PUB - Publikationen an der Universität Bielefeld

During the last decade, the shifting from petroleum based fuel to a greener bio based one was expedited by the increasing concern of global warming. The growing demand oriented firstly to the use of food crops as raw material for producing so called first-generation biofuel. And then converted to the second-generation biofuel, using non-food biomass resources for instance, lignocellulosic raw materials. Although these generations of biofuel offered CO2 emission benefits and improved domestic energy security, they also caused several environmental impacts. Such as high requirement for pesticide and fertilizer and conversion of agricultural land for food to energy crops. Consequently, a new raw material, algae, which store high amounts of energy in form of carbohydrates contributed to eliminate these drawbacks. The algal biomass based fuel production was known as the third-generation biofuel.
In general, algal storage polysaccharides are composed of glucose subunits. Paramylon from Euglena gracilis, a linear β-1,3-glucan with very high level of polymerization contributes up to 85% of cell dry weight. This glucan is supposed to be a superb potential raw material for sustainable production of bioethanol. The production of bioethanol from algal polysaccharides requires efficient hydrolysis in order to generate fermentable monosaccharides. The hydrolysis is generally carried out by chemical or enzymatic methods. The chemical hydrolysis efficiently yields high concentrations of fermentable sugars, however requires environment harmful chemicals and generates byproducts inhibiting fermentation process. While the enzymatic hydrolysis processes under mild conditions without accumulating inhibitory byproducts.
Hence, searching for enzymes which could convert paramylon to glucose became the fundamental purpose of this work. As the first part, E.gracilis intracellular proteins were extracted for investigating the hydrolyzing ability on paramylon. The cell free extract prepared from late stationary phase rapidly hydrolyzes alkali treated paramylon. After partial purification, one protein potentially belonging to glycoside hydrolase family 22 was found, and followed with trail of obtaining the gene sequence from E.gracilis cDNA pool by degenerate PCR.
In the second part of this work, recombinant enzymes of four endo-β-1,3-glucanases (TrGH16, TrGH55, TrGH64, TrGH81), one exo-β-1,3-glucanase (TrGH17) from Trichoderma reesei and one exo-β-1,3-glucanase (PpGH5) from Pichia pastoris were successfully expressed in P.pastoris GS115. Moreover, their activities towards alkali treated paramylon were confirmed by measuring hydrolysis products of reducing groups and glucose, respectively. In these two parts, alkali was involved for pretreating paramylon, which was not fully compatible with the purpose of direct hydrolysis of paramylon granule by enzymes without chemical pretreatment. However, no enzymatic method to deconstruct the highly crystallized paramylon granule has been reported by now. The newly discovered lytic polysaccharide monooxygenases (LPMOs), which could break glycosidic linkages of recalcitrant polysaccharides by oxidation and introduce new chain ends for hydrolytic enzymes, might become the key role to overcome the barrier. In the last part of this work, two LPMOs, TrAA9 from T.reesei and AoAA11 from Aspergillus oryzae were expressed in P.pastoris. The synergistic action assay between LPMO and glucanase revealed the possibility that TrAA9 from T.reesei enhanced oligosaccharide accumulation in enzymatic hydrolysate. The result may open up a new way for depolymerization of recalcitrant paramylon granule in further research.