The Immobilization Of Thermolysin Using Microwave Irradiation Method And Its Application In The Synthesis Of Aspartame Precursor

Aspartame is a dipeptide with intensely sweet taste. Although it is about200times sweeter than sucrose, it has low calorie property. People, who is suffering from obesity, angiocardiopathy and diabetes, can use it as sweetener without any uneasiness. Nowadays, thermolysin is widely used in the synthesis of the precursor of aspartame because it is friendly to the environment. But conventional enzymatic synthesis has a lot of disadvantages, such as long time, low yield, low rate of reusing the enzyme, difficulties in the separation of product, and so on.In this paper, microwave irraidiation was used in the thermolysin immobilization. We dissolved and dispersed thermolysin in an immobilization mixture by salting-in effect and efficiently immobilized it in mesocellular siliceous foam (MCFs) support materials under microwave consecutive irradiation at low temperature. In order to improve the thermal stability of the immobilized enzyme, small molecule was used to quench excessive activated groups on support surface. We also synthesized N-(Benzyloxycarbonyl)-L-aspartyl-L-phenylalanine methyl ester (Z-AspPheOMe), the precursor of aspartame with the immobilized enzyme preparation. The result showed that the yield was increased and the reaction time was shortened. Additionally, the enzyme could be reused and the product could be separated easily.Base on the above research, the main contents and conclusions in our work could be drawn and listed as following:(1) NaCl can improve the solubility of thermolysin and microwave irradiation can dramatically increase reaction rate and yield. When the concentration of sodium chloride was3M and microwave power was40W,93.2%of the enzyme were coupled to the support by3min, and the maximum specific activity of the immobilized enzyme was17925.1U/mg. This was a4.5-fold increase in activity versus enzyme immobilized using conventional techniques, and a1.6-fold increase versus free enzyme. Additionally, the thermal stability and the stability against organic solvent of the immobilized thermolysin was significantly improved, the reaction time was shortened from20h to3min.(2) Excessive active groups on the support surface would cause the possibility of enzyme deactivation by a subsequent slow reaction between the enzyme and those. In this work, to enhance the thermal stability of immobilized enzyme, small molecule reagents such as alanine, valine, tryptophan, tert-amyl alcohol were used to quench and block excessive activated groups on support surface. The thermal stability of immobilized enzyme had been dramatically improved.(3) In the aqueous phase, ZAPM was synthesized from Z-Asp-OH and Phe-OMe.HCl by the natural thermolysin as catalyst under30℃,40W microwave irradiation for10min, the yield was60.96%. It was9percentage points higher than the conventional reaction and the time was also shortened by35times.(4) In ethyl acetate-water two phase system, enzyme immobilized with microwave method had catalyzed synthesized of ZAPM with the assistance of calcium chloride and sodium chloride, the yield reached28.3%. The amount of ZAPM produced by1mg enzyme in1h is0.475, which is19times of that Takeshi Nagayasu etc had reported.