Breeding Of Producing L-threonine Strain And Optimization Of Fermentation Conditions

Threonine is fourth essential amino acid in 8 essential amino acids that body of people and animals need,it close to methionine lysine tryptophane.Threonine is playing more and more important roles, which has been widely used in the food industry, feed industry, and medical treatment, etc. L- threonine fermentation production is affected mainly by L- threonine own feedback inhibition and inhibition and repression of other amino acids feedback in aspartic acid clan. This thesis is Corynebacterium pekinense AS 1.299 as starting strain which was mutagenized by physics and chemistry and screening the drug-resistant drug flat mutations which make producing L-threonine key enzymes improved and large generated and increase output of aspartic acid as precursor,because the drug-resistant drug mutations can relieve inhibition and repression of other amino acids feedback in aspartic acid clan, and then make L-threonine production sharply increased. Finally,the fermentation conditions of obtained mutation strains was optimized and get optimal L-threonine yields. The research contents and the results are as follows:1.Determination on the nature and quantitative determination of L-threonine contents in fermentation liquor was done by TLC and got following conclusions:0.3% ninhydrin as colour-developing agent, Chromatography is TLC and chromatography agent is water: ethanol:glacial acetic acid=1:6:0.5 (volume ratio), which is fit to separate glutamic and threonine,optimal elution time is 30min through the TLC, the optimum absorption wavelength is 512nm through ultraviolet spectrophotometer.The TLC-ultraviolet spectrophotometry is established for natural and quantitative determination of L-threonine contents in fermentation liquor:sample application 1μL every time, sample application spots diameter is in 0.5cm,colour-display is 5min under 105℃, scraping the entire L-threonine spot area to oscillate and wash 30min,and then determination absorbency under the wavelength 512nm within 60min.2.Corynebacterium pekinense AS1.299 as starting strain which was mutagenized by UV and NTG,and then obtain a high producing strain N50-10,its volume of production is 1.150g/L. And then screening out ethionine resistance and sulfaguanidine resistance Strain SG06-10(Ethr+SGr) was on the basis of strain N50-10, which can bring L-threonine up to 2.410g/L,comparing with Corynebacterium pekinense AS1.299 has 2.366g/L with increasing, it showed that go down to 5 posterity had good genetic stability.3.The composition of fermentation culture medium and fermentation technological conditions of producing L-threonine mutant SG06-10 were optimized by the response surface method, and obtained the best recipe for fermentation culture medium:glucose 18.70%,urea 1.25%,MgSO40.10%,KH2PO40.05%, FeSO4·7H2O 0.02%,MnSO4·H2O 0.02%,biotin 8μg/100mL,thiamine 200μg/100mL,pH 7.0-7.2.Best fermentation technological conditions:temperature 33℃,pH 6.8,inoculation quantity 8%,liquid medium volume 61.3 mL,revolving speed 180r/min, fermentation time 72h. Under the optimized combination condition L-threonine production is 4.910 g/L, L-threonine production of optimized fermentation conditions is higher than L-threonine production(2.463 g/L) of initial fermentation conditions,it improved 2.447 g/L.