Suchergebnisse

Commercial utilization of biomass for the production of second generation bioethanol constitutes a sustainable and clean alternative to fossil fuels. This has recently been emphasized by the European Union, which estimated that 56 % of renewable energy generation in 2020 would be derived from biomass. The main constituent of biomass, cellulose, is a simple chemical polymer whose energy-rich component, glucose, can be fermented into ethanol. The breakdown or hydrolysis of cellulose to glucose is facilitated by a broad class of enzymes referred to as Glycoside hydrolases (GH). GH and especially cellobiohydrolases from the GH7 family (Cel7As) hold a crucial role as it constitutes the major part in commercial enzymatic cocktails. Crystalline cellulose is highly resistant toward degradation and even though the presence of enzymes speed the reaction up considerably, the breakdown of the polymer into its fermentable components still constitutes a challenging step in the process making of second generation bioethanol. The major part of this dissertation was dedicated to studying the temperature effect on the kinetics of two native Cel7As; the mesophile two-domain Hypocrea jecorina Cel7A, consisting of a catalytic domain and a carbohydrate binding module, which are connected by a linker region, and the thermophile single domain R. emersonii Cel7A. In addition we studied a truncated version of H.jecorina Cel7A and a chimeric protein composed of the linker and CBM from H.jecorina Cel7A and the R. emersonii enzyme to highlight the role of CBM. We measured the reaction rates of these four enzymes at different substrate loads (crystalline cellulose) and find that the two-domain enzymes are more catalytically efficient at low substrate concentration. Conversely, the single-domain enzymes show a twofold increase in the overall reaction rate compared with the two-domain enzymes at high substrate loads. The reaction temperature also affected the kinetics of the twodomain and single-domain enzymes; at increasing temperatures the interval covering the substrate loads at which two-domain enzymes are more catalytically efficient, grew bigger. This means that the advantage of possessing a CBM (in terms of hydrolytic activity) is much dependent on the substrate load and reaction temperature. Another result which appears from changing the temperature and measuring the disturbed kinetics is that little or no temperature sensitivity of the hydrolytic rates (for both two-domain and single-domain enzymes) was found at low substrate load, while the hydrolytic rate is much accelerated at saturating substrate loads. To get a more detailed description of the kinetic parameters, we applying a steady state model which accounts for the characteristic processive mechanism of Cel7As and measured the degree of processivity. We find that the dissociation rate constant (pkoff) is more accelerated by temperature than the rate constant for the catalytic cycle (pkcat) and the rate constant governing the enzyme substrate association (pkon). The monitored temperature effect on the kinetic constants (pkon, pkcat and pkoff) also II formed the basis for making a free energy diagram for H.jecorina Cel7A hydrolysis of cellulose. The free energy diagram was formed by applying Transition state theory on the kinetic rate constants and by obtaining equilibrium constants for the enzyme-substrate association. The free energy reaction coordinate profile revealed that free activation energy for the enzyme-substrate association is dominated by entropy contributions, while the free energy barrier for dissociation is dominated by enthalpy contributions. This thesis additionally embraces a study of one of the characteristic tunnel forming loops. For these experiments, point mutations were introduced in a loop covering the cellulose strand at the -4 subsite in H.jecorina and R.emersonii. More specifically, two asparagine residues were exchanged with alanine residues in positions 194 and 197 of R.emersonii Cel7A. Replacement of one or both of the asparagine residues led to improved maximum velocity and lowered affinity for crystalline cellulose. Substituting the N197 corresponding asparagine residue in H.jecorina, N200, with 12 different amino acids, additionally led to decreased affinity for cellulose. This site saturation approach in H.jecorina suggests that the role of the asparagine in this position is to facilitate 1) a high degree of processivity and 2) high affinity. In continuation thereof, plots of processivity and pKM for all the N200 variants suggested a possible link between processivity and affinity. Furthermore, the kinetic data for the N200 variants suggest a relationship between the maximum velocity and low affinity.

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Demographie: Alter (klassiert); Geschlecht; Familienstand;
Schulbildung; Beruf; berufliche Position; Berufstätigkeit;
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