Microbial Fermentation Production Of L-arginine Breeding

At present,China has become one of the biggest producer of amino acids, the output of glutamic acid and lysine has grown world leading ranks. But a few kinds of amino acids such as arginine,serine,tryptophan and histidine is still not optimistic and far behind the developed countries, especially arginine. But as the high-yield strains is the most important factors to realize fermentation production of L-arginine, and the yield of L-arginine strains between our country and developed countries such as Japan and others are still exist certain disparity. According to the theory of metabolic control fermentation,the paper focuses on the breeding of L-arginine hyper-producer DHR087, the suitable fermentation condition of shaking flask and so on. The specific results are as follows:1. The L-arginine producer was derived from the original strain Corynebacterium glutamicum CICC20191 (L-Arginine : 0.2g/L) by chemical and physical mutation methods,the plate screening with amino acid analogues:arginine methyl ester HCl,sulfaguanidine,D-arginine.A strain DHR087(AEr,SGr,D-Argr)which could accumulate 2.42g/L L-arginine was acquired in medium using glucose as carbon source and ammonia sulphate as nitrogen source. The yield of L-arginine of Stain DHR087 has been optimized to increased to 4.24 g/L.2. The effects of seed medium compositions on the L-Arginine production in flasks were investigated using orthogonal analysis.The optimum seed medium compositions were determined as follows:glucose 30g/L,(NH4)2SO4 25g/L,corn steep liquor 25g/L,KH2PO4 1g/L,MgSO4·7H2O 0.5g/L,CaCO3 10g/L;and the optimum culture pH was 7.0 and the optimum culture volume was 25mL/250mL.3. Effects of various factors such as sources of carbon,nitrogen,phosphate and growth factors on L-arginine production were investigated . Based on the one-variable-at-a-time experiment of fermentation medium,three main significant factors of the sucrose,ammonium sulfate and corn steep liquor which affect the flask-shaking batch fermentation production of L-arginine were screened .With the help of the software Design Expert,a response surface model was built using the Central Composite Design to optimize the fermentation medium: glucose 112.73g/L,(NH4)2SO4 35.76g/L,corn steep liquor 25.15g/L,KH2PO4 1.5g/L,MgSO4·7H2O 0.5g/L,CaCO3 30g/L.The results indicated that the optimal conditions for degradation were pH 7.0, inoculated time 16h ,inoculum size 10%,and the mutant DHR087 which was cultivated 96h at 30℃on a reciprocating shaker(110r/min)with 30mL medium in the flask(250mL)could produce 4.24g/L of L-Arginine on average,which increased about 73.6%.