The Identification And Fermentation Conditions' Optimizing Of A Strain Of L-lysine Producer

Lysine is a kind of fat amino acid.Its molecular formula is C₆H₁₄N₂O₂,and the molecular weight is 146.19.It widely exists in protein hydrolysate which cannot be synthetized automatically by human beings and animals likecasein,albumen,fibrin,beet molasses,gelatin, haemoglobin,etc.It is one of the eight necessary amino acid.It plays an important role in adjusting the balance of internal metabolism,enhancing the assimilation of internal cereal protein,improving the food nutrition of human being and animal nutrition,and promoting growth and development.At present,it is widely used in industry of medicine,food,and forage and etc.Fermentation is a major method to produce L-Lysine.Ground substance can be used to cultivate microorganism and produce L-Lysine in this method.This study selects L-Lysine producing strains from the soil nearby the thermae of ChangBai Mountain,takes many times of chemical and physical tests on their mutagenizing and hereditary stability,then,gets one L-Lysine strain Ly-H6U79-Z8 of high productivity with the resistance against high temperature and high glucose as well as the metabolism features of AEC⁺ and Thr^-,then carries out the strain identification and optimizing nutritive medium and cultivating condition.During the identification process,the strain Ly-H6U79-Z8,first is identified by morphology experiment as G-Bac without capsule,with gemma,motion,facultative anaerobe; then by amylaceum toco acid fermentation and oxidase experiment as fermentation-type oxidase of negative.In accordance with "Berger's Handbook on the Identification of Bacteria"(the eighth and ninth edition) and "The Handbook on the Identification of Common Bacteria" together with the apparent features the strain Ly-H6U79-Z8 belongs to Enterobacteracear.After a further 20-item experiment on physiological and biochemical features,the strain Ly-H6U79-Z8 is similar to the Escherichia,the Edwardsiella,the Citribacter, Yersinia,and the Irving;among them the strain Ly-H6U79-Z8 is most similar to the Escherichia and the Irving.That is to say,the result of apparent identification is that the strain Ly-H6U79-Z8 belongs to the Escherichia,the Irving or Yersinia.The last step is to firstly extract and test the gene order of the strain Ly-H6U79-Z8 16S rDNA,then input the gene order into the gene bank to make homological comparison,as a result,altogether 178 hits of 99% homology are found;their gene orders are compared one by one,then phyletic evolution tree is drew,and the final result comes out:the strain Ly-H6U79-Z8 is a new strain of the Escherichia in Enterobacteriaceae,which is identical to the result of apparent identification.During the fermentation condition is optimized,first single factor screening method is designed containing carbon source,nitrogen source,inorganic salt,seed quality,temperature, pH,fermentation time and so on.Then orthogonal experiment is designed containing above factors.The relations among each factor are evaluated to attain the optimal nutritional factors and fermentation condition and result is analyzed by SPSS software.Finally the Lysine strain Ly-H6U79-Z8 optimal condition is that 25%glucose,5%Ammonium sulfate,4%com steep liquor,0.5%MgSO4·7H2O,0.05%K₂HPO₄ 0.1%,CaCO₃,threonine 0.04%,methionine 0.03%, bioepiderm 400μg/L,37℃,pH 7,cycle of fermentation is 48h,amount of inoculation is 10%. After fermentation the contents of Lysine is 79.9mg/ml in broth,which reaches much higher lever under the same condition.At present,the producing strains of Lysine mainly concentrate on some strains like Corynebacterium glutamicum,aureous bacillus brevis,and barm etc.whose fermentation temperature is 30℃.30℃is the optimal temperature for most of the bacterium,bacterial invader,and virus,which makes these strains are rather likely to be infected in the process of fermentation;the production,thus,will be influenced.However,the strain Ly-H6U79-Z8 with the resistance against high temperature whose fermentation temperature is 37～39℃can greatly lower the possibility of living contaminants in production,avoid the influence from the instability of output,therefore,higher the producing level while lower the producing cost.In a word,the study and widespread of the strain Ly-H6U79-Z8 can improve the development of Lysine industry,promote the application of Lysine.This will not only increase the quality of agricultural and livestock products,speed up the development of functional food and medicine,also can efficiently higher people's health level,By biotechnology method, Jilin Province's com for high added value can be transformed and a new way for the agro-product processing of Jilin Province is opened.