Breeding Of L-histidine Producer And The Optimization Of Its Fermentation Conditions

The production of L-histidine by fermentation using Corynebacterium glutamicum AJ88 as a parent strain has been studied in this dissertation. Its contents and results are as follows:1) Based on Pauly and ninhydrin reaction of L-histidine, combined with paper chromatography, a method for determining L-histidine in fermentation broth was established. Pauly and ninhydrin reaction were used for qualitative determination of L-histidine, colorimetry of elution after paper chromatography and ninhydrin reaction was used for quantitative determination of L-histidine. The optimum conditions of quantitative determination were as follows: colorimetry wavelength 510nm, elution time 30min, linear range 2μg¹2μg.2) The parent strain was treated by diethyl sulfate (DES) and N-methyl-N'-nitro-N- nitrosoguanidine (NTG). 8-azaguanine (8-AG), 1,6-dicloropurine (DCP), sulfazolepurine (SP), 2-thiouracil (2-TU), 3-Amino-1,2,4-Triazole (AMT), 1,2,4-triazolealanine (TRA) were used as analogs, the producing strain was also marked by histidaseˉ. A strain HZ5013 (TRAR, AMT R, histidaseˉ, DCPR, 2-TUR) that could accumulate 4.63g/L L-histidine was acquired. It produced L-histidine three times as the parent strain in medium using glucose as carbon source and ammonia sulphate as nitrogen source.3) The optimization of medium contents and fermentation conditions for HZ5013 was conducted. The optimum seed medium contains glucose 25 g/L, (NH₄)₂SO₄ 10g/L, corn steep liquor 25g/L, KH₂PO₄ 1.5g/L, CaCO₃ 20g/L, MgSO₄·7H₂O 1g/L,VH 10μg/L The optimum fermentation medium contains glucose 80g/L, (NH₄)₂SO₄ 31 g/L, corn steep liquor 5g/L, KH₂PO₄ 1.3g/L, CaCO₃ 20g/L, MgSO₄·7H₂O 0.5g/L,VH 5μg/L. The optimum fermentation conditions were 250mL contains 15mL broth or 500mL contains 20mL broth, original pH7.0, culturing at 30±1℃on reciprocating shaker, shaking speed 100rpm, seed volume 8%, fermentation time 72h. After optimization, the production of L-histidine was up to 5.5g/L.4) The effects of various substances on L-histidine production had been investigated. Improvements were observed when 0.1g/L L-Leu, L-Ile and L-Pro were added in medium. Addition of other amino acids made decreases in the production of L-histidine. Pyruvate, oxalate, acetate could enhance the L-histidine production while succinate, malate, fumarate had reverse effects. Vitamins didn't affect the production of L-histidine apparently. Lower production of L-histidine was found following the addition of peptone, beef extract, yeast extract. By contrast, more L-histidine was produced after addition of soybean protein hydrolazate. Tween 80, Sodium phytate inhibited the growth of cells and induced decreases in the production of L-histidine. The production of L-histidine was better when glucose and ammonia were flowed.