ABSTRACT
ω-transaminase (ω-TA) is a natural biocatalyst that has good application potential in the synthesis of chiral amines. However, the poor stability and low activity of ω-TA in the process of catalyzing unnatural substrates greatly hampers its application. To overcome these shortcomings, the thermostability of (R)-ω-TA (AtTA) from Aspergillus terreus was engineered by combining molecular dynamics simulation assisted computer-aided design with random and combinatorial mutation. An optimal mutant AtTA-E104D/A246V/R266Q (M3) with synchronously enhanced thermostability and activity was obtained. Compared with the wild- type (WT) enzyme, the half-life t1/2 (35 ℃) of M3 was prolonged by 4.8-time (from 17.8 min to 102.7 min), and the half deactivation temperature (T1050) was increased from 38.1 ℃ to 40.3 ℃. The catalytic efficiencies toward pyruvate and 1-(R)-phenylethylamine of M3 were 1.59- and 1.56-fold that of WT. Molecular dynamics simulation and molecular docking showed that the reinforced stability of α-helix caused by the increase of hydrogen bond and hydrophobic interaction in molecules was the main reason for the improvement of enzyme thermostability. The enhanced hydrogen bond of substrate with surrounding amino acid residues and the enlarged substrate binding pocket contributed to the increased catalytic efficiency of M3. Substrate spectrum analysis revealed that the catalytic performance of M3 on 11 aromatic ketones were higher than that of WT, which further showed the application potential of M3 in the synthesis of chiral amines.
Subject(s)
Transaminases/chemistry , Molecular Docking Simulation , Amines/chemistry , Pyruvic Acid/metabolism , Enzyme StabilityABSTRACT
Background: New enzymes for biotransformations can be obtained by different approaches including directed mutagenesis and in vitro evolution. These mutants have to be efficiently produced for laboratory research on bioreactions as well as for process development. In the framework of a European ERA-IB project, two different types of enzymes (ammonia lyases and aminotransferases) have been selected as biocatalysts for the synthesis of industrially relevant amines. New mutant enzymes have been obtained: a) aspartases able to recognize β-amino acids; b) ω-transaminases with improved activity. The objectives are to find out a common operational strategy applicable to different mutants expressed in E. coli with the same initial genetic background, the development of an integrated process for production and the preparation of stable useful biocatalysts. Results: Mutant enzymes were expressed in E. coli BL21 under the control of isopropylthiogalactoside (IPTG) inducible promoter. The microorganisms were grown in a formulated defined medium and a high-cell density culture process was set up. Fed-batch operation at constant specific growth rate, employing an exponential addition profile allowed high biomass concentrations. The same operational strategy was applied for different mutants of both aspartase and transaminase enzymes, and the results have shown a common area of satisfactory operation for maximum production at low inducer concentration, around 2 μmol IPTG/g DCW. The operational strategy was validated with new mutants and high-cell density cultures were performed for efficient production. Suitable biocatalysts were prepared after recovery of the enzymes. The obtained aspartase was immobilized by covalent attachment on MANA-agarose, while ω-transaminase biocatalysts were prepared by entrapping whole cells and partially purified enzyme onto Lentikats (polyvinyl alcohol gel lens-shaped particles). Conclusions: The possibility of expressing different mutant enzymes under similar operation conditions has been demonstrated. The process was standardized for production of new aspartases with β-amino acid selectivity and new ω-transaminases with improved substrate acceptance. A whole process including production, cell disruption and partial purification was set up. The partially purified enzymes were immobilized and employed as stable biocatalysts in the synthesis of chiral amines.
Subject(s)
Amines/metabolism , Transaminases/metabolism , Ammonia-Lyases/metabolism , Bioreactors , Culture Media , Enzymes, Immobilized , Escherichia coli , Biocatalysis , Batch Cell Culture Techniques , Amines/chemistry , Transaminases/genetics , Transaminases/chemistry , Ammonia-Lyases/genetics , Ammonia-Lyases/chemistry , MutationABSTRACT
The use of fluorescent compounds can be a valuable tool to probe the active site of enzymes. Several examples of this approach are discussed, particularly the use of pyridoxal phosphate analogs. The study of protein-protein interactions by means of fluorescent-labeled proteins is also analyzed
Subject(s)
Adenine Nucleotides/chemistry , Binding Sites , Pyridoxal Kinase/chemistry , Pyridoxal/chemistry , Pyridoxine/chemistry , Transaminases/chemistry , Anisotropy , Protein ConformationABSTRACT
Se da a conocer el primer caso de Drepanocitosis (sickle cell anemia) descrito en Santa Cruz, Bolivia en un paciente de raza negra procedente de los Yungas de La Paz, que simulo un cuadro de artritis. El proposito del articulo es informar que es posible la existencia de mas casos, tanto sintomatico como asintomatico debido a la migracion interna y externa de grupos que racialmente tienen una mayor prevalencia de manera tal que se sospeche precozmente la enfermedad y se adopte la conducta que corresponda