OB.1 PRODUCTION, ISOLATION AND PURIFICATION OF VARIOUS WT- AND MUTANT
PAL and PAM. ∑Wt-PAL from various mesophiles and termophiles will be produced by fermentation. The enzyme activity and stability will be tested and optimal condition for highest reaction rate will be established. Cloning known and novel bacterial useful PALís into the most proper hosts for larger scale productions.
OB2. IMMOBILIZATION OF PAL AND PAM, AND WHOLE CELLS HOSTING PAL/PAM PRODUCTION.
We plan to develop robust immobilization techniques with isolated enzymes as well as with whole cells producing MIO-containing enzymes. The immobilization method can basically determine the reusability and activity of enzymes, moreover it can affect the substrate specificity of the biocatalysts. Several immobilization methods including adsorption binding onto surfaces, entrapment or cross-linking will be performed and compared their feasibility and efficiency.
OB3. DEVELOPMENT OF BIOCATALYTIC PROCEDURES MEDIATED BY PAL/PAM
†The broad substrate tolerance of PAL will be exploited further by using wild-type or mutant native or immobilized mesophile and extermophile enzymes for the efficient production of both enantiomers of various aryl-alanines. Natural or engineered cells will be also checked as potential biocatalysts. Batch and continuous flow processes will be compared as efficiency. A continuous flow sequential procedure will be developed for the simple and efficient production of unnatural β-phenylalanine analogs using as starting material various aryl-cinnamates as shown. In the first stage PAL catalysed ammonia addition (6M NH3 water solution) will provide L-arylalanines. After the reaction will ceased the solution will be degassed and the pH will be adjusted to 8. In the second stage the PAM catalysed izomerization will provide the corresponding β-arylalanine. Finally the unreacted L-arylalanine will be transformed into the starting arylacrylate.†