Method for continuously preparing [14/15N]-L-citrulline by immobilized enzyme

Abstract

The invention discloses a method for continuously preparing [14 / 15N]-L-citrulline by immobilized enzyme, and belongs to the technical field of enzymology and enzyme engineering. The method comprises the following steps of suspending fusion protein containing immobilized enzyme in a packed bed reactor; and enabling a solution containing [14 / 15N]-L-arginine to flow through the packed bed reactor atthe flow rate of 0.3-0.5 BV / h at the temperature of 20-55 DEG C for reaction, and separating and purifying the reaction solution to obtain [14 / 15N]-L-citrulline. According to the technical concept, cipA immobilized fusion protein is adopted as a carrier to immobilize arginine deiminase arc on inclusion body protein cipA, and inclusion body protein cipA-arc with catalytic activity, namely cipA-arcfusion protein, is generated. The immobilized cipA-arginine deiminase cipA-arc fusion protein with the catalytic activity provided by the invention has the advantages that the continuous reaction canbe performed for 560 hours or longer; and meanwhile, the obtained isotopically labeled [14 / 15N] L-citrulline provides an effective way for diagnosis and treatment of prostate diseases, cardiovasculardiseases and the like.

Description

technical field [0001] This application relates to a production of high-purity [ 14 / 15 The method of N]-L-citrulline, especially a method utilizing recombinant arginine deiminase to decompose [ 14 / 15 N]-L-Arginine produces high purity [ 14 / 15 The method for N]-L-citrulline belongs to the technical field of enzymology and enzyme engineering. Background technique [0002] L-citrulline (L-citrulline) is a special amino acid. Participate in a variety of metabolic processes in the body, such as scavenging free radicals, indicators of allogeneic rejection, vasodilation, stabilizing blood pressure and diagnosing rheumatoid arthritis, anti-oxidation, preventing prostate diseases and improving sexual function, anti-aging and enhancing immunity, etc. , the application prospect is very broad. [0003] The methods for producing L-citrulline include chemical method, fermentation method and enzymatic method. The chemical method refers to the hydrolysis of L-arginine under alkaline co...

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Problems solved by technology

The chemical method has a tight bond between the enzyme and the carrier, is not easy to fall off, and has good stability, but the reaction conditions are intense, the operation is complicated, the control conditions are harsh, and the activity loss is relatively large

[0006] In 2008 Zheng Pu (Zheng Pu, Ni Ye, Zhang Wen. Continuous preparation of L-citrulline by immobilized Pseudomonas cells in a packed bed reactor[J]. Journal of Food and Biotechnology, 2008, 27(5): 1673-1689) etc. have reported that immobilized Pseudomonas cells can continuously prepare L-citrulline in a packed bed reactor at 0.0108g (h.g) -1 (grams of citrulline produced per gram of cell per hour) under the condition of continuous 54d operation, but the bacterial fermentation production process is complicated, and the time of immobilized cells is too long, the substrate concentration is low, and the yield is not high. Although the immobilized cells It is simple, but there is still the problem that the cell body is broken to release the impurity protein and other organic substances in the cell, which increases the difficulty of separation and purification of the product in the reaction system, and the cell immobilization also increases the operation steps and increases the production cost.

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