Kinetic resolution of fluoroalcohols catalysed by lipases

Optički čisti spojevi su vrijedni prekursori u farmaceutskoj industriji. Fluorirani spojevi su aktivne tvari u mnogim lijekovima, stoga su nove mogućnosti sinteze fluoriranih kiralnih graĎevnih blokova posebno zanimljive i vrijedne. U ovom radu sintetizirana su tri racemična derivata 1-fluorpropan-2-ola koji su supstituirani u položaju 3 azido (1a), cijano (2a) skupinom i klorom (3a) te su korišteni kao supstrati za kinetičku rezoluciju kataliziranu lipazama. Pripravljeni su i odgovarajući racemični acetati alkohola (1b-3b) kao standardi. U ovim reakcijama ispitano je pet različitih lipaza. Najaktivnije i najselektivnije lipaze za pojedini alkohol testirane su u različitim organskim otapalima. Lipaze su pokazale nisku do srednje visoku enantioselektivnost prema ispitanim supstratima (E = 1 – 44). U preparativnim reakcijama kataliziranim lipazama najviše enantioselektivnosti, pripravljeni su enantiomerno obogaćeni produkti: (R)-1a (ev = 65 – 85 %), (S)-1b (ev = 92 %), (R)-2a (ev = 84 %), (S)-2b (ev = 68 %) te smjesa (S)-3a (ev = 30 %) i (R)-3b (ev = 69 %).Optically pure compunds are valuable precursors in pharmaceutical industry. Active ingridients in many drugs are fluorinated compounds, therefore the new possibilities in synthesis of fluorinated chiral building blocks are especially interesting and valuable. In this thesis three racemic derivatives of 1-fluoropropan-2-ol were synthesized, substituated at position 3 with azide (1a), cyanide (2a) and chloride (3a) and used as substrates in lipasecatalyzed kinetic resolution. Corresponding racemic acetates were prepared (1b-3b) as standards. Five different lipases were tested. Lipase with highest activity and selectivity with each alcohol was tested in different organic solvents. Lipases have shown low to high enantioselectivity toward supstrates (E = 1 – 44). In preparative-scale reactions catalysed by lipases with highest enantioselectivity, enantiomerically enriched products were prepared: (R)-1a (ee = 65 – 85 %), (S)-1b (ee = 92 %), (R)-2a (ee = 84 %), (S)-2b (ee = 68 %), and mixture of (S)-3a (ee = 30 %) and (R)-3b (ee = 69 %)

Iterativno traženje motiva i vreća fraza u genomu i proteomu

Cilj ovog diplomskog rada bio je testirati algoritam iterativnog pretraživanja te analizirati rezultate ovisno o parametrima. Također, važno je bilo usporediti te rezultate s rezultatima sličnog algoritma na analognom problemu. Za analizu smo odabrali genom i proteom krumpira te proteinsku familiju GDSL lipaza krumpira. Na tim nizovima genoma proveli smo postupak traženja specifičnog motiva proteinske familije GDSL lipaza krumpira kojeg smo prethodno preveli u alfabet genoma. Cilj je bio identificirati sve one nizove na genomu koji kodiraju proteinsku familiju GDSL lipaza. Analizirali smo naše rezultate te ih usporedili s rezultatima na proteomu. Zaključili smo kako je ponašanje na našem primjeru poprilično loše. Međutim, postoje indicije kako algoritam sam po sebi nije problem. Problem vjerojatno leži u nedostatku konzistentne anotacije na biološkim nizovima, različitim biološkim procesima zbog kojih naše aproksimacije unose prevelik kaos te u većoj varijabilnosti na genomu naspram proteoma.This thesis is concerned with an iterative search algorithm and the analysis of its results with respect to various choices of parameters. Furthermore, it was important to compare these results with results of a similar algorithm on an analogous problem. For the analysis, we chose the potato genome and proteome and the GDSL lipase protein family. We applied the iterative search algorithm on genome sequences in order to find the specific motif of the GDSL lipase protein family which was translated to the alphabet of the genome earlier in the thesis. The goal was to identify all those genome sequences which code the potato’s GDSL lipase protein family. Finally, we analyzed our results in comparison to those obtained by proteome analysis. We concluded that the results were rather poor. However, there are indications that the algorithm itself is not as problematic as the results would imply. It is likely that the problem lies in the lack of consistent annotation of biological sequences, different biological processes due to which our approximations create far too much chaos, and in the greater variability of the genome when compared to proteom

Cloning and expression of SGNH-hydrolase of Streptomyces coelicolor in heterologous host

Bakterijske SGNH-hidrolaze su značajna grupa enzima koja još uvijek nije dobro istražena, ni strukturalno ni biokemijski. Izvanstanična lipaza iz obitelji SGNH-hidrolaza iz bakterije Streptomyces coelicolor soja A3(2) (Q93J06, SCO7513) je djelomično biokemijski okarakterizirana. Cilj ovog istraživanja bio je izrada konstrukta gena s dodatkom kodona za 6 histidina na 3' kraju gena te kloniranje i ekspresija gena u bakteriji Streptomyces lividans soja TK23 sa svrhom uspostavljanja učinkovite i brze metode za pročišćavanje proteina. Nakon ekspresije rekombinantnog gena u heterolognom sustavu protein je pročišćen taloženjem s amonijevim sulfatom i afinitetnom kromatografijom na nosaču s imobiliziranim metalom. Prisustvo pročišćenog proteina je dokazano Western analizom, a uočeno je da tijekom pročišćavanja dolazi do gubitka specifične aktivnosti. Iako je prema literaturi ovaj protein označen kao izvanstanični, analiza softverom SignalP 3.0 za predviđanje signalnog slijeda proteina nije prepoznala signalni slijed u SGNH-hidrolazi, što upućuje na mogućnost zadržavanja proteina unutar stanica.Bacterial SGNH-hydrolases are an important group of enzymes which are still not well characterised structurally or biochemically. Extracellular SGNH-hydrolase from Streptomyces coelicolor A3(2) (Q93J06, SCO7513) was earlier partially biochemically described. Aim of this investigation was to construct a gene with an additional codons for 6 histidines on 3' end of the gene. Cloning and expression of the gene Streptomyces lividans TK23 was performed with a purpose of establishing an efficient and quick protein purification method. After the expression of the recombinant gene in heterologous system protein was purified applying ammonium sulfate precipitation and immobilized-metal affinity chromatography. Presence of the purified protein was confirmed with Western analysis. The protein lost specific activity during the purification. Although according to the literature this protein was annotated as extracellular, software SignalP 3.0 failed to recognize a signal sequence in SGNH-hydrolase which implicates a possibility that the protein retains inside the cell

Aktivnost nekih enzima lipidnog metabolizma u sjemenkama jele (Abies alba Mill.) tijekom klijanja

The activity of some enzymes of lipid metabolism in silver fir seeds (Abies alba Mill.) varied in different ways in the embryo and the endosperm during germination. The activity was followed in the seeds from the Mašun region, which did not germinate at room temperature, and in the well germinated seeds from the Papuk region. Lipase activity was measured at pH 8.5 (alkaline lipase) and at pH 5.5 (acid lipase). Mature seeds of both proveniences showed acid and alkaline lipase activity. As the activity of alkaline lipase in the endosperm did not change in any appreciable way before and during visible germination, we concluded that the alkaline lipase activity is not a limiting factor in germination. The activity of acid lipase and catalase increased somewhat after the penetration of the radicle in the well germinated seeds, while the activity of isocitrate lyase increased greatly. On the other hand, the activity of all enzymes assayed showed a constant decrease in the endosperm of the seeds from the Mašun region. Acid and alkaline lipase and catalase were active before visible germination. Their activities were almost undetectable after radicle emergence while the activity of the nongerminated seeds from the Mašun region stayed constant (acid lipase, catalase) or showed a slow decrease (alkaline lipase). The embryo showed no isocitrate lyase activity. The presence of microbodies was also examined in the electron microscope. Glyoxysomes were found alredy in the endosperm of mature seeds. Their number increased with germination. Microbodies were also present in the embryo before and during visible germination. It is suggested that the organelles under consideration do not function like glyoxysomes as there was no isocitrate lyase present in embryos. We conclude that in the embryo the lipids are not metabolised via the glyoxylate cycle as is true for the endosperm.Aktivnost nekih enzima, koji u sjemenkama jele (Abies alba Mili.) kataliziraju razgradnju masti, tijekom klijanja u embriju i u endospermu različito varira. Mjerenja smo izvršili na sjemenkama iz Mašuna (Notranjsko, Slovenija), koje kod sobne temperature nisu klijale, i na lako klijavim sjemenkama s Papuka. Aktivnost lipaze mjerili smo kod pH 8,5 (bazična lipaza) i kod pH 5,5 (kisela lipaza). Kisela i bazična lipaza bile su aktivne već u suhim sjemenkama obiju provenijencija. Aktivnost bazične lipaze se u endospermu prije i tijekom vidljivog klijanja nije bitno promijenila. Iz toga smo zaključile da bazična lipaza nije ograničavajući faktor klijanja. Pri prodoru korjenčića kroz testu je međutim u lako klijavim sjemenkama aktivnost kisele lipaze i katalaze prilično porasla, pogotovu pak aktivnost izocitrat-lijaze. U endospermu sjemenki iz Mašuna, koje pri sobnoj temperaturi nisu klijale, aktivnost je svih spomenutih enzima konstantno opadala. Kisela i bazična lipaza te kataliza bile su aktivne u embriju i prije vidljivog klijanja. Nakon prodora korjenčića aktivnost je tih enzima naglo pala, dok je kod sjemenki iz Mašuna ostala konstantna (kisela lipaza, katalaza), ili pak samo sporo padala (bazična lipaza). Izocitrat-lijaza u embriju nije bila aktivna. Prisutnost nespecijaliziranih peroksisoma i glioksisoma utvrđivale smo elektronskim mikroskopom. Glioksisomi su bili prisutni već u endospermu suhih sjemenki. Njihov se broj povećao u sjemenkama koje su klijale. Prije i tijekom vidljivog klijanja ušle smo u trag mikrotjelešcima i u embriju. Budući da ondje izocitrat-lijaza nije bila aktivna, zaključile smo da mikrotjelešca u embriju ne djeluju kao glioksisomi. Prema svemu sudeći, razgradnja masti u embriju ne odvija se preko glioksilatnog ciklusa, kao što je to slučaj pri razgradnji masti u endospermu

Izolovanje izoformi lipaze iz Candida rugosa

The yeast Candida rugosa is a convenient source of lipases for science and industry. Crude preparation of Candida rugosa lipase (CRL) consists of several extracellular lipases. Isoenzyme profile depends on the culture or fermentation conditions. All isoforms are coded by the lip pseudogene family; they are monomers of 534 amino acids and molecular weight of about 60 kDa. They share the same catalytic mechanism and interfacial mode of activation. Isoenzymes differ in isoelectric points, post-translational modifications, substrate specificity and hydrophobicity. The presence of different lipase isoforms and other substances (i.e., inhibitors) in crude preparation leads to lack of their productivity in biocatalytic reactions. Purification of specific isoform improves its overall performance and stability. This paper provides an overview of different methods for purification of CRL isoenzymes up to date, their advantages and disadvantages.Lipaze (hidrolaze estara glicerola, E.C.3.1.3.3) su važna grupa enzima, široko rasprostranjenih u prirodi. Mogu se izolovati iz materijala biljnog, životinjskog ili mikrobnog porekla. Zahvaljujući svojim karakteristikama, pobuđuju sve više pažnje kao efikasni biokatalizatori u različitim sintetičkim i hidrolitičkim procesima. Među lipazama, poreklom iz mikroorganizama, posebno su značajne one koje produkuje kvasac Candida rugosa. Komercijalni preparat lipaza iz C. rugosa može sadržati 5-7 izoformi ekstracelularnih lipaza. Sve te izoforme kodirane su od strane lip familije pseudogena, a na njihovu ekspresiju utiču uslovi u kojima se mikroorganizam gaji (sastav hranljive podloge je najvažniji). Ekstracelularne lipaze, koje proizvodi C. rugosa su monomerni glikoproteini, molekulske mase od oko 60 kDa, sa 534 aminokiseline. Za sve izoforme je karakterističan isti složeni mehanizam aktivacije na granici faza i mehanizam katalize, kakav se sreće i kod serin-proteaza. Izoenzimi se međusobno razlikuju po post-translacionim modifikacijama (udelu ugljohidratne komponente), supstratnoj specifičnosti, izoelektričnim tačkama i hidrofobnosti. Prisustvo više izoformi lipaza u komercijalnom preparatu utiče na njihovu produktivnost u reakcijama koje katalizuju. Takvi preparati često sadrže i druge supstance koje mogu uticati na aktivnost enzima (na primer inhibitore). Razdvajanjem pojedinačnih izoformi iz komercijalnog preparata poboljšavaju se njihova enantioselektivnost, specifična aktivnost i stabilnost enzima, što je od izuzetnog značaja za njihovu dalju primenu. U ovom radu su predstavljeni različiti pristupi u razdvajanju pojedinačnih izoformi vanćelijskih lipaza iz komercijalnog preparata C. rugosa, njihove prednosti i nedostaci

Lipase from Pseudomonas fluorescens and Burkholderia cepacia as Biocatalysts in Biodiesel Synthesis

U današnjoj industrijskoj proizvodnji biodizela kao katalizator koristi se natrijev hidroksid, no ovaj način proizvodnje ima brojne nedostatke. Stoga se sve više istražuju alternativni oblici proizvodnje biodizela, među kojima se ističe upotreba biokatalizatora ‐ lipaza. U usporedbi s kemijski kataliziranom, reakcija katalizirana lipazama je jednostavnija i ekološki prihvatljivija. U ovom radu provedena je biotehnološka karakterizacija Amano lipaza iz bakterija Burkholderia cepacia i Pseudomonas fluorescens primjenom dva različita testa, spektrofotometrijskog uz p‐nitrofenil palmitat kao supstrat i titrimetrijskog testa uz maslinovo ulje kao susptrat te je procijenjeno kako se primjena eksperimentalno određenog optimalnog pH i optimalne temperature odražava na učinkovitost biokatalitičke sinteze biodizela u kotlastom bioreaktoru uz svježe suncokretovo ulje i metanol kao supstrate u reakcijama transesterifikacije navedenim lipazama. Potom je udio metilnih estera masnih kiselina u sirovom biodizelu određen na plinskom kromatografu te je na temelju dobivenih rezultata procijenjeno da lipaza iz Burkholderia cepacia pokazuje veću efikasnost u proizvodnji biodizela.In today's industrial production of biodiesel, sodium hydroxide is being used as a catalyst, but this production method has numerous disadvantages. Therefore, alternative forms of biodiesel production are being explored, among which the use of lipase as biocatalysts is emphasized. Compared to chemically catalyzed, the lipase catalyzed reaction is simpler and more environmentally friendly. In this master thesis, biotechnological characterization of Amano lipases from Burkholderia cepacia and Pseudomonas fluorescens bacterias was conducted, by using two different tests, spectrophotometric with p‐nitrophenyl palmitate as a substrate and a titrimetry test with olive oil as a substrate, and it was estimated that using experimentally specified optimal pH and optimal temperature reflects on efficiency of biocatalitic sinthesys of biodiesel in batch bioreactor with fresh sunflower oil and methanol as supstrates in transesterification reaction with listed lipases. Afterwards, the proportion of fatty acid methyl esters in crude biodiesel was determined on a gas chromatograph and, based on the obtained results, it was estimated that lipase from Burkholderia cepacia showed a higher efficiency in biodiesel production

The Most Common Pancreatic Diseases

Akutni i kronični pankreatitis te karcinom gušterače tri su najčešće bolesti gušterače. U ovome preglednom članku prikazani su najvažniji epidemiološki, patofi ziološki i etiopatogenetski, ali i klinički, dijagnostički i terapijski elementi navedenih bolesti. U posljednjih desetak godina u pankreatologiji je učinjen velik napredak, a neka od novih otkrića omogućila su da se pristup bolesniku s bolešću gušterače promijeni u svakodnevnoj kliničkoj praksi. Cilj je ovog članka ponuditi ponajprije liječnicima opće medicine informacije koje se odnose na suvremen pristup bolestima gušterače.Acute pancreatitis, chronic pancreatitis and pancreatic cancer are three most frequent pancreatic diseases. This overview provides the most important epidemiologic, pathophysiologic and etiopathogenetic, as well as clinical, diagnostic and therapeutic elements of these diseases. Important advances have been made in pancreatology in the last decade, and some of them have changed the approach to the patient with pancreatic disease in clinical practice. The aim of this paper is to provide practicing doctors with information regarding the current diagnostic and therapeutic approach to pancreatic diseases

Transgenic animals for production of therapeutic porteins

Proizvodnja terapeutskih proteina pomoću transgeničnih životinja započela je još 80-ih godina prošlog stoljeća, a vrlo je popularna i korištena tehnika i dan danas. Od transgeničnih životinja u tu svrhu najčešće se koriste miševi, koze i kokoši. Životinje se drže na izoliranim farmama ili u laboratoriju kako bi odredeni faktori poput bolesti i infekcija bili visoko kontrolirani. Postupak dobivanja transgeničnih životinja izrazito je delikatan i osjetljiv proces umetanja željenog transgena u genom životinje, kontroliranih križanja u svrhu dobivanja homozigotnih transgeničnih jedinki kao i process pročišćavanja dobivenog proteina. Popularna je proizvodnja proteina (antitijela i enzima) u mlijeku tj. bjelanjku jajeta zbog dobivanja velikih količina proteina, te modulacije posttranslacijskih modifikacija. Pročišćavanje proteina se kroz više koraka filtracija i afinitetne kromatografije, praćenih imunološkim mikrobiološkim i virusnim analizama. Konačna svrha proizvedenog terapeutskog proteina je primjena u medicinskog praksi. Stoga svaki rekombinantni protein mora proći klinička ispitivanja i evaluaciju. Danas je proizvodnja terapeutskih proteina i dalje vrlo skup proces, što predstavlja prepreku u bržem napredovanju kliničkih i ranijih faza razvoja proteina. Lijekove koji su na koncu uspjeli doći na tržište (ATryn, Kanuma) možemo smatrati pionirima koji će stvoriti utaban put za nova istraživanja i poboljšanja u polju proizvodnje terapeutskih proteina pomoću transgeničnih životinja.The production of therapeutic proteins in transgenic animals started in the 1980s and it is a very popular and widely used technique until today. Mouse, goats and chickens are mostly used as transgenic animals. These animals are kept in isolated farms or laboratories so that disease and infection could be controlled and minimised. The procedure of getting transgenic animals is a very delicate and sensitive process of implanting a desired transgene in the animal's genom, as well as the process of purification of that produced protein. A popular method of protein production (mostly antibodies and enzymes) is by directing it syintheis in the milk or the albumen (egg-white) of the egg of the transgenic animal. In that way large amounts of recombinant protein can be produced including the posttranslational modifications. The purification of the protein is done by many filtration steps and affinity chromatography paralleled with immunologic, microbiological and virus analysis. The final purpose of the produced therapeutic proteins is the use in the clinic. To achieve that, the protein has to pass clinical testing and evaluation. Even today, the production of a protein is an expensive process, which still presents an obstacle for faster development of new candidate proteins. Medication that have reach the market so far (e.g. Atryn, Kanuma) are considered pioneers which will pave the patht for new research and improvement of the field of therapeutic protein production in transgenic animal