阅读说明：本技术 一种固定化酶法制备三磷酸腺苷的方法 (Method for preparing adenosine triphosphate by immobilized enzyme method ) 是由 邓壮梅 徐超男 王宏 傅得响 张燕 于 2019-12-04 设计创作，主要内容包括：本发明涉及生物技术领域,尤其涉及一种固定化酶法制备三磷酸腺苷的方法,具体包括以下步骤：(1)制备ATP生产酶、(2)固定化ATP生产酶、(3)分离产物；本发明采用腺苷激酶(EC 2.7.1.20,Ark)、腺苷酸激酶(EC 2.7.4.3,Adk)、多聚磷酸-腺苷酸磷酸转移酶(EC 2.7.4.-,Pap)三种ATP生产酶,只需两步酶促反应即可合成ATP,反应过程简单,反应容易控制,产品质量更加稳定；采用固定化酶催化的方法制备ATP,固定化酶可连续、反复多次使用,大幅的降低了生产成本；同时避免了使用酵母引入的大量色素和其他类型核苷酸等杂质,更加易于纯化；该方法适用于大规模生产ATP。(The invention relates to the technical field of biology, in particular to a method for preparing adenosine triphosphate by an immobilized enzyme method, which specifically comprises the following steps: (1) preparing ATP producing enzyme, (2) immobilizing ATP producing enzyme, (3) separating product; the invention adopts three ATP production enzymes of adenosine kinase (EC2.7.1.20, Ark), adenylate kinase (EC 2.7.4.3, Adk) and polyphosphate-adenylate phosphotransferase (EC 2.7.4-Pap), and can synthesize ATP by two-step enzymatic reaction, the reaction process is simple, the reaction is easy to control, and the product quality is more stable; the ATP is prepared by adopting an immobilized enzyme catalysis method, and the immobilized enzyme can be continuously and repeatedly used for many times, so that the production cost is greatly reduced; meanwhile, a large amount of pigment and other types of nucleotide and other impurities introduced by using yeast are avoided, and the purification is easier; the method is suitable for large-scale production of ATP.)

1. A method for preparing adenosine triphosphate by an immobilized enzyme method is characterized by comprising the following steps:

(1) preparation of ATP-producing enzyme: fixing the ATP-producing enzyme on an immobilized carrier, and synthesizing the ATP-producing enzyme by three enzymatic reactions of adenosine kinase (EC2.7.1.20, Ark), adenylate kinase (EC 2.7.4.3, Adk) and polyphosphate-adenylate phosphotransferase (EC2.7.4 < - >, Pap);

(2) immobilized ATP-producing enzyme: preparing adenosine triphosphate reaction liquid by using immobilized ATP production enzyme through catalysis of adenosine, filtering and collecting a carrier to obtain immobilized ATP production enzyme;

(3) and (3) separating a product: directly separating the immobilized ATP producing enzyme in a reaction tank, recovering the immobilized ATP producing enzyme from the separated reaction liquid through a filter bag, and obtaining adenosine triphosphate dry product (ATP) after chromatographic separation, crystallization and drying of the permeation liquid.

2. The method for preparing adenosine triphosphate according to claim 1, wherein the preparation of ATP-producing enzyme: preparing high-expression Ark, Adk and Pap strains, and centrifugally collecting thalli after fermentation is completed; respectively taking 1.0-2.0kg of thallus containing Pap, 1.0-2.0kg of thallus containing Adk and 0.5-1.0kg of thallus containing Ark, mixing and suspending with 10L of 0.1M phosphate buffer solution with pH of 7.0, crushing the bacteria with a high-pressure homogenizer, centrifuging and collecting the supernatant to obtain the ATP-producing enzyme.

3. The method for preparing adenosine triphosphate according to claim 1, wherein the immobilized ATP-producing enzyme: adding agarose-IDA-Ni 2+ chelating carrier into a constant-temperature stirring reaction tank, mixing with the ATP producing enzyme 10L, and stirring at 150rpm for 4-6h at room temperature; the carrier was collected by filtration and washed 2 to 4 times with 0.1M phosphate buffer (containing 1mol/L sodium chloride) at pH7.0 to obtain the immobilized ATP-producing enzyme.

4. The immobilized enzyme method of claim 1, wherein the separation product is: preparing reaction liquid with the total volume of 10L in a 20L reaction tank, wherein the reaction liquid contains 300g of Ar 200-; adjusting pH of the reaction solution to 7.0 with NaOH, adding 0.5-0.8kg of the immobilized ATP-producing enzyme, stirring at 37 deg.C and 150rpm for reaction for 4-6h, and detecting ATP generation amount by high performance liquid chromatography; recovering immobilized ATP producing enzyme from the reacted reaction liquid through a filter bag, and obtaining Adenosine Triphosphate (ATP) after chromatographic separation, crystallization and drying of the permeation liquid.

Technical Field

The invention relates to the technical field of biology, in particular to a method for preparing adenosine triphosphate by an immobilized enzyme method.

Background

The glycolysis pathway of yeast is utilized, adenylic acid is taken as a substrate, and the substrate level phosphorylation is the most common method for synthesizing ATP, and is also the method generally adopted by the current industrial production of Adenosine Triphosphate (ATP). However, the reaction process for synthesizing ATP by catalysis of yeast cell enzyme system is complex, the enzyme systems participating in catalytic reaction are numerous, the reaction process is not easy to control, and the quality difference between product batches is large. Meanwhile, the quality of yeast enzyme systems is greatly different due to different suppliers, different batches and even different seasons. The yeast bacterial enzyme system has unstable quality, fast enzyme activity reduction and short service life, and is generally used for one time. In the reaction process, a large amount of yeast cell enzyme liquid is required to be added, and a large amount of pigment and other impurities such as nucleotide existing in the yeast are introduced, so that great difficulty is brought to later purification. At present, the ATP production level in China is generally lower, and the aspects of cost control, product quality and the like of products are also deficient. In view of the above technical problems, there is an urgent need to develop a novel and stable reaction process, simplify the reaction process and improve the product quality.

The patent reports that three ATP production enzymes including novel PPK, Adk and Pap are adopted and immobilized, ATP can be synthesized only through two-step enzymatic reaction, the reaction process is simpler, the reaction is easier to control, and the product quality is more stable. However, this method uses AMP as a substrate, and the cost of the substrate is much higher than that of adenosine (Ar).

Disclosure of Invention

The invention aims to solve the defects in the prior art, and provides the method for preparing the adenosine triphosphate by the immobilized enzyme method.

In order to achieve the purpose, the invention adopts the following technical scheme: a method for preparing adenosine triphosphate by an immobilized enzyme method specifically comprises the following steps:

(1) preparation of ATP-producing enzyme: fixing the ATP-producing enzyme on an immobilized carrier, synthesizing by adopting three enzymatic reactions of adenosine kinase (EC2.7.1.20, Ark), adenylate kinase (EC 2.7.4.3, Adk) and polyphosphate-adenylate phosphotransferase (EC2.7.4 < - >, Pap), and preparing the immobilized ATP-producing enzyme from ATP;

(2) immobilized ATP-producing enzyme: preparing adenosine triphosphate reaction liquid by using immobilized ATP production enzyme through catalysis of adenosine, filtering and collecting a carrier to obtain immobilized ATP production enzyme;

(3) and (3) separating a product: directly separating the immobilized ATP producing enzyme in a reaction tank, recovering the immobilized ATP producing enzyme from the separated reaction liquid through a filter bag, and obtaining adenosine triphosphate dry product (ATP) after chromatographic separation, crystallization and drying of the permeation liquid.

Preferably, the preparing an ATP producing enzyme: preparing high-expression Ark, Adk and Pap strains, and centrifugally collecting thalli after fermentation is completed; respectively taking 1.0-2.0kg of thallus containing Pap, 1.0-2.0kg of thallus containing Adk and 0.5-1.0kg of thallus containing Ark, mixing and suspending with 10L of 0.1M phosphate buffer solution with pH of 7.0, crushing the bacteria with a high-pressure homogenizer, centrifuging and collecting the supernatant to obtain the ATP-producing enzyme.

Preferably, the immobilized ATP producing enzyme: adding agarose-IDA-Ni 2+ chelating carrier into a constant-temperature stirring reaction tank, mixing with the ATP producing enzyme 10L, and stirring at 150rpm for 4-6h at room temperature; the carrier was collected by filtration and washed 2 to 4 times with 0.1M pH7.0 phosphate buffer (containing 1mol/L sodium chloride) to obtain the immobilized ATP-producing enzyme.

Preferably, the isolated product: preparing reaction liquid with the total volume of 10L in a 20L reaction tank, wherein the reaction liquid contains 300g of Ar 200-; adjusting pH of the reaction solution to 7.0 with NaOH, adding 0.5-0.8kg of the immobilized ATP-producing enzyme, stirring at 37 deg.C and 150rpm for reaction for 4-6h, and detecting ATP generation amount by high performance liquid chromatography; recovering immobilized ATP producing enzyme from the reacted reaction liquid through a filter bag, and obtaining Adenosine Triphosphate (ATP) after chromatographic separation, crystallization and drying of the permeation liquid.

The invention has the following beneficial effects:

1. the invention adopts three ATP production enzymes of Ark, Adk and Pap, can synthesize ATP by only two-step enzymatic reaction, and has simpler reaction process, easier reaction control and more stable product quality compared with the traditional process for producing ATP by beer yeast. Compared with the method for preparing the adenosine cyclophosphate by taking AMP as a substrate, the method greatly reduces the cost of raw materials.

2. The ATP is prepared by adopting the method of immobilized enzyme catalysis, and the immobilized enzyme can be continuously and repeatedly used for many times, thereby greatly reducing the production cost. Meanwhile, pigment and other types of nucleotide and other impurities introduced by using yeast are avoided, the purification is easier, and the method is suitable for large-scale production of ATP.

Detailed Description

The technical solutions in the embodiments of the present invention are clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments.