CN114875011A

CN114875011A - AMP phosphotransferase mutant, coding gene thereof and application of AMP phosphotransferase mutant in ATP synthesis

Info

Publication number: CN114875011A
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: 2022-08-09
Anticipated expiration: 2042-05-10
Also published as: CN114875011B

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 is represented by SEQ: ID: the sequence shown in NO. 1 is obtained by one or more times of mutation. Comprises one or more mutation sites of three mutation sites of 124 (A124L), 312 (G312M) and 393 (F393K). The invention also provides a composite biocatalyst comprising the adenosine kinase and the AMP phosphotransferase mutant and its use in ATP synthesis. The mutant of the invention has higher enzyme activity, higher stability and higher ATP generation ratio, the repeated batch after immobilization is more than 100 batches, the cost of ATP production by the holoenzyme method can be further reduced, and the invention can reduce the enzyme types to 2, can complete the reaction of synthesizing ATP by integral adenosine phosphorylation, and is beneficial to large-scale industrial application.

Description

AMP phosphotransferase mutant, coding gene thereof and application of AMP phosphotransferase mutant 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 is an unstable high-energy compound, and is transformed with ADP in cells to realize energy storage and energy release, so that energy supply of various vital activities of the cells is ensured. ATP plays an important role in energy metabolism in the human body, and is involved in metabolism of sugars, proteins, nucleic acids, fats, and the like in the living body as an intermediate of metabolism and a coenzyme. In clinical application, has good treatment and adjuvant therapy effects on muscular atrophy, apoplexy sequelae, myocardial infarction, coronary arteriosclerosis, hepatitis and other diseases.

The synthesis of ATP mainly comprises chemical methods and biological methods, and the biological methods comprise microbial fermentation methods and biological enzyme catalysis methods. The microbial fermentation method is to synthesize ATP by glycolysis and substrate level phosphorylation by using yeast enzyme system and yeast internal enzyme as 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 difference of the quality among product batches, complex separation and purification and the like.

The biological enzyme catalysis method takes biological enzyme as a catalyst, and the biological enzyme directly contacts with a substrate, so that the substrate conversion rate is high, the production cost is low, the production process is more efficient and stable, the control is easy, and the energy is saved and the environment is protected. In the aspect of raw material cost, besides a substrate and a certain amount of inorganic salts such as magnesium salt, a fermentation method needs to consume a large amount of yeast paste, glucose and phosphate, and a biological enzyme method needs to add a certain amount of phosphate donor and corresponding catalytic enzyme to complete the fermentation.

Adenosine Kinase (AK) belongs to the family of ribokinases, catalyzes the phosphorylation of Adenosine to AMP, and also catalyzes the reaction using ATP as a phosphate donor, and AK is the first enzyme in the synthetic pathway utilized by Adenosine, and thus plays a key role in regulating intracellular and extracellular Adenosine levels. AMP phosphotransferase (AMP-PPT) is an enzyme that phosphorylates AMP to generate ADP using polyphosphoric acid as a phosphate donor. AMP phosphotransferase mutant (AMP-PPTM) is obtained by mutating the gene of AMP phosphotransferase mutant by PCR technique based on the original strain, and screening out mutant with high enzyme activity, stability and ATP ratio, thereby changing the original function and having the dual functions of ADP synthesis by AMP phosphorylation and ATP synthesis by ADP phosphorylation.

Compared with fermentation, the enzyme catalysis method has the advantages of high efficiency, stability, simple reaction system, easy operation of reaction process and the like. For example, in 2001, Akihiko MARUYAMA et al reported that ATP is synthesized under the catalysis of amino acid-producing bacteria by taking adenine as a substrate, the ATP production amount is 70.6mg/mL after 28h reaction, and the substrate conversion rate is 82%. Patent CN104762347A proposes a method for producing Adenosine Triphosphate (ATP). The process is complex, the reaction process is not easy to control, the cost is high, the activity of the enzyme system is reduced quickly, the concentration of adenosine serving as a substrate is 30g/L, the reaction lasts 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%. CN110777180A discloses a method for preparing adenosine triphosphate by an immobilized enzyme method, which adopts three enzyme combinations for immobilization, the concentration of adenosine is 30g/L, the reaction is carried out for 6h, and 445g of adenosine triphosphate dry product is obtained by separation and purification. The reaction time is relatively long, the enzyme combination has a plurality of types, 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 process for synthesizing ATP with participation of multiple enzymes. The process takes adenosine as a substrate, and adopts four enzymes of AK enzyme, Ppk2 enzyme, AK enzyme and Pap enzyme to synthesize ATP through combined reaction, the concentration of the substrate is 30g/L, the reaction time is 8 hours, the adenosine conversion rate is 85 percent, the ATP generation rate is 87.71 percent, the generation amount is about 50g/L, and the total yield is 80 percent. The enzyme combination of the process consists of four enzymes, so that the cost is increased, side reactions are increased along with the increase of the types of the enzymes, and great influence is brought to later-stage separation and purification, and the reaction is inhibited to a certain extent by the increase of the types of the enzymes, so that a series of problems of incomplete substrate conversion and the like are caused.

In conclusion, 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, research and development of a catalytic enzyme with better activity and stronger stability and a new simple and efficient ATP production process are needed.

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 purpose of the invention is realized by the following technical scheme:

(I) selection and evolutionary screening of AMP-PPTM enzymes

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 no: ID: on the basis of the amino acid sequence shown in NO 1, Ala at the 124 th position is mutated into Leu;

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

mutant 6, said mutant 6 being a mutant as set forth in SEQ: ID: 1, mutating Ala at the 124 th position of the amino acid sequence into Leu and mutating Gly at the 177 th position of the amino acid sequence into Ala;

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

mutant 8, said mutant 8 being a mutant as set forth in SEQ id no: ID: 1, mutating Ala at the 124 th position to Leu, Gly at the 177 th position to Ala, and Phe at the 393 th position to Lys;

mutant 9, said mutant 9 being a mutant as set forth in SEQ id no: ID: 1, mutating Ala at the 124 th position to Leu, Gly at the 312 th position to Met, and Phe at the 393 th position to Lys;

mutant 10, said mutant 10 being a mutant as set forth in SEQ: ID: 1, the 177 th Gly thereof is mutated to Ala, the 312 th Gly thereof is mutated to Met, and the 393 th Phe thereof is mutated to Lys.

The present invention also provides a gene encoding the above-described AMP phosphotransferase mutant.

The invention selects Pseudomonas aeruginosa (Paeruginosa pseudomonas) species, NCBI sequence number: 553898144 is the original sequence, and forward mutation points with better activity are obtained by a method of activity screening after random mutation. And (3) carrying out accumulated mutation on the mutant with high initial screening activity, namely overlapping the forward mutation point by a molecular biology means to improve the original ATP synthesis activity. Among the many mutations, 10 representative forward mutants were screened. Found in the late cumulative experiments of these 10 representative mutants. The mutant 9 containing the three mutation sites of 124 th (A124L), 312 th (G312M) and 393 th (F393K) can improve the ATP synthesis activity by about 80 times, improve the temperature stability by 20 times at 50 ℃ for 15min, achieve the effect of synthesizing ATP by a single enzyme with high energy, and is renamed to AMP-PPTM.

(II) preparing a composite biocatalyst: the composite biocatalyst comprises adenosine kinase and the above-mentioned AMP phosphotransferase mutant, preferably AMP-PPTM mutant.

Sources of AK enzyme: adenosine kinase genome (GenBank: GHM90234.1) is extracted from the saccharomyces cerevisiae genome and PCR amplification is carried out, and the recovered enzyme gene fragment is connected to pET28a plasmid vector to obtain recombinant plasmid pET28 a-AK. The recombinant plasmid is transferred into Escherichia coli DH5 alpha competent cells for preservation. The recombinant plasmid is transferred into an Escherichia coli BL21(DE3) competent cell, positive clones are screened, and AK enzyme is obtained by culture, induction, centrifugation and crushing.

AMP-PPTM enzyme source: the gene fragment after mutant amplification is connected to pET28a plasmid vector to obtain recombinant plasmid pET28 a-AMP-PPTM. The recombinant plasmid is transferred into Escherichia coli DH5 alpha competent cells for preservation. The recombinant plasmid is transferred into an Escherichia coli BL21(DE3) competent cell, positive clones are screened, cultured, induced, centrifuged and crushed to obtain the AMP-PPTM enzyme.

The composite biocatalyst used was a combination of adenosine kinase (AK enzyme) and AMP phosphotransferase mutant enzyme. Carrying out genetic engineering modification, shake flask pilot test, microbial liquid fermentation, protein crushing and enzyme activity ratio combination according to 0.6:1 to obtain a novel composite biocatalyst; the composite biological catalyst can participate in catalytic reaction in the form of free enzyme or freeze-dried powder, and can also be immobilized to prepare immobilized enzyme for participating in reaction.

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

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

The concentration of adenosine as a substrate is 20-30g/L, ATP salt is 0.5-1.5g/L, magnesium ions are 5-15g/L, sodium hexametaphosphate is 20-40g/L, and immobilized composite biocatalyst is 0.3-0.5 kg/L; the reaction pH is 6.0-8.0, the temperature is 30-40 ℃, and the reaction time is 3-5 h. The substrate, enzyme and salt added in the invention can be added at one time or 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 in a centrifugal and filtering mode;

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

(V) preparing a finished product:

and (4) carrying out enzyme separation (after enzyme separation) on the reaction liquid containing ATP obtained in the step (IV), and carrying out decolorization, ion exchange chromatography, concentration, crystallization, drying and other treatment to obtain an ATP product.

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

(1) a novel ATP composite biocatalyst is developed, and only two enzymes (AK enzyme + AMP-PPTM enzyme) are adopted;

(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 generation rate 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 ATP purification is easy.

Drawings

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

FIG. 2: example 3 reaction for 2h each material change.

FIG. 3: example 3 was run for 3h with various material changes.

Detailed Description

The following examples serve to illustrate the invention in further detail, but without restricting it in any way. Procedures and methods not described in detail in the following examples are conventional methods well known in the art, and reagents used in the examples are commercially available or prepared by methods well known to those of ordinary skill in the art. The following examples all achieve the objects of the present invention.

Example 1 method for obtaining AMP phosphotransferase mutants

Random mutation is introduced through 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 mutation monoclonal strain, extracting plasmid, sequencing to obtain mutation points, and obtaining 4 single-point mutations (see mutants 1-4). And (3) introducing a site-directed mutation mode through primer design to construct a mutant 5-10 at the later stage, and testing the activity.

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

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

Example 2: determination of the Properties of AMP-PPTM enzymes

Respectively transforming the AMP-containing phosphotransferase and mutant plasmids thereof into Escherichia coli BL231(DE)) to obtain mutants, inoculating the mutants into LB culture medium until the thallus OD ₆₀₀ When the value reached 0.6, AMP phosphotransferase and mutants thereof were induced to express by adding IPTG at a final concentration of 0.1mM and induced overnight under the induction condition of 20 ℃. The amount of the collected bacteria at the shake flask level was 5 g/L. The thalli is crushed to prepare a crude enzyme solution.

Determining the high-quality strain according to the activity value of the AMP-PPTM enzyme. Enzyme activity test 1mL reaction system: substrate adenosine (13.5mM/L), ATP disodium salt (5mM/L), magnesium ions (50mM/L) and sodium hexametaphosphate (50mM/L) were reacted at 37 ℃ for 30min at pH7.0, and ATP production and decrease in substrate adenosine were measured by high performance liquid chromatography, and AMP-PPTM enzyme activity was evaluated by calculation. Definition of enzyme activity: under specific conditions, the amount of enzyme required to convert 1 micromole of substrate in 1 minute is one activity unit (U).

Example 3: determination of detection method of each substance in catalytic reaction

And detecting the generation conditions of the residual catalytic reaction substrate and the product by utilizing high performance liquid chromatography. Chromatographic conditions are as follows: c18 reversed phase column, mobile phase phosphate buffer salt-methanol, flow rate of 1.2mL/min, column temperature of 30 deg.C, wavelength of 254 nm. Testing a 100mL reaction system, reacting for 30min, and detecting the conditions of all substances in the system, as shown in figure 1: the ATP production rate is 33%, and a little AMP and ADP are produced; and reacting for 2h, and detecting the generation condition of ATP, as shown in figure 2: the ATP production rate is 81%, and AMP is less; after 3 hours of reaction, the amount of ATP produced was measured, and as shown in FIG. 3, the substrate was almost completely consumed, and the ATP production rate was 97%.

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

Culture of AMP-PPTM enzyme engineering bacteria seed liquid

1. Cloning AMP-PPTM enzyme gene into pET28a expression vector to obtain plasmid for expressing AMP-PPTM enzyme, naming the plasmid as pET28a-AMP-PPTM plasmid, transforming the plasmid into BL21(DE3) cells to obtain genetically engineered bacterium BL21(DE3) [ pET28a-AMP-PPTM ] with positive AMP-PPTM enzyme gene.

2. Taking the prepared genetically engineered bacterium BL21(DE3) [ pET28a-AMP-PPTM ], inoculating the genetically engineered bacterium BL21(DE3) on a culture dish plate to activate strains, and culturing for 12 h; a single colony is picked from the plate and inoculated into LB culture medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L), and cultured in a constant temperature shaking table at 37 ℃ and 220r for 12h to obtain fermented seed culture solution.

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

AMP-PPTM enzyme high-density fermentation was carried out in a 5L fermenter with a liquid loading of 3L. Inoculating the seed liquid obtained by the above culture into a fermentation medium (30 g/L peptone, 30g/L yeast powder, 50g/L glycerin, Na) according to the inoculation amount of 5% ₂ HPO ₄ 16.4g/L，KH ₂ PO ₄ 8g/L, 0.6g/L of anhydrous magnesium sulfate and 10g/L of NaCl), controlling the temperature at 37 ℃, keeping the dissolved oxygen at 25% by the rotation speed and ventilation linkage, and keeping the pH value at 6.5-7.5 by automatically feeding ammonia water. After about 8 hours of fermentation, when the dissolved oxygen is mutated, feeding a feed medium (50% (v/v) of glycerol, 40g/L of peptone and 40g/L of yeast powder) is started, and the feeding rate is 30 mL/h; when cultured for about 6h, OD ₆₀₀ When the temperature is 24 ℃, adjusting the culture temperature to 20 ℃, adding 0.1mM IPTG for induction, and inducing for 10h (sampling every 1h for subsequent protein expression analysis); after the fermentation, the cells OD ₆₀₀ Finally, the wet weight of the obtained cells was 110 g/L.

The high-density fermentation culture method of the 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; suspending with PBS buffer solution of pH7.020mM according to the ratio of 1:10, crushing with a high-pressure homogenizer, centrifuging at high speed to obtain crude enzyme solution, and then mixing AK enzyme and AMP-PPTM enzyme according to the ratio of 0.6:1 proportion to prepare the composite biocatalyst.

And (II) adding 250g of adenosine, 10g of ATP sodium salt, 100g of magnesium chloride hexahydrate and 300g of sodium hexametaphosphate into a 10L reaction system in 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 2000U/L of composite biocatalyst into the reaction system to start reaction. During the reaction, the pH was controlled at 7.0 and the temperature at 37 ℃. After 3 hours of reaction, the conversion of adenosine was 98%, the generation of ATP was 95.47%, and the amount of ATP generated was 45.3 g/L.

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

And (5) measuring the enzyme residual activity in the concentrated solution, detecting that the enzyme residual activity is reduced by 12 percent compared with that before reaction, and repeating the operation of the step (II) by adding a proper amount of new enzyme.

(IV) ATP separation: and (3) decoloring, performing ion exchange chromatography, concentrating, crystallizing and drying the penetrating fluid obtained in the step (III) to obtain an ATP finished product, wherein the purity can reach over 99 percent, and the total yield is 85.92 percent.

Example 6: effect of pH8.0 on the preparation of adenosine triphosphate by the free enzyme method

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

And (II) adding 200g of adenosine, 10g of ATP sodium salt, 150g of magnesium chloride hexahydrate and 400g of sodium hexametaphosphate into a 10L reaction system in 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 8.0, and adding 2500U/L of a composite biocatalyst into the reaction system to start reaction. The pH was controlled at 8.0 and the temperature at 37 ℃ during the reaction. After 5 hours of reaction, the adenosine conversion was 95%, the ATP formation was 90.62%, and the amount of ATP formed was 34.4 g/L.

And (5) measuring the enzyme residual activity in the concentrated solution, detecting that the enzyme residual activity is reduced by 13 percent relative to the enzyme residual activity before reaction, and repeating the operation of the step (II) by adding a proper amount of new enzyme.

(IV) ATP separation: and (3) decoloring, performing ion exchange chromatography, concentrating, crystallizing and drying the penetrating fluid obtained in the step (III) to obtain an ATP finished product, wherein the purity can reach more than 97 percent, and the total yield is 84.27 percent.

Example 7: influence of pH6.0 on preparation of adenosine triphosphate by free enzyme method

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

And (II) adding 250g of adenosine, 10g of ATP sodium salt, 50g of magnesium chloride hexahydrate and 400g of sodium hexametaphosphate into a 10L reaction system in 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 6.0, and adding 2000U/L of composite biocatalyst into the reaction system to start reaction. The pH was controlled at 6.0 and the temperature at 37 ℃ during the reaction. After 4 hours of reaction, the conversion of adenosine was 96%, the production of ATP was 92.94%, and the amount of ATP produced was 44.1 g/L.

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

And (IV) separating ATP, namely decoloring the penetrating fluid obtained in the step (III), performing ion exchange chromatography, concentrating, crystallizing and drying to obtain an ATP finished product, wherein the purity can reach over 98 percent, and the total yield is 83.65 percent.

Example 8: preparation of adenosine triphosphate by novel enzyme method at 30 DEG C

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

And (II) adding 250g of adenosine, 10g of ATP sodium salt, 150g of magnesium chloride hexahydrate and 300g of sodium hexametaphosphate into a 10L reaction system in 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 3000U/L of a composite biocatalyst into the reaction system to start reaction. During the reaction, the pH was controlled at 7.0 and the temperature at 30 ℃. After 6 hours of reaction, the ATP production rate was 94.20%, the production amount was 44.7g/L, and the adenosine conversion rate was 97%.

And (5) measuring the enzyme residual activity in the concentrated solution, detecting that the enzyme residual activity is reduced by 11 percent relative to the enzyme residual activity before reaction, and repeating the operation of the step (II) by adding a proper amount of new enzyme.

(IV) ATP separation: and (3) decoloring, performing ion exchange chromatography, concentrating, crystallizing and drying the penetrating fluid obtained in the step (III) to obtain an ATP finished product, wherein the purity can reach over 99 percent, and the total yield is 84.78 percent.

Example 9: preparation of adenosine triphosphate by novel enzyme method at 40 DEG C

The method for preparing the adenosine triphosphate by the novel enzyme method at the temperature of 40 ℃ specifically comprises the following steps:

And (II) adding 300g of adenosine, 10g of ATP sodium salt, 150g of magnesium chloride hexahydrate and 300g of sodium hexametaphosphate into a 10L reaction system in 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 a composite biocatalyst into the reaction system to start reaction. During the reaction, the pH was controlled at 7.0 and the temperature at 40 ℃. After 3 hours of reaction, the ATP production rate was 94.93%, the production amount was 54.05g/L, and the adenosine conversion rate was 98%.

And (3) determining the enzyme residual activity in the concentrated solution, detecting that the enzyme residual activity is reduced by 12 percent relative to that before the reaction, and adding a proper amount of new enzyme to repeat the operation of the step (II).

(IV) ATP separation: and (3) decoloring, performing ion exchange chromatography, concentrating, crystallizing and drying the penetrating fluid obtained in the step (III) to obtain an ATP finished product, wherein the purity can reach over 99 percent, and the total yield is 85.44 percent.

Example 10: preparation method of immobilized enzyme

AK enzyme and AMP-PPTM enzyme are prepared into 20L mixed solution according to the proportion of 0.6: 1. Adding 5kg of epoxy immobilized carrier LX-1000EP wet carrier into a constant temperature reaction kettle, mixing with the enzyme solution, controlling the temperature to be 28 ℃ and shaking slowly for 24h by a shaking table, filtering and collecting the carrier, and washing respectively for 2-3 times by pH7.0, 20mM phosphate buffer solution and water to obtain the immobilized composite biocatalyst.

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

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

250g of substrate adenosine, 10g of ATP salt, 100g of magnesium chloride hexahydrate, 300g of sodium hexametaphosphate and pure water are added into a 10L system in a reaction kettle. Adjusting pH to 7.0 with 10M sodium hydroxide, adding 0.4kg of the above immobilized ATP novel composite biocatalyst, reacting at 37 deg.C under 300r for 4h, sampling every 1h, and detecting substrate residue and product generation by 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 centrifugation, ultrafiltration, decolorization, ion exchange chromatography, concentration, drying and other steps to obtain 410g of ATP dry powder, wherein the total yield is 85.3%.

Example 12: influence of increasing immobilized enzyme quantity on preparation of adenosine triphosphate

Observing the influence of increasing the immobilized enzyme quantity on the preparation of adenosine triphosphate, the method specifically comprises the following steps:

200g of substrate adenosine, 15g of ATP salt, 150g of magnesium chloride hexahydrate, 400g of sodium hexametaphosphate and pure water in a 10L system are added into a 20L constant-temperature reaction kettle. Adjusting pH to 7.0 with 10M sodium hydroxide, adding 0.5kg of the above immobilized ATP novel composite biocatalyst, reacting at 37 deg.C under 300r for 3h, sampling every 1h, and detecting substrate residue and product generation by 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 centrifugation, tangential flow treatment, decolorization, ion exchange chromatography, concentration, drying and other steps to obtain 320.2g of ATP dry powder, wherein the total yield is 84.2%.

Example 13: influence of reduction of immobilized enzyme quantity on preparation of adenosine triphosphate

250g of substrate adenosine, 5g of ATP salt, 50g of magnesium chloride hexahydrate, 200g of sodium hexametaphosphate and pure water in a 10L system in a 20L constant-temperature reaction kettle. Adjusting pH to 7.0 with 10M sodium hydroxide, adding 0.3kg of the above immobilized composite biocatalyst, reacting at 37 deg.C under 300r for 5h, sampling every 1h, and detecting substrate residue and product formation by 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 centrifugation, tangential flow treatment, decolorization, ion exchange chromatography, concentration, drying and other steps to obtain 386g of ATP dry powder with the total yield of 83.6 percent.

Example 14: repeated batch experiments with 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 pure water are added into a 10L system. Adjusting pH to 7.0 with 10M sodium hydroxide, adding 0.4kg of the above immobilized ATP novel composite biocatalyst, reacting at 37 deg.C under 300r for 4h, sampling every 1h, and detecting substrate residue and product generation by high performance liquid chromatography. After the reaction is finished, the reaction solution is filtered to recover the ATP immobilized novel composite biocatalyst, and 492g of ATP dry powder can be obtained by the steps of centrifugation, ultrafiltration, decoloration, ion exchange chromatography, concentration, drying and the like of the filtrate, wherein the total yield is 85.3%.

And putting the recovered novel immobilized composite biocatalyst into the reaction again, repeating the experiment for 100 times, and obtaining 380g of dry powder on average by separation and purification, wherein the average total yield is 84%.

Example 15: ATP separation and purification process

A process for the separation and purification of ATP, the process comprising the sequential steps of:

(I) ATP preparation by a conversion method: ATP is prepared by the above enzyme method, and the conversion reaction solution is collected.

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

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

and (IV) carrying out ion exchange chromatography treatment on the ATP destaining solution to remove ions and other impurities in the destaining solution.

And (V) concentrating and crystallizing ATP chromatographic solution in vacuum: and (4) concentrating the chromatographic solution collected in the step (IV) at 55 ℃ in vacuum, adding 3 times of 95% ethanol at 4 ℃, stirring, cooling and crystallizing, and collecting crystals.

And (VI) 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 over 80 percent.

Sequence listing

<110> Meibangmei and Biotechnology Ltd

<120> AMP phosphotransferase mutant, coding gene thereof and application thereof 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, which is any one of the following mutants:

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

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. The complex biocatalyst of claim 3, wherein said complex biocatalyst is a free or immobilized enzyme consisting of said adenosine kinase and AMP phosphotransferase mutant.

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

6. Use of the AMP phosphotransferase mutant of claim 1 in ATP synthesis.

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

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

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

10. The application of claim 7, wherein after the synthesis is finished, the obtained reaction solution is subjected to centrifugation, suction filtration, tangential flow treatment, decolorization, ion exchange chromatography, concentration crystallization and drying to obtain a finished product.