle the novel ATP immobilized composite biocatalyst, and the filtered solution is subjected to the steps of centrifugation, ultrafiltration, decolorization, ion exchange chromatography, concentration, drying and the like to obtain 410g of ATP dry powder, wherein the total yield is 85.3%.

Example 12: improving the influence of the immobilized enzyme amount on the preparation of the adenosine triphosphate

Observing the influence of improving the immobilized enzyme amount on the preparation of the adenosine triphosphate, specifically comprising the following steps:

in a 20L constant temperature reaction kettle, 200g of substrate adenosine, 15g of ATP salt, 150g of magnesium chloride hexahydrate, 400g of sodium hexametaphosphate and the balance of purified water are added into a 10L system. Adjusting the pH to 7.0 by using 10M sodium hydroxide, adding 0.5kg of the immobilized ATP novel composite biocatalyst, reacting for 3 hours at 37 ℃ and 300r, and detecting the residual and product generation conditions of the substrate by sampling every 1 hour by using high performance liquid chromatography. After the reaction is finished, the reaction solution is filtered to recover the ATP immobilized novel composite biocatalyst, and the filtered solution is subjected to the steps of centrifugation, tangential flow treatment, decolorization, ion exchange chromatography, concentration and drying and the like to obtain 320.2g of ATP dry powder, wherein the total yield is 84.2%.

Example 13: reducing the influence of the immobilized enzyme amount on the preparation of the adenosine triphosphate

in a 20L constant temperature reaction kettle, 250g of substrate adenosine, 5g of ATP salt, 50g of magnesium chloride hexahydrate, 200g of sodium hexametaphosphate and the balance of purified water are added into a 10L system. Adjusting the pH to 7.0 by using 10M sodium hydroxide, adding 0.3kg of the immobilized composite biocatalyst, reacting for 5 hours at 37 ℃ and 300r, and detecting the residual and the product generation of the substrate by sampling high performance liquid chromatography every 1 hour. After the reaction is finished, the reaction solution is filtered to recover the ATP immobilized novel composite biocatalyst, and 386g of ATP dry powder can be obtained from the filtered solution through the steps of centrifugation, tangential flow treatment, decolorization, ion exchange chromatography, concentration and drying, and the total yield is 83.6%.

Example 14: repeated batch experiment of immobilized enzyme

The immobilized enzyme repeated batch experiment specifically comprises the following steps:

in a reaction kettle, 300g of substrate adenosine, 10g of ATP salt, 100g of magnesium chloride hexahydrate, 300g of sodium hexametaphosphate and the balance of purified water are added into a 10L system. Adjusting the pH to 7.0 by using 10M sodium hydroxide, adding 0.4kg of the immobilized ATP novel composite biocatalyst, reacting for 4 hours at 37 ℃ and 300r, and detecting the residual and product generation conditions of the substrate by sampling every 1 hour by using high performance liquid chromatography. After the reaction is finished, the reaction solution is filtered to recover the ATP immobilized novel composite biocatalyst, and the filtered solution is subjected to the steps of centrifugation, ultrafiltration, decolorization, ion exchange chromatography, concentration, drying and the like to obtain 492g of ATP dry powder, wherein the total yield is 85.3%.

The recovered immobilized novel composite biocatalyst is put into the reaction again, the experiment is repeated for 100 times, 380g of dry powder can be obtained after separation and purification on average, and the average total yield is 84%.

Example 15: ATP separation and purification process

The ATP separating and purifying process includes the following steps:

conversion method to prepare ATP: ATP was prepared by the above-mentioned enzymatic method, and the conversion reaction solution was collected.

(II) pretreatment of ATP conversion reaction liquid: centrifuging the conversion reaction liquid to remove large-particle impurities such as denatured proteins in the reaction, taking supernatant, carrying out suction filtration by using a Buchner funnel, and collecting filtrate; desalting the filtrate by using a tangential flow system to obtain adenosine triphosphate concentrated solution and diluting;

(III) decoloring the ATP pretreatment liquid: stopping the reaction of the concentrated dilute adenosine triphosphate solution collected in the step (II) by using 6N hydrochloric acid, adding active carbon powder according to the addition amount of 2%, stirring for 30min at 30 ℃, carrying out suction filtration by using a Buchner funnel, and collecting filtrate for later use;

and (IV) performing ion exchange chromatography treatment on the ATP decolorization solution to remove ions and other impurities in the decolorization solution.

(V) vacuum concentration and crystallization of ATP chromatographic liquid: and (3) concentrating the chromatographic liquid in vacuum at 55 ℃, adding 95% ethanol with 3 times of volume at 4 ℃, stirring, cooling and crystallizing, and collecting crystals.

Drying the ATP crystals, namely drying the collected crystals in an oven at 80 ℃ for 6 hours to obtain ATP finished products, wherein the total yield is more than 80%.

Sequence listing

<110> Meibaomei and Biotech Co., ltd

<120> AMP phosphotransferase mutant, coding gene thereof and use in ATP synthesis

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 495

<212> PRT

<213> Pseudomonas aeruginosa (Paeruginosa pseudomonas)

<400> 1

Val Arg Ile Arg Gly Ser Trp Pro Gln His Arg Gln Gly His Leu Arg

1 5 10 15

Glu Gly Arg His Arg Val Ala Arg Ser Ala Ala Arg Gly Ala Val Arg

20 25 30

Ala Gln Ala Ala Gly Ala Leu Pro Gly Asp His Pro Asp Gln Arg His

35 40 45

Arg Gly Arg Arg Gln Gly Arg Asp Gly Gln Ala Ala Gln Arg Val Asp

50 55 60

Gly Pro Ala Pro Asn Arg Gly Ala Glu Leu Pro Pro Ser Phe Arg Arg

65 70 75 80

Gly Ala Gly Ala Ala Ala Ala Val Ala Leu Leu Ala Ala Pro Ala Ala

85 90 95

Gln Gly Ala Asp Arg Tyr Leu Leu Arg Gln Leu Val Gln Pro Asp Ala

100 105 110

Leu Arg Ala Gly Arg Gly Ala Tyr Gln Gly Gly Gln Ala Gly Pro Gly

115 120 125

His Arg Cys Arg Arg Thr Leu Arg Ala His Ala Leu Arg Arg Arg Arg

130 135 140

Ala Ala Leu Gln Val Leu Val Pro Ser Leu Gln Glu Thr Val Glu Gly

145 150 155 160

Ala Ser Gln Gly Ala Gly Glu Gly Pro Ala Ala Gln Leu Glu Ala Gln

165 170 175

Ser Ala Gly Leu Glu Ala Glu Arg Gly Leu Arg Pro Leu Arg Ala Leu

180 185 190

Arg Arg Ala Cys Ala Ala Pro Tyr Gln Pro Gly Leu Arg Ala Leu Val

195 200 205

Arg Gly Gly Arg Arg Gly Arg Ala Leu Pro Arg Pro Asp Arg Arg Pro

210 215 220

His Pro Ser Arg Arg Val Ala Gly Arg Ala Gly His Gln Gly Ala Arg

225 230 235 240

Gln Ala Pro Ala Ala Arg Arg Thr Ala Gly Val Glu Pro Gly Gln Pro

245 250 255

Trp Pro Ala Gly Leu Pro Gly Pro Gly Pro Val Pro Gly Gln Gly Arg

260 265 270

Leu Gln Gly Ala Ala Arg Arg Arg Ala Gly Ala Pro Gly Arg Ala Asp

275 280 285

Pro Arg Gln Ala Leu Pro Pro Ala Phe Ala Gly Arg Gly Val Arg Gly

290 295 300

Gln Arg Arg Gly Arg Gln Gly Arg Arg His Pro Pro Cys His Arg Arg

305 310 315 320

Ala Gly Pro Ala Pro Val Pro Tyr Arg Ala Asp Arg Arg Ala Asp Arg

325 330 335

Arg Gly Ala Cys Ala Ala Leu Ser Leu Ala Leu Leu Ala Ala His Ser

340 345 350

Gly Ala Ser Pro Val His His Leu Arg Pro Phe Leu Val Arg Pro Arg

355 360 365

Ala Gly Gly Ala His Arg Gly Leu Leu Arg Thr Gly Arg Leu Ala Thr

370 375 380

Arg Leu Trp Arg Asp Gln Leu Arg Gly Ala Ala Gln Arg Val Arg Asp

385 390 395 400

His Arg Gly Glu Val Leu Ala Gly Asp Arg Gln Ala Asp Pro Asp Gly

405 410 415

Ala Leu Gln Gly Thr Arg Glu Asn Pro Leu Gln Ala Leu Gln Asp His

420 425 430

Arg Gly Arg Leu Ala Gln Pro Arg Gln Val Gly Pro Val Arg Gly Arg

435 440 445

Gly Gly Arg Tyr Gly Arg Pro Tyr Gln His Arg Asp Arg Ala Leu Asp

450 455 460

Pro Gly Arg Ser Gln Arg Gln Ala Leu Arg Pro Gly Gln Gly Ala Ala

465 470 475 480

His His Gln Arg Arg His Arg Gly Gly Val Gln Glu Gly Gln Val

485 490 495

Claims

1. An AMP phosphotransferase mutant, characterized in that it is any one of the following mutants:

2. A gene encoding the AMP phosphotransferase mutant of claim 1.

3. A composite biocatalyst comprising an adenosine kinase and the AMP phosphotransferase mutant of claim 1.

4. A complex biocatalyst according to claim 3, characterized in that said complex biocatalyst is a free or immobilized enzyme consisting of said adenosine kinase and AMP phosphotransferase mutant.

5. The composite biocatalyst according to claim 4, wherein the enzyme activity ratio of the adenosine kinase and the AMP phosphotransferase mutant is 0.6:1.

6. Use of an AMP phosphotransferase mutant according to claim 1 in ATP synthesis.

7. Use of a composite biocatalyst according to any one of claims 3-5 in the synthesis of ATP.

8. The use according to claim 7, wherein in the synthesis system, the substrate adenosine concentration is 20-30g/L, the ATP salt is 0.5-1.5g/L, the magnesium ion is 5-15g/L, the sodium hexametaphosphate is 20-40g/L, and the free composite biocatalyst is 2000-3000U/L; the reaction pH is 6.0-8.0, the temperature is 30-40 ℃ and the time is 3-5h.

9. The use according to claim 7, wherein the concentration of substrate adenosine in the synthesis system is 20-30g/L, the ATP salt is 0.5-1.5g/L, the magnesium ion is 5-15g/L, the sodium hexametaphosphate is 20-40g/L, and the immobilized complex biocatalyst is 0.3-0.5kg/L; the reaction pH is 6.0-8.0, the temperature is 30-40 ℃ and the time is 3-5h.