阅读说明：本技术 一种固定化酶法制备三磷酸胞苷的方法 (Method for preparing cytidine triphosphate by immobilized enzyme method ) 是由 邓壮梅 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胞苷的方法,具体包括以下步骤：(1)制备CTP生产酶、(2)固定化CTP生产酶、(3)分离产物；本发明采用PPK、HY两种CTP生产酶,只需两步酶促反应即可合成CTP,与传统啤酒酵母生产CTP繁琐的工艺过程相比,反应过程更简单,反应更容易控制,产品质量更加稳定；采用固定化酶催化的方法制备CTP,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色素及其他类型的核苷酸等杂质,更加易于纯化；且该方法适用于大规模生产CTP。(The invention relates to the technical field of biology, in particular to a method for preparing cytidine triphosphate by an immobilized enzyme method, which specifically comprises the following steps: (1) preparing a CTP production enzyme, (2) immobilizing the CTP production enzyme, and (3) separating a product; according to the invention, two CTP (phospho-pyruvate carboxylase) enzymes PPK and HY are adopted to produce the enzyme, and the CTP can be synthesized only by two-step enzymatic reaction, so that compared with the traditional process for producing CTP by beer yeast, the reaction process is simpler, the reaction is easier to control, and the product quality is more stable; the CTP is prepared by adopting an immobilized enzyme catalysis method, the immobilized enzyme can be continuously and repeatedly used, and the production cost is greatly reduced. Meanwhile, pigment and other types of nucleotide and other impurities introduced by using yeast are avoided, and the purification is easier; and the method is suitable for large-scale production of CTP.)

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

(1) preparation of CTP-producing enzyme: the CTP producing enzyme is fixed on an immobilized carrier and can be synthesized by two enzymatic reactions of polyphosphate kinase (EC2.7.4.1, PPK) and uridylate kinase (EC2.7.4.22, Hy) to prepare the immobilized CTP producing enzyme;

(2) immobilized CTP produces enzyme: preparing cytidine triphosphate reaction liquid by catalysis by utilizing the immobilized CTP production enzyme, and filtering and collecting a carrier to obtain the immobilized CTP production enzyme;

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

2. The method for preparing cytidine triphosphate according to claim 1, wherein said preparation of CTP-producing enzyme: preparing high-expression polyphosphate kinase (EC2.7.4.1, PPK) and uridylic acid kinase (EC2.7.4.22, Hy) strains, and centrifugally collecting thalli after fermentation; respectively taking 1.0-2.0kg of thallus containing PPK and 1.0-2.0kg of thallus containing Hy, mixing and suspending with 10L of 0.1M phosphate buffer solution with pH of 7.0, crushing the bacteria with a high-pressure homogenizer, and centrifugally collecting the supernatant to obtain the CTP production enzyme.

3. The method for preparing cytidine triphosphate according to claim 1, wherein said immobilized enzyme produces said enzyme: adding agarose-IDA-Ni 2+ chelating carrier into a constant-temperature stirring reaction tank, mixing with the CTP production enzyme 10L, and stirring at 150rpm for 4-6h at room temperature; filtering and collecting the carrier, and washing for 2-4 times by using 0.1M phosphate buffer solution (containing 1mol/L sodium chloride) with pH7.0 to obtain the immobilized CTP production enzyme.

4. The method for preparing cytidine triphosphate through an immobilized enzyme method according to claim 1, wherein the separation product: preparing reaction liquid with the total volume of 10L in a 20L reaction tank, wherein the reaction liquid contains 250g of CMP 150-; adjusting the pH value of a reaction solution to 7.0 by using NaOH, adding 0.5-0.8kg of the immobilized CTP production enzyme, starting stirring at the temperature of 37 ℃ and at the speed of 150rpm for reaction for 4-6h, and detecting the generation amount of CTP by using high performance liquid chromatography; recovering the immobilized CTP from the reaction solution after the reaction through a filter bag to produce enzyme, and obtaining a cytidine triphosphate dry product (CTP) after chromatographic separation, crystallization and drying of the permeation solution.

Technical Field

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

Background

The glycolysis pathway of yeast is utilized, cytidylic acid is taken as a substrate, and matrix-level phosphorylation is carried out to synthesize CTP (cytidine triphosphate) (CTP) which is the most common method and is commonly adopted in the current industrial production of Cytidine Triphosphate (CTP). However, the reaction process of synthesizing CTP catalyzed by 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 among 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. In practical application, the cost of the enzyme is high, the enzyme activity is reduced rapidly by using free enzyme to produce CTP, the CTP cannot be effectively recycled, the production cost is high, and the practical application value is influenced.

At present, the CTP production level in China is generally low, and the product is still deficient in aspects such as cost control, product quality and the like. 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.

Disclosure of Invention

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

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

(1) preparation of CTP-producing enzyme: the CTP producing enzyme is fixed on an immobilized carrier and can be synthesized by two enzymatic reactions of polyphosphate kinase (EC2.7.4.1, PPK) and uridylate kinase (EC2.7.4.22, Hy) to prepare the immobilized CTP producing enzyme;

(2) immobilized CTP produces enzyme: preparing cytidine triphosphate reaction liquid by catalysis by utilizing the immobilized CTP production enzyme, and filtering and collecting a carrier to obtain the immobilized CTP production enzyme;

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

Preferably, said preparing a CTP-producing enzyme: preparing high-expression polyphosphate kinase (EC2.7.4.1, PPK) and uridylic acid kinase (EC2.7.4.22, Hy) strains, and centrifugally collecting thalli after fermentation; respectively taking 1.0-2.0kg of thallus containing PPK and 1.0-2.0kg of thallus containing Hy, mixing and suspending with 10L of 0.1M phosphate buffer solution with pH of 7.0, crushing the bacteria with a high-pressure homogenizer, and centrifugally collecting the supernatant to obtain the CTP production enzyme.

Preferably, said immobilized CTP-producing enzyme: adding agarose-IDA-Ni 2+ chelating carrier into a constant-temperature stirring reaction tank, mixing with the CTP production enzyme 10L, and stirring at 150rpm for 4-6h at room temperature; filtering and collecting the carrier, and washing for 2-4 times by using 0.1MpH7.0 phosphate buffer solution (containing 1mol/L sodium chloride) to obtain the immobilized CTP production enzyme.

Preferably, the isolated product: preparing reaction liquid with the total volume of 10L in a 20L reaction tank, wherein the reaction liquid contains 250g of CMP 150-; adjusting the pH value of a reaction solution to 7.0 by using NaOH, adding 0.5-0.8kg of the immobilized CTP production enzyme, starting stirring at the temperature of 37 ℃ and at the speed of 150rpm for reaction for 4-6h, and detecting the generation amount of CTP by using high performance liquid chromatography; recovering the immobilized CTP from the reaction solution after the reaction through a filter bag to produce enzyme, and obtaining a cytidine triphosphate dry product (CTP) after chromatographic separation, crystallization and drying of the permeation solution.

The invention has the following beneficial effects:

1. according to the invention, two CTP (phospho-pyruvate carboxylase) enzymes PPK and HY are adopted to produce the enzyme, and the CTP can be synthesized only by two-step enzymatic reaction.

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

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.