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OBJECTIVES 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 PAL’s 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. |
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