Biological preparation method of agmatine sulfate

A technology for agmatine sulfate and biological preparation, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc., can solve the environmental pollution of agmatine sulfate and the lack of agmatine sulfate Seeing and unfavorable for energy saving and emission reduction of enterprises, achieving the effect of low cost, high production efficiency and simple preparation process

Inactive Publication Date: 2015-09-16
CHANGXING PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the preparation of agmatine sulfate by the above method is easy to cause larger pollution to the environment and the yield is low, which is unfavorable for ...

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  • Biological preparation method of agmatine sulfate
  • Biological preparation method of agmatine sulfate
  • Biological preparation method of agmatine sulfate

Examples

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Embodiment 1

[0021] Example 1: Small-scale preparation process of recombinant L-arginine decarboxylase

[0022] (1) Pilot fermentation of recombinant L-arginine decarboxylase

[0023] Take the 5L fermentation charge in a 7L small fermentation tank as an example:

[0024] Spread the recombinant E. coli glycerol tube or oblique interview tube containing the L-arginine decarboxylase gene in an LB agar oblique interview tube, and the medium contains 100 mg / L kanamycin. Activate and culture at 37°C for 13h. Add 6 mL of sterile normal saline to the cultured LB oblique tube to make a bacterial suspension. Take 1 mL of the bacterial suspension into a 100 mL TB shake flask seed solution, and add 10 mg of kanamycin at the same time. The seed liquid was cultivated to OD at 35℃, 220rpm shaker 600 When it reaches 4 to 5, it is connected to 5L TB fermentation broth. The culture conditions are as follows: Inoculation volume: 100mL / 5L (2.0%); Ventilation volume: 1vvm (5 L / min); Tank pressure: 0.04MPa; Stirri...

Embodiment 2

[0026] Example 2: Reaction monitoring method

[0027] HPLC method for detecting substrates and products: quickly dilute the reaction solution 250 times with frozen deionized water to terminate the enzymatic reaction, mix well and filter it with a 0.45μm microporous membrane, and wait for sample injection. Chromatographic conditions are: HILIC amino column: 3μm, 100?, 250mm×4.6mm; mobile phase: water (pH adjusted to 2.5 by phosphoric acid); flow rate: 0.5mL / min; column temperature: 35°C; detection wavelength: 205nm; sample injection Quantity: 10μL. The specific map is shown in the attached drawings of the manual Figure 2 ~ Figure 6 .

Embodiment 3

[0028] Example 3: Enzyme activity verification method

[0029] Preparation of citric acid phosphate buffer solution with pH 6.0: 2g citric acid monohydrate, 12g Na 2 HPO 4 ·12H 2 Dissolve O in deionized water and dilute to 200 mL, set aside. Preparation of 10g / 99mL arginine sulfate buffer solution: Dissolve 10g arginine in 50mL pH 6.0 citrate phosphate buffer solution and adjust the pH to about 6.0 with sulfuric acid (40%, V / V), and continue to use pH The volume of 6.0 citrate phosphate buffer solution is adjusted to 99mL. Add 99mL of the above-mentioned arginine sulfate buffer solution, 0.02g of coenzyme PLP (pyridoxal phosphate), 1mL of wet bacteria concentration of 1g / 10mL of crude enzyme solution after breaking the wall into a 250mL reaction flask, 150rpm water bath shaker 30 After shaking the reaction at ℃ for 5 minutes, when the arginine content is reduced by about 20g / L by HPLC, it can be judged that the enzyme can be used for normal production.

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Abstract

The invention belongs to the technical field of biological catalysis, and relates to a biological preparation method of agmatine sulfate, which comprises the following steps: by using an L-arginine sulfate solution as a substrate raw material, carrying out decarboxylation reaction in the presence of a biocatalyst and a coenzyme to obtain agmatine sulfate, and discharging gas CO2, wherein the biocatalyst adopts L-arginine decarboxylase, and the coenzyme adopts phosphopyridoxal. The L-arginine sulfate solution used as the substrate is stirred and mixed with the L-arginine decarboxylase and the coenzyme (phosphopyridoxal) to perform decarboxylation reaction under certain conditions, thereby obtaining the agmatine sulfate. The conversion rate is greater than or equal to 98%, and the continuous production yield can reach 90%. The technical route has the advantages of simple preparation technique, high production efficiency, low cost and the like. The fermentation and conversion techniques of the recombinant L-arginine decarboxylase can achieve the industrial preparation level, thereby basically solving the problems of high chemical synthesis cost, low yield, and great environmental hidden danger and the like.

Description

technical field [0001] The invention belongs to the technical field of biocatalysis and relates to a biological preparation method of agmatine sulfate. Background technique [0002] In vivo, agmatine is a product formed by the decarboxylation of arginine by L-arginine decarboxylase (L-ADC). As a supplement for maintaining sports nutrition, agmatine is superior to arginine in many aspects of its function. It can help users improve the body's endurance during exercise, accelerate the body's recovery after exercise, and improve the body composition, including Improve muscle mass and reduce fat fat. Agmatine also has a variety of pharmacological activities, and is a detoxification drug with great development value. Especially in the form of sulfate, it is widely used in the pharmaceutical and health care products market. Its structural formula is as follows. [0003] [0004] In recent years, there have been some reports on the preparation of agmatine sulfate at home and ...

Claims

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Application Information

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IPC IPC(8): C12P13/00C12N9/88C12R1/19
Inventor 肖延铭谈聪
Owner CHANGXING PHARMA
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