Breeding Of L-Isoleucine High-Yielding 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 thin-layer chromatograpHy and HPLC. The quantitative determination condition and computing method were decided. Compared with high-speed amino acid analyzer, the determination value of L-isoleucine concentration with HPLC is more accurate than with thin-layer chromatograpHy.(2) Brevibacterium flavum T6-13 as a starting strain was stepwise mutated with protoplast UV and Diethyl sulfate. One L-isoleucine high-producing strain AJS406 (Met-+α-ABr)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 9.2g/L after fermentation under the fermentation condition without being optimized. Genetic character of strain AJS406 was very stable.(3) The protoplast formation rate and regeneration and fusion condition of strain AJS406 and F9114 was studied in this dissertation. One L-isoleucine high-producing strain IA412 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 14.3g/L after fermentation under the fermentation condition without being optimized. Genetic character of strain IA412 was very stable.(4)The batch fermentation conditions of strain IA412 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 IA412 could produce L-isoleucine 20.7g/L after fermentation for 96 hours by flask-shaking batch fermentation.(5) Base on the above optimum condition, the batch fermentation was performed in 20000-liter fermentor. And the optimal fermentation conditions were got.