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Method for preparation of L-citrulline with arginine as raw material

A technology of arginine and citrulline, applied in the biological field, can solve the problems of low arginine deiminase activity, difficult to meet industrial production by the enzymatic method, affecting the production of citrulline enzymatic method, etc., and achieve product purity. High, overcoming the bottleneck of citrulline production and saving dosage

Inactive Publication Date: 2015-12-23
滨州市生物技术研究院有限责任公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] (2) The difficulty of fermentation production is that the yield of L-citrulline per unit volume is low, the highest is only 1.7g / L, and the cost of extracting L-citrulline from the fermentation broth is relatively high
But its disadvantage is that the current enzymatic method is difficult to meet the requirements of industrial production. The reason is that the activity of arginine deiminase obtained from animal offal is low, the conversion rate is low, and the price of arginine raw materials is high, which affects Enzymatic production scale-up of citrulline

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  • Method for preparation of L-citrulline with arginine as raw material
  • Method for preparation of L-citrulline with arginine as raw material

Examples

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Effect test

Embodiment 1

[0035] Example 1 A bacterial strain MQO-153 producing arginine deiminase, the present invention through repeated chemical mutagenesis and ultraviolet mutagenesis to Streptococcus faecalis (Streptococcus faecalis purchased from Beijing Microorganism Culture Collection Center), And after screening a large number of strains, a strain MQO-153 with high arginine deiminase activity was obtained, and its preservation number is CGMCCNo.10726; the preservation unit is the General Microbiology Center of China Microbiological Culture Collection Management Committee ; The preservation date is April 21, 2015; the preservation address is No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing.

Embodiment 2

[0036] Example 2 The method for preparing arginine deiminase by using bacterial strain MQO-153, the specific steps are as follows:

[0037] (1) Slant culture: inoculate the bacterial strain MQO-153 on the slant medium under aseptic conditions for cultivation, the cultivation temperature is 30° C., and the cultivation time is 24 hours. At this time, the bacterial colony is large and smooth without pigment accumulation;

[0038] Slant medium: Slant medium (g / L): beef extract 5, peptone 10, sodium chloride 5, agar 20; pH is 7.0-7.3; preferably 7.2; the sterilization temperature of the slant medium is 115 ° C, sterilized The time is 15 minutes.

[0039] (2) Expansion culture: pick bacterium colony from the slant culture medium of step (1), add water and dilute to obtain thalline solution, the concentration after dilution is 3 * 10 5 -5×10 5 cells / mL, inoculate the bacterial cell solution onto the seed medium and place it in a shaker for expanded culture, the inoculum size is 10%...

Embodiment 3

[0044] Example 3 The method for preparing L-citrulline from L-arginine through the conversion of arginine deiminase, the specific steps are as follows:

[0045] (1) Obtaining the substrate: the arginine ceramic membrane filtrate was adsorbed by 732 ammonia resin, and eluted with 2.0N ammonia water to obtain arginine positive column purified solution;

[0046] (2) Transformation culture: the bacteria containing arginine deiminase were washed once with normal saline, and the bacteria were collected after centrifugation. Transfer the bacteria to the transformation solution, which includes 0.45mol / L acetate buffer and 0.5g / L CTAB, add 10% L-arginine purification solution, and incubate at 37°C for 24h to obtain L-citrulline Amino acid conversion solution.

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Abstract

The invention relates to a method for preparation of L-citrulline with arginine as the raw material. The method specifically includes the steps of: (1) acquisition of a substrate: subjecting an arginine ceramic membrane filtrate to adsorption by 732 amino type resin, conducting elution with 2.0N ammonia water to obtain an arginine positive column purified liquid; and (2) transformation culture: washing a arginine deiminase-containing thallus with normal saline once, performing centrifugation, then collecting the thallus, and transferring the thallus into a transformation liquid, which contains a 0.45mol / L acetic acid buffer solution and 0.5g / L CTAB, adding a 10% L-arginine purified liquid, and conducting heat preservation at 37DEG C for 24h, thus obtaining an L-citrulline transformation liquid. The method provided by the invention adopts an arginine fermented purified liquid as the transformation raw material, overcomes the bottleneck in production of citrulline by arginase transformation method, and has the advantages of cheap and easily available raw materials, simple process, and high product purity.

Description

technical field [0001] The invention relates to a method for preparing L-citrulline by using arginine as a raw material, belonging to the field of biotechnology. Background technique [0002] In 1914, Uga Taro and others isolated citrulline for the first time from watermelon juice, and it was recognized as an amino acid by Wada Mitsutoku afterwards. Citrulline exists in the seeds of Cucurbitaceae plants in a free state. So far, people have successfully isolated citrulline H1 from watermelon juice, wild watermelon leaves, walnut kernels, walnut seedlings and seeds. Citrulline is a non-protein amino acid. Foreign scientists have done more research on citrulline, especially in Japan, the United States and Europe. In China, the research on citrulline is still in the stage of understanding, there is no corresponding report on the production method, and there are not many researches on its detection method and physiological function, only a small number of literature reports. Yu...

Claims

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Application Information

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IPC IPC(8): C12P13/10C12N9/78C12R1/46
Inventor 高法民王威闫洪波
Owner 滨州市生物技术研究院有限责任公司
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