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Aeromonas yq and its application in enzymatic preparation of l-citrulline

A technology of Aeromonas and citrulline, applied in the direction of bacteria, microorganism-based methods, biochemical equipment and methods, etc., can solve the problems that there is no Aeromonas to produce L-citrulline

Active Publication Date: 2019-02-01
河北远大九孚生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] So far there is no report on the production of L-citrulline by Aeromonas sp.

Method used

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  • Aeromonas yq and its application in enzymatic preparation of l-citrulline
  • Aeromonas yq and its application in enzymatic preparation of l-citrulline
  • Aeromonas yq and its application in enzymatic preparation of l-citrulline

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Screening of L-citrulline strains produced by enzymatic transformation

[0020] (1) Strain screening

[0021] Soil samples were collected from watermelon greenhouses in Gouzhuang, Hangzhou City, Zhejiang Province, made suspensions with normal saline, and diluted with normal saline gradients, spread on screening medium plates, placed in an incubator, and incubated at 37°C for 24 hours to produce a red color The strain with larger color change circle is the object of preliminary screening. The above strains were inoculated into 100ml of fermentation culture, cultured at 37°C and 180r / min for 24 hours, and the bacterial cells were collected by centrifugation and transferred to 60ml of acetic acid buffer solution with a final concentration of 100g / L L-arginine at pH 6, placed at 37 ℃ shaker, 180r / min transformation 1d. The concentration of L-citrulline in the transformation solution was measured by HPLC, and the ability of the bacteria to transform substrate L-...

Embodiment 2

[0027] Activation and fermentation of embodiment 2 bacterial strains

[0028] Aeromonas YQ was inoculated into the slant medium and cultured at 37°C for 24 hours to obtain slant cells. Inoculate one loop of slant bacteria into a 250 mL Erlenmeyer flask filled with 50 mL of seed medium, and cultivate at 37° C. for 24 hours at 180 rpm to obtain seed liquid. Then inoculate the seed liquid in 100ml fermentation medium with an inoculation amount of 4% volume concentration, culture at 37°C and 180r / min for 24h, centrifuge the fermentation liquid at 6000rpm for 10min, collect the bacteria and wash them with sterilized physiological saline for 1-2 times , to obtain wet cells.

[0029] Slant medium composition: peptone 10g / L, beef extract 5g / L, NaCl 3g / L, KH 2 PO 4 2g / L, the solvent is deionized water, agar 20g / L, pH=7.4.

[0030] Seed medium composition: peptone 10g / L, beef extract 5g / L, NaCl 3g / L, KH 2 PO 4 2g / L, the solvent is deionized water, pH=7.4.

[0031] Fermentation ...

Embodiment 3

[0032] Example 3: Preparation of Aeromonas YQ transformation liquid and derivatization of L-citrulline

[0033]Add 0.621 g of the wet bacteria prepared in Example 2 into 60 ml of acetic acid buffer solution at pH = 6 containing a final concentration of 100 g / L L-arginine, repeatedly pipette and beat evenly, with a liquid volume of 60 mL, place at 37 ° C, 180 r / min Start the conversion reaction. After the transformation reaction for 3 days, take the transformation solution and centrifuge it at 10000r / min for 5min every 24h, dilute the supernatant 30 times with distilled water, take 1mL and put it into a 10mL brown volumetric flask (because the derivative is easy to decompose when exposed to light), add the prepared pH Na of 9 2 CO 3 -NaHCO 3 Add 1 mL of buffer solution, then add 1 mL of 2,4-dinitrofluorophenylacetonitrile solution with a volume concentration of 1.5%, shake well, heat in a water bath at 60°C for 1 h, and use K 2 HPO 4 -KH 2 PO 4 Buffer to volume. The der...

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Abstract

The invention discloses aeromonas sp. YQ and its application in the enzymatic preparation of L-citrulline. The aeromonas sp. YQ is preserved in China Center for Type Culture Collection in Wuhan University in Wuhan, China under CCTCC No: M2015271 on 3th May, 2015, with postcode being 430072. The aeromonas sp. YQ obtained by screening is able to convert the substrate L-arginine to produce L-citrulline; whole-cells of the aeromonas sp. YQ are able to act for 3 days in pH 6 acetate buffer at 30 to 37 DEG C at a speed of 180r / min and able to convert 100g / L of the substrate L-arginine to produce 75g / L of L-citrulline, a product is about 47.8% in purity, the whole process has a total product yield of 40%, and basis is laid for industrialization of the enzymatic production of L-citrulline.

Description

(1) Technical field [0001] The invention relates to an L-citrulline-producing strain-Aeromonas sp.YQ and a process for producing L-citrulline by using the strain to transform substrate L-arginine. (2) Background technology [0002] L-citrulline (L-citrulline) is an important metabolite in the human urea cycle, which can absorb and remove harmful free radicals to play an antioxidant role. Citrulline can relax human blood vessels and is used to enhance male sexual function and treat sexual dysfunction. Maintain healthy lung function, improve mental clarity, and help brain nerve cells have the function of storing and recalling information. L-citrulline can be produced by deiminolation of L-arginine under the action of arginine deiminase. At present, the methods for producing L-citrulline mainly include: fermentation method, chemical synthesis method, extraction method and enzymatic conversion method. The disadvantage of fermentation production is the low yield of L-citrullin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12P13/10C12R1/01
Inventor 吴石金戴安迪邱乐泉钟卫鸿钟莉
Owner 河北远大九孚生物科技有限公司
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