Compost metagenome as a source of biotechnologically interesting enzymes

Zaradi negativnih vplivov uporabe fosilnih goriv je potrebno raziskati načine pridobivanja obnovljivih virov energije. Sem spadajo biogoriva, pridobljena iz lignocelulozne biomase. Za obdelavo lignocelulozne biomase so potrebni encimi, ki katalizirajo razgradnjo lignoceluloznih polimerov, med katere spadajo glikozid hidrolaze. Nove encime iz okolja lahko med drugim pridobivamo z metagenomskim pristopom. Ta temelji na analizi celotne DNA mikrobne združbe vzorca iz nekega okolja, izbiri ustreznih genov ter izražanju teh genov v heterolognih ekspresijskih sistemih. V magistrski nalogi smo analizirali 5 zapisov, ki domnevno kodirajo encime, vpletene v razgradnjo lignoceluloznih substratov ter jih poskusili heterologno izraziti v različnih sevih bakterije Escherichia coli. Med tarčnimi geni, ki smo jih poskusili heterologno izraziti smo uspešno proizvedli 40 kDa veliko ksilanazo iz družine glikozid hidrolaz 10. Tarčni gen smo vnesli v ekspresijski sev E. coli in sprožili njegovo izražanje. Potrdili smo ksilanolitično aktivnost produkta tarčnega gena, na ksilanu in na odpadnih pivskih tropinah. Ugotovili smo, da je encimska aktivnost tako grobega encimskega pripravka iz bakterijske kulture kot očiščenega tarčnega encima na pivskih tropinah približno dvakrat višja pri 25 °C kot pri 37 °C. Da bi neposredno preverili biotehnološki potencial nove ksilanaze za pretvorbo pivovarskih odpadkov v bioplin, smo izvedli tudi test biometanskega potenciala. Primerjali smo proizvodnjo biometana oz. bioplina iz pivskih tropin predobdelanih z encimom Y006 z neobdelanimi pivskimi tropinami.Due to the negative effects associated to fossil fuel use more research needs to be done on renewable energy sources. Biofuels synthesized from lignocellulosic biomass represent one of the alternatives to fossil fuels. Enzymes that catalyze the degradation of lignocellulosic polymers are crucial for processing of lignocellulosic biomass to bioenergy. Glycoside hydrolases represent the most important group of these enzymes. New enzymes from the environment can be obtained without isolating new bacteria by using the metagenomic approach. This approach is based on the analysis of the whole DNA isolated from an environmental sample, bioinformatic analysis of the genes and/or expression of target genes in a heterologous expression system. In this work we analysed 5 ORFs encoding putative enzymes involved in lignocellulose degradation on compost metagenome, which we expressed in various Escherichia coli strains. Among these putative enzymes, we successfully expressed a 40 kDa xylanase from glycoside hydrolase family 10. In addition, we also tested biotechnological potential of the target enzymes for valorization of brewer\u27s spent grain. The activity of crude as well as isolated enzyme on brewer’s spent grain was approximately two times higher at 25 °C then at 37 °C. Finally, we conducted a biomethane potential test (BMP), and compared methane production from brewer\u27s spent grain that was pre-processed with Y006 and methane production from unprocessed brewer\u27s spent grain