Arginine deiminase high-yielding Bacillus cereus and application thereof

An arginine deiminase, Bacillus cereus technology, applied in bacteria, microorganism-based methods, microorganisms, etc., can solve the problem that the enzymatic activity does not reach the ideal industrial level, etc., and achieves short fermentation culture time, separation The effect of low purification cost and high product concentration

Active Publication Date: 2015-07-22
WUHAN GRAND HOYO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] It can be seen that at present, citrulline is mainly biosynthesized by the resting cells of arginine deiminase fermented by Enterococcus faecalis, Pseudomonas putida, Pseudomonas mutans, and Kurtia spp. The report of Bacillus cereus, and the current level can only convert more than 80% of the arginine substrate into citrulline, and the enzyme activity has not yet reached the ideal industrial level

Method used

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  • Arginine deiminase high-yielding Bacillus cereus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Obtaining of mutant strains

[0035] 1. Preparation of Type-Based Strains

[0036] Take a strain of Bacillus cereus obtained from the soil as the starting strain, insert it into a medium containing 100ml, cultivate it at 30-37°C, 100-200r / min for 5-30h, then take 1ml of the bacterial solution and add it to 9ml of sterile water, mix well, dilute the concentration to 10 times, continue to dilute 10 times in this way -2 , 10 -3 and 10 -4 times, and the bacterial solution is set aside.

[0037] The composition of the culture medium is: 0.1-10% peptone, 0.1-5% yeast powder, 0.1-10% sodium chloride, the balance is deionized water, and the pH is 6.0-7.5.

[0038] 2. N ion beam mutagenesis

[0039] Take 0.5ml of the bacterial solution prepared in step 1, apply it to a sterile petri dish and dry it, and use a TITAN ion beam implanter to perform N ion beam implantation on the bacteria with an energy of 30keV and an injection dose of 1-5×10 15 ion / cm 2 , after inj...

Embodiment 2

[0049] The production method of embodiment 2 L-citrulline

[0050] 1. Culture of strains

[0051] Including seed culture and fermentation culture, the specific steps are as follows:

[0052] (1) Primary seed cultivation:

[0053] Primary seed medium: 1% peptone, 0.5% yeast powder, 1% sodium chloride, 1% glycerin, the balance is deionized water, and the pH is 7.

[0054] Cultivation method: Take a glycerol tube to store the bacteria, place it at room temperature until it melts, draw 500ul of the bacteria solution into the first-level seed shake flask, and culture at 37°C for 15 hours at 200r / min.

[0055] (2) Secondary seed cultivation:

[0056] Secondary seed medium: 1% glycerin, 4% corn steep liquor, 0.5% arginine, the balance is deionized water, pH is 7.

[0057] Cultivation method: Inoculate the primary seeds into a 30L secondary seed tank at a volume ratio of 5%, and cultivate for 15 hours at 200r / min.

[0058] (3) Fermentation culture

[0059] 2% glycerin, 2% corn s...

Embodiment 3

[0072] The production method of embodiment 3 L-citrulline

[0073] 1. Culture of strains

[0074] (1) Primary seed cultivation:

[0075] Primary seed medium: 0.1% peptone, 5% yeast powder, 0.1% sodium chloride, 0.1% glycerol, the balance is deionized water, pH 6.

[0076] Cultivation method: Take a glycerol tube to store the bacteria, place it at room temperature until it melts, draw 100ul of the bacteria solution into the first-level seed shaker flask, and cultivate at 100r / min at 30°C for 16h.

[0077] (2) Secondary seed cultivation:

[0078] Secondary seed medium: 5% glycerin, 5% corn steep liquor, 5% arginine, the balance is deionized water, pH 6.

[0079] Cultivation method: inoculate primary seeds into secondary seed tanks at a volume ratio of 1%, and cultivate for 16 hours at 100 r / min.

[0080] (3) Fermentation culture

[0081] 0.1% glycerin, 5% corn steep liquor, 0.1% arginine, 0.1% magnesium sulfate, the balance is deionized water, the pH is 6.

[0082] Cultiva...

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Abstract

The invention discloses an arginine deiminase high-yielding Bacillus cereus, which is preserved in China Center for Type Culture Collection with a preservation number of CCTCC NO:M 2015047. The invention also discloses application of the Bacillus cereus in production of L-citrulline and a production method of L-citrulline. The Bacillus cereus provided by the invention can transform over 98% of arginine to L-citrulline, and L-citrulline with purity of more than 99% can be prepared. The method provided by the invention also has the characteristics of low production cost, short period and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a strain of Bacillus cereus capable of high-yielding arginine deiminase, and the invention also relates to the application of the Bacillus cereus in the production of L-citrulline And a method of producing L-citrulline. Background technique [0002] L-citrulline has the functions of relaxing blood vessels, treating sexual dysfunction, and improving brain power. In recent years, it has attracted more and more attention in the fields of food and medicine, and has broad development and application prospects. At present, the production methods of L-citrulline mainly include plant extraction method, chemical synthesis method, fermentation method and enzyme conversion method. Among them, the plant extraction method has low content and high extraction cost, which is not suitable for large-scale production; the chemical organic synthesis method causes great environmental polluti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/20C12P13/10C12R1/085
CPCC12P13/10C12N1/205C12R2001/085
Inventor 苏海霞王君英邢盼盼王炯
Owner WUHAN GRAND HOYO
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