阅读说明：本技术 一种固定化酶法制备三磷酸鸟苷的方法 (Method for preparing guanosine triphosphate by immobilized enzyme method ) 是由 李晓燕 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鸟苷的方法,具体包括以下步骤：(1)制备GTP生产酶、(2)固定化GTP生产酶、(3)分离产物；本发明采用PPK、GMPK两种GTP生产酶,只需两步酶促反应即可合成GTP,与传统啤酒酵母生产GTP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GTP,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；该方法适用于大规模生产GTP。(The invention relates to the technical field of biology, in particular to a method for preparing guanosine triphosphate by an immobilized enzyme method, which comprises the following steps: (1) preparing GTP production enzyme, (2) immobilizing GTP production enzyme, and (3) separating a product; the method adopts two GTP production enzymes of PPK and GMPK, can synthesize GTP only by two-step enzymatic reaction, and has the advantages of simpler reaction process, easier control of the reaction and more stable product quality compared with the traditional process for producing GTP by using beer yeast; the GTP 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, 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 GTP.)

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

(1) preparation of GTP-producing enzyme: immobilizing GTP production enzyme on an immobilized carrier, and performing enzymatic reaction by using polyphosphate kinase (EC2.7.4.1, PPK) and guanylate kinase (EC 2.7.4.8, GMPK) to synthesize the immobilized GTP production enzyme;

(2) immobilized GTP-producing enzyme: preparing guanosine triphosphate reaction liquid by catalysis by using the immobilized GTP production enzyme, filtering and collecting a carrier to obtain the immobilized GTP production enzyme;

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

2. The method for preparing guanosine triphosphate according to claim 1, wherein said preparation of GTP producing enzyme: preparing high-expression PPK and GMPK strains, and centrifugally collecting thalli after fermentation is finished; respectively taking 1.0-2.0kg of thallus containing PPK and 1.0-2.0kg of thallus containing GMPK, 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 GTP production enzyme.

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

4. The method for preparing guanosine triphosphate according to claim 1, wherein said separation product is: preparing reaction liquid with the total volume of 10L in a 20L reaction tank, wherein the reaction liquid contains 200-300g of GMP, 200g of sodium hexametaphosphate, 130g of magnesium chloride 110-; adjusting pH of the reaction solution to 7.0 with NaOH, adding 0.4-0.6kg of the immobilized GTP production enzyme, starting stirring at 37 ℃ and 150rpm for reaction for 4-6h, and detecting the generation amount of GTP by high performance liquid chromatography; recovering immobilized GTP production enzyme from the reaction liquid after reaction through a filter bag, and obtaining a guanosine triphosphate dry product (GTP) 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 guanosine triphosphate by an immobilized enzyme method.

Background

The glycolysis pathway of yeast is utilized, guanylic acid is taken as a substrate, and the synthesis of GTP through matrix level phosphorylation is the most common method and is also the method generally adopted in the industrial production of Guanosine Triphosphate (GTP) at present. However, the reaction process of catalytically synthesizing GTP by a yeast cell enzyme system is complex, a plurality of enzyme systems participating in catalytic reaction are provided, 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. At present, the GTP production level in China is generally low, and the cost control, the product quality and the like of the product 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.

In addition, in practical application, the cost of the enzyme is high, the GTP is produced by using free enzyme, the enzyme activity is reduced rapidly, the GTP cannot be effectively recycled, the production cost is high, and the practical application value is influenced.

Disclosure of Invention

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

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

(1) preparation of GTP-producing enzyme: immobilizing GTP production enzyme on an immobilized carrier, and performing enzymatic reaction by using polyphosphate kinase (EC2.7.4.1, PPK) and guanylate kinase (EC 2.7.4.8, GMPK) to synthesize the immobilized GTP production enzyme;

(2) immobilized GTP-producing enzyme: preparing guanosine triphosphate reaction liquid by catalysis by using the immobilized GTP production enzyme, filtering and collecting a carrier to obtain the immobilized GTP production enzyme;

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

Preferably, said preparing a GTP-producing enzyme: preparing high-expression PPK and GMPK strains, and centrifugally collecting thalli after fermentation is finished; respectively taking 1.0-2.0kg of thallus containing PPK and 1.0-2.0kg of thallus containing GMPK, 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 GTP production enzyme.

Preferably, the immobilized GTP-producing enzyme: adding agarose-IDA-Ni 2+ chelating carrier into a constant-temperature stirring reaction tank, mixing with 10L GTP production enzyme, 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 GTP 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 200-300g of GMP, 200g of sodium hexametaphosphate, 130g of magnesium chloride 110-; adjusting pH of the reaction solution to 7.0 with NaOH, adding 0.4-0.6kg of the immobilized GTP production enzyme, starting stirring at 37 ℃ and 150rpm for reaction for 4-6h, and detecting the generation amount of GTP by high performance liquid chromatography; recovering immobilized GTP production enzyme from the reaction liquid after reaction through a filter bag, and obtaining a guanosine triphosphate dry product (GTP) after chromatographic separation, crystallization and drying of the permeation liquid.

The invention has the following beneficial effects:

1. the invention adopts two GTP production enzymes of PPK and GMPK, can synthesize GTP only by two-step enzymatic reaction, and has simpler reaction process, easier control of the reaction and more stable product quality compared with the traditional process for producing GTP by beer yeast.

2. The GTP 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, 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 GTP.

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.