The use of purified recombinant enzymes produced in E. coli for the bioconversion of small aromatic compounds is a well-established approach in this branch of the biotech industry. In contrast to chemical conversion, use of these enzymes ensures correct chirality and improves overall quality of the final product. In this work, we have optimized culturing conditions in a small-scale bioreactor allowing production of relatively high yields of soluble and active recombinant alcohol dehydrogenase (ADH) and formate dehydrogenase (FDH) in E. coli. These enzymes are essential for the proposed two-enzyme bioconversion system used for biotransformation of trans-2-hexenal to more stable trans-2-hexanol, an aromatic compound often used in food and cosmetic industry. Although ADH is a tetrameric complex protein, which predominantly aggregates into insoluble inclusion bodies in E. coli cytoplasm, by careful optimization of cultivation conditions a high degree of solubility can be achieved. We were able to produce ADH in the scale of 30 – 50 kU per liter of culture and 500 – 800 U of FDH per liter of culture, respectively. Both enzymes were purified using affinity chromatography utilizing engineered histidine-tag and their activity has been determined by standard procedures.
This work was supported by the Slovak Research and Development Agency grant APVV-0061-11 and is also the result of the „Comenius University in Bratislava Science Park“ (ITMS 26240220086) project implementation supported by the Research and Development Operational Program funded by the ERDF.
trans-2-hexenol
Dobrý deň,
pekná práca. Chcem sa spýtať či trans-2-hexenol je cieľová látka - pri zelených arómach sa váčšinou ide po trans-2-hexenale a cis-3-hexenole.
Aká môže byť fyziologická príčina vyššej rozpustnosti ADH produkovanej pri zníženej hladine kyslíka?
Re: trans-2-hexenol
Dobry den,
dakujem Vam za otazku,trans-2-hexenol je cielom z dovodu nizsej prchavosti oproti aldehydu, nasim primarnym cielom je (bola) konstrukcia a optimalizacia expresnych systemov a purifikacia enzymov. Fyziologicka pricina vyssej rozpustnosti ADH pri znizenej hladine kyslika je pravdepodobne v znizeni specifickej urovne expresie, ktora stale zostava na vysokej urovni ale menej zatazuje bunkovy proteosynteticky aparat. Ide skor o doplnok efektu znizenej teploty. V tomto pripade by sa teoreticky dalo uvazovat aj o pouziti nizkokopiovych plazmidov alebo slabsich promotorov, ale zatial sme sa tomu nevenovali.
trans-2-hexenol
drobné chybičky:
Názov produktu Vám trochu nevyšiel v abstrakte ani v príspevku.
Re: trans-2-hexenol
Dakujem za upozornenie, dame si na to pozor.