Pridobivanje ksilanaze iz družine GH10 iz metagenoma komposta in ugotavljanje njenih lastnosti

Uporaba fosilnih goriv je zaradi sproščanja toplogrednih plinov vse večji okoljevarstveni problem, zato je pomembno iskanje novih, trajnostnih in okolju prijaznejših virov energije. Biogoriva druge generacije imajo velik potencial za razvoj trajnostne ekonomije. Pridobivajo jih z razgradnjo in fermentacijo lignocelulozne biomase, ki je najbolj razširjen in cenovno ugoden obnovljiv substrat. Za razvoj proizvodnje biogoriv je ključno iskanje novih encimov za razgradnjo biomase, saj ti zaenkrat zaradi visoke cene, premajhne učinkovitosti in slabe stabilnosti v procesnih razmerah še ne dosegajo povpraševanja. V magistrski nalogi smo v metagenomski DNA komposta identificirali ksilanazo iz družine GH10 z dvema vezavnima domenama za ogljikove hidrate (CBM). Tarčni gen (xylM_3) smo prenesli v ekspresijski sev in analizirali izražanje produkta pri različnih pogojih. Ugotovili smo, da se produkt tarčnega gena optimalno izraža ob dodatku 0,75 mM IPTG, največ aktivnega encima pa dobimo po 20-urni inkubaciji pri 18 °C. Tarčni encim smo poskušali očistiti z afinitetno kromatografijo (Ni-NTA) v nativnih in denaturirajočih pogojih, vendar izoliranega nismo uspeli pridobiti v aktivni obliki. Kljub vsemu smo z analizo encimskih frakcij ugotovili, da je encimski ekstrakt aktiven v temperaturnem območju med 20 in 60 °C in pri pH 5-8, najvišjo aktivnost pa dosega pri temperaturi 50 °C in pH = 6. Pokazali smo tudi, da encimski ekstrakt lahko razgrajuje bukov ksilan in arabinoksilan. Aktivnost encimskega ekstrakta na ksilanu se je povečala ob dodatku 1, 5 in 10 mM K+ in 1 mM Co2+ ter 0,2 mM detergenta Triton-X 100. Ob dodatku ostalih testiranih ionov in kemikalij nismo ugotovili sprememb v encimski aktivnosti ali pa se je ta znižala. The use of fossil fuels is an increasing environmental problem due to the release of greenhouse gases. It has therefore become increasingly important to find new sustainable and more environmentaly friendly energy resources. Second-generation biofuels have great potential for development of bioeconomy as they are obtained by decomposition and fermentation of lignocellulosic biomass, the most widespread and affordable renewable substrate. In recent years a lot of effort has been put into finding novel enzymes, which are the key for the development of cost-effective biofuels production. In the following work, the product of a gene encoding putative xylanase (xylM_3), obtained from metagenomic library of compost microbial community was heterologically expressed and biochemicaly characterized. Bioinformatic analysis of the target gene revealed an enzyme with a single catalytic site belonging to glycoside hydrolase family 10 (GH10) and two carbohydrate-binding modules (CBM 2 and CBM 60). Enzyme was heterologically expressed in E. coli BL21 (DE3). The optimum conditions for the enzyme production were determined. Largest amounts of active enzyme were produced at 0.75 mM IPTG, after 20 hours incubation at 18 °C. The enzyme extract was active in the temperature range between 20 and 60 °C and pH 5-8, with the highest activity measured at 50 °C and pH = 6. The activity of the enzyme extract on beechwood xylan and arabinoxylan was confirmed. The addition of 1, 5 and 10 mM K+, 1 mM Co2+ and 0.2 mM Triton-X 100 increased the xylanolytic activity of the enzyme extract. On the other hand, the enzyme extract retaind the original activity in the presence of of 20 % formic acid, while the addition of other tested organic solvents (methanol, ethanol and acetone) reduced its activity.