Tselluloosi ensümaatilise hüdrolüüsi mehhanismi uurimine madalmolekulaarsete mudelsubstraatide abil

Väitekirja elektrooniline versioon ei sisalda publikatsiooneTselluloos on vees lahustumatu biopolümeer, mis koosneb lineaarsetest ahelatest, kus glükoosi jäägid on omavahel ühendatud glükosiidsete sidemetega. Looduses on tselluloos oluline energia- ja süsinikuallikas mitmetele baketri- ja seeneliikidele. Tselluloosi lagundamiseks kasutavad need organismid komplekti erinevatest hüdrolüütilisest ja oksüdatiivsetest ensüümidest, mida kokkuvõtvalt nimetatakse tsellulolüütiliseks süsteemiks. Kõige olulisemad komponendid tsellulolüütilistes süsteemides on tsellobiohüdrolaasid. Neid ensüüme iseloomustab protsessiivsus, see tähendab, et olles seostunud tselluloosiahelale need ensüümid hüdrolüüsivad glükosiidsidemeid järjest edasi liikudes ilma vahepeal dissotsieerumata. Pehmemädanikseen Trichoderma reesei on üks paremini uuritud tselluloosi lagundav organism. Ehkki Trichoderma reesei tsellulaase on uuritud aastakümneid, ei mõisteta tselluloosi hüdrolüüsi lõplikult tänini. Tsellulaasidele ei ole suudetud üheselt määratleda klassikalises ensümoloogias laialdaselt kasutatavat kineetilist parameetrit katalüütiline konstant kcat. Raskused katalüütilise konstandi määramisel on tingitud ühelt poolt substraadist – tselluloos on vees lahustumatu ning sellest tulenevalt ei jaotu ruumis ühtlaselt. Teiselt poolt on see tingitud ensüümist – tsellulaasid on võimelised seostuma tselluloosiga nii produktiivselt kui ka mitteproduktiivselt. Samuti on teada, et tselluloosi hüdrolüüs tsellobiohüdrolaasidega ei järgi klassikalist Michaelis Menteni kineetikat vaid hüdrolüüsi kiirus langeb ajas järsult. Käesolevas doktoritöös töötati välja meetod ensümaatilise tselluloosi hüdrolüüsi katalüütilise konstandi määramiseks. Meetod põhineb mudelsubstraadi hüdrolüüsil ning võimaldab eristada produktiivselt seostunud ensüümi mitteproduktiivsest. Samuti võimaldab see meetod jälgida produktiivselt seostunud ensüümi osakaalu hüdrolüüsi kulgedes ning uurida näiliste kineetiliste parameetrite muutumist ajas. Püstitatud hüpoteesi kohaselt on tsellobiohüdrolaaside kiiruse langus tingitud ensüümimolekulide „kinni jäämisest“ tselluloosi pinnale. Protsessiivsed tsellobiohüdrolaasid liiguvad mööda tselluloosi ahelat kuni nende teele jääb takistus ning nende liikumine peatub. Kuna tsellobiohüdrolaaside dissotsiatsiooni kiirus on madal jäävad ensüümimolekulid takistuse taha pidama ning mitteproduktiivselt seostunud ensüümi oskaal suureneb. Tsellobiohüdrolaaside aktiivsust saab tõsta, kui soodustada mitteproduktiivselt seostunud ensüümide dissotsiatsiooni. Saadud tulemused annavad eelduse tootmaks paremaid ensüümisegusid tselluloosi tööstuslikuks hüdrolüüsiks.Cellulose is a water-insoluble polysaccharide that consists of linear chains of glucose residues. Cellulose is an important energy and carbon source for many species of fungi and bacteria. These organisms secrete a set of hydrolytic and oxidative enzymes also called cellulolytic system. The major components of these cellulolytic systems are cellobiohydrolases. These enzymes are processive which means that enzyme, once bound productively to the substrate, performs several consecutive catalytic steps on a single polysaccharide chain before it dissociates. The best described cellulolytic system is that of the soft rot fungus Trichoderma reesei. While Trichoderma reesei cellulases have been subject of intensive study for decades, the mechanism of cellulase catalyzed cellulose hydrolysis is still not fully understood. One of the biggest shortcomings is the difficulty to measure the rate constant of cellulases acting on cellulose. Problems arise from the heterogeneous insoluble substrate that renders the reaction non-uniform as well as from modular structure of the enzyme that enables both productive and non-productive binding of the enzyme. Also, it is well known that the rate of enzymatic cellulose hydrolysis drops rapidly in time. The initial burst of activity is followed by a rapid decrease in the hydrolysis rate. This work introduces novel methods for determining the rate constants of cellobiohydrolase catalyzed cellulose hydrolysis. These methods are based on the hydrolysis of small soluble model substrates and enable the determination of productively bound enzyme. The methods were used to investigate the mechanism behind the decrease in activity of cellulases during the cellulose hydrolysis. Our hypothesis is that the decline of cellulose hydrolysis rate is caused by obstacles on the path of the processive movement of the enzyme. According to this model, the newly formed productive enzyme-substrate complex moves along the cellulose chain at a constant rate. Once the complex encounters an obstacle it is stalled. Since the dissociation rate constant is low, the enzyme remains “stuck” on the substrate and the proportion of non-productively bound enzyme increases. The action of cellobiohydrolases could be enhanced by promoting recruitment of the enzyme. The results provide a basis for producing better enzyme cocktails for industrial degradation of cellulose

Prisoner Reentry into Society Viewed Through the Lens of Women

In the 1970s, President Richard Nixon declared a War on Drugs and initiated a series of policies that were intended to combat the use of illegal drugs by implementing severe sentences for drug-related crimes. As a result, the focus of the criminal justice system veered away from rehabilitation efforts. Today, the U.S. criminal justice system has considerably high rates of recidivism. This paper examines the importance of successful reentry into society for formerly incarcerated women, especially those who must resume a caregiver role to their children. However, a relapse into criminal behavior seems imminent due to societal barriers in obtaining housing and employment, both of which are critical for a formerly incarcerated individual to be a productive and law-abiding citizen and not resort to illegal methods of earning an income. The implementation of reentry programs as well as risk and needs assessments are critical tools that can aid in a successful transition and provide the individual with personalized support so that they have a chance of a fruitful life outside of incarceration

Screening for nitrite producing Bacillus licheniformis from various origins : a thesis presented in partial fulfilment of the requirements for the degree of Master of Food Technology, Massey University, New Zealand

Nitrite, which has been considered a chemical danger, can cause infant methemoglobinemia. Bacillus licheniformis is one of the spores forming nitrite-producing bacteria that serves as a potential risk in the dairy industry based on its roles in foodborne illness and dairy spoilage. This study was carried out to assess the nitrite production by 10 strains of B. licheniformis under different environmental conditions. B. licheniformis isolated in this research comes from various sources, including a pasteurisation system, tuber feed, and milk samples obtained from the Massey University Microbiology Lab. This research focused on five different temperatures (25°C, 30°C, 37°C, 55°C and 60°C) approximate processing conditions found in the dairy industry checking the B. licheniformis growth and measuring optical density at 570nm at all five temperatures. This study also examined the production of nitrite in aerobic and anaerobic conditions. All isolated strains were able to convert nitrate into nitrite at all 5 temperatures at both aerobic and anaerobic conditions except some isolates from spring (15, 17) and winter (36, 43, 50,55) convert nitrite into some other nitrogenous compounds under 60°C anaerobic conditions, suggesting that there is a potential variability in metabolic pathways. However, oxygen availability does not affect nitrate reduction. It was observed that optimal growth occurred between 30°C and 55°C. These findings have shown the likely health hazards associated with B. licheniformis, as the organism can convert nitrate into nitrite. The finding suggests a need for further research on the metabolic pathways of these isolates to understand their behaviour and mitigate associated risks

Gaasikaitse

Digiteeritud Euroopa Regionaalarengu Fondi rahastusel, projekti "Eesti teadus- ja õppekirjandus" (2014-2020.12.03.21-0848) raames.https://www.ester.ee/record=b1515812*es

Isiku õigus tervisekaitsele ja selle realiseerimise piirangud Eestis

http://www.ester.ee/record=b5141969*es

Innovatsiooni toetamine väikese ja keskmise suurusega ettevõtetes Ettevõtluse Arendamise Sihtasutuse innovatsiooniosakute toetusmeetme näitel

http://tartu.ester.ee/record=b2611627~S1*es

Bifunctional nanoparticles decorated Ni_1-xMn_xCo₂O₄ ultrathin nanoflakes-like electrodes for supercapacitor and overall water splitting

Synthesizing triple transition metal oxide (TTMO) is an extraordinary strategy to develop electrodes for efficient energy storage and conversion devices, owing to their unique nanostructure with high porosity and specific surface area. The cobalt-based mixed-valence oxides have attracted great attention due to their facile synthesis, low cost, and excellent electrochemical performance. However, less attention is paid to investigating the effect of different substitutions on the physico-chemical properties of TTMO. In this study, nanoparticles (NPs) decorated ultrathin Ni1-xMnxCo2O4 nanoflakes (NPs@NFs) are synthesized by tuning the molar ratio between Mn and Ni via facile deep eutectic solvents (DESs) method. Unique and highly porous NPs@NFs nanostructures aid to increase the overall surface area of the materials, whereas Mn, Ni, and Co ions participate in their redox-active capacity, improving the electrochemical activity of the material. This Ni0.8Mn0.2Co2O4 hybrid nanostructure exhibited excellent supercapacitive performance with a high specific capacity (Cs) of 761 mAh g−1 at a higher current density of 30 mA cm−2 and superior cycling retention of 92.86% after 10 000 cycles. Further, a hybrid asymmetric supercapacitor (Ni0.8Mn0.2Co2O4//AC) device exhibited an extended potential window of 1.5 V, which results in an ultrahigh energy density of 66.2 W kg−1 by sustaining a power density of 1519 Wh kg−1. The electrocatalytic activity of the optimized Ni0.8Mn0.2Co2O4 shows the outstanding performance toward hydrogen evolution reaction (HER) (150 mV/ 161 mV dec−1) and oxygen evolution reaction (OER) (123 mV/47 mV dec−1) with a lower voltage of 1.51 V (@10 mA cm−2) for overall water splitting, with outstanding stability up to 25 hours. These results indicate that chemically synthesized ultrathin NPs@NFs-like nanostructure is a capable electrode for multiple applications, such as supercapacitors, and overall water splitting. Highlights: Facial synthesis of nanoflakes of Ni1-XMnXCo2O4 thin films by a DES method. Fully decorated NPs on the interconnected NFs provided a higher surface area. Nanostructures with a maximum specific capacity of 761 mAh g−1 at 30 mA cm−2, with excellent stability of 92.86%. The solid-state device shows an excellent energy density of 66.2 Wh kg−1 at a power density of 1519 W kg−1. The assembled Ni0.8Mn0.2Co2O4 /Ni-based water splitting exhibited low voltage (1.51 V) and superb stability up to 25 hours.</p

Free energy diagram for the heterogeneous enzymatic hydrolysis of glycosidic bonds in cellulose

Kinetic and thermodynamic data have been analyzed according to transition state theory and a simplified reaction scheme for the enzymatic hydrolysis of insoluble cellulose. For the cellobiohydrolase Cel7A from Hypocrea jecorina (Trichoderma reesei), we were able to measure or collect relevant values for all stable and activated complexes defined by the reaction scheme and hence propose a free energy diagram for the full heterogeneous process. For other Cel7A enzymes, including variants with and without carbohydrate binding module (CBM), we obtained activation parameters for the association and dissociation of the enzyme-substrate complex. The results showed that the kinetics of enzyme-substrate association (i.e. formation of the Michaelis complex) was almost entirely entropy-controlled and that the activation entropy corresponded approximately to the loss of translational and rotational degrees of freedom of the dissolved enzyme. This implied that the transition state occurred early in the path where the enzyme has lost these degrees of freedom but not yet established extensive contact interactions in the binding tunnel. For dissociation, a similar analysis suggested that the transition state was late in the path where most enzyme-substrate contacts were broken. Activation enthalpies revealed that the rate of dissociation was far more temperature-sensitive than the rates of both association and the inner catalytic cycle. Comparisons of one- and two-domain variants showed that the CBM had no influence on the transition state for association but increased the free energy barrier for dissociation. Hence, the CBM appeared to promote the stability of the complex by delaying dissociation rather than accelerating association

Probing substrate interactions in the active tunnel of a catalytically deficient cellobiohydrolase (Cel7)

Cellobiohydrolases break down cellulose sequentially by sliding along the crystal surface with a single cellulose strand threaded through the catalytic tunnel of the enzyme. This so-called processive mechanism relies on a complex pattern of enzyme-substrate interactions, which need to be addressed in molecular descriptions of processivity and its driving forces. Here, we have used titration calorimetry to study interactions of cellooligosaccharides (COS) and a catalytically deficient variant (E212Q) of the enzyme Cel7A from Trichoderma reesei. This enzyme has ∼10 glucopyranose subsites in the catalytic tunnel, and using COS ligands with a degree of polymerization (DP) from 2 to 8, different regions of the tunnel could be probed. For COS ligands with a DP of 2–3 the binding constants were around 10(5) m(−1), and for longer ligands (DP 5–8) this value was ∼10(7) m(−1). Within each of these groups we did not find increased affinity as the ligands got longer and potentially filled more subsites. On the contrary, we found a small but consistent affinity loss as DP rose from 6 to 8, particularly at the higher investigated temperatures. Other thermodynamic functions (ΔH, ΔS, and ΔC(p)) decreased monotonously with both temperature and DP. Combined interpretation of these thermodynamic results and previously published structural data allowed assessment of an affinity profile along the length axis of the active tunnel