| Abstract | S obzirom na sva dosadašnja postignuća u području biokatalize, proteinskog inženjerstva i računalnog modeliranja, posljednjih 20 godina halogenhidrin-dehalogenaze (HHDH) su doživjele su nagli razvoj i sve širu primjenu u industriji. Jedna od najznačajnijih je farmaceutska industrija, u kojoj su HHDH svojom visokom kemo-, regio- i stereoselektivnošću napravile veliki iskorak. U reakcijama dehalogeniranja halogenalkohola i nukleofilnog otvaranja epoksida ovi enzimi pokazali su visoku aktivnost te visoku enantioselektivnost, pogotovo C-tip HHDH iz Agrobacterium radiobacter AD1 (HheC). Iz tog razloga su se u ovom diplomskom radu ispitivali preci HheC enzima dobiveni rekonstrukcijom filogenetskog stabla velikog novootkrivenog broja HHDH enzima. U radu su ispitani direktni predak N122, daljnji predak N124 (zajednički predak C- i A-skupina HHDH) i N134 koji je direktni predak svih HHDH enzima. Svi enzimi su sintetizirani u rekombinantnim stanicama E. coli, izolirani i pročišćeni, ali nisu svi pokazali očekivane rezultate. Točnije, preci N122, N124 i N134 nisu pokazali ni približno dobre rezultate kao ranije provedeno istraživanje s istim enzimima. Katalitička aktivnost im je u reakcijama dehalogeniranja para-nitro-2-brom-1-feniletanola (PNSHH) bila čak 20 puta (N134), 55 puta (N124) odnosno 220 puta (N122) manja u odnosu na HheC. Ovi rezultati nisu u skladu s prethodnim istraživanjima gdje su ovi preci pokazali značajno višu aktivnost u odnosu na HheC. Zato je potrebno provesti daljnja istraživanja da bi se ustanovilo što se tijekom vremena dogodilo unutar rekombinantne kulture E. coli što je moglo uzrokovati ovakav gubitak aktivnosti. U reakciji otvaranja epoksida 2-benzil-oksirana ovi enzimi pokazali su vrlo nisku (N134) do nepostojeću aktivnost (N122 i N134), te time i enantioselektivnost, što je dodatno potvrdilo gubitak aktivnosti ovih enzima. Uz pretke HheC, ispitivao se i predak skupine D HHDH – N81 koji je direktni predak HheD12 iz arktičke morske bakterije Halopseudomonas pelagia. On je, s druge strane, pokazao izvanredno dobru katalitičku aktivnost u obje ispitivane reakcije. U reakciji otvaranja PNSHH pokazao je specifičnu aktivnost od čak 1,73 U mg⁻¹ što je čak 15 puta više od N134, 50 puta više od N124 i 171 puta više od N134. Većina enzima iz skupine D nije do sad pokazivalo veliku aktivnost prema ovom tipu supstrata, izuzev HheD (iz Dechloromonas aromatice) i HheD2 (iz gammaproteobacterium HTCC2207 soja) čiji je N81 direktni predak. U otvaranju epoksida N81 je pokazao visoku konverziju od čak 88% što je dvostruko više od divljeg tipa HheC s konverzijom od 41%. Ovaj predak pokazao je u ovoj reakciji osrednju enantioselektivnost, s evs vrijednosti u kinetičkoj rezoluciji epoksida od čak 78% i evp vrijednosti prilikom sinteze (R)-1-azido-fenil-2-propanola od 20%. Ovi rezultati su, iako osrednji, bolji od do sad dobivenih rezultata za divlje D-tipove, što ukazuje na dodatne prilike u istraživanju ovog pretka. Ovo istraživanje upućuje na to da je potrebno provesti daljnja istraživanja sa širim rasponom supstrata, ali tek nakon što se ustanovi razlog gubitka aktivnosti predaka HheC, odnosno pripreme enzimi koristeći svježe pripremljene plazmide. Također, veću pažnju treba dati enzimima skupine D i njihovim precima zbog njihove visoke katalitičke aktivnosti. |
| Abstract (english) | Given the significant advancements in the fields of biocatalysis, protein engineering, and computational modeling over the past 20 years, halohydrin dehalogenases (HHDHs) have experienced rapid development and increasingly widespread application in industry. One of the most significant areas is the pharmaceutical industry, where HHDHs have made significant strides due to their high chemo-, regio-, and stereoselectivity. In dehalogenation and nucleophilic epoxide ring-opening reactions, these enzymes have demonstrated high activity and enantioselectivity, especially the C-type HHDH from Agrobacterium radiobacter AD1 (HheC). This is why, in this study, ancestral enzymes of HheC, obtained by reconstructing the phylogenetic tree of a large number of newly discovered HHDH enzymes, were examined. The study examined the direct ancestor N122, the further ancestor N124 (the common ancestor of the C- and A-group HHDHs), and N134, which is the ancestor of all HHDH enzymes. All enzymes were synthesized in recombinant E. coli cells, isolated, and purified, but not all showed the expected results. Specifically, the ancestors N122, N124, and N134 did not show nearly as good results as previously conducted research with the same ancestral HheC cells. Their catalytic activity in the dehalogenation reactions of
para-nitro-2-bromo-1-phenylethanol (PNSHH) was up to 20 times (N134), 55 times (N124), and 220 times (N122) lower compared to the wild-type HheC. These results do not align with previous studies where these ancestors showed significantly higher activity compared to HheC. Therefore, further research is needed to determine what might have happened within the recombinant E. coli culture over time that could have caused such a loss of activity. In the epoxide ring-opening reaction of 2-benzyloxirane, these enzymes exhibited very low (N134) to non-existent activity (N122 and N134), and therefore enantioselectivity, further confirming the loss of activity in these enzymes. In addition to HheC ancestors, the ancestor of a large number of D-type HHDHs—N81, the direct ancestor of HheD12 from the Arctic marine bacterium Halopseudomonas pelagia—was also studied. This enzyme, on the other hand, demonstrated exceptionally good catalytic activity in both tested reactions. In the PNSHH opening reaction, it showed a specific activity of as much as 1.73 U mg⁻¹, which is 15 times higher than N134, 50 times higher than N124, and 171 times higher than N134. Most D-types have not shown significant activity towards this type of substrate so far, except for HheD (from Dechloromonas aromatica) and HheD2 (from Gammaproteobacterium HTCC2207 strain), of which N81 is the direct ancestor. In the epoxide opening, N81 showed a high conversion rate of as much as 88%, which is double that of the wild-type HheC with a conversion of 41%. This ancestor demonstrated moderate enantioselectivity in this reaction, with ees values in the kinetic resolution of the epoxide of up to 78% and eep values during the synthesis of (R)-1-azido-3-phenyl-2-propanol of 20%. These results, though moderate, are better than those obtained so far for wild D-types, indicating further opportunities for research into this ancestor. This research suggests that further studies with a broader range of substrates are needed, but only after determining the cause of the activity loss in HheC ancestors and preparing the enzymes using freshly prepared plasmids. Additionally, more attention should be given to D-types of HHDHs and their ancestors due to their high catalytic activity. |
