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Insulin was discovered 100 years ago and has been well studied from the perspectives of life and biomedical sciences. This paper reports chemical and biothermodynamic properties of biosynthesis of insulin. This paper reports for the first time the molecular and empirical formulas, biosynthesis reactions, and thermodynamic properties of molecules and their biosynthesis for human preproinsulin, proinsulin, insulin chain A, insulin chain B, insulin, signal peptide and intermediate peptide (C-peptide). Based on these, metabolic reactions were formulated for conversion of preproinsulin to insulin and their thermodynamic feasibility was analyzed.

The main objective of this research is the development of catalytic materials based on a combination of metal-oxides with extremely low-loading of Pt for oxygen reduction reactions (ORR). Materials compared were La2O3 with 5 mass% loading of Pt (La2O3-Pt) and La2O3/MnO2 multioxide based material with 5 mass% loading of Pt (LMO-Pt). The electrochemical characterization of the catalytic materials has been done using cyclic voltametry (CV), linear sweap voltammetry (LSV) and galvanostatic electrochemical impedanse spectroscopy (GEIS). It was proven that both synthesized catalytic materials were ORR-active. The ORR behavior indicated that La2O3-Pt electrode is significantly less active than LMO-Pt. The GEIS results showed that the ORR is complex electrochemical reaction, being electrochemically and diffusion controlled. Diffusion is of mixed character due to transport of O2 molecule and chemical reaction of oxygen reduction reaction. O2 diffusion is the dominant process for LMO-Pt catalytic materials, while chemical reaction is dominant process for La2O3-Pt electrolytic material.

The main objective of this research was a systematic development of advanced micro/nanostructured materials based on the most used metal-oxides for ORR and metal-oxides with an extremely low-loading of Pt for comparison. Hybrid composites compared were: MnO2, La2O3, mixed lanthanum manganese oxides (LMO), and mixed lanthanum manganese oxides with reduced platinum load (LMO-Pt). The influence of the reduced amount of noble metal, as well as single oxide activity toward ORR, was analyzed. The complete electrochemical performance of the hybrid materials has been performed by means of CV, LSV, and EIS. It was shown that all synthesized catalytic materials were ORR-active with noticeable reduction currents in O2 saturated 0.1 M KOH. The ORR behavior indicated that the La2O3 electrode has a different mechanism than the other tested electrode materials (MnO2, LMO, and LMO-Pt). The EIS results have revealed that the ORR reaction is of a mixed character, being electrochemically and diffusion controlled. Even more, diffusion is of mixed character due to transport of O2 molecules and the chemical reaction of oxygen reduction. O2 diffusion was shown to be the dominant process for MnO2, LMO, and LMO-Pt electrolytic materials, while chemical reaction is the dominant process for La2O3 electrolytic materials.

Cellobiose dehydrogenase (CDH) can be used in industry for lactobionic acid production, as a part of biosensors for disaccharides and in wound healing. In fungi it is involved in lignocellulose degradation. CDH gene from Phanerochaete chrysosporium has been cloned in pYES2 plasmid for extracellular expression and protein engineering in yeast Saccharomyces cerevisiae InvSC1 for the first time. A CDH gene library was generated using error-prone PCR and screened by spectrophotometric enzymatic assay based on 2,6-dichloroindophenol reduction detection in microtiter plates. Several mutants with increased activity and specificity towards lactose and cellobiose were found, purified and characterized in detail. Recombinant CDH enzymes showed a broad molecular weight between 120 and 150 KDa due to hyper-glycosylation and the best S137 N mutant showed 2.2 times increased k cat and 1.5 and 2 times increased specificity constant for lactose and cellobiose compared to the wild type enzyme. pH optimum of mutants was not changed while thermostability of selected mutants improved and S137 N mutant retained 30% of it's original activity after 15 min at 70 °C compared to 10% of activity that the wild type enzyme retained. Mutants M65S and S137 N showed also 1.6 and 1.5 times increased productivity of hydrogen peroxide in the presence of 30 mM lactose compared to the wild type.

We investigated the removal of phenol from water solutions (200 mg L-1) using two varieties of lettuce (Lactuca sativa L) and their hairy roots. Experiments were done in a hydroponic system where adult plants were grown in phenol solutions for 10 days. The solution was refreshed every two days in order to maintain the constant concentration of phenol. Hairy roots were also cultivated in a solution containing phenol at concentrations varying from 25 to 125 mg L-1 in order to determine the maximum concentration of phenol that can be removed by hairy roots. Both varieties of lettuce reduced the concentration of phenol below the detection limit after six days at the initial phenol concentration of 200 mg L-1. Transformed roots completely removed phenol at the initial concentrations of 100 mg L-1, but were not able to remove phenol at constant concentration above 25 mg L-1. Lettuce plants and hairy roots are excellent candidates for the process of phenol removal from wastewaters. This plant is good choice for bioremediation of water and represents a potentially efficient and inexpensive system for water purification. The performance of lettuce plants and hairy roots to remove phenol from water solutions under real conditions, depleted nutrients or presence of other compounds should be examined further.

In this study we investigated physiological parameters of stress (enzymatic and non-enzymatic) in lettuce (Lactuca sativa L.) and its hairy roots induced by water solution of phenol. Two varieties of lettuce were examined, Ljubljanska ledenka and Nansen. Plants were grown in water with phenol concentration of 200 mgL(-1), during 10 days. We monitored activity of peroxidases, catalases, polyphenol oxidase and superoxide dismutase, as well as proline and chlorophyll content. We observed a decrease in peroxidases, and increase in activity of catalase, polyphenol oxidase and superoxide dismutase compared to control plants. The concentration of praline was constantly increasing in both lettuce varieties over the course of the experiment. We detected an increase in activity of all monitored enzymes, except polyphenol oxidases, in hairy roots. The hydroponic system provides a useful framework for studying the effect of different harmful substances and its elimination. In such a system, as used in this work for the study of physiological processes in antioxidant protection activated when plant was exposed to phenol, lettuce and its hairy roots can be viewed as tools for water remediation.

The aim of this study was to determine the enzymatic profile of esterases and peroxidases during early stages of somatic embryogenesis of Fritillaria meleagris L. Somatic embryogenesis was induced using the leaf base as explant on a medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D). Zymography showed the presence of different moieties, six isoforms of esterases and peroxidases, during morphogenesis as compared to control explants. One isoform of esterases was detected only during the process of somatic embryogenesis, and one isoform was detected in control explants. Analysis of esterases with 1-naphthyl butyrate proved that esterases, which participate in somatic embryogenesis of F. meleagris, belong to the family of aryl esterases. For the first time it was proved that five isoforms of esterases, which are involved in morphogenesis of F. meleagris, belong to the family of aryl esterases, while two isoforms are carboxyl esterases. One isoform of carboxyl esterases was visible in control explants. This is also the first description of peroxidases during the morphogenetic process, and of the difference between aryl and carboxyl esterases. More isoforms of esterases during morphogenesis as compared to control explants are probably responsible for some early physiological process during somatic embryogenesis of F. meleagris.

We have cloned the gene for carbohydrate oxidase (CHO) from Lactuca sativa in two species of yeasts (Saccharomyces cerevisiae and Pichia pastoris). The synthetic gene for the carbohydrate oxidase (1821 bp) from L. sativa cloned into the vector pUC57 and inserted into plasmids pYES2 and pGAP using Escherichia coli DH5 alpha strain. The P. pastoris strain X-33 and the S. cerevisiae strain InvSC1 were used for extracellular expression of CHO. After transformation of P. pastoris X-33 with CHO-pGAP construct none of the colonies showed CHO activity. Two samples displayed a band which did not exist in the sample with the empty vector similar to the molecular weight of CHO. The S. cerevisiae strain InvSC1 has been also transformed with CHO-pYES constructs. Three colonies grew on the plate with cells transformed with the construct. One of the samples showed a band corresponding to about 110 kDa, but no CHO activity was recorded in this case either. Cloning of the foreign genes and heterologous expression in yeasts is widely used in biotechnology, but sometimes can be very dependent on the gene sequence and strain used. In order to obtain active CHO enzyme the further studies on purification and refolding of expressed protein are necessary., Ugljeni hidrat-oksidaza (CHO) iz zelene salate (Lactuca sativa) je enzim koji je do danas nedovoljno ispitan. Vrlo se malo zna o njegovoj strukturi i funkciji. CHO pripada velikoj familiji ugljenihidrat-oksidaza, koje oksiduju šećere. Svaki od članova ove velike familije dobio je ime po supstratu koji oksiduje. Oksidaze iz ove familije enzima imaju kako sličnu ulogu tako i sličnu strukutru. Sve ili većina ovih enzima su monomeri, čiji se polipeptidni lanac uvija u dva domena. Jedan od domena vezuje flavinski kofaktor, a drugi domen je supstrat vezujući. Većina njih oksidaciju supstrata vrši po takozvanom ping-pong mehanizmu. Sve oksidaze iz karbohidrat-oksidaza familije, pa među njima i enzim koji je predmet ove studije (CHO), danas su našle veliku primenu u industriji. CHO se može primenjivati kako u medicinskoj djagnostici, konkretno u biosenzorima za određivanje glukoze u krvi, u prehrambenoj industriji, poljoprivredi, proizvodnji hleba, deterdženata i u raznim drugim industrijskim oblastima. Problem sa ovim enzimom, kao i sa ostalim članovima ove familije, jeste niska koncentracija u prirodnim izvorima. Zato su danas razvijene različite metode rekombinantne tehnologije, kojima se dobijaju ovi enzimi. U ovom radu opisano je kloniranje gena za CHO iz zelene salate u dve vrste kvasaca (Saccharomyces cerevisiae i Pichia pastoris). Sintetički gen za CHO (1821 bp) iz zelene salate kloniran je u vektor pUC57. Escherichia coli soj DH5α korišćen je za kloniranje gena i održavanje plazmida. P. pastoris soj X-33 i S. cerevisiae soj InvSC1 korišćeni su za ekstracelularnu ekspresiju CHO. Aktivnost CHO određena je ABST esejom, a promena absorbance merena je na 405 nm. Potvrda prisustva enzima rađena je na DNK agaroznoj elektroforezi i SDS-PAGE. Posle transformacije P. pastoris X-33, nijedan od klonova nije pokazivao aktivnost CHO. Posle prve fermentacije, kolonije su testirane na SDS-PAGE. Kako su dva uzorka pokazala trake, koje ne postoje na praznom vektoru, ove trake bi mog

The main goal of this paper is the overview of the scope and dynamics of biomass production as a renewable energy source for substitution of coal in the production of electrical energy in the Kolubara coal basin. In order to successfully realize this goal, it was necessary to develop a dynamic model of the process of coal production, overburden dumping and re-cultivation of dumping sites by biomass planting. The results obtained by simulation of the dynamic model of biomass production in Kolubara mine basin until year 2045 show that 6870 hectares of overburden waste dumps will be re-cultivated by biomass plantations. Biomass production modeling point out the significant benefits of biomass production by planting the willow Salix viminalis cultivated for energy purposes. Under these conditions, a 0.6% participation of biomass at the end of the period of intensive coal production, year 2037, is achieved. With the decrease of coal production to 15 million tons per year, this percentage steeply rises to 1.4% in 2045. This amount of equivalent tons of coal from biomass can be used for coal substitution in the production of electrical energy.

U ovoj doktorskoj disertaciji ispitivana je mogućnost uklanjanja fenola iz rastvora, korišćenjem različitih sorti zelene salate, sa ciljem njihove upotrebe u procesu fitoremedijacije. Ispitivano je 11 različitih sorti zelene salate, čija semena su klijala na podlogama sa različitim koncentracijama fenola (0-400 mgL-1) tokom 20 dana. Pokazano je da dve sorte zelene salate mogu klijati na visokim koncentracijama fenola. Sorta Ljubljanska ledenka (LJL) klija na koncentraciji fenola do 350 mgL-1 i sorta Nansen (N) podnosi koncentraciju fenola do 300 mgL-1. Dalja istraživanja aktivnosti antioksidativnih enzima peroksidaza (POX), katalaza (CAT), kao polifenol oksidaza (PPO) rađena su na klijancima koji su gajeni na inhibitronim koncentracijama fenola (IK50), odnosno onim koncentracijama fenola, specifičnim za svaku sortu, na kojima klija 50% semena. Rezultati su prikazani za četiri sorte zelene salate i pokazano je da je aktivnost POX relativno niska, dok je aktivnost CAT i PPO viša u odnosu na klijance koji su gajeni bez prisustva fenola. Uporedo je praćena i morfologija klijanaca koji su gajeni na IK50 fenola. Praćen je tempo uklanjanja fenola iz otpadne vode i analiziran je tokom gajenja odrasle zelene salate u hidroponičnim uslovima. Odabrane su dve sorte zelene salate (LJL i N) koje su pokazale najveću otpornost na prisustvo fenola, i gajene su na koncentraciji fenola u rastvoru od 200 mgL-1. Ova koncentracija fenola je najviša koncentracija ispod IK50 na kojoj klija 100% semena. Pomenuta koncentracija fenola dodavana je u hidroponični rastvor na početku eksperimenta (početna koncentracija fenola) ili svaka dva dana tokom trajanja eksperimenta (konstantna koncentracija fenola). Praćena je promena koncentracije fenola u rastvoru posle 2, 4, 6, 8 i 10 dana gajenja, kao i aktivnost enzima (POX, CAT, SOD i PPO) u korenu i listovima zelene salate. Obe sorte zelene salate uklanjaju fenol iz rastvora za osam dana, a brzina uklanjanja fenola je veća kod sorte LJL u odnosu na, In this doctoral dissertation, the possibilities of use of various cultivars of lettuce have been investigated for removal of dissolved phenol in the process of phytoremediation. Seeds of 11 different cultivars of lettuce germinated on media with different phenol concentrations (0-400 mgL-1) over 20 days. It has been shown that seeds of two cultivars of lettuce can germinate even on high phenol concentrations: Seeds of Ljubljanska ledenka (LJL) can germinate on 350 mgL-1, while Nansen (N) tolerates as high as 300 mgL-1. Further investigation of antioxidant enzyme proxidases (POX), cataleses (CAT) and polyphenol oxidase (PPO) activity was carried out on seedlings grown on inhibitory concentrations of phenol (IC50), that is, the cultivar-specific concentration of phenol reducing the germination efficiency to 50% for the seeds of a given cultivar. Results are shown for four lettuce cultivars, revealing that POX activity is relatively low, while CAT and PPO activity show higher values compared to seedlings grown without phenol. Morphological features of seedlings grown on IC50 of phenol were also recorded. Dynamics of phenol removal from waste water were recorded in adult lettuce plants grown hydroponically. Two lettuce cultivars that showed the highest tolerance to phenol, were chosen for hydroponic growth at 200 mgL-1 phenol, which is the highest concentration on which 100% germination has been recorded for these cultivars. This concentration of phenol was either established in the hydroponic solution once, at the beginning of the experiment (this was called the "starting phenol concentration") or maintained by supplying additional phenol to the solution every two days (which was called the "constant phenol concentration"). Changes in phenol concentration in the solution, as well as enzyme activity (POX, CAT, SOD and PPO) in lettuce roots and leaves were recorded after 2, 4, 6, 8 and 10 days of growth. Both lettuce cultivars are able to completely remove the starting

Morphogenesis in vitro is a complex and still poorly defined process. We investigated esterase and peroxidase isoforms detected in bulb scale, during Fritillaria meleagris morphogenesis. Bulbs were grown either at 4 degrees C or on a medium with an increased concentration of sucrose (4.5%) for 30 days. After these pre-treatments, the bulb scales were further grown on nutrient media that contained different concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) or thidiazuron (TDZ). Regeneration of somatic embryos and bulblets occurred at the same explant. The highest numbers of somatic embryos and bulblets were regenerated on the medium containing 2,4-D and KIN (1 mg/L each), while morphogenesis was most successful at a TDZ concentration between 0.5 and 1 mg/L. Monitoring of esterases and peroxidases was performed by growing bulb scales on a medium enriched with 2,4-D and KIN or TDZ (1 mg/L), and the number and activity of isoforms were followed every 7 days for 4 weeks. In control explants, six isoforms of esterase were observed. Three isoforms of peroxidase were not detected in the control bulb scale, which has not begun its morphogenesis process. 2015 Academie des sciences. (C) Published by Elsevier Masson SAS. All rights reserved.

The essential oil from aerial parts of Tanacetum larvatum (gris.) Kanitz, an endemic perennial herb collected at three various natural habitats in Montenegro (Planinica [Sample a ], Visitor [Sample b ] and Sinjajevina [Sample c ]), were analyzed using GC and GC/MS. Sixty-four components were identified, representing 83.1%, 96.6% and 89.4% of the total oils content in the Samples a, b and c , respectively. The major constituent in Samples a and b , was oxygenated monoterpene, trans-sabinyl acetate (38.1% and 55.8% respectively). Monoterpene hydrocarbons, β-pinene (13.5%) and santolinatriene (30.6%), were found to be the dominant components in Sample c . The toxic trans-sabinyl acetate was present only in traces in this sample. trans-Chrysanthenyl acetate, as one of major components in feverfew essential oil, has not been previously identified in the investigated essential oils.