Development of a biotechnological enzyme cascade process for the production of optically pure beta amino acids
Final Report Abstract
The aim of the project was to develop an enzyme cascade process for the production of chiral β3-amino acids starting from racemic (aryl-) substituted dihydropyrimidines (e.g., phenyldihydrouracil, PheDU). For this purpose, 13 hydantoinases/dihydropyrimidinases or putative cyclic amidases were studied. For seven of these enzymes, hydrolysis of PheDU to N-carbamoyl-βphenylalanine (NCβPhe) was detected. Three enzymes from Aminobacter sp. DSM24754 and DSM 24755 were characterized in more detail. The highest activity measured with PheDU was 1128 mU/mg for dihydropyrimidinase from Aminobacter sp. DSM24754 (pM24754.2), but it has no stereoselectivity towards the substrate. In contrast, the hydantoinase from Geobacillus stearothermophilus (pBGwtHyd) is (S)-selective for PheDU and showed very good activity with this substrate. In addition, activity with para-chloro-PheDU was detected for four dihydropyrimidinases. In the second part of the project, enzymes for stereoselective decarbamolyation of NCβPhe to βPhe were explored. A screening of wild-type strains revealed indications of this activity. Subsequently, 12 enzymes of enzyme class (EC) 3.5 were examined. No enzyme was able to hydrolyze NCβPhe, but almost all were able to catalyze the hydrolysis of N-carbamoyl-β-alanine (NCβAla). Therefore, the project goal was adapted to address the question of: What factors in the molecular structure of an NC amino acid (NCAS) are affecting its acceptability as a substrate? Substrates (including N carbamoyl-β-homo amino acids, NCβHAS) were synthesized and analytical methods were developed. Indication for activity of several β-ureidopropionases (βUP) with NCβHAS was found. In addition, in silico enzyme models were calculated and ligand docking studies were performed. Finally, the βUP from Pseudomonas aeruginosa PAO1 was characterized in detail for the first time. The enzyme converts the natural βUP substrates NCβAla and N-carbamoyl-α-aminoisobutyric acid (NCAib) and is L-specific for N-carbamoyl-α-serine, but showed no activity with NCβHAS. The development of an enzyme cascade could not be comprehensively addressed due to the lack of a decarbamoylating enzyme. However, all active enzymes were immobilizable with their MBP tag, and for some their reusability was demonstrated. Overall, the project was successful. Suitable dihydropyrimidinases were found and characterized in detail and the in-depth investigation of decarbamoylating enzymes led to valuable data for further studies.
Publications
-
Development of an enzyme cascade process for the production of chiral β-amino acids. VAAM
Lohmann & Engel
-
Screening, purification and characterization of decarbamoylizing enzymes for the production of chiral βamino acids. 4th International Summer School - Biotransformations
Lohmann
-
Characterization of dihydropyrimidinases for biocatalytic production of chiral β-amino acids within an enzyme cascade process. 9th International Congress on Biocatalysis
Lohmann & Engel
-
Development of an enzyme cascade process for the production of chiral β-amino acids. VAAM
Lohmann& Engel
-
Screening, purification and characterization of decarbamoylizing enzymes for the production of chiral β-amino acids. 9th International Congress on Biocatalysis
Lohmann & Engel
-
Development of an enzyme cascade process for the production of chiral β-amino acids. DECHEMA - Frühjahrstagung der Biotechnologen
Lohmann& Engel
-
Screening, characterization and biotechnological use of decarbamoylizing enzymes. VAAM
Lohmann & Engel
-
Development of an enzyme cascade process for the production of chiral β-amino acids VAAM
Rudat & Engel
-
Development of an enzyme cascade process for biocatalytic production of chiral beta amino acids. European Federation of Biotechnology
Engel
-
Characterization of dihydropyrimidinases for the biocatalytic synthesis of chiral beta amino acid. VAAM
Marcaux & Engel
-
Enzymatic synthesis of non-canonical amino acids by carbamoylases. VAAM
Schwab & Engel
