Method for preparing cytidine triphosphate (CTP) by immobilized enzyme method

A technology of cytidine triphosphate and immobilized enzyme, which is applied in the field of preparation of cytidine triphosphate by immobilized enzyme method, which can solve the problems of high cost of enzyme, rapid decline of enzyme activity, short service life, etc., and achieve stable product quality and smooth reaction process Simplicity and the effect of reducing production costs

Pending Publication Date: 2020-02-21
美亚药业海安有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The quality of the yeast enzyme system is unstable, the enzyme activity declines rapidly, and the service life is short. Generally, it is for one-time use.
The reaction process needs to add a large amount of yeast cell enzyme solution, and the introduction of a large amount of pigments and other impurities such as nucleotides in the yeast itself brings great difficulties to the later purification
In practical applications, the cost of enzymes is expensive. Using free enzymes to produce CTP, the enzyme activity declines rapidly and cannot be effectively recycled, which will inevitably lead to high production costs and affect the actual application value.
[0003] At present, the overall level of CTP production in my country is still relatively low, and there are still some deficiencies in product cost control and product quality.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] A method for preparing cytidine triphosphate by an immobilized enzyme method, specifically comprising the following steps:

[0018] (1) Preparation of CTP-producing enzyme: immobilize the CTP-producing enzyme on an immobilized carrier, and use two kinds of enzymes, polyphosphate kinase (EC 2.7.4.1, PPK) and uridylate kinase (EC 2.7.4.22, Hy) The reaction can be synthesized, and the immobilized CTP production enzyme can be prepared;

[0019] (2) Immobilized CTP production enzyme: use the immobilized CTP production enzyme to catalyze the preparation of cytidine triphosphate reaction solution, filter and collect the carrier, and obtain the immobilized CTP production enzyme;

[0020] (3) Isolation product: the above-mentioned immobilized CTP production enzyme is directly separated in the reaction tank, and the separated reaction solution is recovered through a filter bag to recover the immobilized CTP production enzyme, and the permeate is chromatographically separated, cry...

Embodiment 2

[0025] A method for preparing cytidine triphosphate by an immobilized enzyme method, specifically comprising the following steps:

[0026] (1) Preparation of CTP-producing enzyme: immobilize the CTP-producing enzyme on an immobilized carrier, and use two kinds of enzymes, polyphosphate kinase (EC 2.7.4.1, PPK) and uridylate kinase (EC 2.7.4.22, Hy) The reaction can be synthesized, and the immobilized CTP production enzyme can be prepared;

[0027] (2) Immobilized CTP production enzyme: use the immobilized CTP production enzyme to catalyze the preparation of cytidine triphosphate reaction solution, filter and collect the carrier, and obtain the immobilized CTP production enzyme;

[0028] (3) Isolation product: the above-mentioned immobilized CTP production enzyme is directly separated in the reaction tank, and the separated reaction solution is recovered through a filter bag to recover the immobilized CTP production enzyme, and the permeate is chromatographically separated, cry...

Embodiment 3

[0033] A method for preparing cytidine triphosphate by an immobilized enzyme method, specifically comprising the following steps:

[0034] (1) Preparation of CTP-producing enzyme: immobilize the CTP-producing enzyme on an immobilized carrier, and use two kinds of enzymes, polyphosphate kinase (EC 2.7.4.1, PPK) and uridylate kinase (EC 2.7.4.22, Hy) The reaction can be synthesized, and the immobilized CTP production enzyme can be prepared;

[0035] (2) Immobilized CTP production enzyme: use the immobilized CTP production enzyme to catalyze the preparation of cytidine triphosphate reaction solution, filter and collect the carrier, and obtain the immobilized CTP production enzyme;

[0036] (3) Isolation product: the above-mentioned immobilized CTP production enzyme is directly separated in the reaction tank, and the separated reaction solution is recovered through a filter bag to recover the immobilized CTP production enzyme, and the permeate is chromatographically separated, cry...

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Abstract

The invention relates to the technical field of biology and particularly relates to a method for preparing cytidine triphosphate (CTP) by an immobilized enzyme method. The method specifically comprises the following steps: (1) preparing CTP production enzymes, (2) immobilizing the CTP production enzymes, and (3) separating a product. According to the method, two kinds of CTP production enzymes, i.e., PPK and HY are employed, and the CTP can be synthesized by only two steps of enzymatic reactions; compared with the traditional tedious process flows for producing the CTP by a brewer's yeast, thereaction process is simpler, reactions are easier in control, and the product quality is more stable; and the CTP is prepared by adopting an immobilized enzyme catalyzing method, immobilized enzymescan be continuously and repeatedly used, and thus, the production cost is reduced greatly. Simultaneously, pigments and impurities such as nucleotides of other types introduced by using yeasts are avoided, and purification is easier; and the method is applicable to large-scale production of the CTP.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for preparing cytidine triphosphate by an immobilized enzyme method. Background technique [0002] Utilizing the glycolysis pathway of yeast and using cytidylic acid as a substrate to phosphorylate CTP at the matrix level is the most commonly used method, and it is also the method commonly used in the industrial production of cytidine triphosphate (CTP). However, the reaction process of synthesizing CTP catalyzed by yeast cell enzyme system is complicated, and there are many enzyme systems involved in the catalytic reaction, the reaction process is difficult to control, and the quality of products varies greatly among batches. At the same time, the quality of yeast enzymes often varies greatly due to different suppliers, different batches, and even different seasons. The quality of yeast enzymes is unstable, the enzyme activity decreases rapidly, and the service life is sho...

Claims

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Application Information

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IPC IPC(8): C12P19/30C12N9/12C12N11/14C12N11/10
CPCC12P19/305C12N9/1229C12Y207/04001C12Y207/04022C12N11/14C12N11/10
Inventor邓壮梅王宏傅得响张燕
Owner美亚药业海安有限公司