Breeding Of L-Isoleucine High Producing Strain And Studies On Its Fermentation Conditions

According to the theory of metabolic control fermentation, the dissertation focuses on the breeding of L-isoleucine high-yielding strain and its fermentation conditions. The main research contents and results are as follows:(1) L-isoleucine concentration in fermented broth was quantitatively determined with methods of paper chromatography-mottle elution colorimetry and chemical colorimetry. The quantitative determination condition and computing method were decided. Compared with high-speed amino acid analyzer, the determination value of L-isoleucine concentration with chemical colorimetry is more accurate than with paper chromatography-mottle elution colorimetry.(2) Brevibacterium flavum HL41 as a starting strain was stepwise mutated with protoplast UV and Diethyl sulfate. One L-isoleucine high-producing strain ISW330 with genetic marker was screened through shake tube primary screening, shake flask compound screening, genetic marker test and single clone isolation and continuous passage cultivation This strain could produce L-isoleucine 9.18g/L after fermentation under the fermentation condition without being optimized. Genetic character of strain ISW330 was very stable.(3) The protoplast formation rate and regeneration and fusion condition of strain ISW330 and AS 1.495 was studied in this dissertation. One L-isoleucine high-pro.ducing strain SW0370 was screened through protoplast fusion, shake flask compound screening, genetic marker test and single clone isolation and continuous passage cultivation. This strain could produce L-isoleucine 20.7g/L after fermentation under the fermentation condition without being optimized. Genetic character of strain SW0370 was very stable.(4)The batch fermentation conditions of strain SW0370 in shake flask were studied in this dissertation. The proportions of seed medium and fermentation medium components were optimized by uniform design experiment and MATLAB software .The seed culture conditions and fermentation conditions were also studied.Under the optimum conditions, strain SW0370 could produce L-isoleucine 20.7g/L after fermentation for 96 hours by flask-shaking batch fermentation.(5) Based on the above optimum condition, the batch fermentation was performed with strain SW0370 in 7-liter fermentor. The L-isoleucine batch fermentation kinetics was studied by genetic algorithms based on the experimental data from 7-liter fermentor's batch fermentation. Three kinetic models were constructed which could reflect the regularity of growth, product formation and substrate consumption in the process of batch fermentation.(6) Fed batch fermentation was studied with SW0370 according to the optimum condition of batch fermentation. The optimum initial glucose concentration was 80g/L in shake flask fed batch fermentation. The intermittent feeding was performed at the 24th, 48lh, 60th, 80th hour respectively. The production of L-isoleucine reached 21.96g/L after fermentation for 96 hours. Based on the above experiment, fed batch fermentation in 7-liter fermentor was performed. The production was improved by 7.29% than that of fermentation in shake flask fed batch.Because of the technology of low glucose, low amino and fed batch, production of L-isoleucine reached 24.82g/L after fermentation for 96 hours.The innovation of this dissertation in the research field of L-isoleucine metabolic control fermentation is that protoplast fusion technique was successfully applied to breed L-isoleucine high-yielding strain.which has two defective mutations(Leu"+Met") An L-isoleucine high-yielding strain SW0370 was obtained, which could produce L-isoleucine 24.82g/L after fed batch fermentation in 7-liter fermentor for 96 hours, which has reached the advanced technique level of the same research in China. The chemical colorimetric method was built to detect L-isolecuine,which was quick , efficient and .In addition ,this method has importantly instrumental function in industrial production of L-isoleucine to assay its content...