Breeding Of L-Isoleucine Producing Strain By Protoplast Fusion And Studies On Its Key Enzyme And Fermentation Conditions

According to the theory of metabolic control fermentation, the thesis focuses on the breeding of L-isoleucine high-yielding strain and its fermentation conditions. The main research contents and results are as follows:(l)L-isoleucine concentration in fermented broth was quantitatively determined with Partial Least-Squares colorimetry method. The quantitative determining conditions and computing methods were decided. Compared with high-speed amino acid analyzer, the determination value of L-isoleucine concentration with Partial Least-Squares colorimetry method was more accurate than with paper chromatography-mottle elution colorimetry.(2)The protoplast formation and regeneration and fusion conditions of strain ISW330 and AS 1.495 were studied in this thesis. One L-isoleucine high-producing strain SW0370 was screened through protoplast fusion, shake flask screening, genetic marker test and single clone isolation and continuous cultivation for generations. This strain could produce L-isoleucine 14.27g/L after fermentation under the fermentation condition without being optimized. Genetic character of strain SW0370 was very stable.(3)The methods of extracting and purificating and adjusting properties of the key enzyme Threonine Dehydratase in strain SW0370 were studied. On the base of those,the effects of different L-isoleucine concentration or different amino acids concentration on that enzyme activity were also reviewed.(4)The batch fermentation conditions of strain SW0370 in shake flask were studied in this thesis. The proportions of fermentation medium components were optimized by rotation composite design of quadratic regression.The culture conditions of batch fermentation and fed batch fermentation were also studied.Under the optimum conditions, strain SW0370 could produce L-isoleucine 21.96g/L after fermentation for 96 hours by flask-shaking fed batch fermentation.The innovation of this thesis in the research field of L-isoleucine metabolic control fermentation was that protoplast fusion technique had been successfully applied to breed an L-isoleucine high-yielding strain SW0370, whose key enzyme Threonine Dehydratase' s adjusting properties were studied,which would be good at more researches on L-isoleucine breeding. The strain SW0370 could produce L-isoleucine 21.96g/L after fed batch fermentation by flask-shaking for 96 hours, which had reached the advanced technique level of the same research in China. The strain SW0370 had had a potential of producing in fermentor.