【作者】 何俊锋；

【导师】 张伟国；

【作者基本信息】 江南大学 ， 发酵工程， 2006， 硕士

【摘要】 本文以谷氨酸棒杆菌AJ88为出发菌株,对L-组氨酸的发酵法生产进行了研究。主要内容及结果如下:1)以组氨酸的茚三酮显色和Pauly特征显色为根据,结合纸层析法,确定利用Pauly显色和茚三酮显色相结合对发酵液中的L-组氨酸进行定性分析,采用点样纸层析茚三酮显色后洗脱再比色来定量测定发酵液中的L-组氨酸,茚三酮显色洗脱液最佳吸收波长为510nm,最佳洗脱时间为30min,当点样组氨酸含量在2μg¹2μg范围内时呈现较好的线性。2)对出发菌用DES和NTG逐级诱变处理,采用8-氮鸟嘌呤（8-AG）,2,6-二氯嘌呤（DCP）,硫唑嘌呤（SP）,2-硫尿嘧啶（2-TU）,氨基三氮唑（AMT）以及1,2,4-三唑丙氨酸（TRA）作为抗性药物进行选育,并使菌株带上组氨酸酶缺陷型标记,最终获得一株能够较多积累L-组氨酸的菌株HZ5013（TRAR, AMTR, histidase-, DCPR, 2-TUR）。在以葡萄糖为碳源、硫酸铵为氮源的培养基中发酵3d,产酸可达4.63g/L,比出发菌株产酸提高了近2倍。3)对菌株HZ5013进行了培养基和发酵条件的优化。得到最佳的种子培养基为（g/L）:葡萄糖25,（NH₄）₂SO₄ 10,玉米浆25,KH₂PO₄ 1.5,MgSO₄·7H₂O 1,CaCO₃ 20,VH 10μg/L;最佳发酵培养基为（g/L）:葡萄糖80,（NH₄）₂SO₄ 31,玉米浆5,KH₂PO₄ 1.3,MgSO₄·7H₂O 0.5,CaCO₃ 20,VH 5μg/L;摇瓶发酵最佳培养条件为:培养基初始pH7.0,接种量8%,装液量为15mL/250mL摇瓶或20mL/500mL摇瓶,往复式摇床30±1℃培养,摇床转速100次/min,发酵周期72h。优化后最终产酸可达5.5g/L。4)考察了添加氨基酸、有机酸、维生素、各种营养源等以及改变培养手段对L-组氨酸代谢的影响。实验发现添加0.1g/L L-亮氨酸、L-异亮氨酸和L-脯氨酸时,产酸有所提高。添加其它氨基酸均使产酸有所下降。添加丙酮酸、草酸、乙酸能使产酸明显有所提高,添加琥珀酸、苹果酸、富马酸使产酸有所下降。添加各种维生素对L-组氨酸产量的影响很小,少量添加产酸略高。添加蛋白胨、牛肉膏和酵母膏会导致产酸降低,而添加豆饼水解液对产酸有一定的促进作用。在培养基中添加表面活性剂吐温80和稳定剂植酸钠均会抑制菌体生长并导致产酸下降。流加葡萄糖和氨水产酸均优于不流加时的产酸。更多还原

【Abstract】 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.更多还原