Enzymatic Synthesis Of Alanyl-glutamine

In this paper, enzymatic synthesis of Ala-Gln which is an important abenteric nutritious drug was considerd as the core of the research. The synthetic route including protection, enzymatic synthesis of depeptide and deprotection was successfully developed. On that basis, CLEA technology was applied to the papain and PGA, both of them are key enzymes in synthetizing Ala-Gln. The conditions of preparing CLEA were optimized, the properties of CLEA were studied and the structrue of CLEA was also characterized. For synthesizing Ala-Gln with high yield and low cost, CLEA microcapsules have also been explored and studied. The conclusions can be drawn as follows:Enzymatic synthesis of Ala-Gln: Ala-Gln was successfully synthesized by using papain and PGA. The reactions can be divided into 3 steps including protection, peptide synthesis by papain and unprotection by PGA. The yields of the 3 steps were 86.41%, 30.5% and 94.8% respectively. The final yield was 24.98%. HPLC-MS and NMR were used for detecting and identifying products. Meanwhile, the preparative chromatography for purifying the final product has been also developed. Synthesis of phac-Ala-Gln by papain: The reaction is kinetically controlled.The optimum condition was pH8.8, 25℃, 1.5mL ethanol, 0.02 g/mL enzyme, 0.5mol/L Gln, 0.1mol/L KCl. The yield was 30.5%, and reaction time was 160min. Papain-CLEA was also used. The reaction time of synthesis was 15h and the yield was 9.32%. The particle of CLEA is too small to be used in industry, and a new ideal of microcapsulation was proposed. 6 kinds of Papain-CLEA microcapsules were prepared, and the solid microcapsule had the highest activity retention, which was 53.42%.Synthesis of Ala-Gln via deprotection by PGA: The reaction time using free PGA was 0.33h and the yield was 94.8%. PGA-CLEA was prepared. Saturated ammonium sulfate was used as the ideal precipitant and the optimum volume was 2.3:1 compared with enzyme solution. Glutaraldehyde was the ideal cross-linker and the optimum concentration was 0.35%. The optimum time and temperature were determined respectively as 2h and 4℃, and the activity retention was 52.61%. The optimum temperature of CLEA was higher by 10℃. The suitable pH of CLEA was wider. The CLEA thermal stability has been improved under high temperature. The reaction time of deprotection using PGA-CLEA was 1.0h and the yield was 95.6%. 2 kinds of PGA-CLEA microcapsulates were prepared, and the activity retention of hollow microcapsule was 16.84% with the highest mechanical strength(7.95 N). The reaction time using PGA-CLEA hollow microcapsule was 2.2h and the yield was 95.2%.