| Summary: | For multiple decades enzymes have been essential to industrial sectors such as health, food, agriculture, cosmetics, energy, and others. However, many naturally occurring enzymes cannot stay active under the harsh conditions needed in those processes. On the search for more stable enzymes, scientists started looking at microorganism living under extreme conditions. Chile is home to many almost uninhabitable environments hosting very-well adapted extremophiles. The aim of the project was to produce and purify two proteases (subtilisin and trypsin) and a glycosidase (xylanase) whose sequences all originate from bacteria isolated from the Chilean Antarctica or Atacama Desert. The sequences were inserted into the genome of Pichia pastoris KM71 via homologous recombination utilizing the vector pPIC9K. The vector generates His+ MutS Pichia strains. Though all sequences could be confirmed inside the yeast’s genome, only trypsin was successfully expressed. Proper conditions for the expression xylanase could not be found while there was no time left to work on the expression of subtilisin. An attempt was made to purify trypsin by utilizing the propeptides’ His-tag. However, the zymogen trypsinogen did not bind to the column while active trypsin was found in the flow-through. Since the cleaved propeptide did bind to the HisTrap column it is likely that the tertiary structure of trypsinogen occludes the histidine tag.
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