Enzýmová hydrolýza α-substituovaných akrylonitrilov

Abstract

Cilem diplornové préce bylo ovéiit schopnost kmene A4 néleiejiciho do rodu Rhodococcus transformovat chirélni on-substitutovane' akrylonitrily, které jsou prekursory stavebnich blokfi pro syntetickou organickou chemii. Zminénjz kmen produkoval komplex enzymfi - nitrilhydratasu a amidasu -- umoifiujici transformaci nitrilfi na karboxylové kyseliny s amidy jako intermediéty. Transformace oc-substitutovanjrch akrylonitrilfi byla provedena pomoci celjzch bunék této baktérie nebo éésteéné purifikované nitrilhydratasy. Reakéni produkty byly izolovény a identifikovény pomoci spektrélnich metod jako amidy, kyseliny nebo lakton. Koncentrace substrétfi a produktfi V reakénich smésich byly sledovémy pomoci HPLC na reverzni fézi. Pro tento fiéel byly vypracovény originélni metody, podobné jako methody chirélni I-IPLC, které byly vyuiity ke stanoveni enantiomerni éistoty chirélnich produktfi. Katalyzétory obsahujici celé bufiky nebo éésteéné purifikovanou nitrilhydratasu byly vhodné pro piipravu wsubstitutovanjrch akrylamidfi a akrylovjzch kyselin, z nichi nékteré jsou intermediéty pro syntézu biologicky aktivnich létek.

The aim of the diploma work was to examine the ability of strain A4 belonging to Rhodococcus to transform chiral on-substituted acrylonitriles, which are the precursors of building blocks for synthetic organic chemistry. The aforementioned strain produced a complex of enzymes - nitrile hydratase and amidase - enabling to transform nitriles into carboxylic acids with amides as intermediates. The transformation of ot-substituted acrylonitriles were carried out using whole cells of the bacterium or the partially purified nitrile hydratase. The reaction products were isolated and identified by spectral methods as the corresponding amides, acids or a lacton. The concentrations of the substrates and products in the reaction mixtures were monitored by reversed-phase HPLC. Original HPLC methods were worked out for this purpose, as well as the chiral HPLC methods, which were used to determined the enantiomeric purity of the chiral products. The catalysts consisting of whole cells or the partially purified nitrile hydratase proved to be suitable for the preparation ofon-substituted acrylamides and acrylic acids, some ofwhich are intermediates for the synthesis ofbiologically active compounds.