阅读说明：本技术 一种酶催化精氨酸制备l-瓜氨酸的方法 (Method for preparing L-citrulline by catalyzing arginine through enzyme ) 是由 汪大顺 于 2018-08-13 设计创作，主要内容包括：本发明涉及一种酶催化精氨酸制备L-瓜氨酸的方法,其特征在于包含以下步骤：1)确定酶类；2)酶重复利用；3)实验生产。本发明一种酶催化精氨酸制备L-瓜氨酸的方法克服了精氨酸酶转化法生产L-瓜氨酸的瓶颈,价格低廉易得,工艺简单,产品纯度高。(The invention relates to a method for preparing L-citrulline by catalyzing arginine through enzyme, which is characterized by comprising the following steps: 1) determining enzymes; 2) recycling the enzyme; 3) and (5) experimental production. The method for preparing L-citrulline by catalyzing arginine with enzyme overcomes the bottleneck of producing L-citrulline by an arginase conversion method, and has the advantages of low price, easy obtainment, simple process and high product purity.)

1. A method for preparing L-citrulline by catalyzing arginine through enzyme is characterized by comprising the following steps:

1) determining enzyme classes: adopting arginine deiminase, and performing directional design and rational evolution on catalytic active sites of enzyme molecules according to the three-dimensional space structure of the enzyme molecules to improve the catalytic activity of the enzyme;

2) and (3) recycling the enzyme: immobilizing the constructed engineering bacteria of the escherichia coli, and increasing the permeability of cells and the hardness of immobilized cells by adding a surfactant, other matrixes and the like;

3) experimental production: and pilot test experimental production is carried out on the completed small test work, and scientific and reasonable parameters are provided for large-scale production.

Technical Field

The invention relates to a method for preparing L-citrulline by catalyzing arginine through enzyme.

Background

In 1914, citrulline was first isolated from watermelon juice by the right hertailang et al, and thereafter recognized as an amino acid by hitachi. Citrulline exists in a free state in seeds of cucurbitaceae plants, and to date, citrulline H1 has been successfully separated from watermelon juice, wild watermelon leaves, walnut kernels, walnut seedlings and seeds. Citrulline is a non-protein amino acid and foreign scientists have made more research on citrulline, especially in japan, the united states and europe. In China, the research on citrulline is still only in the recognition stage, and the production method has no corresponding report on the research on the detection method and the physiological function, and only a small amount of literature reports exist. Yuerei et al measured citrulline content in Trichosanthis radix by dual wavelength thin layer scanning method. When the induction and culture of the mutant strain of the botrytis crassa are researched, the citrulline has an obvious growth promoting effect on the mutant strain. Studies of Zhengchunfu and the like find that increasing the L citrulline exogenous to cells can obviously improve the NO yield induced by activated macrophages, further reduce the infection rate of the activated macrophages and inhibit the proliferation of Toxoplasma gondii tachyzoites in the cells. The small peaks and the like discover that the anti-cyclic citrullinated peptide antibody has an important role in the diagnosis of rheumatoid arthritis.

In terms of the physiological functions of citrulline, foreign scientists have researched and found that citrulline has some very important pharmacological functions, such as free radical scavenging action, and has very important significance in the health care aspect of human body; vasodilatation, which is expected to be a good medicine for treating endotoxemia and sepsis. A scientist of Japan has developed a novel citrullinated polypeptide and developed it into an anti-AIDS drug. Meanwhile, citrulline is considered as a very effective antioxidant, and the function is applied to various fields of cosmetics, medicines, health-care foods and the like. In recent years, many foreign researches show that citrulline has many important physiological functions, such as free radical removal, foreign body rejection effect indicators, vasodilation, blood pressure stabilization, rheumatoid arthritis diagnosis, oxidation resistance and the like, and has very wide application prospects.

The preparation method of citrulline mainly comprises three methods, wherein fermentation method and enzyme method production are mainly adopted abroad, and ion exchange and other common separation methods are mostly used for separation and purification.

(1) The chemical method comprises the following steps: the method is characterized in that L-arginine is hydrolyzed under alkaline conditions to obtain L-citrulline, the process control is difficult, optical antipode D-citrulline is contained in the product, the product quality is influenced, a large amount of waste water is generated in the production process, and the environment is polluted.

(2) The difficulty of the fermentation production is that the yield of the L-citrulline in unit volume is low, the highest yield is only 1.7g/L, and the cost for extracting the L-citrulline from the fermentation liquor is high. The fermentation method has the advantages of low cost, high yield, high product purity, no toxic substances generated in the production process of the product, convenience for post-processing of the product and cost reduction. At present, the country which has been most studied on citrulline production by fermentation is Japan, and the research on the citrulline production is available in the last 30 s and reaches a certain level in the last 60 s.

(3) An enzyme method comprises the following steps: under the action of arginine deiminase, L-arginine is converted into L-citrulline, the production condition is mild, no toxic substance is generated, impurities in a conversion system are less, the extraction process is simple, and the pollution is less. The method takes microbial somatic cells with strong specificity and high conversion rate as a catalyst to catalyze the deimination of arginine to generate L citrulline. The enzymatic method is used for synthesizing the citrulline, and a one-step enzymatic reaction is mostly adopted, so that the complex feedback regulation effect in the complete synthesis path of the citrulline can be avoided, and the citrulline can be accumulated to a higher concentration.

In 1973, IchifoChibata et al' 261 reported that Pseudomonas putida ATCC4359, Pseudomonas fluorescens IFO3081, Pseudomonas valisiAM1002, Leuconostoc ceritrovororum TCC8081 and the like could produce arginine deiminase, and L-arginine or DL-arginine was used as a substrate, and could produce citrulline at a concentration of 80 or more. The enzymatic synthesis of citrulline has the advantages of high product concentration, less purification steps, no D-type optical antipode in the product, simple production process and low cost. However, the method has the defects that the enzyme method at present hardly meets the requirements of industrial production because the arginine deiminase obtained by depending on animal internal organs has low activity and low conversion rate, and simultaneously, the arginine raw material has high price, thereby influencing the enzyme method production amplification of citrulline. Therefore, the price of the cheap arginine raw material and the acquisition of the arginine deiminase are the key points for the success of the enzyme method production.

Disclosure of Invention

The invention aims to provide a method for preparing L-citrulline by catalyzing arginine through enzyme.

The invention relates to a method for preparing L-citrulline by catalyzing arginine through enzyme, which is characterized by comprising the following steps:

1) determining enzyme classes: adopting arginine deiminase, and performing directional design and rational evolution on catalytic active sites of enzyme molecules according to the three-dimensional space structure of the enzyme molecules to improve the catalytic activity of the enzyme;

2) and (3) recycling the enzyme: immobilizing the constructed engineering bacteria of the escherichia coli, and increasing the permeability of cells and the hardness of immobilized cells by adding a surfactant, other matrixes and the like;

3) experimental production: and pilot test experimental production is carried out on the completed small test work, and scientific and reasonable parameters are provided for large-scale production.

The method for preparing L-citrulline by catalyzing arginine with enzyme overcomes the bottleneck of producing L-citrulline by an arginase conversion method, and has the advantages of low price, easy obtainment, simple process and high product purity.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the invention are not limited thereto.