@misc{Latacz_Gniewomir_Research_2010,
 author={Latacz, Gniewomir},
 address={Kraków},
 howpublished={online},
 year={2010},
 school={Wydział Farmaceutyczny},
 language={pol},
 abstract={D-hydantoinase is a commercially enzyme used for the preparation of optically pure D-amino acids from D,L-5-monosubstituted hydantoins via hydantoinase method. It catalyzes the reversible hydrolytic ring cleavage of hydantoin to yield N-carbamoyl-D-amino acids, which can be further chemically or enzymatically converted to D-amino acids. D-amino acids are important chiral building blocks for biologically active compounds including: pesticides, peptidomimetics and semisynthetic antibiotics. Also, several enantiomers hydroxy- or carboxy- derivatives of phenylglycine or phenylalanine have been described as potent agonists or antagonists at glutamate receptors of the central nervous system. In recent years industrial demand for D-hydantoinases and N-carbamoylases with enhanced activity and substrates specificity have been growing rapidly. Hence, up to now natural habitats of the enzymes involved in hydantoinase method have been intensively screened for the presence of microbes having high levels of D-hydantoinase or N-carbamoylase activity. Moreover, the introduction of D-hydantoinase and N-carbamoylase gene into Escherichia coli by employing recombinant DNA techniques and next their expression or co-expression lead to enhanced activity, increased enantioselectivity and enlarged substrate specificity of examined enzymes. Additionally, the immobilization technique offers many advanta},
 abstract={ges including: the easy separation of the latter from the reaction products, the elevation of enzyme concentrations per unit volume, and even the enhancement of enzyme stability and activity. In the present work, we report the use of two D-hydantoinases: D-Hydantoinase, recombinant, immobilized from E. coli (rD-Hyd) and D-hydantoinase from Vigna angularis (V.a.D-Hyd) for the synthesis of N-carbamoyl derivatives of several ring-monosubstituted and ring-disubstituted D-phenylalanine analogs, which are next easily converted to the corresponding D-amino acids. The required 21 racemic (R,S) phenyl ring-substituted 5-benzylhydantoins were synthesized using at first Knoevenagel’s condensation and then either the catalytic hydrogenation of non-saturated bond in 5 position of the hydantoin ring in the presence of Pd/C catalyst or by reduction of appropriate 5-arylidenehydantoin with 57 % hydroiodic acid in the presence of red phosphorus. To study the activity of D-hydantoinase towards phenyl ring-substituted 5 benzylhydantoins the calibration curves for each substrate were estimated by capillary electrophoresis. Obtained regression equations were used to calculate the concentrations of substrates remaining at the time in the reaction mixtures. The efficiencies of the enzymes were expressed in terms of k (reaction rate constant) and t0,5 (substrate half-life). Enzymatic activities of b},
 abstract={oth D-hydantoinases were compared and substrate specificity towards all 21 obtained (R,S) phenyl ring-substituted 5 benzylhydantoine derivatives were determinated. Reaction rate constant and substrate half-life in different pH conditions were also estimated. High pressure liquid chromatography was used for determinating enantiomeric ratio (%ee) of the obtained D-phenylalanine derivatives. The enantioseparations were performed using a ChiroSil (RCA+) column containing the chiral stationary phase prepared by a covalent trifunctional bonding of (+) or(-)-(18-Crown-6)-tetracarboxylic acid. For the first time the enantioselectivity of D-hydantoinase, recombinant, immobilized from E. coli towards novel phenyl ring-substituted 5 benzylhydantoine derivatives was successfully evaluated. The enantioseparations of a few obtained D-phenylalanine derivatives using Capillary Electrophoresis were also performed. In order to accomplish enantioseparation the optical isomers of amino acids have to come into contact with a chiral environment to form two diastereomeric complexes. In the present work were used highly sulfated cyclodextrins (HS-CDs) as a chiral selectors. To determine enantiomeric purity, enantiomeric ratio (%ee) for each examined compound was calculated. Several obtained D-phenylalanine derivatives possess structural similarity to the described glutamate receptor active phenyla},
 abstract={lanine or phenylglycine derivatives. Using Amberlite 120 H+ ion-exchange resin and next liophilisation process were purified and isolated proper D-amino acids from the reaction mixtures. The compounds were next examined for their activity against glutamate receptors. Reported was also an enzymatic route which enables the subsequent hydrolysis of N-cabamoyl-D-amino acid to the corresponding D-phenylalanine derivative, mediated by N-carbamoylase (rN-Kar). Plasmid pAH71 containing the N-carbamoylase gene used in this study was kindly donated by Prof. Yun-Peng Chao from the Department of Chemical Engineering, Feng Chia University, Taiwan. For inducing N-carbamoylase gene 1 mM of IPTG (Isopropyl β-D-1-thiogalactopyranoside) was added to transformed, recombinant Escherichia coli strains. The presence of N-carbamoylase was confirmed by SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis. Recombinant, inducted E.coli strains were used for biotransformation of the obtained previously N-carbamoyl-D-amino acids to the corresponding D-amino acids. The reactions were monitored by capillary electrophoresis. In this work capillary electrophoresis was also used as a tool for DNA separations. The comparison of CE DNA separation method with conventional agarose gel electrophoresis was also performed.},
 title={Research on obtaining nonnatural amino acids by hydantoinase method},
 type={Praca doktorska},
 keywords={hydantoinase method, D-hydantoinase, N-carbamoylase, D-amino-acids, capillary electrophoresis},
}