| Abstract | Biokataliza predstavlja upotrebu enzima za ubrzavanje kemijske reakcije iz koje sami enzimi izlaze nepromijenjeni. Enzimi su izrazito važne molekule u farmaceutskoj industriji, jer posjeduju značajnu regio-, kemo- i stereoselektivnost te omogućuju dobivanje čistih aktivnih farmaceutskih komponenti. Jedni od zanimljivijih enzima u području dobivanja ovih komponenti su halogenhidrin-dehalogenaze (HHDH). Ovi enzimi pripadaju skupini liaza i izrazito su važni industrijski enzimi koji se mogu primijeniti kod sinteze optički aktivnih epoksida, β-supstituiranih alkohola, kao i kiralnih tercijarnih alkohola. U današnje vrijeme ovi se enzimi uglavnom dobivaju prekomjernom ekspresijom u rekombinatnim stanicama Escherichia coli. Kako bi se poboljšala svojstva enzima, oni se podvrgavaju procesu imobilizacije na nosiocu ili bez nosioca umrežavanjem. Imobilizirani enzimi pokazali su se izrazito učinkoviti za komercijalnu upotrebu zbog ekonomičnosti procesa u kojem se koriste, veće stabilnosti i mogućnosti recikliranja enzima. Jedna od dostupnih metoda uključuje imobilizaciju enzima ili cijele stanice u polimernu maticu kalcijeva alginata, koja posjeduje različite prednosti poput blagih uvjeta polimeriziranja, netoksičnosti, biokompatibilnosti, ekonomičnosti i otpornosti na kontaminaciju različitim mikroorganizmima. Organski fluorirani spojevi izrazito su važne građevne molekule u farmaceutskoj, agronomskoj i industriji funkcijskih materijala. Oni pokazuju različita jedinstvena fizikalna, kemijska i biološka svojstva u odnosu na nefluorirane spojeve, međutim njihova dostupnost proizlazi gotovo u potpunosti iz umjetne sinteze. Zbog ove potrebe, kao i zbog potrebe za zelenim pristupom ovom problemu, u njihovo dobivanje uključeni su enzimi, čija upotreba dovodi do uštede energije, pridonosi zaštiti okoliša, sigurnosti, visokoj selektivnosti procesa itd. U ovom radu uspješno je provedena imobilizacija rekombinantnih stanica E. coli koje sadrže HheC-ISM4 enzim na alginatnim zrnima, te je s istima potom uspješno provedena reakcija sinteze
(S)-2-(4-fluorfenil)-3-hidroksipropanonitrila u reaktoru s rotirajućim slojem. Pritom je dobiveno izrazito visoko iskorištenje na produktu, 98,87 %, visoka konverzija supstrata, 75,61 %, kao i visoka optička čistoća koja je bila izražena preko enantiomernog suviška supstrata i iznosila je 87,61 %. |
| Abstract (english) | Biocatalysis represents the application of enzymes to accelerate a chemical reaction from which the enzymes themselves exit unchanged. Enzymes are extremely important biological molecules in the pharmaceutical industry, because they possess significant regio-, chemo- and stereoselectivity and allow obtaining pure active pharmaceutical components. Halohydrin-dehalogenase (HHDH) is one of the more interesting enzymes in the field of obtaining these components. These enzymes, which belong to the group of lyases, are extremely important industrial enzymes that can be used in the synthesis of optically active epoxides, β-substituted alcohols, as well as chiral tertiary alcohols, and nowadays are mostly obtained by overexpression in recombinant Escherichia coli cells. In order to improve properties of the enzymes, enzymes are subjected to the process of immobilization on a carrier or without a carrier by cross-linking. Immobilized enzymes have proven to be extremely effective for commercial use due to lower costs of the process, greater stability and the possibility of recycling the enzyme. One of the available immobilization methods is the immobilization of enzymes or whole cells in a polymer matrix of calcium alginate, which has various advantages such as mild polymerization conditions, non-toxicity, biocompatibility, economical advantages and resistance to contamination by various microorganisms. Organic fluorinated compounds are extremely important building molecules in the pharmaceutical, agronomic and functional materials industry. They show different unique physical, chemical and biological properties compared to non-fluorinated compounds, however their availability results almost entirely from artificial synthesis. Due to this, as well as due to the need for development of green industry, enzymes are included in their production, the use of which leads to energy savings, contributes to environmental protection, safety, high process selectivity, etc. In this work, the process of immobilization of recombinant Escherichia coli cells containing the HheC-ISM4 enzyme on alginate beads was successfully carried out, with which the synthesis reaction of (S)-2-(4-fluorophenyl)-3-hydroxypropanonitrile was successfully carried out in a rotating bed reactor. High product yield was obtained, 98.87%, as well as high conversion of the substrate, 75.61%, and high optical purity expressed through the enantiomeric residue of the substrate, 87.61%. |
