Irene Fraga

Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi

Wheat straw was submitted to a pre-treatment by the basidiomycetous fungi Euc-1 and Irpex lacteus, aiming to improve the accessibility of cellulose towards enzymatic hydrolysis via previous selective bio-delignification. This allowed the increase of substrate saccharification nearly four and three times while applying the basidiomycetes Euc-1 and I. lacteus, respectively. The cellulose/lignin ratio increased from 2.7 in the untreated wheat straw to 5.9 and 4.6 after the bio-treatment by the basidiomycetes Euc-1 and I. lacteus, respectively, thus evidencing the highly selective lignin biodegradation. The enzymatic profile of both fungi upon bio-treatment of wheat straw have been assessed including laccase, manganese-dependent peroxidase, lignin peroxidase, carboxymethylcellulase, xylanase, avicelase and feruloyl esterase activities. The difference in efficiency and selectivity of delignification within the two fungi treatments was interpreted in terms of specific lignolytic enzyme profiles and moderate xylanase and cellulolytic activities.

Influence of ligninolytic enzymes on straw saccharification during fungal pretreatment.

Solid state and submerged fermentations in the presence of white-rot basidiomycetes (Bjerkandera adusta, Fomes fomentarius, Ganoderma resinaceum, Irpex lacteus, Phanerochaete chrysosporium, Trametes versicolor and basidiomycete Euc-1) and the litter-decomposing basidiomycete Lepista nuda were evaluated as a pretreatment to increase enzymatic saccharification of wheat straw. Enzymatic hydrolysis of holocellulose after solid state pretreatment showed a significant (P<0.05) increase of saccharification process for T. versicolor, Euc-1, G. resinaceum and I. lacteus, being T. versicolor (strain Tv2) the best one with a sugar yield increase of 91% compared with untreated straw. In submerged medium the pretreatment with I. lacteus, Euc-1 and P. chrysosporium enhanced saccharification but at a lesser extent. Covariance analysis was used to evaluate the relationships between ligninolytic enzymes (lignin peroxidase, manganese-dependent peroxidase and laccase) and saccharification increase. Results showed that only the presence of lignin peroxidase during pretreatment can lead to a significant (P<0.05) increase in the saccharification yield.

Influence of culture medium growth variables on Ganoderma lucidum exopolysaccharides structural features

In this work the effect of carbon and nitrogen levels and initial pH of the wheat extract culture medium of submerged culture of Ganoderma lucidum on the amount, purity and structural features of exopolysaccharides (EPS) were studied. A low peptone level (1.65 g L(-1)) favored mycelium biomass, EPS purity, but a higher supply of peptone (4.80 g L(-1)) is needed for maximum EPS production. The carbohydrate composition of the EPS and structural features also changed significantly according to the different growing conditions, being observed significant differences in the (1 → 3)/(1 → 4)-Glcp ratio and also on the branching degree of EPS. As the biological activities of EPS are highly dependent on the polysaccharide structural features, this variability can have implications on the EPS biological activities, but can also be used advantageously to produce tailor made polysaccharides with specific applications.

Utilization of integrated Michaelis-Menten equations for enzyme inhibition diagnosis and determination of kinetic constants using Solver supplement of Microsoft Office Excel

Enzyme kinetic parameters are usually determined from initial rates nevertheless, laboratory instruments only measure substrate or product concentration versus reaction time (progress curves). To overcome this problem we present a methodology which uses integrated models based on Michaelis-Menten equation. The most severe practical limitation of progress curve analysis occurs when the enzyme shows a loss of activity under the chosen assay conditions. To avoid this problem it is possible to work with the same experimental points utilized for initial rates determination. This methodology is illustrated by the use of integrated kinetic equations with the well-known reaction catalyzed by alkaline phosphatase enzyme. In this work nonlinear regression was performed with the Solver supplement (Microsoft Office Excel). It is easy to work with and track graphically the convergence of SSE (sum of square errors). The diagnosis of enzyme inhibition was performed according to Akaike information criterion.

Simulaaneous ethanol and cellobiose inhibition of cellulose hydrolysis studied with integrated equations assuming constant or variable substrate concentration

The integrated forms of the Michaelis-Menten equation assuming variable substrate (depletion) or constant substrate concentration were used to study the effect of the simultaneous presence of two exoglucanase Cel7A inhibitors (cellobiose and ethanol) on the kinetics of cellulose hydrolysis. The kinetic parameters obtained, assuming constant substrate (Km=21 mM, Kic=0.035 mM; Kicl=1.5×1015mM; kcat=12 h−1) or assuming variable substrate (Km=16 mM, Kic=0.037 mM; Kicl=5.8×1014 mM; kcat=9 h−1), showed a good similarity between these two alternative methodologies and pointed out that bothethanol and cellobiose are competitive inhibitors. Nevertheless, ethanol is a very weak inhibitor, as shown by the large value estimated for the kinetic constant Kicl. In addition, assuming different concentrations of initial accessible substrate present in the reaction, both inhibition and velocity constants are at the same order of magnitude, which is consistent with the obtained values. The possibility of using this kind of methodology to determine kinetic constants in general kinetic studies is discussed, and several integrated equations of different Michaelis-Menten kinetic models are presented. Also examined is the possibility of determining inhibition constants without knowledge of the true accessible substrate concentration.

Enzyme inhibition studies by integrated Michaelis-Menten equation considering simultaneous presence of two inhibitors when one of them is a reaction product

To determine initial velocities of enzyme catalyzed reactions without theoretical errors it is necessary to consider the use of the integrated Michaelis-Menten equation. When the reaction product is an inhibitor, this approach is particularly important. Nevertheless, kinetic studies usually involved the evaluation of other inhibitors beyond the reaction product. The occurrence of these situations emphasizes the importance of extending the integrated Michaelis-Menten equation, assuming the simultaneous presence of more than one inhibitor because reaction product is always present. This methodology is illustrated with the reaction catalyzed by alkaline phosphatase inhibited by phosphate (reaction product, inhibitor 1) and urea (inhibitor 2). The approach is explained in a step by step manner using an Excel spreadsheet (available as a template in Appendix). Curve fitting by nonlinear regression was performed with the Solver add-in (Microsoft Office Excel). Discrimination of the kinetic models was carried out based on Akaike information criterion. This work presents a methodology that can be used to develop an automated process, to discriminate in real time the inhibition type and kinetic constants as data (product vs. time) are achieved by the spectrophotometer.

Dataset on ammonia, nitrous oxide, methane and carbon dioxide fluxes from two soils fertilized amended with treated and non-treated cattle slurry

The current data article presents a set of fluxes of ammonia (NH3), nitrous oxide (N2O), methane (CH4), and carbon dioxide (CO2) measured from two different soils under a Mediterranean double-cropping system (oat in autumn/winter followed by maize in spring/summer). The two soils were fertilized using four different treatments: (i) Injection of raw cattle slurry (100 mm depth), (ii) application of raw cattle slurry followed by soil incorporation (20 mm depth), (iii) band application of acidified (pH=5.5) cattle slurry followed by soil incorporation (20 mm depth), and (iv) band application of acidified (pH=5.5) cattle slurry without soil incorporation. A non-amended soil was also considered as control treatment. The data presented here were obtained over a three years experiment between 2012 and 2015. Fluxes were measured in a period between slurry applications to soil (before plant seeding) till crop harvest. The data presented here are supporting the research article “Band application of acidified slurry as an alternative to slurry injection in a Mediterranean double-cropping system: Agronomic effect and gaseous emissions” (Fangueiro et al., 2018).