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Strain capable of producing L-arginine and method for producing L-arginine by same

A technology of arginine and bacterial strains, applied in the field of L-arginine-producing bacterial strains and using the strains to produce L-arginine, can solve the problem that the level of acid production is not high, not enough for practical production, and the level of expression is low And other issues

Active Publication Date: 2012-05-09
FUJIAN GUTIAN PHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these engineering bacteria often modify a single gene, the expression level is very low, and the acid production level is not high enough to be used in actual production

Method used

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  • Strain capable of producing L-arginine and method for producing L-arginine by same
  • Strain capable of producing L-arginine and method for producing L-arginine by same
  • Strain capable of producing L-arginine and method for producing L-arginine by same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Preparation of histidine-deficient, succinic acid-deficient Brevibacterium flavum mutant strain HX1009 (His - ,Suc - )

[0039] Using the slant strain of the starting strain ATCC14067, pick a ring and inoculate it in the seed medium (glucose 30-60 g / L, corn steep liquor 10-30 g / L, (NH 4 ) 2 SO 4 10~30g / L,KH 2 PO 4 1~5 g / L, MgSO 4 ·7H 2 O 0.5~1 g / L, urea 1.5~5 g / L, pH 7.0~7.2, sterilized at 121°C for 20 minutes, liquid volume 30 mL / 250 mL), on a reciprocating shaker, temperature 30°C, speed 100 RPM, after culturing for 20 hours, take 10 mL of the culture solution and aseptically centrifuge to collect the bacteria, wash with 0.1 mol / L, pH 6.0 phosphate buffer for 2-3 times, and transfer to the Erlenmeyer flask with glass beads Shake to disperse to prepare bacterial suspension (cell concentration 3.2×10 8 individual / mL).

[0040]Take 0.8mL bacterial suspension, add 0.2mL pre-prepared nitrosoguanidine acetone solution with a concentration of 1.0 mg / mL, shake a...

Embodiment 2

[0044] Preparation of D-arginine-resistant, S-methylcysteine-resistant Brevibacterium flavum mutant strain HX1009 (His - ,Suc - , D-Arg r , SMC r )

[0045] HX0901 (His - ,Suc - ) according to the method of Example 1, cultivated to the middle and late logarithmic growth period, and then transferred to fresh medium with 5% inoculum size and cultivated for 5 hours. Then take 10 mL of the culture solution and aseptically centrifuge to collect the thallus. According to the method of Example 1, nitrosoguanidine is used for mutagenesis. After the mutagenesis is completed, it is suspended with 0.5 mL of sterile saline. Finally, take the bacterial suspension and spread it on the resistance screening medium plate, and incubate at a constant temperature of 30°C for 3-5 days. During this period, large colonies are selected according to the growth of the colonies for shake flask screening.

[0046] Resistance screening basal medium (g / L): Glucose 10, (NH 4 ) 2 SO 4 3, KH 2 PO ...

Embodiment 3

[0056] Fermentation of Brevibacterium flavum mutant strain HX1009 to produce arginine in 5L and 15L fermenters

[0057] Screen the mutant strain HX1009 cultivated according to Example 1 and Example 2, inoculate the seeds it has cultivated in the fermentation medium of 5L and 15L tanks by 10% seed quantity and ferment, fermentation medium: glucose 100 g / L, urea 1 g / L, corn steep liquor 25 g / L, (NH 4 ) 2 SO 4 40 g / L, KH 2 PO 4 1.1 g / L, K 2 HPO 4 ·3H 2 O 0.5 g / L, MgSO 4 ·7H 2 O 0.5 g / L, L-His 1 mg / L, biotin 0.05 mg / L, thiamine hydrochloride 0.2 mg / L, and the initial pH of the medium was 7.0-7.2.

[0058] The fermentation tank automatically controls the fermentation temperature at 30±1°C, adjusts the pH to 6.5~7 with 25% ammonia water, the stirring speed during the fermentation process is 300r / min~850 r / min, the ventilation rate is 1:0.5~1:3 (v / v), Adjust according to dissolved oxygen conditions. During the fermentation process, glucose and corn steep liquor were fed ...

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Abstract

The invention relates to a strain capable of producing L-arginine and a method for producing the L-arginine by the strain and belongs to the technical field of biological engineering. For the strain, Brevibacterium flavum ATCC 14067 is used as a starting strain; nitrosoguanidine is adopted to carry out mutagenesis step by step; mutant strains with histidine and succinic acid auxotrophic strains are screened out so as to cut off a competitive metabolic pathway; and mutant strains (His-, Suc-, D-Argr, SMCr) with resistances of arginine structural analogs and cysteine structural analogs are screened out. The strain is named as Brevibacterium flavum HX1009, is preserved in the China general microbiological culture collection center, has the preservation number of CGMCC No.4464 and has the genetic characters of histidine auxotroph His-, succinic acid auxotroph Suc-, D-arginine resistance D-Argr and S-methyl cysteine resistance SMCr for improving the yield of the L-arginine. Under the optimized condition, the L-arginine is produced by fermentation on a fermentation tank with the volume of 5L to 5M3 and the arginine production level achieves 50 to 70g / L.

Description

technical field [0001] The present invention relates to a kind of method utilizing Brevibacterium flavum to produce L-arginine, relates to using Brevibacterium flavum ( Brevibacterium flavum ) ATCC14067 was the starting strain, and nitrosoguanidine was used for stepwise mutagenesis to screen for mutants with histidine and succinic acid auxotrophic strains that cut off the competitive metabolic pathway, and screened for mutants with arginine structural analogs, cysteine Mutant strain HX1009 (His - ,Suc - , D-Arg r , SMC r ) to increase the production of L-arginine. It belongs to the technical field of bioengineering. Background technique [0002] L-arginine is a semi-essential basic amino acid in human body and animals. It is the raw material for the synthesis of protein and creatine. It is also an important intermediate metabolite in the urea cycle of organisms. Improving immunity, preventing and treating cardiovascular diseases, and enhancing the activity of male sper...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/20C12N15/01C12P13/10C12R1/13
Inventor 李少平方佳茂陈伟滨许正宏郑璞孙志浩
Owner FUJIAN GUTIAN PHARMA
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