Synthesis Of L-isoleucine By Yeast And Optimization Of Its Conditions

L-isoleucine belongs to hydrophobic branched chain amino acid（BCAA）,which is one of the eight essential amino acids for human body.It has a wide range of applications in food,medicine,feed,etc.Nowadays,with the continuous expansion of the application scope of amino acids,people’s demand for amino acids has gradually increased.Japan has occupied the main market share of the international amino acid market,and has entered the ranks of amino acid powers.However,there is still a gap between domestic amino acid supply and demand,the fermentation time and the average acid production in China is still a certain gap compared with the developed countries.The production of L-isoleucine by using Corynebacterium glutamicum as engineering strain is often used in domestic industry.However,there are still some problems such as long fermentation period and phage pollution.It is necessary to develop a new way of synthesizing L-isoleucine.In this experiment,Yeast was selected as the engineering bacteria,a new reaction system was established,the fermentation conditions of L-isoleucine synthesis by Yeast were optimized,and the colorimetric method was explored to detect the content of L-isoleucine in fermentation broth.The main contents and conclusions are as follows:1.Investigated the effect of reaction time and metal ions on fermentation:in a 100ml system,set blank control group and experimental group,added 0.2 g Fe²⁺,Mg²⁺,Ca²⁺,Zn²⁺,Ba²⁺,Fe³⁺,Mn²⁺,Cu²⁺to the experimental group respectively and compared with the blank control group.It was found that the optimal reaction time of the blank control group was 48 h.The L-isoleucine production in the four groups with Mg²⁺,Ca²⁺,Zn²⁺,and Mn²⁺added was different from the control group,and showed an increasing trend.We set the three concentration conditions of each ion as:1.5 g/L,3 g/L,4.5 g/L,and accorded this to designed a L₉（3⁴）orthogonal experiment to study the best combination of these four ions.the results showed that:When the concentration of Mg²⁺added to the fermentation broth was 4.5 g/L,the concentration of Ca²⁺was 1.5 g/L,the concentration of Zn²⁺was 4.5 g/L,and the concentration of Mn²⁺was 3 g/L,L-isoleucine had the maximum yield.2.The single factor experiment was designed to determine the two better conditions of each factor in the fermentation process.The effects of fermentation p H and temperature,the amount of yeast,the ratio between the CTAB and the yeast,the reaction time in the cell,the different concentrations of citric acid,and the concentration of supplemented glucose on fermentation were investigated.The experimental results showed that:in a 100 m L system,the two better fermentation conditions were as follows:p H was 5.0 and 5.5,temperature was 40°C and 45°C,The yeast in the fermentation broth was 12 g and 14 g,citric acid was 2 g/L and 3 g/L,sugar was 2 g/L/h and 3 g/L/h,CTAB was added after 3 h and 6 h,and the addition of CTAB was 12.5%and 15.0%of yeast.3.Designed an orthogonal experiment to determine the optimal fermentation conditions for L-isoleucine.According to the results of the previous single-factor experiment,we selected the best two conditions for each factor,designed an orthogonal experiment of L₈（2⁷）,and added the best ratio of metal ions obtained in the second step to the group.After the orthogonal experiment results were obtained,the optimal fermentation conditions were obtained throughed the analysis of the experimental results.The results show that:in a 100 ml system,when the shaking table temperature was set at 40℃,the p H of the reaction solution was 5.5,the amount of yeast was 14 g,the amount of citric acid was 3 g/L,the way of sugar supplement was 3 g/L/h,CTAB was added after 6 h,and the amount of CTAB was 12.5%of the amount of yeast,the maximum yield of L-isoleucine was obtained,and the average yield of L-isoleucine was 6.87 g/L...