Studies On The Preparation And Extraction Of L-Arginine

L-arginine, one kind of alkaline amino acid, is widely used in the pharmacy and foodindustry. At present, it has been produced successfully through the fermentation in Japan,American and Germany,while in china, it is mainly made through hydrolysis method fromnatural protein in China. So it is of great urgence to study the L-arginine fermentation productionmethod in our country. In this thesis, the method of L-arginine determination and the breeding ofL-arginine producing strain were conducted. The fermentation and purification process were alsoinvestigated systemically.In this paper, a paper choromatography method was established to measure the amount ofthe L-arginine of the broth with conditions is: 77% ethanol: diethylamine=100:1 as thespreading reagent, and Sakaguchi reagent as spraying reagent. Furthermore, The optimalconditions of quantitative determination of L-arginine were as follows: 40g/L NaOH, 80g/L1-naphthol-propanol and 0.5mL/L Diacetyl-propanol were added sequently as the indicator, eachof which was 1 mL. The reaction was carried out in the water bath at 30℃ for 15 minutes.A L-Arginine producing mutant UNC90-41(SGr 0.4 mg/mL,D-Argr 6mg/mL,AEr6mg/mL) was selected out by sulfaguanidine and D-arginine and arginine-methylester resistanceafter combination stepwise mutagenic treatment with UV (ultra violetrays), NTG(N-menthyl-N-nitro-N-nitrosoguanidine) and 60Co from Corynebacterium glutamicumATCC138761. And also this mutant has good stability of descendiblity of L-arginine.Effects of various factors such as sources of carbon, nitrogen and phosphate on L-arginineproduction were investigated. Fermentation conditions for over-production of L-arginine in aflask of 500mL containing 15mL medium was optimized. The strain was cultured with 96r/m ofto-and-fro shaker at 30℃ for 96h. It could accumulate 15.0g/L L-arginine in an optimal medium,containing glucose 100g/L, (NH4)2SO4 20g/L, MgSO4 0.5g/L, KH2PO4 1g/L, corn steep liquor15g/L, biotin 80μg/L, CaCO3 30 g/L.The fed batch cultivation with urea and ammonia and L-glutamic acid were alsoinvestigated preliminarily. Among these, the fed batch cultivation with ammonia has animprovement on L-arginine by supplying 0.5mL 1.34mol/L ammonia at 24h and one time every12h in the whole culture period. Result suggests that this fed batch cultivation could accumulate16.4g/L L-arginine.The pretreatment, extraction and discoloring process of L-arginine fermentation broth werealso preliminarily studied. Experiment results indicated that the best flocculant waspolypropylene-acyl-amine, and the optimum flocculantion conditions were: flocculant dosage400mg/L, pH 10.0, temperature 30℃ . The L-arginine was separated through the ion-exchangeresin and the optimal conditions were as follows: Adjusting fermentation liquor to pH2.0, theadsorption velocity of flow 2mL/min, the elution concentration of ammonia 0.1mol/L. Theoptimum conditions of activated carbon powder discoloring were as follows: activated carbonpowder dosage 10g/L, temperature 80℃ , pH 5.0. Adopting the whole technology of recovery,the total recovery ratio was 91.2%.