| L-tyrosine is one of the 20 kinds of amino acids that consisted of proteins and plays an important role in the growth and metabolism of humans and animals.It is widely used in food,medicine and cosmetic fields.Tyrosine Phenol Lyase?TPL?could catalyze the substrates as phenol,ammonia and pyruvate to synthesize L-tyrosine.However,the efficiency of enzymatic synthesis is still to be strengthened.Immobilized cell technology has the advantages of reusing strains,easy preparation and separation.Combined with the whole cell catalytic method,it could greatly reduce the loss of enzyme activity,thereby achieve efficient synthesis of target products and reduce costs.In order to synthesize L-tyrosine efficiently,a recombinant Esherichia coli was used as the research object.The conditions for the whole cell-catalyzed conversion,the immobilization conditions and the manner of addition of the substrate were optimized.The main findings were as follows:1)The concentrations of substrates?phenol,ammonia and sodium pyruvate?and the the coenzyme pyridoxal phosphate?PLP?were optimized during the catalytic synthesis proess of L-tyrosine with immobilized cells.The results showed that the titer of L-tyrosine could reach27.6 g·L-1 after reacting 10 h,when the concentration of phenol was 0.08 mol·L-1,ammonium chloride was 0.65 mol·L-1,sodium pyruvate was 0.1 mol·L-1 and the concentration of coenzyme PLP was 0.15?mol·L-1.Surfactants?such as triazone,Tween-80 and sodium lauryl sulfate?were added to the reaction system and effects of the three surfactants on L-tyrosine production were analyzed.0.1%Tween-80 has a certain role in promoting L-tyrosine production.The titer of L-tyrosine reached 31.6 g·L-1,which was increased by 120%compared with that of no adding added surfactant.2)The effects of different common immobilized carriers,their concentrations,and immobilization conditions on the L-tyrosine accumulation were studied.Results showed that when using a mixed carrier of sodium alginate?4%?and gelatin?6%?,CaCl2?4%?as a forming agent,and calcium carbonate?0.6%?as an adjuvant,the staility of immobilized cells was improved.The immobilized cell microspheres could be reused nine times.The total titer of L-tyrosine was 62.6 g·L-1.The effects of different metal ions on the phosphate tolerance of immobilized cell microspheres were investigated due to the immobilized cell microspheres were easy to be broken under phosphate condition.Results show that positive effects of metal ions were successively aluminum ions,manganese ions,calcium ions and barium magnesium ions.Adding 1%aluminum sulfate in the reaction system,the immobilized cells could be reused 12 times and the total titer of L-tyrosine reached 115.6 g·L-1.3)A strategy of reaction system reusing-substrate adding was established in order to increase the conversion rate of the substrate.The adding amounts of substrates?phenol,sodium pyruvate and ammonium chloride?were studied during the catalytic synthesis proess of L-tyrosine with immobilized cells.When the adding amounts of substrates were as follows(40 mmol·L-1 phenol,75 mmol·L-1 sodium pyruvate and 65 mmol·L-1 ammonium chloride),the immobilized cells could be reused 16 times and the total titer of L-tyrosine was 111.6g·L-1 with 90.7%conversion rate on phenol.However,the accumulation of L-tyrosine presented a trend?first increasing slowly,decreasing slowly and then rapidly?during the entire conversion process.Therefore,the amounts of adding substrates in different rounds in the process were optimized.When the adding substrates kept the above amounts in the first 5rounds(40 mmol·L-1 phenol,75 mmol·L-1 sodium pyruvate and 65 mmol·L-1 ammonium chloride)and then changed to the following amounts(50 mmol·L-1 phenol,35 mmol·L-1sodium pyruvate and 25 mmol·L-1 ammonium chloride),the immobilized cells could be reused for 17 rounds.The total titer of L-tyrosine reached 132.6 g·L-1 and the conversion rate of phenol was 87.2%. |
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