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