Optimizing reaction conditions for an LPMO-enzyme from Trichoderma reesei with a downscaled TTC-assay

• Main Author: Karjalainen, M. (Marika)
• Format: Dissertation
• Language: English
• Published: University of Oulu 2017
• Subjects: MDP in Protein Science and Biotechnology
• Online Access: http://urn.fi/URN:NBN:fi:oulu-201711293191; http://nbn-resolving.de/urn:nbn:fi:oulu-201711293191

<div lang="en" class="abs"> Abstract The increasing awareness of the causes and consequences of climate chance has led to actions to reduce the dependency on oil and other finite energy and raw material sources. Plant biomass is used in increasing amounts as a resource for biofuel, biochemical and fiber production. Carbohydrate enzymology has provided new ways to utilize and modify renewable carbon sources, especially the lignocellulolytic systems of fungi. Cellulolytic enzymes work in a synergistic manner on recalcitrant structure of cellulose, hydrolyzing it into soluble oligosaccharides, and eventually, glucose. Lytic polysaccharide monooxygenases (LPMOs) contribute to this system by oxidizing either C1- or C4-carbon from the carbohydrate chain on a crystalline cellulose with the help of copper-core induced radicals, thus creating available substrates for the other cellulolytic enzymes. Since their discovery in 2010, the research on their activities and specificities have increased rapidly, but the analytical methods to investigate this diverse group of enzymes is mostly limited to short and soluble products, which are only a fraction of the oxidation products. In addition, most of the methods require special equipment, wide range of standards and expertise to interpret the results. In this study, HPLC and HPAEC-PAD were tested, unsuccessfully, to quantify soluble products from LPMO-catalysis. A TTC-method, in which 2,3,5-triphenyl-2H-tetrazolium chloride is reduced into red and spectrophotometrically quantifiable formazan by reducing ends from insoluble LPMO-products, was successfully optimized and downscaled, and used to optimize reaction conditions for a type 3 LPMO from Trichoderma reesei, TrAA9A, with Whatman filter paper 1 as a substrate. Experiments were conducted to investigate the effects of pH, temperature, donor, time and the presence/absence of H₂O₂ to the accumulation of reducing ends. The results did not show any substantial differences in the accumulation of aldehydes in different reaction conditions. This study showed that cellulose degrades in the presence of TrAA9A and an electron donor. The greatest effects were observed with longer reaction times and the addition of H₂O₂, both increasing the amount of measured aldehydes in the insoluble products. The highest yield was recorded from the reactions with gallic acid as a donor at pH 6, and in the presence of 0.7 mM H₂O₂. The results from this study could lead to understanding the rate-limiting factors of the LPMOs and further improve the utilization of this enzyme in the degradation of lignocellulosic biomass.